AU737893B2 - Diagnosis of migraine with aura, depression and anxiety from allelic variations in dopaminergic genes - Google Patents

Description

Diagnosis of Migraine with Aura, Depression and Anxiety from Allelic Variations in Dopaminergic Genes Technical Field The present invention relates generally to the diagnosis and treatment of a syndrome characterized by symptoms of migraine with aura, depression and/or anxiety.

Background Migraine headaches are a type of vascular headaches. Migraine headaches are characterized in part as recurrent attacks of headaches, with or without associated visual and gastrointestinal disturbances. Symptoms of migraine headaches usually follow a o1 pattern in each patient. Attacks may be daily or only once in several months. Untreated attacks may last for hours or even days. Nausea, vomiting, photophobia and sonophobia are common. The extremities can become cold and cyanosed, and the patient can become irritable and seek seclusion. In the United States alone, approximately 18 million females and 5.7 million males have been estimated to suffer form severe migraine annually.

Migraines are believed to be a leading cause of lost time from the workplace.

A classification of migraines based on patient 9* r* 8 0** a a [N:\LIBF]08161:bav J, WO 98/07426 PCT/US97/14830 -2symptoms has been proposed by the Committee on the Classification of Headache of the International Headache Society, Cephalalgia 8:1-96 (1988). The committee proposes that migraines be classified a migraine without aura (formerly known as common migraine), migraine with aura (formerly known as classic migraine) and hemiplegic migraine (formerly known as complicated migraine).

Migraine with aura and migraine without aura are the two most frequent forms of migraine.

A locus for familial hemiplegic migraine has been reported to occur on chromosome 19, Joutel et al., Nature Genetics 5, 40-45 (1993); Joutel et al., Am. J. Hum.

Genet. 55, 1166-1172 (1994).; Ophoff et al. Genomics 22, 21-26 (1994); Elliott et al., Ann. Neurol. 39, 100-106 (1996)). However, the genetic bases and corresponding biochemical mechanisms underlying other forms of migraine disease, such as migraine with aura and migraine without aura, have not been reported and it is unknown whether the different types of migraine are different in kind or only in degree.

At present no specific genetic or biochemical tests are available for the positive diagnosis of migraine with or without aura. Diagnosis and treatment is presently based solely on patient self-reporting and symptom description. Although a wide variety of agents, such as anti-inflammatory drugs, ergots, 5-HTi receptor agonists and antiemetics, have been reported to have some benefit for treatment of migraine, their use is complicated by the clinical heterogeneity associated with migraine, side effects of drugs, and the limitations of patient reporting (Caviness et al., N. Engl. J. Med. 302, 446-450 (1980); Wilkinson, Cephalalgia 3, 61-67 (1983); Peatfield, Handbook of Clinical Neurology (ed Rosem Raven Press, New York, 1986) pp. 173-216; Welch, N.E.J.M. 329, 1476-1483 (1993); Peroutka, The Pharmacological Basis of Therapeutics (eds. Hardman et al., McGraw-Hill, New York, 1996) pp. 487-502. For example, there have been several reports that antiemetics such as domperidone, prochlorperzine, and metoclopramide have beneficial WO 98/07426 PCT/US97/14830 -3effects in some migraine patients. However, because the subtype of migraine patient in which such drugs may be effective is not known and because possible side effects of these drugs mitigate against administration without a clear expectation of benefit, these drugs have not been widely administered and have not been approved by the FDA for treatment of migraine. Thus, it has been concluded that, at present, migraine is neither efficiently diagnosed nor managed (Cephalalgia 8, 96 (1988)).

Similarities exist between the epidemiological characteristics of migraine, anxiety and depression (Robins et al., Arch. Gen. Psychiatry 41:949-958(1984); Stewart et al., JAMA 267:64-69 (1992); Rasmussen Eisen, J Clin Psychiatry 55:5-14 (1994); Kessler et al., Arch.

Gen. Psych. 51:8-19 (1994), Marazziti et al., Biol.

Psychiatr. 31:125-129 (1995). All three disorders afflict approximately 10 to 25% of the general population at some point in life and are approximately twice as common in females than males. Prophylactic medications for all three disorders have a subacute onset of action, requiring 3 to 6 weeks of therapy to measure clinical improvement.

