http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, 2014; 34(1): 38–43 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2013.848893
ORIGINAL ARTICLE
Mustafa Yilmaz1, Tuba Gokdogan Edgunlu2, Nigar Yilmaz3, Esin Sakalli Cetin4, Sevim Karakas Celik5, Gu¨lser Karadaban Emir1, and Ays e So¨zen1 1
Department of Neurology, Faculty of Medicine, 2Mugla School of Healthy Sciences, Mugla University, Mugla, Turkey, 3Department of Biochemistry, Department of Medical Biology and Genetic, Faculty of Medicine, Mugla University, Mugla, Turkey, and 5Department of Medical Genetic, Faculty of Medicine, Bulent Ecevit University, Mugla, Turkey
4
Abstract
Keywords
Background: The aim of this study was to analyze the role of the genetic variants of two synaptic vesicle proteins (VAMP2 and Synaptotagmin XI) and two presynaptic plasma membrane proteins (Syntaxin 1A and SNAP-25) in patients with idiopathic generalized epilepsy (IGE). Method: Eighty-five patients with IGE and 93 healthy subjects were included in the study. We analyzed the functional polymorphisms of VAMP2, Synaptotagmin XI, Syntaxin 1A and SNAP-25 genes with polymerase chain reaction and restriction fragment length polymorphism methods. Results: In the patients with IGE, significant differences alleles and genotypes of 26 bp Ins/Del polymorphism of the VAMP2 gene and the 33-bp promoter region of Synaptotagmin XI were observed, however no associaton was found regarding Intron 7 rs1569061 of Syntaxin 1A gene, MnlI rs3746544 and DdeI rs1051312 polymorphisms of SNAP25 gene compared with healthy subjects. Carriers of the C allele of Synaptotagmin XI had worse measures compared with the T allele of Synaptotagmin XI. In the haplotype analysis, the frequency of the T alleles of rs1569061 and of the C alleles of the 33-bp promoter region of Synaptotagmin XI was found to be significantly higher in patients with IGE as compared with the healthy subjects. Conclusion: The genetic variations of VAMP2, Synaptotagmin XI might be indication of the relationship between these genes and IGE.
Idiopathic generalized epilepsy, SNAP-25, Synaptotagmin XI, Syntaxin 1A, VAMP2
Introduction Epilepsy is a common disease characterized by recurrent spontaneous seizures arising from abnormal neuronal electrical over-activity (1). Idiopathic generalized epilepsies (IGEs) are present in nearly 40% of all epilepsies (2). The etiology of IGE is thought to predominantly depending on genetic variance that leads to the defect of synaptic processes (3–7). The variation in genes codifying for receptors in synapse, especially modulating Ca2þ channels, has been associated with IGE (8). Intracellular Ca2þ mediates changes in membrane proteins to promote the transmitter release and ion channel opening; it also enhances binding receptor sites that alter neuronal sensitivity (9). The variety of genetic information changes in excitability can occur as a result of the Ca2þ influx lead to depolarized synaptic site which is triggered by the vesicle-associated membrane protein (VAMP/synaptobrevin), as well as the syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25). These proteins Address for correspondence: Mustafa Yilmaz, Department of Neurology, Faculty of Medicine, Mugla University Medical School, 48000 Mugla, Turkey. Tel: ++90 252 211 4839. Fax: ++90 252 211 1345. E-mail: [email protected]
History Received 5 September 2013 Revised 17 September 2013 Accepted 23 September 2013 Published online 29 October 2013
then bind together in a lock and key fashion to form a tripartite structure. The synaptotagmin gene encodes a protein known as calcium sensors and mediates the calcium-dependent regulation of membrane trafficking in synaptic transmission. Synaptotagmin XI is implicated in the potential role of neuronal plasticity, so that Synaptotagmin XI has a crucial role in the presence of changes in membrane trafficking resulting epileptic seizures. A defect in the synaptic function is thought to underlie epilepsy. It was reported that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a fundamental role in neurological disorders, especially in epilepsy (10). VAMP2/synaptobrevin is involved in the synaptic vesicles of the presynaptic membrane (11). One of the isoforms identified, the VAMP2 protein, is a synaptic vesicle protein of the type that has been implicated in the pathogenesis of the IGE, encodes VAMP2 gene. VAMP2 triggers SNARE complex formation and fusion between synaptic vesicles which is critical for neurotransmission (12). SNAP-25 is regulated by the SNAP-25 gene and is expressed in the cytoplasmic face of a membrane in neuron (13,14). The genes interaction is required for synaptic formation (15). The Syntaxin 1A and SNAP-25 complex is catalyzed through the recognition of Caþ in neurons and
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Genetic variants of synaptic vesicle and presynaptic plasma membrane proteins in idiopathic generalized epilepsy
Genetic variants of synaptic vesicle and presynaptic plasma membrane proteins
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DOI: 10.3109/10799893.2013.848893
neuroendocrine cells. In addition, the free vesicular synaptobrevin might quickly rearrange with the SNAP 25 and syntaxin end of membrane fusion, especially under highfrequency synaptic activity. The well-characterized group of the SNARE complex, including VAMP, Syntaxin 1A and SNAP-25, regulates the processes of docking, priming and fusion between the synaptic vesicle membrane and specific regions of the plasma membrane. The group also initiates the subsequent release of neurotransmitters. Analyzing the role of the genetic variants of two synaptic vesicle proteins (VAMP2 and Synaptotagmin XI) and of two presynaptic plasma membrane proteins (Syntaxin 1A and SNAP-25) in IGE patients is crucial to show the possible effective factors of IGE.
