A Dopamine D4 Receptor Exon 3 VNTR
Allele Protecting against Migraine
Sara Campos de Sousa, BSc,1Andreas Karwautz, MD,2Christian Wo ¨ber, MD,3Gudrun Wagner, MRN,2
Gerome Breen, PhD,1Heidi-Elisabeth Zesch, MD,2Andrea Konrad, MA,2Arno Zormann, MD,2
Christian Wanner, MD,2Christian Kienbacher, MD,2David A. Collier, PhD,1and C ¸ic ¸ek Wo ¨ber-Bingo ¨l, MD2
Objective: As dopamine plays an important role in the pathophysiology of migraine and antimigraine drugs have an effect on
the dopamine system, the objective of this study was to examine the dopamine D4 receptor gene for involvement in the cause
Methods: We tested a VNTR-polymorphism in the dopamine D4 receptor gene, the exon 3 VNTR, in a sample of 190 family
trios each with a proband with childhood migraine by using transmission disequilibrium test tests.
Results: We found a trend for transmission distortion of this marker in migraine, with the common seven-repeat allele of the
VNTR transmitted 58 times and not transmitted 82 times (global likelihood ratio score (LRS) ? 12.27, degress of freedom
(DF) ? 6, p ? 0.06; for the 7-repeat allele: ?2? 5.1, p ? 0.02). This effect came only from migraine without aura (145 trios),
with the common 7-repeat allele transmitted 45 times and not transmitted 69 times (global LRS ? 15.18; DF ? 6, p ? 0.019;
for the 7-repeat allele: ?2? 6.4, p ? 0.01; odds ratio, 0.47), whereas in migraine with aura (45 trios) there was no transmission
distortion of the 7-repeat allele.
Interpretation: We conclude that seven-repeat allele of the dopamine D4 receptor VNTR is a protective factor for migraine
without aura. Because migraine is a common disorder, this protective effect may have contributed to the positive selection acting
on the dopamine D4 receptor exon 3 VNTR seven-repeat allele in recent human history. We speculate that dopamine function
in the lateral parabrachial nucleus is involved in migraine without aura.
Ann Neurol 2007;61:574–578
Genetic epidemiological twin studies demonstrated a
significant heritability for migraine, with 61% of liabil-
ity coming from additive genetic factors and 39% from
nonshared environmental factors in migraine without
aura (MO)1,2and 65% additive genetic effects in mi-
graine with aura (MA).3In these two subtypes of mi-
graine, a complex genetic pattern of inheritance has
been suggested involving several susceptibility genes,
most consistent with a polygenic-multifactorial model
involving both genetic and environmental factors.
Dopamine, a neurotransmitter in the central nervous
system, is involved in the regulation of a variety of be-
havioral and visceral functions, including reward-
related behavior, movement, nociception, vasoregula-
tion, and autonomic responses, as well as in various
pathological conditions such as Parkinson’s disease,
attention-deficit disorder, and drug abuse.4
A role for dopamine in the pathophysiology of mi-
graine has been proposed,5–8based on evidence that
the density of dopamine D3 (DRD3) and D4 recep-
tors (DRD4) on lymphocytes is increased in mi-
graineurs9and that dopamine receptors are hypersensi-
tive, demonstrated by the observation that low doses
(10?g/kg) of apomorphine may activate both presyn-
aptic and postsynaptic receptors in migraine patients.10
Furthermore, several drugs used in the treatment of
migraine act on the dopamine system. Randomized,
controlled trials have shown that dopamine antagonists
such as metoclopramide,11prochlorperazine,12and hal-
operidol13are active in reducing migraine headaches,
and open trials suggested that dopamine agonists such
as bromocriptine and lisuride are effective in prevent-
ing migraine.14,15It has not been proved, however,
that the efficacy of these substances in migraine is ex-
plained by their action on the dopamine system.
In the intravital model of trigeminal activation, it
appears that dopamine receptors do not play a major
role and may not present an acute treatment option.16
From the1Social, Genetic and Developmental Centre (SGDP) at the
Institute of Psychiatry, University of London, London, United King-
Outpatient Centre, Medical University of Vienna; and3Department
of Neurology, Medical University of Vienna, Vienna, Austria.