Identification of inheritance pattern(s) and genetic bases for migraine, depression and anxiety would greatly facilitate the diagnosis and treatment of these diseases.

The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION In one aspect, the invention is directed to methods of diagnosing a patient for susceptibility to a syndrome characterized by symptoms of migraine with aura, depression and/or anxiety. The methods entail detecting a variant allele of one or more dopaminergic genes in the patient. Dopaminergic genes correlated with the syndrome include DRD1, DRD2, DRD3 and DAT. For example, the presence of homozygous Al alleles of the DRD2 NcoI Al gene indicates increased susceptibility to the syndrome relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence of homozygous A2/A2 alleles. In some methods, variant alleles are detected in two or more of the DRD 1, DRD2, DRD3 and DAT, and risk factors associated with the presence of each variant allele detected are combined to indicate susceptibility to the syndrome.

Accordingly, a first particular aspect of the present invention provides a method of diagnosing a patient for susceptibility to a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms of anxiety, or a combination of these symptoms, comprising detecting a variant allele of two or more dopaminergic genes in the patient, wherein risk factors associated with the presence of each variant allele are io combined to indicate susceptibility to the syndrome.

A second aspect of the present invention provides a method for determining the suitability of a patient suffering from a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety for treatment with an agent that antagonises binding of dopamine to DRD l, DRD2, DRD3 and/or DAT comprising the steps of detecting a Ii variant allele of one or more dopaminergic genes in the patient.

The invention further provides methods of treating a patient suffering from a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety comprising administering to a patient a therapeutically effective amount of an agent that antagonises binding of dopamine to DRD 1, DRD2, DRD3 and/or DAT. Some such agents 20 lack specific binding to DRD4 and/or DRD5. Exemplary agents are shown in Table 1.

Some agents are incapable of permeating the blood-brain barrier. Agents can be administered intravenously, orally or intramuscularly. Agents can be administered therapeutically or prophylactically. Patients amenable to treatment with such methods include those suffering from migraine with aura and having homozygous DRD2 NcoI Al S 25 alleles.

i A related aspect of the present invention provides an agent that antagonises binding of dopamine to DRD 1, DRD2, DRD3 and/or DAT when used in treating a patient o suffering from the syndrome characterised by symptoms of migraine with aura, depression and/or anxiety.

A further aspect of the present invention provides a method of treating a patient suffering from migraine with aura and having homozygous DRD2 NcoI Al alleles comprising administering to the patient a therapeutically effective amount of an agent that antagonises binding of dopamine to DRD2.

PIn another aspect, the invention provides methods of screening for a drug effective to treat the syndrome described above. Such drugs are screened by determining their [I:\)ayLib\LIBA103616.doc:nss capacity to antagonise binding of dopamine to a dopaminergic receptor or DAT.

Optionally, such drugs can be screened for lack of specific binding to DRD4 and/or In another aspect, the invention provides for the use of an agent that antagonises S binding of dopamine to DRD1, DRD2, DRD3 and/or DAT for the manufacture of a medicament for use in the treatment of a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety in patients having a variant allele of one or more dopaminergic genes. Such dopaminergic genes include DRD1, DRD2, DRD3 an DAT. Some suitable agents are listed in Table 1. For some agents, the blood-brain I0 barrier is impermeable to passage of the agent. Some agents antagonise DRDI, DRD2, DRD3 and/or DAT without binding to DRD4 or DRD5. In some uses, the variant allele is DRD2 Ncol Al. A related aspect of the present invention provides for an agent that antagonises binding of dopamine to DRD2 when used in treating a patient suffering from migraine with aura and having homozygous DRD2 Ncol Al alleles. The presence of homozygous Al alleles indicates increased susceptibility to the syndrome relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence of homozygous A2/A2 alleles.

Thus, a patient having homozygous DRD2 Ncol Al alleles has a relatively high susceptibility to the syndrome. In some patients, the syndrome is manifested by 20 symptoms of migraine with aura. In some of the uses, the medicament is administered intravenously, orally, or intramuscularly. In some uses noted above, the medicament is administered prophylactically.