Materials and methods Participants This study was performed in 2012 and included a multidisciplinary project on epilepsy genetics in Mugla Sitki Kocman University. The protocol was approved by the regional ethical committee, and procedures were performed according to the principles of The Helsinki Declaration. A total of 85 patients (39 females/46 males) with IGE and 93 healthy subjects (46 females/47 males), from the neurology outpatient clinic, were enrolled in the study (Table 1). The study fulfilled the following inclusion criteria: Diagnostic Criteria of the Commission on Classification and Terminology of the International League against Epilepsy were used to diagnose probands with IGE (16). There was no evidence of developmental and neurological abnormalities, or of global cognitive impairment on the Mini-Mental State Examination (17); at least one electroencephalography (EEG) examination showed the generalized spike wave; there were neither abnormal nor unusual findings on conventional magnetic resonance (MR) images. Patients who were subject to pregnancy, drug abuse, alcoholism, migraine or other health problems such as a history of severe major psychiatric disorders or moderate to severe intellectual disability were excluded. Clinical information, the EEG finding, as well as data on antiepileptic therapy were recorded on data collection forms. The spectrums of Table 1. Characteristics of patients. Patients Age (year) (mean SD) 30.8 13.6 Gender n (%) Male 46 (54) Female 39 (56) Age of epilepsy onset 18.1 15.9 Disease duration 14.2 9.8 Frequency of seizures n (%) 52 month 32 (37.2) 2 month 53 (62.3) Family of history n (%) 51 (61) AED n (%) 79.1 (93) Monotherapy n (%) 52 (61.1) Polytherapy n (%) 33 (38.8) 2 drugs 24 (28.2) 3 drugs 9 (10) No of AED n (%) 5.9 (7)
Healthy subjects p Values 31.9 7.5
SD, Standart deviation; AED, antiepileptic drug.
0.26 0.33
39
antiepileptic drugs used in patients with IGE were carbamazepine, lamotrigine, valproic acid, clonazepam, topiramate, levetiracetam, phenytoin and oxcarbazepine. The healthy subjects had no history of seizures or other neurological disease, no drug abuse or alcoholism, no pregnancy and no other health problems and did not take any drug with an effect on cortical excitability. The mean of age in patients with IGE was 30.87 13.61 years, and the mean of age in healthy subjects was 31.9 7.56 years. This study was approved by the local ethics committee of Mugla University, and all participants provided written, informed consent. Genotyping After written, informed consent was obtained, venous blood samples were collected into vacutainer plastic tubes containing sodium/potassium ethylene diamine tetra acetic acid. DNA was extracted from Genejet Genomic DNA purification kit (Thermo K0772). We used polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) technique. We determined alleles and the genotypes of polymorphism of VAMP2, synapsin III, SNAP-25 and Syntaxin 1A genes. PCR was performed in a 25 -mL volume with 100 ng DNA, 100 mm dNTPs, 20 pmol of each primer, 1.5 mM MgCl2, 1 PCR buffer with (NH4)2SO4 (Fermentas, Vilnius, Lithuania), 10% dimethyl sulfoxide (DMSO) and 2 U Taq DNA polymerase (Fermentas, Vilnius, Lithuania). Amplification was performed on an automated Thermal Cycler (Techne Flexigene, Cambridge, UK). These genes polymorphisms were determined by fragment separation at 120 V for 40–50 min on a 3.5% agarose gel containing 0.5 mg/ mL ethidium bromide. A 100-bp DNA Ladder (Fermentas Vilnius, Lithuania) was used as a size standard for each gel lane. The gel was visualized under UV light using a gel electrophoresis visualizing system (Vilber Lourmat). PCRRFLP conditions of polymorphisms of VAMP2, Synaptotagmin XI, Syntaxin 1 A and SNAP-25 genes have been shown in Figure 1 and Table 2. Statistical analysis A case-healthy subjects study was performed and allelic and genotypic frequency of the polymorphisms was calculated both in cases and healthy subjects. The Hardy–Weinberg equilibrium was verified using the chi-square ( 2) test and by estimating the expected genotypic frequencies on the basis of the development of the square of the binomial for these polymorphisms. The 2 test was used to compare genotype and allele frequency of the VAMP2, Synaptotagmin XI, Syntaxin 1A, SNAP-25 polymorphisms between patients and healthy subjects. The association between polymorphisms and epilepsy was modeled through binary logistic regression analysis and odds ratio (OR) and 95% confidence interval (CI) were calculated to compare epilepsy risk around genotypes. Also, haplotype analysis was used to determine the effect of the genetic correlation between two polymorphic regions.
Results The demographic characteristics of the patients are given in Table 1. There was no difference between the groups regarding age and gender (p50.05). A family of history of
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J Recept Signal Transduct Res, 2014; 34(1): 38–43
Figure 1. (A) Genotyping analysis of SNAP-25 gene rs1051312 polymorphism; 1 and 5 numbers of subjects have TC genotype, 2 number of subject has TT genotype, and 3 and 4 numbers of subjects have CC genotypes. (B) Genotyping analysis of SNAP-25 gene rs3746544 polymorphism; 2 and 4 numbers of subjects have TT genotypes, 1, 3 and 5 numbers of subjects have TG genotypes and 6 number of subject has GG genotype. (C) Genotyping analysis of Synaptotagmin XI (Syt11) gene 33-bp repeats in promoter region polymorphism; 2 and 4 numbers of subjects have CC genotypes (431 bp (2 repeats)), 3 number of subject has TC genotype (477 bp (2 repeats) and 431 bp (2 repeats)), and 1 number of subject has CC genotype (464 bp (3 repeats) and 431 bp (2 repeats)). (D) Genotyping analysis of Syntaxin 1A gene rs1569061polymorphism; 1 and 5 numbers of subjects have TC genotypes, 2 and 3 numbers of subjects have TT genotypes and 5 number of subject has CC genotype. (E) Genotyping analysis of VAMP2 gene 26 bp Ins/Del polymorphism; 1, 3 and 4 numbers of subjects have Ins/Ins genotypes and 2 number of subject has Ins/Del genotype. Table 2. PCR-RFLP conditions of polymorphisms of VAMP2, Synaptotagmin XI, Syntaxin 1A and SNAP-25 genes.
