Abstract
Several studies suggest a role of oxidative stress in the physiopathology of migraine, particularly in the form with aura. In a case-control study, we investigated the association between migraine and superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) genes in a cohort of 490 consecutive unrelated Caucasian migraineurs (migraine with aura [MwA], n=107; migraine without aura [MwoA], n=246; chronic migraine [CM], n=137) and 246 healthy controls recruited at our Headache and Pain Unit and stored in the Interinstitutional Multidisciplinary BioBank (BioBIM). Migraine phenotype was carefully detailed using face-to-face interviews. We examined polymorphisms of SOD1 gene (A/C substitution—rs2234694) and SOD2 gene (C/T transition—rs4880—Ala16Val). The rs4880 TT (Val/Val) genotype was associated (p=0.042) with the presence of unilateral cranial autonomic symptoms (UAs) in MwA patients. We also found a mild correlation between SOD2 rs4880 genotype and the type of acute migraine treatment (p=0.048) in MwA patients. Our findings suggest that SOD2 is a disease-modifier gene influencing oxidative mechanisms in MwA. These observations lead to the hypothesis that SOD2 polymorphism may cause a defective control of the oxidative phenomena linked to cortical spreading depression, the neurophysiological hallmark of migraine aura, causing an overstimulation of trigeminal neurons and UAs triggering. Antioxid. Redox Signal. 22, 275–279.
Introduction
Migraine is a primary brain dysfunction characterized by a dysregulation of cortical excitation–inhibition balance, leading to activation and sensitization of the trigeminovascular system with consequent meningeal inflammation (9). Increased oxidative stress is a key event in migraine physiopathology (2, 7, 8), and generation of diffusible reactive oxygen species (ROS) in the cerebral cortex activates nociceptive transmission during the attack in migraine with aura (MwA) (6). Therefore, antioxidants and ROS scavengers have been suggested as promising therapeutic strategy for migraine (6).
Innovation.
Experimental models reveal that oxidative stress has a significant role in cortical spreading depression, the neurophysiological event underlying migraine aura, and in trigeminal nociceptive activation. This is the first study investigating the association between superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) genetic polymorphisms and migraine. The strength of our study includes the detailed demographic and clinical characterization of migraineurs recruited by headache specialists, and the use of a specific migraine biorepository. We found that a common polymorphism of SOD2 gene contributes to the clinical phenotype of migraine with aura.
Oxidative stress is characterized by an impairment in balance between free-radical production that leads to damage of DNA, lipids, and proteins and levels of biological antioxidant systems (6). One of the most efficient antioxidant defense system of cells exposed to ROS is the superoxide dismutase (SOD) system. SODs are enzymes that catalyze the dismutation of superoxide (O2−) into oxygen (O2) and hydrogen peroxide (H2O2). Among the different SOD isoforms identified, SOD1 is localized in the intracellular cytoplasmic compartments, and SOD2 plays an essential role as a primary mitochondrial antioxidant enzyme. The SOD1 maps to chromosome 21q22 (#147450; OMIM) and presents in intron 3, 34 base pairs downstream the GT splice donor site of exon 3, a polymorphism resulting in an A-to-C substitution (rs2234694). This polymorphism is related to SOD1 activity, resulting in higher activities in subjects carrying AA genotype. Several studies have suggested an association of this SNP with different diseases such as diabetes, autism, and amyotrophic lateral sclerosis (#147450; OMIM). SOD2 gene, mapping to chromosome 6q25.3 (#147460; OMIM), is characterized by a common polymorphism at codon 16 in exon 2, a C-to-T transition (rs4880) resulting in an Alanine (GCT) to Valine (GTT) substitution (Ala16Val). The presence of Valine (T allele) leads to the production of instable mRNA and reduces transport of the SOD2 enzyme into the mitochondrial matrix and then its antioxidant function. Several studies have previously demonstrated the association between the SOD2 Ala16Val polymorphism and breast, prostate, and lung cancers, and cardiovascular, liver, and neurological diseases (#147460; OMIM).