In another aspect, the invention provides at least one method for determining the suitability of a patient suffering from a syndrome characterised by symptoms of migraine S 25 with aura, depression and/or anxiety for treatment with an agent that antagonises binding i of dopamine to DRDI, DRD2, DRD3 and/or DAT. The method entails detecting a variant allele of one or more dopaminergic genes in the patient. Some exemplary agents are listed in Table 1. For some agents the blood-brain barrier is impermeable to passage of the agent. Some agents antagonise DRDI, DRD2, DRD3 and/or DAT without binding to DRD4 or DRD5. Such dopaminergic genes include DRDI, DRD2, DRD3 and DAT.

In some methods the variant allele is DRD2 Ncol Al and the presence of homozygous Al alleles indicates increased suitability relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous Al/A2 alleles indicates increased susceptibility A relative to the presence of homozygous A1/A2 alleles. Thus, a patient having S homozygous DRD2 NcoI Al alleles has a relatively high susceptibility to the syndrome.

I :\DavLib\LIH A]03616.doc:nss 23. JUL. 2001 15:06 SPRUSON AND FERGUSON 61292615486 NO. 0843 P. 6 6 In some patients, the syndrome is manifested by symptoms of migraine with aura. In some methods, the agent is administered intravenously, orally or intramuscularly.

The invention further provides for use of an agent that aritagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT for the manufacture of a medicament for therapy. In such uses, a patient is diagnosed for susceptibility to a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety by a method comprising the step of detecting a variant allele of one or more dopaminergic genes in the patient. Often, the one or more dopaminergic genes for which a variant allele is detected are selected from DRD1, DRD2, DRD3 and/or DAT. In some such uses, the variant 1t allele is DRD2 Ncol Al and the presence ofhomozygous Al alleles indicates increased susceptibility to the syndrome relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous Al/A2 allele indicates increased susceptibility relative to the presence of homozygous A2/A2 alleles. Thus, a patient having homozygous DRD2 NcoI Al alleles has a relatively high susceptibility to the syndrome. In some patients, the 15 syndrome is manifested by symptoms of migraine with aura. After diagnosis of the patient, if appropriate, a therapeutically effective amount of an agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT is then administered to the patient. Some exemplary agents are listed in Table 1. For some agents, the blood-brain barrier is impermeable to passage of the agent. Some agents antagonise DRD1, DRD2, DRD3 and/or DAT without binding to DRD4 or DRD5. In some of the above uses, the agent is administered intravenously, orally or intramuscularly. In some uses, the agent is administered prophylactically.

The invention further provides a diagnostic agent or diagnostic agents allelespecific probes and primers), for detecting a variant allele of two or more dopaminergic genes when used in therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms of anxiety, or a combination of these symptoms. Such dopaminergic genes can be selected from DRD1, DRD2, DRD3 and DAT. For example, some diagnostic reagents are used to detect the NcoI Al allele of DRD2.

The invention further provides an agent for detecting a variant allele of one or more dopaminergic genes when used in therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms of migraine with aura. The invention further provides for use of such agent in the therapy, prophylaxis or diagnosis of migraine with aura.

The invention further provides for the use of an agent or agents for detecting a C variant allele of two or more dopaminergic genes for the manufacture of a diagnostic for II. abyLibLBAJ03616.dI...~ 7 use in therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms of anxiety, or a combination of these symptoms. Preferred dopaminergic genes are selected from DRD1, DRD2, DRD3 and DAT. For example, a suitable variant allele is DRD2 Ncol Al. The diagnostic is typically used in the therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety. Further, the diagnostic can be used in the therapy, prophylaxis or diagnosis of migraine with aura.

The invention further provides for the use of an agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT for the manufacture of a medicament for use in the treatment of a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety associated with the presence of a variant allele of one or more dopaminergic genes. In some such uses, a variant allele is present in one or more dopaminergic genes selected from DRD1, DRD2, DRD3 and DAT. In some such uses, the variant allele is DRD2 NcoI Al and the presence of homozygous Al alleles indicates increased susceptibility to the syndrome relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence ofhomozygous A2/A2 alleles. Thus, a patient having homozygous DRD2 Ncol Al alleles has a relatively high susceptibility to the syndrome.