Polymorphism
Primer
Temperature of Annealing
Restriction Endonuclease
P1 P2
57 C
–
33-bp repeats in promoter region
P3 P4
53 C
HphI
Syntaxin 1A
Intron 7 (rs1569061)
57 C
TaiI
SNAP-25
MnlI (rs3746544)
P5 P6 P7 P8 P9 P10
58 C
MnlI
VAMP2
26 bp Ins/Del polymorphism
Synaptotagmin XI
DdeI (rs1051312)
DdeI
PCR products Ins allele:116 bp Del allele:90 bp T allele: 510 bp (three repeats), 477 bp (2 repeats), 444 bp (1 repeat), C allele: 464 bp (3 repeats), 431 bp (2 repeats), 398 bp (1 repeat) T allele: 312 bp C allele: 186,126 bp T allele: 256, 5 bp G allele: 210, 46, 5 bp T allele: 261 bp C allele: 228, 33-bp
P1: F50 -ACAAAGTGCGCCTTATACGC-30 . P2: R50 -GGGATTTTCCTTGACGACACTC-30 . P3: F50 -TCTACCTATGCTTCTTACCC-30 . P4: R50 -TG TCGTAATCAGAGGCTGTTGCT-30 . P5: F50 -CAATGCTGCTGCTGAACTC-30 . P6: R50 -CGCTGACATTTATGTGACC-30 . P7: F50 -TTCTCCTCC AAATGCTGTCG-30 . P8: R50 -CCACCGAGGAGAGAAAATG-30 . P9: R50 -CCACCGAGGAGAGAAAATG-30 . P10: F50 -TTCTCCTCCAAATGCTG TCG-30 . Significant results of statistical analysis are shown in bold.
epilepsy was found in 61% in patients with IGE. We found significant differences alleles and genotypes of 26-bp Ins/Del polymorphism of VAMP2 gene, 33-bp repeats in promoter region of Synaptotagmin XI (Table 2). Also, we have not found any significant difference between the groups when they were compared according to whether they have rs1569061 of Syntaxin 1A gene, rs3746544 and rs1051312 polymorphisms of SNAP-25 gene. There was a significant association between the CC genotype in Synaptotagmin XI gene and IGE (p50.002; OR 4.93; 95% CI 2.01–12.1). There
was a significant association between the ins/del genotype in VAMP2 gene and IGE (p50.042; OR 0.474; 95% CI 0.23– 0.978). The C allele in Synaptotagmin XI gene was found significant different in patients with IGE compared with healthy subjects (p50.001; OR 2.31; 95% CI 1.5–3.57). We have found that individuals who have Synaptotagmin XI C/C alleles are 4.9 times more at risk of developing IGE (Table 3). Individuals who have C alleles in Synaptotagmin XI gene are 2.31 times more at risk of developing IGE compared with healthy subjects (Table 4). We found increased occurrence of
Genetic variants of synaptic vesicle and presynaptic plasma membrane proteins
DOI: 10.3109/10799893.2013.848893
epilepsy via the haplotype analysis for rs1569061 of Syntaxin 1A gene and 33-bp promoter region of Synaptotagmin XI gene. We determined that CC, TC haplotypes for rs1569061 Syntaxin 1A gene, 33-bp promoter region of Synaptotagmin
XI gene could be risk factors for all types of epilepsy disease (respectively, OR ¼ 2.254 95% CI ¼ 1.229–4.137, OR ¼ 4.794 95% CI ¼ 2.312–9.940) (Table 5). There was no correlation between the frequency of seizures and polymorphisms of the genes.
Table 3. Haplotypes distributations of Syntaxin 1A, Synaptotagmin XI and SNAP-25 genes in controls and IGE (n ¼ number of individuals).
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Haplotype
Healthy controls n (%)
Cases n (%)
Syntaxin 1A/Synaptotagmin XI CT 59 (31.7%) 30 TT 70 (37.6%) 54 CC 41 (22.0%) 47 TC 16 (8.6%) 39 Snap-25 Mnll/Snap-25 Ddel TT 95 (51.1%) 95 GT 46 (24.7%) 27 TC 31 (16.7%) 24 GC 14 (7.5%) 24
OR
95% CI
(17.6%) (31.8%) (27.6%) (22.9%)
1 (reference) 1.517 2.254 4.794
0.862–2.669 1.229–4.137 2.312–9.940
(55.9%) (15.9%) (14.1%) (14.1%)
1 (reference) 0.587 0.774 1.714
0.337–1.021 0.423–1.416 0.836–3.514
Discussion We analyzed the genetic variants of the presynaptic plasma membrane proteins such as SNAP-25 and Syntaxin 1A, two synaptic vesicle proteins such as the VAMP2 and Synaptotagmin XI which contributes to the formation of the fusion complex are necessary for the docking and/or fusion of synaptic vesicles in patients with IGE for the first time to the best of our knowledge. Extensive studies have been performed using several biochemical and immunohistochemical methods, suggesting the existence of SNARE mechanisms virtually in all regions of the central nervous system (CNS) (18–21). SNARE complexes may be used as a biological marker in seizure generation for the reason of roles in synaps. In some studies, the GABRG2 rs211037’s
Significant results of statistical analysis are shown in bold.
Table 4. Alleles of VAMP2, SNAP-25, Syntaxin 1A and Synaptotagmin XI genes (n ¼ number of individuals).