Despite oxidative stress being implicated in the pathophysiology of migraine, to date, the association between migraine and SOD genetic polymorphisms has not been investigated. Since we established a biorepository specifically dedicated to migraine patients (Interinstitutional Multidisciplinary BioBank [BioBIM]) in this study, we aimed at exploring the association between SOD1 and SOD2 specific genetic variants and migraine in a cohort of carefully clinical characterized Caucasian patients.
Results and Discussion
We consecutively enrolled 490 migraineurs affected by MwA (n=107), migraine without aura (MwoA, n=246), and chronic migraine (CM, n=137) (5) and 246 healthy control subjects. Sociodemographic and clinical characteristics of the study population, gathered with face-to-face interviews, are presented in Table 1.
Table 1.
Baseline Characteristics of the Migraine Patients and Controls
| Variable | Controls (246) | All patients with migraine (490) | Chronic migraine (137) | Migraine with aura (107) | Migraine without aura (246) |
|---|---|---|---|---|---|
| Sex, F/M, (F%) | 175/71 (71.1) | 369/74 (83.1) | 122/15 (89.0) | 82/25 (76.6) | 201/45 (81.7) |
| Age at observation, mean (SD) | 42.1 (12.56) | 41.4 (12.72) | 41.8 (12.50) | 41.1 (12.38) | 41.4 (12.36) |
| Age at onset, mean (SD) | 20.3 (11.67) | 20.3 (11.26) | 19.8 (10.60) | 20.1 (11.17) | |
| Family history of migraine, (%) | 348 (71.0) | 93 (67.8) | 78 (72.8) | 175 (71.1) | |
| UAs | 191 (38.9) | 63 (45.9) | 35 (32.7) | 93 (37.8) | |
| Migraine acute treatment | |||||
| NSAIDs | 126 (25.4) | 28 (20.4) | 30 (28.0) | 68 (27.6) | |
| Triptans | 146 (29.7) | 35 (25.5) | 26 (24.2) | 85 (34.5) | |
| NSAIDs+Triptans | 136 (27.7) | 60 (43.7) | 17 (15.8) | 59 (23.9) | |
| Others | 82 (16.7) | 14 (10.2) | 34 (31.7) | 34 (13.8) | |
NSAIDs, nonsteroidal anti-inflammatory drugs; SD, standard deviation; UAs (unilateral cranial autonomic symptoms), conjunctival injection, lacrimation, nasal congestion/rhinorrhea, ptosis, eyelid swelling or forehead/facial sweating, singly or combined.
Genotypes and the allele frequencies of the SOD1 and SOD2 polymorphisms between migraineurs and controls did not significantly differ from those predicted by the Hardy-Weinberg equilibrium (Table 2). We found no association between SOD1 and SOD2 polymorphisms and migraine susceptibility, migraine type (MwoA, MwA, and CM), sociodemographic characteristics, or concomitant diseases. However, when focusing in detail on migraine clinical phenotype, in patients with MwA we observed a correlation between SOD2 TT (Val/Val) genotype and the presence of unilateral cranial autonomic symptoms (UAs) (1) (p=0.04, effect size [ES]=0.243) (Fig. 1A). We also found a mild association between SOD2 genotype and acute migraine treatment type in the same patients' group (p=0.04, ES=0.249): CC (Ala/Ala) genotype carriers used more triptans (45.83%), TT (Val/Val) carriers used more nonsteroidal anti-inflammatory drugs (NSAIDs, 35.13%), while heterozygous CT (Ala/Val) carriers used both triptans and NSAIDs (45.65%) (Fig. 1B).
Table 2.