2Department of Child and Adolescent Psychiatry, Headache
Received Dec 14, 2006, and in revised form Feb 16, 2007. Ac-
cepted for publication Feb 26, 2007.
(www.interscience.wiley.com). DOI: 10.1002/ana.21140
Address correspondence to Prof Dr Karwautz, Department of
Child and Adult Psychiatry, Wa ¨hringer Gu ¨rtel 18-20, A-1090 Vi-
enna, Austria. E-mail: firstname.lastname@example.org
© 2007 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Thus, a direct role for the dopamine system in mi-
graine remains controversial, not least because evidence
for involvement of the serotonin system is much stron-
ger; however, there is substantial interaction between
the dopamine and serotonin systems. Because of this
and the evidence outlined earlier, involvement of the
dopamine system in migraine is plausible, and dopa-
mine system genes are good candidate genes for caus-
Several studies have examined dopamine receptor
genes in migraine; del Zompo and colleagues17exam-
ined the DRD2, DRD3, and DRD4 genes in family
trios from Sardinia and found weak evidence for asso-
ciation with DRD2. Peroutka and colleagues18,19ana-
lyzed a DRD2 “NcoI” C to T silent polymorphism
exon 6 of DRD2 and found a significantly increased
incidence of MA in carriers of DRD2 NcoI C/C ge-
notype, which Dichgans and coworkers20failed to rep-
licate. Lea and colleagues21examined the dopamine
?-hydroxylase gene and the DRD2 gene and found
?-hydroxylase microsatellite marker. Maude and co-
workers22examined a 2bp deletion in the promoter of
the DRD2 gene and found no association with mi-
graine. Shepherd and coworkers23examined polymor-
phisms in DRD1, DRD3, and DRD5, but found no
association with migraine. Stochino and colleagues24
examined polymorphisms in DRD1, DRD3, DRD5,
and DRD2, including the DRD2 “NcoI” C to T silent
polymorphism, and found no association; Rebaudengo
and coworkers25likewise found no association between
the DRD2 “NcoI” polymorphism and migraine or its
Specifically regarding DRD4, Mochi and col-
leagues26examined three functional polymorphisms in
dopaminergic genes: a 48bp VNTR in DRD4, a 40bp
tandem repeat in the 5-untranslated region of the do-
pamine transporter (DAT) gene (5?-UTR VNTR), and
a dinucleotide repeat in the dopamine ?-hydroxylase
gene. They found no association except for a signifi-
cantly different distribution of alleles for the DRD4
gene in MO compared with MA and control groups,
with the seven-repeat allele underrepresented and the
four-repeat overrepresented in the MO cases.
In summary, a number of genes of the dopamine
system have been tested for association with migraine;
the only dopamine gene polymorphism showing previ-
ous association was the DRD4 exon 3 VNTR, in
which the seven-repeat allele appears to be underrepre-
sented in patients. In this study, we used the transmis-
sion disequilibrium test (TDT) to examine the involve-
ment of this polymorphism in migraine. The TDT
approach has several advantages over case–control stud-
ies, for instance, the absence of stratification bias. In
addition, the studies described earlier have not analyzed
children with well-characterized MO and MA.
Subjects and Methods
All patients (n ? 190) from the family trios were assessed by
a face-to-face interview screening to determine International
Classification of Headache Disorders II (ICHD-II) criteria
because they are superior to ICHD-I in identifying migraine
in children.27,28All diagnoses were made by one experienced
child neurologist. All probands met ICHD-II criteria for
MO (n ? 145) or MA (n ? 45). Forty-eight percent were
male subjects and 52% female subjects; age ranged from 6 to
19 years with a mean age of 10.8 years (standard deviation,
This study was part of a project on association of migraine
with genetic markers29approved by the Ethics Committee of
the Medical University of Vienna. We obtained informed
written consent from all participants and parents, and we
kept data anonymous to ensure subject confidentiality.