Allele VAMP ins del Snap-25 Mnll T G Snap-25 Ddel T C Syntaxin 1A T C Synaptotagmin XI T C
Healthy controls n (%)
Cases n (%)
146 (78.5%) 40 (21.5%)
135 (79.4%) 35 (20.6%)
0.897
Reference 0.946 (0.568–1.577)
126 (67.7%) 60 (32.3%)
119 (70.0%) 51 (30.0%)
0.731
Reference 0.9 (0.574–1.411)
141 (75.8%) 45 (24.2%)
122 (71.8%) 48 (28.2%)
0.400
Reference 1.233 (0.768–1.979)
86 (46.2%) 100 (53.8%)
93 (54.7%) 77 (45.3%)
0.113
Reference 0.712 (0.469–1.081)
129 (69.4%) 57 (30.6%)
84 (49.4%) 86 (50.6%)
50.001
Reference 2.317 (1.503–3.573)
X2 p Value
OR (95% CI)
Significant results of statistical analysis are shown in bold. Table 5. Genotypes of VAMP2, SNAP-25, Syntaxin 1A and Synaptotagmin XI genes (n ¼ number of individuals). Genotype VAMP2 ins/ins ins/del del/del Snap-25 Mnll T/T T/G G/G Snap-25 Ddel T/T T/C C/C Syntaxin 1A T/T T/C C/C Synaptotagmin XI T/T T/C C/C
41
Healthy controls n (%)
Cases n (%)
X2 p Value
OR (95% CI)
56 (60.2%) 34 (36.6%) 3 (3.2%)
58 (68.2%) 19 (22.4%) 8 (9.4%)
0.042
Reference 0.474 (0.230–0.978) 2.024 (0.4778–0.593)
41 (44.1%) 44 (47.3%) 8 (8.6%)
44 (51.8%) 31 (36.5%) 10 (11.8%)
0.327
Reference 0.725 (0.363–1.447) 1.193 (0.385–3.694)
53 (57.0%) 35 (37.6%) 5 (5.4%)
46 (54.1%) 30 (35.3%) 9 (10.6%)
0.432
Reference 0.926 (0.470–1.825) 2.116 (0.592–7.562)
18 (19.4%) 50 (53.8%) 25 (26.9%)
28 (32.9%) 37 (43.5%) 20 (23.5%)
0.114
Reference 0.531 (0.241–1.169) 0.521 (0.210–1.294)
47 (50.5%) 35 (37.6%) 11 (11.8%)
26 (30.6%) 32 (37.6%) 27 (31.8%)
0.002
Reference 2.043 (0.978–4.268) 4.939 (2.015–12.106)
Significant results of statistical analysis are shown in bold.
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association with IGE and febrile seizures was examined. They showed a significant association of GABRG2 rs211037 with febrile seizures, but any significant association was found in patients with IGE (22–24). In addition, it was reported that genetic background is present in the etiology of IGE and in other variants contributing to IGE, as such, future studies must be performed (24). SNARE complex modulates calcium dynamics in response to depolarization, so that it has an essential role in neurotransmitter release via the formation (15). In an experimental study that involved a pentylenetetrazole (PTZ)-induced epilepsy rat model, the expressional levels of synaptic vesicle protein 2A, SNARE complexes were evaluated. It was reported that PTZ did not affect the hippocampal level of SNARE complexes, and also it was mentioned that the hippocampus is a key structure underlying seizure generation (25,26). In contrast, in another experimental study, SNARE complexes were found increase in amygdala kindling. It was reported that the asymmetric accumulation of SNARE complexes occurs during kindlinginduced epileptogenesis (26). The SNARE gene may have a role in discrete structure expression, and/or it may function as contributing directly to neurological disorders as a consequence. Synaptotagmins comprise a large family of transmembrane proteins. Synaptotagmin XI is found in the brain and in minimally studied protein compared with other isoforms. In addition, other isoforms are expressed in a wide variety of tissue outside of the brain. Synaptotagmin XI is thought to be one of the primary calcium sensors for the exocytosis of synaptic vesicles, which is crucial in Ca2þ-triggered vesicle fusion (27). It was well documented that synaptotagmin regulates membrane trafficking and also has an important role in the synaptic plasticity in epilepsy (28). It was reported that synaptotagmin plays a crucial role in refractory epilepsy (29). In our study, synaptotagmin expression was found to be significantly higher than that in the healthy subjects and non-refractory epilepsy groups. The patients with the IGE group, carriers of the CC genotype in Synaptotagmin XI gene, might have a high risk of carrying IGE than carriers of other genotypes. One of the SNARE complexes, SNAP-25, is essential for the exocytosis that plays a key role in transferring information from photoreceptor cells to the CNS (18). The SNAP-25 protein has been studied in psychiatric diseases (10). The expression of SNAP-25 could arrange the synaptic homeostasis resulting neuronal activity. It may be required to describe the potential mechanisms through which alterations in SNAP-25 may play a direct role in the etiology as well as contribute to the pathology of IGE. In recent studies, it was reported that the deficiency of the SNAP-25 gene contributes to neuropsychiatric and neurological disorders (10,30). In addition, studies including experimental rat models showed that the deficiency of SNAP-25 caused the disturbance of the presynaptic voltage-gated Ca2þ channels and resulted in seizures (31). In this study, SNAP-25 MnlI (res3746544) and Ddel (rs1051312) gene polymorphism were not found to be different in patients with IGE compared to that of healthy subjects. It may be possible that SNAP-25 is not related to IGE etiology. As such, another protein in the rolling modulation of neurotransmitter release and synaptogenesis
J Recept Signal Transduct Res, 2014; 34(1): 38–43
process may act in etiology. Conversely, the transmission of SNAP-25 alleles was determined to increase in patients with adult attention deficit hyperactivity disease. In another study, the SNAP-25 gene demonstrated that the subject was a strong candidate for IGE. Therefore, the association between SNAP-25 gene polymorphisms and IGE is currently unclear. Syntaxin 1A is particularly a key molecule in synaptic exocytosis. It was detected that Syntaxin 1A has a potential role in epileptogenesis (32). Syntaxin 1A acts as a potential molecular mechanism for the alteration of exocytotic process and neural plasticity. It was reported that Syntaxin 1A provides evidence of serving for serve as an intrinsic enhancer molecular mechanism in epileptogenesis. In the study, there were no significant differences in the Intron 7 rs1569061 of Syntaxin 1A gene for patients with IGE compared to that of healthy subjects. The study accomplished an analysis concerning about the genetic variants of VAMP2, Synaptotagmin XI, Syntaxin 1A and SNAP-25 in patients with IGE. We observed mainly significant the differences in alleles and genotypes of 26 bp Ins/Del polymorphism of VAMP2 gene and 33-bp promoter region of Synaptotagmin XI. There was no significant difference in the Intron 7 rs1569061 of Syntaxin 1A gene, MnlI rs3746544 and DdeI rs1051312 polymorphisms of SNAP-25 gene in patients with IGE compared with healthy subjects. Consequently, the differences alleles and genotypes of 26 bp Ins/Del polymorphism of VAMP2 gene and 33-bp promoter region of Synaptotagmin XI could be effective in occurrence in patient with IGE. The genetic study indicates that polymorphisms in VAMP2 gene and Synaptotagmin XI might be associated with IGE. In future studies, the expression of these genes and their effect on IGE should be analyzed.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of this article.