Distributions of Genotype and Allele Frequencies of SOD1 rs2234694 and SOD2 rs4880 Polymorphisms Observed in Patients and Controls
| rs2234694 genotypes (%) | rs2234694 alleles (%) | |||||
|---|---|---|---|---|---|---|
| n | AA | AC | CC | A | C | |
| Controls | 246 | 222 (40.25) | 19 (45.20) | 1 (14.55) | 406 (62.85) | 240 (37.15) |
| All patients | 490 | 200 (39.84) | 223 (44.42) | 79 (15.74) | 623 (62.05) | 381 (37.95) |
| MwA | 107 | 98 (39.81) | 9 (42.59) | 0 (17.59) | 132 (61.11) | 84 (38.89) |
| MwoA | 246 | 217 (40.63) | 28 (46.09) | 0 (13.28) | 326 (63.67) | 186 (36.33) |
| CM | 137 | 127 (38.41) | 9 (42.75) | 1 (18.84) | 165 (59.78) | 111 (40.22) |
| rs4880 genotypes (%) | rs4880 alleles (%) | |||||
|---|---|---|---|---|---|---|
| n | CC | CT | TT | C | T | |
| Controls | 246 | 45 (40.25) | 110 (45.20) | 77 (14.55) | 406 (62.85) | 240 (37.15) |
| All patients | 490 | 200 (39.84) | 223 (44.42) | 79 (15.74) | 623 (62.05) | 381 (37.95) |
| MwA | 107 | 24 (39.81) | 46 (42.59) | 37 (17.59) | 132 (61.11) | 84 (38.89) |
| MwoA | 246 | 54 (40.63) | 114 (46.09) | 76 (13.28) | 326 (63.67) | 186 (36.33) |
| CM | 137 | 33 (38.41) | 67 (42.75) | 37 (18.84) | 165 (59.78) | 111 (40.22) |
Values are given as No. (%).
CM, chronic migraine; MwA, migraine with aura; MwoA, migraine without aura; SOD1, superoxide dismutase 1; SOD2, superoxide dismutase 2.
FIG. 1.
Genotypic frequencies of SOD2 rs4880 displayed according to the presence of unilateral cranial autonomic symptoms (A) and acute migraine treatment (B) in patients with MwA. Percentage values of relative frequencies are reported. MwA, migraine with aura; NSAIDs, nonsteroidal anti-inflammatory drugs; SOD2, superoxide dismutase 2.
This is the first study investigating the association between specific SOD1 and SOD2 gene polymorphisms and migraine. We found no direct relationship between the SOD1 and SOD2 analyzed polymorphisms and migraine susceptibility. However, we report an interesting correlation between SOD2 Ala16Val variant and the clinical phenotype of MwA, namely the presence of UAs (1).
To date, studies on SOD activity in migraine have provided conflicting results. A low concentration of platelet SOD levels and a decreased SOD activity in MwA have been reported, suggesting a higher vulnerability to oxidative stress in these patients (7). Higher SOD activity in red cells in patients with MwA compared with those with MwoA, probably due to a relative increase in oxidative stress due to vasoconstriction induced by ROS and associated with occurrence of aura, has also been described (8). Conversely, no changes in SOD concentration in neutrophils and platelets of MwA patients have been described (2), and a not considerable decrease in erythrocytes from 34 young patients suffering from MwoA or MwA was found (2). These discrepancies are probably due to the different parameters and assays used in determining SOD activity and level, especially across different ages (adults vs. children), and to the evidence that the enzyme activities may change in different cellular compartments (e.g., platelet vs. erythrocytes) (2).
For this reason, we decided to perform our study using molecular methods and examining SNPs of genes encoding SOD1 and SOD2.
The association between SOD2 Ala16Val polymorphism and the occurrence of UAs during the attack in MwA is the main result of our study. This is in agreement with current knowledge in the pathophysiology of MwA and UAs (1, 4, 6). Experimental data reveal that oxidative stress has a significant role in cortical spreading depression (CSD), the neurophysiological event underlying migraine aura, and in trigeminal nociceptive activation (6). During CSD, production of ROS in the brain cortex, meninges, and trigeminal ganglia may induce nociceptive activation and release of calcitonin gene-related peptide from peptidergic nerves, inducing the firing of peripheral trigeminal neurons and meningeal inflammation (6). Experimental examination of more than 1176 genes demonstrated that CSD involves the expression of genes implied in responses to oxidative stress such as major prion protein, glutation-S-transferase-5, and apolipoprotein E (4). UAs, the hallmark of trigeminal autonomic cephalgias that also appear during the attack in approximately 45.8% of migraine patients (1), are known to occur in those migraineurs whose attacks are characterized by an overstimulation of trigeminal afferents that triggers the efferent parasympathetic arm of the trigeminoautonomic reflex (1). Based on the findings mentioned earlier, we hypothesize that patients with MwA and UAs carrying the SOD2 TT (Val) genotype have a defective control of the CSD oxidative phenomena. This could magnify CSD-induced activation of trigeminal nociceptive firing, leading to an overstimulation of trigeminal neurons, which, in turn, trigger the trigemino-autonomic reflex, giving rise to UAs.