S: In some patients, the syndrome is manifested by symptoms of migraine with aura. Some 20 exemplary agents are listed in Table 1. For some agents, the blood-brain barrier is impermeable to passage of the agent. Some agents antagonise DRDI, DRD2, DRD3 and/or DAT without binding to DRD4 or DRD5. In some uses, the agent is administered intravenously, orally or intramuscularly. In some uses, the agent is administered prophylactically.

oo I :\I.)avi \I BAl 103616, doc: nss WO 98/07426 PCTIUS97/14830 -8- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-1D show the percentage of individuals having migraine with aura for different DRD1, DRD2, DRD3 and DAT alleles.

Figure 2 shows a risk factor analysis of migraine with aura.

DETAILED DESCRIPTION I. General The present invention provides methods of diagnosing and treating a syndrome (mada) characterized by symptoms of, or susceptibility to, migraine with aura, depression and/or anxiety, and resulting, at least in part, from variation in one or more dopaminergic genes. These methods are premised in part on the insight that variations in the dopaminergic genes DRD1, DRD2, DRD3 and DAT are associated with increased susceptibility to migraine with aura, depression and/or anxiety.

DRD1, DRD2 and DRD3 are three of five G proteincoupled receptors (DRD1-DRD5) for which dopamine appears to be the primary neurotransmitter. O'Dowd et al., "Dopamine Receptors," in Handbook of Receptors and Channels (ed. Peroutka, CRC Press, Boca Raton, 1994).

DRD1 is encoded by an intronless gene (Dearry et al., Nature 347:72-76 (1990); Zhou et al., Nature 347:76-80 (1990); Sunahara et al., Nature 347:80-83 (1990)) and is expressed most abundantly in the caudate, nucleus accumbens and olfactory tubercle. DRD1 receptors are thought to act as pre-synaptic autoreceptors modulating neurotransmitter release.

The DRD2 gene has a length of about 270 kb, including six introns, the first of which accounts for about 200 kb of the gene. A cDNA sequence of 2500 bp has been reported, which includes some flanking sequences.

Grandy et al., Proc. Natl. Acad. Sci. 86, 9762-9766 (1989). DRD2s are localized in numerous anatomical locations that are believed to play a major role in the pathogenesis of migraine. The highest density of DRD2s WO 98/07426 PCT/US97/14830 -9are in the substantia nigra and basal ganglia. In the substantia nigra, the DRD2s act as presynaptic autoreceptors which modulate dopamine release (Mengod et al., Neurochem. Int. 20, 33S-43S (1992)). In addition, dopamine receptors have been located directly in vascular beds on cerebral arteries) that are believed to be critical in the pathogenesis of migraine. For example, dopamine receptors have been localized to pial vessels (Oudart et al., Arch. Int. Pharmacodyn. 252, 196-209 (1981); Edvinsson et al., Br. J. Pharmac. 85, 403-410 (1985)) the site of neurogenic inflammation that is believed to play a major role in the headache component of migraine (Moskowitz, Trends Pharmacol. Sci. 13, 307-311 (1992)). DRD2s are also located in the peripheral and/or central sympathetic nervous system.

DRD2s are also located on presynaptic noradrenergic sympathetic ganglia, where they act to inhibit the release from the sympathetic nerve terminals (Clark et al., Acta Endocrine., Suppl. 216 88, 75-81 (1978); Ziegler et al., Clin. Pharmacol. Ther. 25, 137-142 (1979); Mercuro, Eur. J. Clin. Pharmacol. 27, 671-675 (1985); Montastruc et al. Eur. J. Pharmacol. 166, 511-514 (1989)).

The DRD3 gene has a high degree of sequence identity with the DRD2 gene (O'Dowd et al., Handbook of Receptors and Channels: G Protein-coupled Receptors (ed Peroutka, 95-123 (CRC Press, Boca Raton, 1994); Sokoloff et al., Nature 347:146-151 (1990)). Like DRD2, the DRD3 receptor acts as both a postsynaptic receptor and an autoreceptor which inhibits dopamine release (Tang et al., J. Pharmacol. Exp. Ther. 270:475-476 (1994)).