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ORIGINAL ARTICLE
Mustafa Yilmaz1, Tuba Gokdogan Edgunlu2, Nigar Yilmaz3, Esin Sakalli Cetin4, Sevim Karakas Celik5, Gu¨lser Karadaban Emir1, and Ays e So¨zen1 1
Department of Neurology, Faculty of Medicine, 2Mugla School of Healthy Sciences, Mugla University, Mugla, Turkey, 3Department of Biochemistry, Department of Medical Biology and Genetic, Faculty of Medicine, Mugla University, Mugla, Turkey, and 5Department of Medical Genetic, Faculty of Medicine, Bulent Ecevit University, Mugla, Turkey
4
Abstract
Keywords
Background: The aim of this study was to analyze the role of the genetic variants of two synaptic vesicle proteins (VAMP2 and Synaptotagmin XI) and two presynaptic plasma membrane proteins (Syntaxin 1A and SNAP-25) in patients with idiopathic generalized epilepsy (IGE). Method: Eighty-five patients with IGE and 93 healthy subjects were included in the study. We analyzed the functional polymorphisms of VAMP2, Synaptotagmin XI, Syntaxin 1A and SNAP-25 genes with polymerase chain reaction and restriction fragment length polymorphism methods. Results: In the patients with IGE, significant differences alleles and genotypes of 26 bp Ins/Del polymorphism of the VAMP2 gene and the 33-bp promoter region of Synaptotagmin XI were observed, however no associaton was found regarding Intron 7 rs1569061 of Syntaxin 1A gene, MnlI rs3746544 and DdeI rs1051312 polymorphisms of SNAP25 gene compared with healthy subjects. Carriers of the C allele of Synaptotagmin XI had worse measures compared with the T allele of Synaptotagmin XI. In the haplotype analysis, the frequency of the T alleles of rs1569061 and of the C alleles of the 33-bp promoter region of Synaptotagmin XI was found to be significantly higher in patients with IGE as compared with the healthy subjects. Conclusion: The genetic variations of VAMP2, Synaptotagmin XI might be indication of the relationship between these genes and IGE.
Idiopathic generalized epilepsy, SNAP-25, Synaptotagmin XI, Syntaxin 1A, VAMP2
Introduction Epilepsy is a common disease characterized by recurrent spontaneous seizures arising from abnormal neuronal electrical over-activity (1). Idiopathic generalized epilepsies (IGEs) are present in nearly 40% of all epilepsies (2). The etiology of IGE is thought to predominantly depending on genetic variance that leads to the defect of synaptic processes (3–7). The variation in genes codifying for receptors in synapse, especially modulating Ca2þ channels, has been associated with IGE (8). Intracellular Ca2þ mediates changes in membrane proteins to promote the transmitter release and ion channel opening; it also enhances binding receptor sites that alter neuronal sensitivity (9). The variety of genetic information changes in excitability can occur as a result of the Ca2þ influx lead to depolarized synaptic site which is triggered by the vesicle-associated membrane protein (VAMP/synaptobrevin), as well as the syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25). These proteins Address for correspondence: Mustafa Yilmaz, Department of Neurology, Faculty of Medicine, Mugla University Medical School, 48000 Mugla, Turkey. Tel: ++90 252 211 4839. Fax: ++90 252 211 1345. E-mail: [email protected]
History Received 5 September 2013 Revised 17 September 2013 Accepted 23 September 2013 Published online 29 October 2013
then bind together in a lock and key fashion to form a tripartite structure. The synaptotagmin gene encodes a protein known as calcium sensors and mediates the calcium-dependent regulation of membrane trafficking in synaptic transmission. Synaptotagmin XI is implicated in the potential role of neuronal plasticity, so that Synaptotagmin XI has a crucial role in the presence of changes in membrane trafficking resulting epileptic seizures. A defect in the synaptic function is thought to underlie epilepsy. It was reported that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a fundamental role in neurological disorders, especially in epilepsy (10). VAMP2/synaptobrevin is involved in the synaptic vesicles of the presynaptic membrane (11). One of the isoforms identified, the VAMP2 protein, is a synaptic vesicle protein of the type that has been implicated in the pathogenesis of the IGE, encodes VAMP2 gene. VAMP2 triggers SNARE complex formation and fusion between synaptic vesicles which is critical for neurotransmission (12). SNAP-25 is regulated by the SNAP-25 gene and is expressed in the cytoplasmic face of a membrane in neuron (13,14). The genes interaction is required for synaptic formation (15). The Syntaxin 1A and SNAP-25 complex is catalyzed through the recognition of Caþ in neurons and
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Genetic variants of synaptic vesicle and presynaptic plasma membrane proteins in idiopathic generalized epilepsy
Genetic variants of synaptic vesicle and presynaptic plasma membrane proteins
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Universitaet Zuerich on 05/05/15 For personal use only.
DOI: 10.3109/10799893.2013.848893
neuroendocrine cells. In addition, the free vesicular synaptobrevin might quickly rearrange with the SNAP 25 and syntaxin end of membrane fusion, especially under highfrequency synaptic activity. The well-characterized group of the SNARE complex, including VAMP, Syntaxin 1A and SNAP-25, regulates the processes of docking, priming and fusion between the synaptic vesicle membrane and specific regions of the plasma membrane. The group also initiates the subsequent release of neurotransmitters. Analyzing the role of the genetic variants of two synaptic vesicle proteins (VAMP2 and Synaptotagmin XI) and of two presynaptic plasma membrane proteins (Syntaxin 1A and SNAP-25) in IGE patients is crucial to show the possible effective factors of IGE.
