Investigation of the functional brain-derived neurotrophic factor gene variant Val66MET in migraine
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- Marziniak, M., Herzog, A., Mössner, R. et al. J Neural Transm (2008) 115: 1321. doi:10.1007/s00702-008-0056-1
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Experimental studies and investigations of the cerebrospinal fluid in migraineurs have suggested an involvement of brain-derived neurotrophic factor (BDNF) in migraine pathophysiology. In a case–control study approach, the functional Val66MET polymorphism (rs6265) of the BDNF gene was investigated in 265 migraine patients and 153 controls. Genotype and allele frequencies did not differ between healthy subjects and migraineurs. A subgroup analysis for the occurrence of aura or clinical characteristics, including the number of attacks, did not reveal a positive association for the investigated polymorphism. Our data argue against a role of this well characterized BDNF gene variant as a risk factor in migraine.
KeywordsBrain-derived neurotrophic factor Polymorphism Migraine Calcitonin-gene-related peptide
Migraine severely impairs the patient’s quality of life and ranks among the most frequent neurological diseases. Genetic factors seem to play a major role in migraine but, at present, the number and types of genes responsible for migraine with aura and migraine without aura are still not clearly understood. There is major evidence that brain-derived neurotrophic factor (BDNF) plays a critical role in the modulation of nociceptive function. BDNF is synthesized by tyrosine kinase (trk) A-positive sensory neurons, acts through trkB receptors, and is elevated in experimental pain models and chronic daily headache patients (Pezet and Malcangio 2004; Sarchielli and Gallai 2004). BDNF belongs to a neurotrophin family of closely related peptides that also includes nerve growth factor (NGF), neurotrophin-3, and neurotrophin-4/5. The synthesis of BDNF appears to be potentiated by NGF, which is also upregulated in hyperalgesia and experimental pain models (Lewin et al. 1994). BDNF gives trophic support to dopaminergic and serotonergic neurons (Hyman et al. 1991; Mamounas et al. 1995) that are involved in the pathogenesis of migraine and in activity-dependent plasticity in nociceptive pathways, that may lead to pain chronification (Pezet and McMahon 2006). Second, BDNF is widely distributed in the central nervous system, including hippocampus, amygdala, hypothalamus, and the sensory and trigeminal ganglia, key regions in the regulation of pain, mood, and behavior. Additionally, animal models have confirmed that BDNF is involved in the response to drugs such as antidepressants, lithium, and antipsychotics (Angelucci et al. 2005). In a B lymphoblast model, which has several molecular and functional similarities to serotonergic neurons, BDNF treatment was found to decrease serotonin uptake by serotonin transporters, thereby increasing extracellular serotonin levels (Mössner et al. 2000). Furthermore, BDNF is coexpressed with calcitonin gene-related peptide (CGRP) (Buldyrev et al. 2006), which is expressed by trigeminal nociceptors and has recently been identified as one of the key players in the mechanism of migraine headaches. The intravenous administration of the CGRP-receptor antagonist BIBN 4096 BS was effective in the treatment of migraine (Olesen et al. 2004) and the development of oral formulas is on their way. CGRP potently enhances BDNF release from cultured trigeminal neurons, and this effect is dose-dependent and abolished by pretreatment with a CGRP receptor antagonist. Using transmission electron microscopy, it could be shown that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors (Buldyrev et al. 2006). Interestingly, levels of BDNF were decreased in platelets of migraine patients in comparison to healthy controls (Blandini et al. 2006) and increased in the cerebrospinal fluid of patients with chronic daily headache (Sarchielli et al. 2002), suggesting a potential role in the pathogenesis of migraine.
BDNF maps to human chromosome 11p13 (Maisonpierre et al. 1990) and is organized in 13 exons. The entire BDNF open reading frame is contained within the last exon and, due to alternative splicing, encodes two BDNF protein variants (GenBank accession no. AF411339). The long form of BDNF, with 247 amino acids, consists of a 5′ pro-BDNF sequence and is proteolytically cleaved to form the mature protein (Seidah et al. 1996). The short BDNF form, which is 153 amino acids long, lacks the 5′ pro-BDNF region. A single nucleotide polymorphism (SNP), G to A at nucleotide 196, which results in a valine (Val) 66-to-methionine (Met) (V66M; 11350.0002; SNP database [dbSNP] rs6265) change, is located in the 5′ pro-BDNF sequence. The Val66Met variant has been extensively studied through linkage and association approaches in several psychiatric disorders, as well as measures of cognitive function. A meta-analysis of case-control studies confirmed association of the functional Val66Met polymorphism to substance-related disorders, eating disorders, and schizophrenia (Gratacos et al. 2007).
Taken together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity. In this study, we tested the hypothesis whether a positive association exists between the functional Val66MET polymorphism and migraine. We performed a case-control association study in two tertiary Headache Centers, and in healthy individuals without migraine, to evaluate the effects of the Val66MET polymorphism of the BDNF gene on the prevalence and the clinical characteristics of migraine.
Patients and methods
265 consecutive unrelated migraineurs (222 women, 43 men, 43.6 ± 13 years) were diagnosed according to the 2nd International Headache Society (IHS) criteria and were recruited from the Headache Clinics in Würzburg and Homburg/Saar after informed consent and approval by the local ethics committee. 122 patients had migraine with aura (MA), while 143 patients had migraine without aura (MO). Patients completed a standardized headache questionnaire and were subject to a full neurological examination. Patients who reported migraine with aura attacks and co-occurence of migraine attacks without aura were classified as migraineurs with aura. Depression or other comorbid psychiatric disorders were excluded by history and with a score in the Beck depression index lower than ten, in patients and controls. One hundred and fifty three control subjects (110 women, 43 men, 64.5 ± 9.4 years) older than 55 years were chosen, to be confident that they were migraine free, and migraine and a positive family history for migraine were excluded by personal interview. All subjects were of German Caucasian descent. The same population had previously been used for genetic association studies in migraine (Marziniak et al. 2005, 2007; Hohoff et al. 2007).