Expression of DRD3 has been localized to the limbic areas of the brain (Mengod et al., Neurochem. Int. 20:33S-43S (1992); Sokoloff et al., Nature 347:146-151 (1990)) suggesting that it may be associated with cognitive, emotional and endocrine functions. Most drugs that interact with DRD2 also interact with similar affinity with DRD3 (Sokoloff et al., Nature 347:146-151 (1990)).

However, the density of expressed DRD3 receptors is low, SWO 98/07426 PCTIUS97/14830 estimated at about 1% of that of DRD2 (Accili et al., Proc. Nat. Acad. Sci. 93:1945-1949 (1996)).

The dopamine transporter (DAT) gene is a key regulatory protein in the dopamine pathway that modulates the amount of dopamine release and re-uptake (Shimada et al., Science 254:576-577 (1991)).

Variant alleles of the dopaminergic genes DRD1, DRD2, DRD3 and DAT probably cause increased susceptibility to migraine with aura, depression and/or anxiety as a result of increased dopaminergic transmission. The proposed role of dopaminergic genes in migraine with aura is consistent with several clinical features of the disease. For example, nausea and/or vomiting are common features of migraine in which dopamine stimulation is likely. Gastrokinetic changes, hypotension and other autonomic nervous system changes are additional migraine symptoms that are consistent with disturbances in dopaminergic neurotransmission. The proposed mechanism is also consistent with previous reports of exaggerated autonomic responses to dopamine agonists in migraine patients. For example, apomorphine has been reported to induce symptoms of migraine, as well as the associated phenomenon of nausea, vomiting, yawning, hypotension and syncope (DelZompo et al., Headache 35, 222-224 (1995)).

II. Analysis of Polymorphisms in Dopaminergic Genes The methods of diagnosis and treatment described below usually require knowledge of the genotype of an individual with respect to polymorphisms in the genes DRD1, DRD2, DRD3, and/or DAT. Exemplary polymorphisms within each of these genes that correlate with migraine with aura, depression and/or anxiety are described in the Examples, as are methods for their detection.

Specifically, these polymorphisms include: an A to G polymorphism in the 5' untranslated region of DRD1 having alleles designated B1 and B2 as described by Cichon et al., Hum. Mol. Genet. 3:209-209 (1994); a C to T WO 98/07426 PCT/US97/14830 -11polymorphism at codon 313 of DRD2 having alleles designated NcoI Al and NcoI A2 (Sarker et al., Genomics 11:8-14 (1991)); an A to G substitution 25 bp downstream from the start codon in DRD3 having alleles designated Al and A2 (Rietschel et al., Psychiatr. Res. 46:253-259 (1993)); and a polymorphic 40 bp repeat in the 3' untranslated region of DAT having alleles designated 9 and 10 (Vandenbergh et al., Genomics 14:1104-1106 (1992)). It is expected that the DRD2 TaqI Al allele (Noble, Science Medicine 3, 52-61 (1996)), which is known to be in linkage disequilibrium with DRD2 NcoI Al, can be used in diagnostic methods in a similar manner to DRD2 NcoI Al.

Other variant forms of the DRD1, DRD2, DRD3 and DAT genes that correlate with migraine with aura, anxiety and/or depression can be identified as follows. The first step is to identify additional polymorphic sites within one of these genes. Such polymorphic sites can be identified either by comparative sequencing of these genes in a population of individuals or from the published literature and databases. For example, several additional polymorphic sites in the DRD2 gene have been published including TaqlB within intron 2, FokI B at position 1105, HphI at position 3208, C311S at position 3413, NcoI at position 3420 and TaqIA within the 3' untranslated region. (Residues in genomic DNA are assigned the same number as the corresponding nucleotide in cDNA when the two are maximally aligned, and nucleotides in the cDNA are numbered according to the convention of Dal Toso, EMBO J. 8, 4025-4034 (1989)).

Having identified the location of a polymorphism and the nature of its polymorphic forms, a correlation is performed between type of polymorphic form and presence or absence of migraine with aura, depression, and/or anxiety in a population. Optionally, the correlation can be determined with respect to combinations of two or more polymorphisms within the same gene. For example, individuals having the NcoI Al allele are subdivided into two classes respectively having Al and A2 alleles of the SWO 98/07426 PCTIUS97/14830 -12- FokI polymorphism. Thus, for example, one can determine whether the NcoI Al/ FokI Al genotype correlates significantly more strongly with migraine with aura than the NcoI Al/FokI A2 genotype.