Materials and methods Participants This study was performed in 2012 and included a multidisciplinary project on epilepsy genetics in Mugla Sitki Kocman University. The protocol was approved by the regional ethical committee, and procedures were performed according to the principles of The Helsinki Declaration. A total of 85 patients (39 females/46 males) with IGE and 93 healthy subjects (46 females/47 males), from the neurology outpatient clinic, were enrolled in the study (Table 1). The study fulfilled the following inclusion criteria: Diagnostic Criteria of the Commission on Classification and Terminology of the International League against Epilepsy were used to diagnose probands with IGE (16). There was no evidence of developmental and neurological abnormalities, or of global cognitive impairment on the Mini-Mental State Examination (17); at least one electroencephalography (EEG) examination showed the generalized spike wave; there were neither abnormal nor unusual findings on conventional magnetic resonance (MR) images. Patients who were subject to pregnancy, drug abuse, alcoholism, migraine or other health problems such as a history of severe major psychiatric disorders or moderate to severe intellectual disability were excluded. Clinical information, the EEG finding, as well as data on antiepileptic therapy were recorded on data collection forms. The spectrums of Table 1. Characteristics of patients. Patients Age (year) (mean SD) 30.8 13.6 Gender n (%) Male 46 (54) Female 39 (56) Age of epilepsy onset 18.1 15.9 Disease duration 14.2 9.8 Frequency of seizures n (%) 52 month 32 (37.2) 2 month 53 (62.3) Family of history n (%) 51 (61) AED n (%) 79.1 (93) Monotherapy n (%) 52 (61.1) Polytherapy n (%) 33 (38.8) 2 drugs 24 (28.2) 3 drugs 9 (10) No of AED n (%) 5.9 (7)
Healthy subjects p Values 31.9 7.5
SD, Standart deviation; AED, antiepileptic drug.
0.26 0.33
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antiepileptic drugs used in patients with IGE were carbamazepine, lamotrigine, valproic acid, clonazepam, topiramate, levetiracetam, phenytoin and oxcarbazepine. The healthy subjects had no history of seizures or other neurological disease, no drug abuse or alcoholism, no pregnancy and no other health problems and did not take any drug with an effect on cortical excitability. The mean of age in patients with IGE was 30.87 13.61 years, and the mean of age in healthy subjects was 31.9 7.56 years. This study was approved by the local ethics committee of Mugla University, and all participants provided written, informed consent. Genotyping After written, informed consent was obtained, venous blood samples were collected into vacutainer plastic tubes containing sodium/potassium ethylene diamine tetra acetic acid. DNA was extracted from Genejet Genomic DNA purification kit (Thermo K0772). We used polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) technique. We determined alleles and the genotypes of polymorphism of VAMP2, synapsin III, SNAP-25 and Syntaxin 1A genes. PCR was performed in a 25 -mL volume with 100 ng DNA, 100 mm dNTPs, 20 pmol of each primer, 1.5 mM MgCl2, 1 PCR buffer with (NH4)2SO4 (Fermentas, Vilnius, Lithuania), 10% dimethyl sulfoxide (DMSO) and 2 U Taq DNA polymerase (Fermentas, Vilnius, Lithuania). Amplification was performed on an automated Thermal Cycler (Techne Flexigene, Cambridge, UK). These genes polymorphisms were determined by fragment separation at 120 V for 40–50 min on a 3.5% agarose gel containing 0.5 mg/ mL ethidium bromide. A 100-bp DNA Ladder (Fermentas Vilnius, Lithuania) was used as a size standard for each gel lane. The gel was visualized under UV light using a gel electrophoresis visualizing system (Vilber Lourmat). PCRRFLP conditions of polymorphisms of VAMP2, Synaptotagmin XI, Syntaxin 1 A and SNAP-25 genes have been shown in Figure 1 and Table 2. Statistical analysis A case-healthy subjects study was performed and allelic and genotypic frequency of the polymorphisms was calculated both in cases and healthy subjects. The Hardy–Weinberg equilibrium was verified using the chi-square ( 2) test and by estimating the expected genotypic frequencies on the basis of the development of the square of the binomial for these polymorphisms. The 2 test was used to compare genotype and allele frequency of the VAMP2, Synaptotagmin XI, Syntaxin 1A, SNAP-25 polymorphisms between patients and healthy subjects. The association between polymorphisms and epilepsy was modeled through binary logistic regression analysis and odds ratio (OR) and 95% confidence interval (CI) were calculated to compare epilepsy risk around genotypes. Also, haplotype analysis was used to determine the effect of the genetic correlation between two polymorphic regions.
Results The demographic characteristics of the patients are given in Table 1. There was no difference between the groups regarding age and gender (p50.05). A family of history of
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J Recept Signal Transduct Res, 2014; 34(1): 38–43
Figure 1. (A) Genotyping analysis of SNAP-25 gene rs1051312 polymorphism; 1 and 5 numbers of subjects have TC genotype, 2 number of subject has TT genotype, and 3 and 4 numbers of subjects have CC genotypes. (B) Genotyping analysis of SNAP-25 gene rs3746544 polymorphism; 2 and 4 numbers of subjects have TT genotypes, 1, 3 and 5 numbers of subjects have TG genotypes and 6 number of subject has GG genotype. (C) Genotyping analysis of Synaptotagmin XI (Syt11) gene 33-bp repeats in promoter region polymorphism; 2 and 4 numbers of subjects have CC genotypes (431 bp (2 repeats)), 3 number of subject has TC genotype (477 bp (2 repeats) and 431 bp (2 repeats)), and 1 number of subject has CC genotype (464 bp (3 repeats) and 431 bp (2 repeats)). (D) Genotyping analysis of Syntaxin 1A gene rs1569061polymorphism; 1 and 5 numbers of subjects have TC genotypes, 2 and 3 numbers of subjects have TT genotypes and 5 number of subject has CC genotype. (E) Genotyping analysis of VAMP2 gene 26 bp Ins/Del polymorphism; 1, 3 and 4 numbers of subjects have Ins/Ins genotypes and 2 number of subject has Ins/Del genotype. Table 2. PCR-RFLP conditions of polymorphisms of VAMP2, Synaptotagmin XI, Syntaxin 1A and SNAP-25 genes.