DNA and Genotyping
DNA was prepared using established methods from buccal
swabs as described elsewhere.30,31The samples were taken by
the subjects themselves and were either immediately left at
the clinic or sent to the Vienna center by mail. The samples
were coded and labeled anonymously before DNA extraction
and storage at the Social Genetics and Developmental Centre
(SGDP) molecular genetic laboratory of the Institute of Psy-
chiatry. Genotyping of the DRD4 exon 3 VNTR was per-
formed by polymerase chain reaction followed by agarose gel
electrophoresis and staining with ethidium bromide, as de-
The program suite UNPHASED was used to test for associ-
ation between the individual marker locus and the hypothet-
ical disease locus.33UNPHASED can be used for association
analysis unphased genotype data. Statistical analysis of family
trios was performed using the TDTPHASE from UN-
PHASED. TDTPHASE performs TDT and haplotype-based
haplotype relative risk (HHRR) analysis for nuclear families
using the TDT; it is robust to families where genotypes are
not available from both parents.
For 190 family trios with migraine analyzed by the TDT
(total sample of aura, nonaura), the sample has a noncentral-
ity parameter of 8.73 and 84% power at ? ? 0.05 (65% at
? ? 0.01) to detect genetic susceptibility arising from
DRD4 exon 3 VNTR seven-repeat allele (heterozygote rela-
tive risk, 0.5; homozygote relative risk, 0.25; allele present in
15% of people). Power calculation was performed using the
genetic power calculator (http://statgen.iop.kcl.ac.uk/gpc/in-
The exon 3 VNTR was in Hardy–Weinberg equilib-
rium in the population, based on genotypes of unre-
lated parents; cases were not examined because Hardy–
Weinberg equilibrium is not a valid quality-control test
because disease association itself can cause Hardy–
Campos de Sousa et al: DRD4 Polymorphisms in Migraine
Weinberg equilibrium deviation. The results of the
TDT analysis using UNPHASED are shown in the
Table for the sample overall. The DRD4 exon 3
VNTR was not globally significant in the sample over-
all (LRS ? 12.27; DF ? 6; p ? 0.05623), but the
seven-repeat allele was, being transmitted 58 times and
not transmitted 82 times to the probands (p ? 0.02).
The sample was also divided into MO and MA (see
the Table). When the sample was divided in this way,
the significance level increased for the MO subgroup,
being significant overall for MO (LRS ? 15.18; DF ?
6; p ? 0.019), but not for MA (LRS ? 1.726; DF ?
4; p ? 0.786). The effect seen in MO came from a
deficit in transmission of the seven-repeat allele (trans-
mitted 45 times, not transmitted 69 times; odds ratio,
0.47; p ? 0.01).
In this study, we found association between the seven-
repeat allele of the DRD4 exon 3 VNTR and mi-
graine. We found that this allele was underrepresented,
that is, has a protective effect against migraine, al-
though DRD4 alleles were not globally associated with
migraine overall (p ? 0.056). When we divided the
sample into MA (45 trios) and MO (145 trios), we
found that the effect of the DRD4 seven-repeat allele
was confined to the MO group, and was significant
globally (p ? 0.02) and specifically for allele seven
(p ? 0.01), whereas showing no association with MA.
However, because of the relatively small number of
MA trios, we cannot be sure that this apparent differ-
ence is the result of low power in the MA group, even
though there was no transmission trend for any allele.
This finding replicates a previous study in patients with
MO, in which the authors also found that the DRD4
exon 3 VNTR polymorphism was associated with MO
but not MA.26Their association was in the same di-
rection as this study, that is, underrepresentation of the
seven-repeat allele in the MO cases (5.9%) relative to
the control subjects (13.2%) and MA cases (14.5%).
However, one previous small TDT study did not re-
port this association.17Taken together, these studies
suggest that allele seven of the DRD4 exon 3 VNTR is
protective against the development of MO. However,
our sample size is still relatively small and the findings
should be replicated in a larger sample.
The DRD4 exon 3 VNTR has attracted substantial
attention because it is a rare example of a large coding
Table. Transmission Disequilibrium Test Analysis for the Dopamine D4 Receptor Exon 3 VNTR in Overall
Migraine, Migraine without Aura, and Migraine with Aura
DiagnosisAlleleT Frequency-T NT Frequency-NTOR%T
For migraine overall, the likelihood ratio test results were: null ? ?728.8; alternative ? ?722.6; LRS ? 12.27; DF ? 6; p ?
0.05623. For migraine without aura (MO), the likelihood ratio test results were: null ? ?568.5; alternative ? ?560.9; LRS ? 15.18;
DF ? 6; p ? 0.019. For migraine with aura (MA), the likelihood ratio test results were: null ? ?156; alternative ? ?155.1; LRS ?