Our study also suggests that in MwA patients, SOD2 CC genotype is more frequent among triptans users whereas the TT genotype is more common among NSAIDs users, CT genotype carriers behaving midway. These data need to be considered with caution, as in our study patients were asked to report their current acute migraine treatment but not specifically the reason of their choice. One possibility is that SOD2 activity in CC carriers counteracts CSD-induced oxidative pro-nociceptive effects, contributing toward preventing or delaying the development of central sensitization, therefore resulting in a better efficacy of triptans, drugs known to work before central sensitization has occurred (3). Conversely, TT carriers could have more pronounced CSD-induced pain consequences, including rapid central sensitization, which reduces triptans response (3), thus preferring NSAIDs, which are also effective when taken during the late stages of the attack, when the central sensitization has been developed.
Data based on a specifically dedicated migraine biobank, coupled to extremely detailed clinical and demographic features gathered with face-to-face interviews by headache specialists, are the main strength of the study. Limitations are that we recruited patients in a tertiary-referral center and that we cannot exclude possible artefacts due to recall bias. Moreover, we acknowledge the need to enlarge the patients' sample in future studies, hoping in replication studies by future headache-dedicated biorepositories.
In conclusion, our findings suggest that SOD2 is a disease-modifier gene influencing oxidative mechanisms in MwA; SOD2 polymorphisms may impact MwA phenotype through a defective control of CSD-related oxidative phenomenon that amplifies trigeminal nociceptive activation. Further genetic association studies are imperative to understand the SODs gene involvement in migraine physiopathology that may be useful as a biomarker for detecting asymptomatic individuals at increased risk for migraine and for developing effective treatment strategies (1).
Notes
Population study
From 2009 to 2013, 490 Caucasian unrelated patients affected by MwoA, MwA, and CM were consecutively recruited at our Headache and Pain Unit. Migraine was diagnosed according to the International Headache Society (IHS) criteria (5). Detailed information on the patients' demographics and clinical characteristics of migraine, including age of onset, family history of migraine, presence of aura, frequency and duration of attacks, location, side, quality and intensity of pain, presence of UAs (conjunctival injection, lacrimation, nasal congestion/rhinorrhea, ptosis, eyelid swelling, or forehead/facial sweating, singly or combined) (1), duration of CM, preventive and acute treatments, presence and duration of medication overuse, sleep problems, obesity, head or neck injuries, other concomitant diseases, or any other factors currently known to be associated with migraine, was gathered by specifically trained neurologists during the first visit by means of face-to-face interviews using a semi-structured questionnaire. Patients with significant cardiovascular, metabolic, pulmonary, renal, hepatic, endocrine, or other systemic disease were excluded. Informative and consent forms were provided at the time of diagnosis to patients, along with the permission to obtain blood samples for research purposes. As a control, we enrolled 246 healthy subjects that matched with the cases for age, gender, and race-ethnicity. None of the controls had clinical evidence or family history of migraine or other neurological diseases.
Genotyping
All biological samples were stored at the BioBIM of IRCCS San Raffaele Pisana following the biobanking standard operative procedures. DNA was isolated from EDTA anticoagulated whole blood using MagNA Pure LC instrument and the MagNA Pure LC total DNA isolation kit I (Roche Diagnostics, GmbH, Mannheim, Germany) according to the manufacturer's instructions.
The +34A>C intron 3 SOD1 polymorphism (rs2234694) and the exon 2 SOD2 Val16Ala polymorphism (rs4880) were determined by polymerase chain reaction (PCR) amplification on the basis of the Ensembl sequences ENSG00000142168 and ENSG00000112096, respectively, for SOD1 and SOD2 Genes.
Standard PCR was performed using the following primers: F5′-TATCCAGAAAACACGGTGGGCC-3′ R5′-TCCTGTATTAGTTCCCCTTTGGCAC-3′ (SOD1), and F5′-TCTCGTCTTCAGCACCAGCAGG-3′ R5′-TGGTACTTCTCCTCGGTGACG-3′ (SOD2) in a GeneAmp PCR System 9700 (Life Technologies, Carlsbad, CA) using HotStarTaq Master Mix (QIAGEN, Inc., Chatsworth, CA). All Sanger sequencing analyses were carried out in order to exclude preanalytical and analytical errors on both strands using Big Dye Terminator v3.1 Cycle Sequencing kit (Life Technologies), run on an ABI 3130 Genetic Analyzer (Life Technologies) and repeated on PCR products obtained from new nucleic acid extractions.