Polymorphism
Primer
Temperature of Annealing
Restriction Endonuclease
P1 P2
57 C
–
33-bp repeats in promoter region
P3 P4
53 C
HphI
Syntaxin 1A
Intron 7 (rs1569061)
57 C
TaiI
SNAP-25
MnlI (rs3746544)
P5 P6 P7 P8 P9 P10
58 C
MnlI
VAMP2
26 bp Ins/Del polymorphism
Synaptotagmin XI
DdeI (rs1051312)
DdeI
PCR products Ins allele:116 bp Del allele:90 bp T allele: 510 bp (three repeats), 477 bp (2 repeats), 444 bp (1 repeat), C allele: 464 bp (3 repeats), 431 bp (2 repeats), 398 bp (1 repeat) T allele: 312 bp C allele: 186,126 bp T allele: 256, 5 bp G allele: 210, 46, 5 bp T allele: 261 bp C allele: 228, 33-bp
P1: F50 -ACAAAGTGCGCCTTATACGC-30 . P2: R50 -GGGATTTTCCTTGACGACACTC-30 . P3: F50 -TCTACCTATGCTTCTTACCC-30 . P4: R50 -TG TCGTAATCAGAGGCTGTTGCT-30 . P5: F50 -CAATGCTGCTGCTGAACTC-30 . P6: R50 -CGCTGACATTTATGTGACC-30 . P7: F50 -TTCTCCTCC AAATGCTGTCG-30 . P8: R50 -CCACCGAGGAGAGAAAATG-30 . P9: R50 -CCACCGAGGAGAGAAAATG-30 . P10: F50 -TTCTCCTCCAAATGCTG TCG-30 . Significant results of statistical analysis are shown in bold.
epilepsy was found in 61% in patients with IGE. We found significant differences alleles and genotypes of 26-bp Ins/Del polymorphism of VAMP2 gene, 33-bp repeats in promoter region of Synaptotagmin XI (Table 2). Also, we have not found any significant difference between the groups when they were compared according to whether they have rs1569061 of Syntaxin 1A gene, rs3746544 and rs1051312 polymorphisms of SNAP-25 gene. There was a significant association between the CC genotype in Synaptotagmin XI gene and IGE (p50.002; OR 4.93; 95% CI 2.01–12.1). There
was a significant association between the ins/del genotype in VAMP2 gene and IGE (p50.042; OR 0.474; 95% CI 0.23– 0.978). The C allele in Synaptotagmin XI gene was found significant different in patients with IGE compared with healthy subjects (p50.001; OR 2.31; 95% CI 1.5–3.57). We have found that individuals who have Synaptotagmin XI C/C alleles are 4.9 times more at risk of developing IGE (Table 3). Individuals who have C alleles in Synaptotagmin XI gene are 2.31 times more at risk of developing IGE compared with healthy subjects (Table 4). We found increased occurrence of
Genetic variants of synaptic vesicle and presynaptic plasma membrane proteins
DOI: 10.3109/10799893.2013.848893
epilepsy via the haplotype analysis for rs1569061 of Syntaxin 1A gene and 33-bp promoter region of Synaptotagmin XI gene. We determined that CC, TC haplotypes for rs1569061 Syntaxin 1A gene, 33-bp promoter region of Synaptotagmin
XI gene could be risk factors for all types of epilepsy disease (respectively, OR ¼ 2.254 95% CI ¼ 1.229–4.137, OR ¼ 4.794 95% CI ¼ 2.312–9.940) (Table 5). There was no correlation between the frequency of seizures and polymorphisms of the genes.
Table 3. Haplotypes distributations of Syntaxin 1A, Synaptotagmin XI and SNAP-25 genes in controls and IGE (n ¼ number of individuals).
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Haplotype
Healthy controls n (%)
Cases n (%)
Syntaxin 1A/Synaptotagmin XI CT 59 (31.7%) 30 TT 70 (37.6%) 54 CC 41 (22.0%) 47 TC 16 (8.6%) 39 Snap-25 Mnll/Snap-25 Ddel TT 95 (51.1%) 95 GT 46 (24.7%) 27 TC 31 (16.7%) 24 GC 14 (7.5%) 24
OR
95% CI
(17.6%) (31.8%) (27.6%) (22.9%)
1 (reference) 1.517 2.254 4.794
0.862–2.669 1.229–4.137 2.312–9.940
(55.9%) (15.9%) (14.1%) (14.1%)
1 (reference) 0.587 0.774 1.714
0.337–1.021 0.423–1.416 0.836–3.514
Discussion We analyzed the genetic variants of the presynaptic plasma membrane proteins such as SNAP-25 and Syntaxin 1A, two synaptic vesicle proteins such as the VAMP2 and Synaptotagmin XI which contributes to the formation of the fusion complex are necessary for the docking and/or fusion of synaptic vesicles in patients with IGE for the first time to the best of our knowledge. Extensive studies have been performed using several biochemical and immunohistochemical methods, suggesting the existence of SNARE mechanisms virtually in all regions of the central nervous system (CNS) (18–21). SNARE complexes may be used as a biological marker in seizure generation for the reason of roles in synaps. In some studies, the GABRG2 rs211037’s
Significant results of statistical analysis are shown in bold.
Table 4. Alleles of VAMP2, SNAP-25, Syntaxin 1A and Synaptotagmin XI genes (n ¼ number of individuals).