Variant genes can be detected, for example, by sequencing, allele-specific amplification (Gibbs, Nucleic Acid Res. 17, 12427-12448 (1989)), restriction enzyme analysis, allele-specific probe hybridization assays (Saiki et al., Nature 324, 163-166 (1986)) or singlestranded conformational analysis (Orita et al., Proc. Natl. Acad. Sci. 86, 2766-2770 (1989)). For example, the NcoI Al and A2 alleles can be distinguished by NcoI digestion. Only the A2 allele is cut. Reagents used for detecting variant alleles, such as allele specific probes and primers can be packaged as diagnostic reagents. The diagnostic reagents can bear labels indicating their suitability for use in diagnosis of the mada syndrome or a symptom thereof.

III. Susceptibility Analysis The present data indicate that analysis of DRD1, DRD2, DRD3 and/or DAT gene in a patient can be used as a measure of susceptibility to migraine with aura, depression and/or anxiety. For example, detection of one or both copies of the NcoI Al allele of DRD2 indicates increased susceptibility to migraine with aura, depression and/or anxiety in the patient, and detection of both copies indicates increased susceptibility relative to one copy. Detection of one or both copies of DRD1 Bl indicates increased risk of migraine with aura relative to homozygous DRD1 B2. Detection of homozygous DRD3 A2 indicates increased risk of migraine with aura relative to homozygous or heterozygous DRD3 Al.

Detection of homozygous DAT 10 indicates increased risk of migraine with aura relative to heterozygous DAT 10/9 and probably 9/9 genotype, although the latter occurs with insufficient frequency to have been included in the present analysis.

Although the probability of an individual having any WO 98/07426 PCTIU~S97/14830 -13one variant allele developing the mada syndrome is low (no more than the individual probabilities combine in an additive fashion. The presence of each variant allele associated with the mada syndrome can be assigned a risk factor related to the probability, as discussed in the Examples. Fig. 2 shows the percentage of individuals having migraine with aura as a function of number of risk factors present. It can be seen that no individuals without any dopaminergic risk factor have symptoms of the mada syndrome and about 75% of individuals with all five dopaminergic risk factors have symptoms of the mada syndrome. Individuals with 1-4 risk factors show intermediate frequencies of symptoms of the mada syndrome in relation to the number of risk factors present.

There are probably other genes besides the dopaminergic genes described above having variant forms associated with risk of the mada syndrome. The existence of variant forms of such genes can be detected and correlated with probabilities of susceptibility to the syndrome in similar fashion to the analysis of dopaminergic genes. Combined statistical analysis of dopaminergic genes with other genes still further increases the predictive value of the diagnosis.

The analysis is useful in identifying a subset of patients having a common genetic basis giving rise to the mada syndrome. Such patients are amenable to treatment with antagonists of dopaminergic genes as discussed below. Treatment with such antagonists may be ineffective in other patients, who exhibit similar symptoms to patients with the syndrome, but due to a different genetic basis. This analysis is also useful in distinguishing migraine with aura from migraine without aura, and others diseases, such as stroke, which may present with similar symptoms. That is, a patient with a high number of risk factors for the mada syndrome is more at risk for migraine with aura than for migraine without aura or stroke.

WO 98/07426 PCTIUS97/14830 -14- IV. Methods of Treatment The invention further provides methods of treating patients suffering from, or susceptible to, the mada syndrome. In these methods, a patient having or susceptible to symptoms of migraine with aura, depression and or anxiety is treated with a therapeutically effective dose of an antagonist to one or more of the dopaminergic genes DRD1, DRD2, DRD3 and DAT. Such a dose is sufficient to prevent, arrest or detectably relieve symptoms of migraine with aura, depression and/or anxiety. The dose can be administered prophylactically or therapeutically. The dose can also be administered to pediatric or handicapped patients but who are unable to articulate their symptoms but are known to have variant forms of one or more variant forms of the dopaminergic genes associated with the disease.