1.726; DF ? 4; p value not significant. Note that UNPHASED does not provide ?2or p values when transmissions are identical in
frequency or rare; thus, these cells have dashes in this table for MA.
OR ? odds ratio.
Annals of NeurologyVol 61No 6June 2007
sequence VNTR34–36; VNTR alleles range from 2 to
11 repeats, resulting in receptors proteins having be-
tween 32 and 176 amino acids at this position. Popu-
lation studies demonstrated wide variation in allele fre-
populations, yet high in the Americas.36The exon 3
VNTR has been associated with a variety of human
traits and diseases, the most notable being attention-
deficit hyperactivity disorder, a highly prevalent (ap-
proximately 3%) childhood psychiatric disorder that is
robustly associated with the seven-repeat allele.37–39
This is the opposite association to this study, in which
the seven-repeat allele was protective against migraine.
Ding and coworkers40resequenced haplotypes of
600 DRD4 alleles from populations across the world,
and showed the seven-repeat allele is not simply related
to the other common alleles, but originated as a rare
mutational event and is at least 5- to 10-fold “younger”
than the common four-repeat allele. Wang and co-
workers41provided evidence that the seven-repeat allele
has increased to high frequency by positive selection,
but the major association of this gene, attention-deficit
hyperactivity disorder, cannot explain this because it is
a deleterious phenotype of early onset, and thus should
be selected against. However, one possible positive se-
lection contributing to the spread of the seven-repeat
allele could be resistance to migraine, because migraine
is common (?10%) in human populations.
The region of the DRD4 receptor protein that con-
tains the amino acid repeats encoded by the exon 3
VNTR couples to G proteins and mediates intercellu-
lar cyclic adenosine monophosphate levels.42,43It ap-
pears that the seven-repeat variant has an attenuated
ability to reduce cyclic adenosine monophosphate lev-
els, compared with the common four-repeat allele.44
However, this is not universally accepted, and the
seven-repeat allele has also been associated with re-
duced expression of DRD4.45
So is DRD4 a plausible causative protein for mi-
graine? The DRD4 protein is expressed in a number of
brain regions, with a high level of expression in the
prefrontal cortex, particularly in the orbital, prelimbic,
cingulate, and rostral agranular insular portions.43,46
Current evidence indicates that cortical spreading de-
pression is the most probable primary event in trigemi-
novascular system activation in migraine.47This pro-
cess appears to involve abnormal cortical activity,
particularly cortical excitability, and it is possible to hy-
pothesize that DRD4 is involved in this process. How-
ever, abnormal cortical excitability appears more rele-
vant to migraine with aura than MO.48In addition to
the prefrontal cortex, DRD4 expression follows meso-
cortical dopamine projections originating in the ventral
tegmental area, as well as three other brain nuclei
known to be innervated by dopaminergic neurons: the
lateral parabrachial nucleus, the ventral pallidum, and
the anterior olfactory nucleus.46The lateral parabra-
chial nucleus together with the rostral agranular insular
cortex are involved in emotional responses to noxious
stimuli and in autonomic antinociception. Both the
lateral parabrachial nucleus and rostral agranular insu-
lar cortex are connected49; because dopamine uptake
inhibition in the rostral agranular insular cortex pro-
duces antinociception,50it is tempting to speculate that
DRD4 stimulation in these regions mediates the devel-
opment of pain, as specifically proposed by Noain and
colleagues.46This, of course, could be extended to mi-
graine, whereby the attenuated seven repeat might in-
crease the propensity to migraine-induced pain.
This study was supported by the Jubila ¨umsfonds of the Austrian
National Bank (10645, C ¸.W.-B.).
1. Gervil M, Ulrich V, Kyvik KO, et al. Migraine without aura: a
population-based twin study. Ann Neurol 1999;46:606–611.
2. Gervil M, Ulrich V, Kyvik KO, et al. The relative role of ge-
netic and environmental factors in migraine without aura. Neu-
3. Ulrich V, Gervil M, Kyvik KO, et al. Evidence of a genetic
factor in migraine with aura: a population-based Danish twin
study. Ann Neurol 1999;45:242–246.