Allele VAMP ins del Snap-25 Mnll T G Snap-25 Ddel T C Syntaxin 1A T C Synaptotagmin XI T C
Healthy controls n (%)
Cases n (%)
146 (78.5%) 40 (21.5%)
135 (79.4%) 35 (20.6%)
0.897
Reference 0.946 (0.568–1.577)
126 (67.7%) 60 (32.3%)
119 (70.0%) 51 (30.0%)
0.731
Reference 0.9 (0.574–1.411)
141 (75.8%) 45 (24.2%)
122 (71.8%) 48 (28.2%)
0.400
Reference 1.233 (0.768–1.979)
86 (46.2%) 100 (53.8%)
93 (54.7%) 77 (45.3%)
0.113
Reference 0.712 (0.469–1.081)
129 (69.4%) 57 (30.6%)
84 (49.4%) 86 (50.6%)
50.001
Reference 2.317 (1.503–3.573)
X2 p Value
OR (95% CI)
Significant results of statistical analysis are shown in bold. Table 5. Genotypes of VAMP2, SNAP-25, Syntaxin 1A and Synaptotagmin XI genes (n ¼ number of individuals). Genotype VAMP2 ins/ins ins/del del/del Snap-25 Mnll T/T T/G G/G Snap-25 Ddel T/T T/C C/C Syntaxin 1A T/T T/C C/C Synaptotagmin XI T/T T/C C/C
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Healthy controls n (%)
Cases n (%)
X2 p Value
OR (95% CI)
56 (60.2%) 34 (36.6%) 3 (3.2%)
58 (68.2%) 19 (22.4%) 8 (9.4%)
0.042
Reference 0.474 (0.230–0.978) 2.024 (0.4778–0.593)
41 (44.1%) 44 (47.3%) 8 (8.6%)
44 (51.8%) 31 (36.5%) 10 (11.8%)
0.327
Reference 0.725 (0.363–1.447) 1.193 (0.385–3.694)
53 (57.0%) 35 (37.6%) 5 (5.4%)
46 (54.1%) 30 (35.3%) 9 (10.6%)
0.432
Reference 0.926 (0.470–1.825) 2.116 (0.592–7.562)
18 (19.4%) 50 (53.8%) 25 (26.9%)
28 (32.9%) 37 (43.5%) 20 (23.5%)
0.114
Reference 0.531 (0.241–1.169) 0.521 (0.210–1.294)
47 (50.5%) 35 (37.6%) 11 (11.8%)
26 (30.6%) 32 (37.6%) 27 (31.8%)
0.002
Reference 2.043 (0.978–4.268) 4.939 (2.015–12.106)
Significant results of statistical analysis are shown in bold.
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association with IGE and febrile seizures was examined. They showed a significant association of GABRG2 rs211037 with febrile seizures, but any significant association was found in patients with IGE (22–24). In addition, it was reported that genetic background is present in the etiology of IGE and in other variants contributing to IGE, as such, future studies must be performed (24). SNARE complex modulates calcium dynamics in response to depolarization, so that it has an essential role in neurotransmitter release via the formation (15). In an experimental study that involved a pentylenetetrazole (PTZ)-induced epilepsy rat model, the expressional levels of synaptic vesicle protein 2A, SNARE complexes were evaluated. It was reported that PTZ did not affect the hippocampal level of SNARE complexes, and also it was mentioned that the hippocampus is a key structure underlying seizure generation (25,26). In contrast, in another experimental study, SNARE complexes were found increase in amygdala kindling. It was reported that the asymmetric accumulation of SNARE complexes occurs during kindlinginduced epileptogenesis (26). The SNARE gene may have a role in discrete structure expression, and/or it may function as contributing directly to neurological disorders as a consequence. Synaptotagmins comprise a large family of transmembrane proteins. Synaptotagmin XI is found in the brain and in minimally studied protein compared with other isoforms. In addition, other isoforms are expressed in a wide variety of tissue outside of the brain. Synaptotagmin XI is thought to be one of the primary calcium sensors for the exocytosis of synaptic vesicles, which is crucial in Ca2þ-triggered vesicle fusion (27). It was well documented that synaptotagmin regulates membrane trafficking and also has an important role in the synaptic plasticity in epilepsy (28). It was reported that synaptotagmin plays a crucial role in refractory epilepsy (29). In our study, synaptotagmin expression was found to be significantly higher than that in the healthy subjects and non-refractory epilepsy groups. The patients with the IGE group, carriers of the CC genotype in Synaptotagmin XI gene, might have a high risk of carrying IGE than carriers of other genotypes. One of the SNARE complexes, SNAP-25, is essential for the exocytosis that plays a key role in transferring information from photoreceptor cells to the CNS (18). The SNAP-25 protein has been studied in psychiatric diseases (10). The expression of SNAP-25 could arrange the synaptic homeostasis resulting neuronal activity. It may be required to describe the potential mechanisms through which alterations in SNAP-25 may play a direct role in the etiology as well as contribute to the pathology of IGE. In recent studies, it was reported that the deficiency of the SNAP-25 gene contributes to neuropsychiatric and neurological disorders (10,30). In addition, studies including experimental rat models showed that the deficiency of SNAP-25 caused the disturbance of the presynaptic voltage-gated Ca2þ channels and resulted in seizures (31). In this study, SNAP-25 MnlI (res3746544) and Ddel (rs1051312) gene polymorphism were not found to be different in patients with IGE compared to that of healthy subjects. It may be possible that SNAP-25 is not related to IGE etiology. As such, another protein in the rolling modulation of neurotransmitter release and synaptogenesis
J Recept Signal Transduct Res, 2014; 34(1): 38–43
process may act in etiology. Conversely, the transmission of SNAP-25 alleles was determined to increase in patients with adult attention deficit hyperactivity disease. In another study, the SNAP-25 gene demonstrated that the subject was a strong candidate for IGE. Therefore, the association between SNAP-25 gene polymorphisms and IGE is currently unclear. Syntaxin 1A is particularly a key molecule in synaptic exocytosis. It was detected that Syntaxin 1A has a potential role in epileptogenesis (32). Syntaxin 1A acts as a potential molecular mechanism for the alteration of exocytotic process and neural plasticity. It was reported that Syntaxin 1A provides evidence of serving for serve as an intrinsic enhancer molecular mechanism in epileptogenesis. In the study, there were no significant differences in the Intron 7 rs1569061 of Syntaxin 1A gene for patients with IGE compared to that of healthy subjects. The study accomplished an analysis concerning about the genetic variants of VAMP2, Synaptotagmin XI, Syntaxin 1A and SNAP-25 in patients with IGE. We observed mainly significant the differences in alleles and genotypes of 26 bp Ins/Del polymorphism of VAMP2 gene and 33-bp promoter region of Synaptotagmin XI. There was no significant difference in the Intron 7 rs1569061 of Syntaxin 1A gene, MnlI rs3746544 and DdeI rs1051312 polymorphisms of SNAP-25 gene in patients with IGE compared with healthy subjects. Consequently, the differences alleles and genotypes of 26 bp Ins/Del polymorphism of VAMP2 gene and 33-bp promoter region of Synaptotagmin XI could be effective in occurrence in patient with IGE. The genetic study indicates that polymorphisms in VAMP2 gene and Synaptotagmin XI might be associated with IGE. In future studies, the expression of these genes and their effect on IGE should be analyzed.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of this article.
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