Venous blood samples of patients and controls were obtained. Genomic DNA was prepared from lymphocytes by standard procedures and diluted to a stock concentration of 25 ng/ml.
For the BDNF gene, the G->A- SNP coding for the Val66Met substitution was genotyped employing a modification of a protocol described by Sen et al. (2003). A 274-bp PCR product containing the SNP was amplified by polymerase chain reaction (PCR) using the following reaction mix: 20 ng of genomic DNA in 75 mM Tris–HCl (pH 9.0), 20 mM ammonium sulfate, 0.01% Tween-20, 1.5 mM magnesium chloride, 0.4 μM of each of the primers, 5′-AAA GAA GCA AAC ATCCGA GGA CAA G and 5′-ATT CCT CCA GCA GAA AGA GAA GAG G, 0.4 mM dNTP, and 1 U Taq polymerase. After an initial denaturation for 5 min at 95°C, 35 cycles of denaturating at 95°C for 30 s, annealing at 55°C for 40 s and extension of 72°C for 50 s were performed, followed by a final extension of 72°C for 5 min. PCR products were digested with BseGI. The digested PCR product with two fragments of 217 and 57 bp carries the G variant, whereas the digested product with three fragments of 57, 77, and 140 bp contains the A allele.
All analyzes were carried out using SPSS 13.0 (SPSS Inc., Chicago, USA). Symptoms and history data were compared for a correlation between MA and MO and for the three genotypes. Statistical analysis consisted of the chi-squared tests for Hardy–Weinberg proportions in the patient and control groups. The Mann–Whitney U test was used for nonparametric variables and the t test for the parametric variables of the clinical symptoms for the comparison between patient groups of MA and MO.
Genotypic distribution and allelic frequencies of the BDNF Val66MET polymorphism
Migraine with aura
Migraine without aura
Clinical characteristics of the migraineurs according to the Val66MET BDNF polymorphism (rs 6265)
Mean age at onset, years
20.1 ± 10.5
20.1 ± 10.3
17.8 ± 8.6
Mean duration of one attack, hours
34 ± 28.2
35.3 ± 27
38.6 ± 29.2
Mean number of attacks per month
2.7 ± 2.7
2.6 ± 2.5
2.1 ± 1
Positive family history of migraine
Average pain intensity (VAS)
7.3 ± 1.7
7.5 ± 1.6
7.1 ± 1.5
The findings of our study do not support our hypothesis that the functional Val66MET polymorphism influences the risk of migraine. Genotype and allele frequencies were similarly distributed in migraineurs and controls. When migraineurs were divided into subgroups with and without aura, no differences could be found. The functional Val66MET polymorphism did not influence the clinical symptoms of migraine.
This is the first study, to our knowledge, that investigated an association between the BDNF gene and migraine. The Val66MET polymorphism is an ideal SNP candidate for the investigation because its functional relevance has been demonstrated in cell culture experiments and in clinical association studies. Whereas, this BDNF polymorphism does not affect mature BDNF protein function, it has been shown to alter the intracellular tracking and packaging of pro-BDNF and, thus, to affect the regulated secretion of the mature peptide (Chen et al. 2004; Egan et al. 2003). We cannot rule out that other potentially functional variants of the BDNF gene influence the migraine pathophysiology, but it is unlikely that polymorphisms within the BDNF coding region substantially contribute to migraine susceptibility and the severity of the disease.
Increased cerebrospinal fluid levels of BDNF and nerve growth factor (NGF) in chronic daily headache patients lead to the hypothesis that BDNF participates in the induction and maintenance of chronic headache (Sarchielli et al. 2002). This assumption is emphasized by an up-regulation of BDNF and an increased expression of trkB in sensitized C- nociceptors (Kerr et al. 1999; Pezet and McMahon 2006). The trkB activation by BDNF leads to phosphorylation and activation of N-methyl-D-aspartate (NMDA) receptor subunits 1 and 2B and increases the probability that these channels, which are strongly recruited in activated nociceptive pathways and mediate C-fiber evoked discharge (Kerr et al. 1999), are in an open state (Suen et al. 1997; Small et al. 1998). A positive correlation emerged between the cerebrospinal fluid levels of BDNF and NGF in chronic daily headache patients (Sarchielli et al. 2002).
The size of our patient group with 265 migraineurs seems to be reasonable. Significant differences are not expected in a larger study population, particularly with regard to the very small differences of the genotype frequencies between migraineurs and controls. Our data may not apply to other ethnic groups and subtypes of migraine. For example, a marked difference could be found in the frequency of the G allele between the Caucasian (80%) and the Asian population (56%), therefore, our results may not be transferred to the Asian population (Gratacos et al. 2007). Furthermore, we recruited our patients from two tertiary headache centers and a large proportion of the patients had a long-standing history of migraine and were unresponsive to many drugs. Thus, our association data may not be transferable to a group of migraineurs with an “uncomplicated”, easy treatable migraine.
In conclusion, we did not find a positive association between the very well characterized Val66MET polymorphism and migraine. These results do not support a role of the investigated functional variant in migraine pathophysiology and argue against a major influence of the BDNF gene in migraine. However, these data should be replicated in an independent sample to elucidate the involvement of BDNF in migraine pathophysiology and, furthermore, a combined analysis of the BDNF serum and platelets levels in relation to the Val66MET gene variants may reveal additional data about the role of BDNF in migraine.