4. Smythies J. The dopamine system. Int Rev Neurobiol 2005;64:
5. Peroutka SJ. Dopamine and migraine. Neurology 1997;49:
6. Mascia A, Afra J, Schoenen J. Dopamine and migraine: a re-
view of pharmacological, biochemical, neurophysiological, and
therapeutic data. Cephalalgia 1998;18:174–182.
7. Fanciullaci M, Rosso AD. Dopamine involvement in the mi-
graine attack. Funct Neurol 2000;15:171–181.
8. Chen SC. Epilepsy and migraine: the dopamine hypotheses.
Med Hypotheses 2006;66:466–472.
9. Barbanti P, Fabbrini G, Ricci A, et al. Migraine patients show
an increased density of dopamine D3 and D4 receptors on lym-
phocytes. Cephalalgia 2000;20:15–19.
10. Cerbo R, Barbanti P, Buzzi MG, et al. Dopamine hypersensi-
tivity in migraine: role of the apomorphine test. Clin Neurop-
11. Friedman BW, Corbo J, Lipton RB, et al. A trial of metoclo-
pramide vs sumatriptan for the emergency department treat-
ment of migraines. Neurology 2005;64:463–468.
12. Brousseau DC, Duffy SJ, Anderson AC, Linakis JG. Treatment
of pediatric migraine headaches: a randomized, double-blind
trial of prochlorperazine versus ketorolac. Ann Emerg Med
13. Honkaniemi J, Liimatainen S, Rainesalo S, Sulavuori S. Halo-
peridol in the acute treatment of migraine: a randomized,
double-blind, placebo-controlled study. Headache 2006;46:
14. Herzog AG. Continuous bromocriptine therapy in menstrual
migraine. Neurology 1997;48:101–102.
15. Soyka D, Frieling B. [Lisuride for the prevention of migraine.
Results of a multicenter study] Fortschr Med 1989;107:
16. Akerman S, Goadsby PJ. The role of dopamine in a model of
trigeminovascular nociception. J Pharmacol Exp Ther 2005;
Campos de Sousa et al: DRD4 Polymorphisms in Migraine
17. Del Zompo M, Cherchi A, Palmas MA, et al. Association be-
tween dopamine receptor genes and migraine without aura in a
Sardinian sample. Neurology 1998;51:781–786.
18. Peroutka SJ, Wilhoit T, Jones K. Clinical susceptibility to mi-
graine with aura is modified by dopamine D2 receptor (DRD2)
NcoI alleles. Neurology 1997;49:201–206.
19. Peroutka SJ, Price SC, Wilhoit TL, et al. Comorbid migraine
with aura, anxiety, and depression is associated with dopamine
D2 receptor (DRD2) NcoI alleles. Mol Med 1998;4:14–21.
20. Dichgans M, Forderreuther S, Deiterich M, et al. The D2 re-
ceptor NcoI allele: absence of allelic association with migraine
with aura. Neurology 1998;51:928.
21. Lea RA, Dohy A, Jordan K, et al. Evidence for allelic associa-
tion of the dopamine beta-hydroxylase gene (DBH) with sus-
ceptibility to typical migraine. Neurogenetics 2000;3:35–40.
22. Maude S, Curtin J, Breen G, et al. The -141C Ins/Del poly-
morphism of the dopamine D2 receptor gene is not associated
with either migraine or Parkinson’s disease. Psychiatr Genet
23. Shepherd AG, Lea RA, Hutchins C, et al. Dopamine receptor
genes and migraine with and without aura: an association study.
24. Stochino ME, Asuni C, Congiu D, et al. Association study be-
tween the phenotype migraine without aura-panic disorder and
dopaminergic receptor genes. Pharmacol Res 2003;48:531–534.
25. Rebaudengo N, Rainero I, Parziale A, et al. Lack of interaction
between a polymorphism in the dopamine D2 receptor gene
and the clinical features of migraine. Cephalalgia 2004;24:
26. Mochi M, Cevoli S, Cortelli P, et al. A genetic association
study of migraine with dopamine receptor 4, dopamine trans-
porter and dopamine-beta-hydroxylase genes. Neurol Sci 2003;
27. Headache Classification Subcommittee of the International
Headache Society. The International Classification of Headache
Disorders. 2nd ed. Cephalalgia 2004;24(suppl 1):1–151.