Statistical analysis
Accordance of allelic frequencies with the Hardy–Weinberg equilibrium was assessed by Chi-square goodness-of-fit test. Data are presented as mean±SD for normally distributed variables. Mean differences were assessed by two-way analysis of variance; while for nominal ones, association was assessed by the Chi-squared test or Fisher–Freeman–Halton exact test where needed. ES was estimated by Cramer's V. Statistical significance was assessed for a p-value less than 0.05. For a minimal observed ES of 0.24 and a sample of 490 (six degrees of freedom), the achieved power of association study was 99.09% for an error probability of 5%.
Abbreviations Used
- BioBIM
Interinstitutional Multidisciplinary BioBank
- CM
chronic migraine
- CSD
cortical spreading depression
- ES
effect size
- MwA
migraine with aura
- MwoA
migraine without aura
- NSAID
nonsteroidal anti-inflammatory drug
- ROS
reactive oxygen species
- SD
standard deviation
- SOD1
superoxide dismutase 1
- SOD2
superoxide dismutase 2
- UAs
unilateral cranial autonomic symptoms
Acknowledgments
This study was partially supported by Grant MERIT RBNE08NKH7 to San Raffaele Foundation Ceglie Messapica (www.fondazionesanraffaele.com) and partially by the grant PO FESR 2007/2013 Linea di Intervento 4.1.1.1—SIASOP.
References
- 1.Barbanti P, Fabbrini G, Pesare M, Vanacore N, and Cerbo R. Unilateral cranial autonomic symptoms in migraine. Cephalalgia 22: 256–259, 2002 [DOI] [PubMed] [Google Scholar]
- 2.Boćkowski L, Sobaniec W, Kułak W, and Smigielska-Kuzia J. Serum and intraerythrocyte antioxidant enzymes and lipid peroxides in children with migraine. Pharmacol Rep 60: 542–548, 2008 [PubMed] [Google Scholar]
- 3.Burstein R, Collins B, and Jakubowski M. Defeating migraine pain with triptans: a race against the development of cutaneous allodynia. Ann Neurol 55: 19–26, 2004 [DOI] [PubMed] [Google Scholar]
- 4.Choudhuri R, Cui L, Yong C, Bowyer S, Klein RM, Welch KM, and Berman NE. Cortical spreading depression and gene regulation: relevance to migraine. Ann Neurol 51: 499–506, 2002 [DOI] [PubMed] [Google Scholar]
- 5.Headache Classification Committee of the International Headache Society (IHS). The International Classification of Headache Disorders, 3rd Edition (beta version). Cephalalgia 33: 629–808, 2013 [DOI] [PubMed] [Google Scholar]
- 6.Shatillo A, Koroleva K, Giniatullina R, Naumenko N, Slastnikova AA, Aliev RR, Bart G, Atalay M, Gu C, Khazipov R, Davletov B, Grohn O, and Giniatullin R. Cortical spreading depression induces oxidative stress in the trigeminal nociceptive system. Neuroscience 253: 341–349, 2013 [DOI] [PubMed] [Google Scholar]
- 7.Shimomura T, Kowa H, Nakano T, Kitano A, Marukawa H, Urakami K, and Takahashi K. Platelet superoxide dismutase in migraine and tension-type headache. Cephalalgia 14: 215–218, 1994 [DOI] [PubMed] [Google Scholar]
- 8.Tuncel D, Tolun FI, Gokce M, Imrek S, and Ekerbiçer H. Oxidative stress in migraine with and without aura. Biol Trace Elem Res 126: 92–97, 2008 [DOI] [PubMed] [Google Scholar]
- 9.Vecchia D. and Pietrobon D. Migraine: a disorder of brain excitatory–inhibitory balance? Trends Neurosci 35: 507–520, 2012 [DOI] [PubMed] [Google Scholar]