In general, the stronger the binding of an antagonist to the dopaminergic protein, the greater the efficacy. Buproprion (2-tert-butylamino-3'chloropropiophenone hydroxide) manufactured by Glaxo Wellcome is an example of a DAT antagonist. A list of DRD2 antagonists and appropriate dosages is provided in Table 1. Many of these antagonists also bind to other dopaminergic receptors, particular DRD3 and DRD4, which are most closely related to DRD2. DRD2 antagonists include phenothiazines (chlorpromazine, fluphenazine, prochlorperazine, promethazine, thioridazine and trifluoperazine), butyrophenomes (droperidol, haloperidol, pimozide, spiperone), thioxanthines (chlorprothixene, thiothixene) and other drugs, such as clozapine. Some antagonists are capable of crossing the blood-brain barrier and therefore capable of antagonizing both central and peripheral dopaminergic receptors.

Other antagonists such as domperidone do not cross the blood-brain barrier and therefore antagonize only peripheral receptors. Some agents such as domperidone, metoclopramide, chlorpromazine, prochloperazine and flunarazine have previously been reported to have some value in treating some migraine patients. However, the WO 98/07426 PCT/US97/14830 mechanism of action was not known, not was it appreciated that the agents are most appropriate for administration to the subset of migraine patients having migraine with aura and variant forms of one or more of the dopaminergic genes DRD1, DRD2, DRD3, DRD4 and DAT.

Antagonists can be used to manufacture medicaments for use in treatment of the syndrome. Antagonists can be mixed with a pharmaceutical carrier, which can be any compatible, non-toxic substance suitable to deliver the antagonist to the patient. Sterile water, alcohol, fats, waxes, and inert solids can be used as the carrier.

Pharmaceutically-acceptable adjuvants, buffering agents, dispersing agents, and the like, can also be incorporated into the pharmaceutical compositions. The concentration of the active agent in the pharmaceutical composition can vary widely, from less than about 0.1% by weight, usually being at least about 1% by weight to as much as by weight or more. Medicaments can be administered intravenously, intramuscularly, subcutaneously, intranasally, cutaneously, via suppository, by inhalation or orally. Methods for preparing parenterally administrable compositions are described in more detail in, for example, Remington's Pharmaceutical Science ed., Mack Publishing Company, Easton, Pennsylvania, 1980) (incorporated by reference in its entirety for all purposes).

For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium WO 98/07426 PCT/US97/14830 -16lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours.

Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

V. Screening Drugs The invention further provides methods of screening for novel antagonists of DRD1, DRD2, DRD3 and/or DAT for treatment of the syndrome. Potential agents are screened for specific binding (Kd gM) to human DRD1, DRD2, DRD3 or DAT, optionally in competition with dopamine.

Preferred agents bind with a Kd less than 10 nM and can therefore usually be used at a dose of about mg/patient. Receptor binding assays can be performed as described by Ison Peroutka, Cancer Treatment Reports 637 (1986); Peroutka Snyder, Am. J. Psychiatry 137, 12 (1980) using cloned and expressed human receptors or human receptors located in post-mortem human tissues.

Some agents are screened for lack of specific binding to at least one of dopaminergic receptors DRD4 or DRD5. The agents can also be screened for lack of specific binding to other receptors to minimize side effects. For example, lack of binding to the a-adrenergic receptor minimizes orthostatic hypotensive side-effects.

Preferred agents have a serum half-life of about 24 hr and can reach peak plasma levels within about 15-60 min of administration.

WO 98/07426 PCTIS97/14830 -17-

EXAMPLES

Correlations Between Migraine with Aura and Dopaminergic Genes This example describes analysis of allelic variants within all five dopamine receptor genes (DRD1, DRD2, DRD3, DRD4 and DRD5) and the DAT gene in control, migraine without aura (MO) and MWA individuals.

A. METHODS 1. Subiects Subjects were identified for this study by physician referral. Individuals were evaluated using the diagnostic criteria for MO and MWA established by the International Headache Society (Cephalalgia 8, 1-96 (1988)). The lifetime presence or absence was determined for each of the criteria in the IHS definition of migraine. Interviews were conducted by physicians, nurses and/or trained interviewers. All interviewers were trained by the present inventor in the use of the IHS criteria and all clinical data were reviewed by the same neurologist. Control group individuals did not meet IHS criteria for migraine (based on direct interview) and were predominantly unaffected spouses of the individuals with migraine. Informed consent was obtained and DNA samples collected. All clinical data were obtained independently of the genotypic data. The average age of the study participants is 53 1 years. No variation was observed between the 3 study groups in terms of age, sex or ethnic origin.