28. Kienbacher C, Woeber C, Zesch HE, et al. Clinical features,
classification and prognosis of migraine and tension-type head-
ache in children and adolescents: a long-term follow-up study.
29. Karwautz AFK, Campos de Sousa S, Wo ¨ber C, et al. Family-
based analysis of serotonin transporter gene polymorphisms in
migraine with & without aura. Cephalalgia (in press).
30. Freeman B, Powell J, Ball D, et al. DNA by mail: an inexpen-
sive and noninvasive method for collecting DNA samples from
widely dispersed populations. Behav Genet 1997;27:251–257.
31. Plomin R, Crabbe J. DNA. Psychol Bull 2000;126:806–828.
32. Shaikh S, Collier D, Kerwin RW, et al. Dopamine D4 receptor
subtypes and response to clozapine. Lancet 1993;341:116.
33. Dudbridge F. Pedigree disequilibrium tests for multilocus hap-
lotypes. Genet Epidemiol 2003;25:115–121.
34. Van Tol HH, Wu CM, Guan HC, et al. Multiple dopamine
D4 receptor variants in the human population. Nature 1992;
35. Lichter JB, Barr CL, Kennedy JL, et al. A hypervariable seg-
ment in the human dopamine receptor D4 (DRD4) gene.
Hum Mol Genet 1993;2:767–773.
36. Chang FM, Kidd JR, Livak KJ, et al. The world-wide distribu-
tion of allele frequencies at the human dopamine D4 receptor
locus. Hum Genet 1996;98:91–101.
37. Brookes K, Xu X, Chen W, et al. The analysis of 51 genes in
DSM-IV combined type attention deficit hyperactivity disorder:
association signals in DRD4, DAT1 and 16 other genes. Mol
38. Smalley SL, Bailey JN, Palmer CG, et al. Evidence that the
dopamine D4 receptor is a susceptibility gene in attention def-
icit hyperactivity disorder. Mol Psychiatry 1998;3427–3430.
39. Swanson J, Oosterlaan J, Murias M, et al. Attention deficit/
hyperactivity disorder children with a 7-repeat allele of the do-
pamine receptor D4 gene have extreme behavior but normal
performance on critical neuropsychological tests of attention.
Proc Natl Acad Sci USA 2000;97:4754–4759.
40. Ding YC, Chi HC, Grady DL, et al. Evidence of positive se-
lection acting at the human dopamine receptor D4 gene locus.
Proc Natl Acad Sci USA 2002;99:309–314.
41. Wang E, Ding YC, Flodman P, et al. The genetic architecture
of selection at the human dopamine receptor D4 (DRD4) gene
locus. Am J Hum Genet 2004;74:931–944.
42. Jovanovic V, Guan HC, Van Tol HH. Comparative pharma-
cological and functional analysis of the human dopamine D4.2
and D4.10 receptor variants.
43. Oak JN, Oldenhof J, Van Tol HH. The dopamine D(4)
receptor: one decade of research. Eur J Pharmacol 2000;405:
44. Asghari V, Sanyal S, Buchwaldt S, et al. Modulation of intra-
cellular cyclic AMP levels by different human dopamine D4
receptor variants. J Neurochem 1995;65:1157–1165.
45. Schoots O, Van Tol HH. The human dopamine D4 receptor
repeat sequences modulate expression. Pharmacogenomics J
46. Noain D, Avale ME, Wedemeyer C, et al. Identification of
brain neurons expressing the dopamine D4 receptor gene using
BAC transgenic mice. Eur J Neurosci 2006;24:2429–2438.
47. Pietrobon D, Striessnig J. Neurobiology of migraine. Nat Rev
48. Chronicle EP, Pearson AJ, Mulleners WM. Objective assess-
ment of cortical excitability in migraine with and without aura.
49. Jasmin L, Burkey AR, Granato A, et al. Rostral agranular insu-
lar cortex and pain areas of the central nervous system: a tract-
tracing study in the rat. J Comp Neurol 2004;468:425–440.
50. Burkey AR, Carstens E, Jasmin L. Dopamine reuptake inhibi-
tion in the rostral agranular insular cortex produces antinoci-
ception. J Neurosci 1999;4169–4179.
Annals of Neurology Vol 61No 6 June 2007