BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE

Investigation of the functional brain-derived neurotrophic factorgene variant Val66MET in migraine

Martin Marziniak Æ Andrea Herzog ÆRainald Mossner Æ Claudia Sommer

Received: 7 January 2008 / Accepted: 20 April 2008 / Published online: 14 May 2008

� Springer-Verlag 2008

Abstract 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.

Keywords Brain-derived neurotrophic factor �Polymorphism �Migraine � Calcitonin-gene-related peptide

Introduction

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 synthe-

sized by tyrosine kinase (trk) A-positive sensory neurons,

acts through trkB receptors, and is elevated in experi-

mental 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 plas-

ticity 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 regu-

lation of pain, mood, and behavior. Additionally, animal

models have confirmed that BDNF is involved in the

response to drugs such as antidepressants, lithium, and

M. Marziniak � C. Sommer

Department of Neurology, University of Wurzburg,

Wurzburg, Germany

M. Marziniak

Department of Neurology, Saarland University,

Homburg/Saar, Germany

M. Marziniak (&)

Department of Neurology, University of Munster,

Albert-Schweitzer-Strasse 33, 48149 Munster, Germany

e-mail: marzinia@uni-muenster.de

A. Herzog � R. Mossner

Department of Psychiatry and Psychotherapy,

University of Wurzburg, Wurzburg, Germany

R. Mossner

Department of Psychiatry and Psychotherapy,

University of Bonn, Bonn, Germany

123

J Neural Transm (2008) 115:1321–1325

DOI 10.1007/s00702-008-0056-1

antipsychotics (Angelucci et al. 2005). In a B lymphoblast

model, which has several molecular and functional simi-

larities to serotonergic neurons, BDNF treatment was

found to decrease serotonin uptake by serotonin trans-

porters, thereby increasing extracellular serotonin levels

(Mossner 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 micros-

copy, 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 (Maisonpi-

erre 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 50 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 50 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 50 pro-BDNF

sequence. The Val66Met variant has been extensively

studied through linkage and association approaches in

several psychiatric disorders, as well as measures of cog-

nitive function. A meta-analysis of case-control studies

confirmed association of the functional Val66Met poly-

morphism 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 hypoth-

esis 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 poly-

morphism of the BDNF gene on the prevalence and the

clinical characteristics of migraine.

Patients and methods

Subjects

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 Wurzburg 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 associa-

tion studies in migraine (Marziniak et al. 2005, 2007;

Hohoff et al. 2007).

Genotyping

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 modi-

fication 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 lM of each of the

primers, 50-AAA GAA GCA AAC ATCCGA GGA CAA G

and 50-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

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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 exten-

sion 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 con-

tains the A allele.

Statistical analysis

All analyzes were carried out using SPSS 13.0 (SPSS Inc.,

Chicago, USA). Symptoms and history data were com-

pared 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.

Results

Hardy-Weinberg equilibrium was verified for the investi-

gated population. Table 1 shows the genotype and allele

frequencies between migraineurs and control subjects.

Genotype and allele frequencies were comparable to the

frequencies reported in other studies (Gratacos et al. 2007).

Genotype frequencies were not significantly different

between migraineurs and controls (p = 0.92, v2 = 0.17,

df = 2) and between migraineurs with and without aura

(p = 0.84, v2 = 0.35, df = 2). Multiple comparisons

between the genotypes (G/G + G/A versus A/A, G/

A + A/A versus G/G) revealed no significant differences.

Table 2 compares the clinical characteristics of migraine

patients according to the different Val66MET genotypes.

The different genotypes had no significant effect on the

examined clinical characteristics: mean age of onset, mean

duration of one migraine attack, mean number of migraine

attacks per month, positive family history for migraine,

average pain intensity on the visual analogue scale (VAS).

The prevalence of nausea, vomiting, photophobia, phono-

phobia, unilateral pain location, pulsating quality of pain,

aggravation by physical activity, and avoidance of routine

physical activity were not effected by the different geno-

types (not shown).

Discussion

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 investi-

gated 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 poly-

morphism 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 poly-

morphisms 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

Table 1 Genotypic distribution and allelic frequencies of the BDNF Val66MET polymorphism

Genotype Allele

N G/G A/G A/A G A

Total sample 418 236 (56.5%) 161 (38.5%) 21 (5.0%) 633 (75.7%) 203 (24.3%)

Controls 153 88 (57.5%) 57 (37.3%) 8 (5.2%) 233 (76.1%) 73 (23.9%)

Female 110 60 (54.6%) 45 (40.9%) 5 (4.5%) 165 (75.0%) 55 (25.0%)

Male 43 28 (65.1%) 12 (27.9%) 3 (7.0%) 68 (79.1%) 18 (20.9%)

Migraineurs 265 148 (55.9%) 104 (39.2%) 13 (4.9%) 400 (75.5%) 130 (24.5%)

Female 222 124 (55.9%) 86 (38.7%) 12 (5.4%) 334 (75.2%) 110 (24.8%)

Male 43 24 (55.8%) 18 (41.9%) 1 (2.3%) 66 (76.7%) 20 (23.3%)

Migraine with aura 122 68 (55.7%) 47 (38.5%) 7 (5.8%) 183 (75.0%) 61 (25.0%)

Migraine without aura 143 80 (55.9%) 57 (39.9%) 6 (4.2%) 217 (75.9%) 69 (24.1%)

J Neural Transm (2008) 115:1321–1325 1323

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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 recrui-

ted 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 fre-

quencies 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 poly-

morphism 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.

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