2. Genotypina A total of 246 DNA samples from unrelated individuals, who were 35 years of age or older, were analyzed (115 control individuals; 77 MO individuals; 54 MWA individuals). Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems, Research Triangle Park, North Carolina). Genotypes were scored independently by two individuals blinded to the clinical status.

WO 98/07426 PCT/US97/14830 -18- 3. DRD1. DRD2. DRD3 and DRD1, DRD2, DRD3 and DRD5 were amplified as follows.

Briefly, 40 ng of genomic DNA was amplified in 10 gL of a solution containing lx Perkin Elmer PCR amplification buffer, 400 AM each dNTP, 0.5 U TaqGold polymerase (Perkin Elmer, Foster City, CA) and 1 AM primers. The enzyme was activated with an initial incubation at 94 0

C

for 10 minutes, followed by 14 cycles of amplification with denaturation at 94 0 C for 20 seconds, Annealing at 63 0 C for 1 minute, elongation at 72 0 C for 30 seconds with a decrease of 0.5 0 C and 3 seconds for each annealing step, and an additional 40 cycles of denaturation at 94 0 C for seconds, annealing at 56 0 C for 30 seconds and elongation at 72 0 C for 1 minute. After amplification, 10 uL of a solution containing 2x restriction enzyme buffer and 4 U of the appropriate restriction enzyme were added directly to the amplification reaction and incubated at 37 0 C for greater than 4 hours. Digested products were separated on SFR agarose gels (Amresco, Solon, OH). Primers used for amplification of each marker, restriction enzymes used to distinguish genotypes and the expected sizes of each allele are listed in Table 2.

ALLELE IDENTIFICATION METHODS [at Refeene 0801 5'D1.0 ATTCAGGGGCTTTCTGGTG Ddel B1 265 35301I.D AGCAGGGAATAGGGGTCAGI 02 223 0802 DR02.35 ATCCTGCAGCCATGG AtcI Al 450 30802.38 AITGTCCGGCTTTACC A2 250 0803 08D3.PCRl.l GCTCTATCTCCAACTCTCACA MscI Al -3D4 38DRD3.PCRl .2 AAGTCTACTCACCTCCAGGTA A2 200 0804 DRD4.SB.PCR3 GTGCACCACGAAGAAGGG 48-hp repeat 4 500 390RD4.SB.I'C84 GCTGCTGCTCTACTGGGC 7- 750 0805 DRD5-3' GGGTTGAGTGAGGAGTTAG Eco57I Al 360 40DR05.S8.PCR2 GACGTGAATGCAGAGAACTG A2 460 OAT T3-5Long TGTGGTGTAGGGAACGGCCTGAG 40-hp repeat 9 440 33T7-3al on, CTTGnAnnTrArnnrTrAAG 10 *480 4. DRD DRD4 genotypes were assessed by amplifying 50 ng of genomic DNA in 10 AL of lx ThermoPol buffer (New England Biolabs), 400 uM dNTP, 1 UM of each primer (see Table 2) and 0.2 U Vent (exo&) Polymerase. Amplification consisted of 35 cycles of denaturation at 98 0 C for 1 WO 98/07426 PCT/US97/14830 -19minute and annealing/elongation at 70 oC for 5 minutes.

Amplification products were analyzed on 1.2% agarose gels.

DMA

DAT genotypes were assessed by amplifying 40 ng of genomic DNA in the presence of 0.5 AM of fluorescencelabelled dUTP (Applied Biosystems, Foster City, CA).

Reactions progressed through 35 cycles of denaturation at 94 OC for 1 minute and annealing/elongation at 72 °C for 1 minute. Amplified products were analyzed on an ABI 373 sequencer using a 6% polyacrylamide gel. Analysis was performed as described previously (Vandenbergh et al., Mol. Brain Res. 15, 161-166 (1992)); Doucette-Stamm et al. Genet. Epidemiol. 12, 303-308 (1995)).

B. RESULTS 1. DRD1 5' UTR B1 and B2 Allele Frequencies An A to G polymorphism has been described in the untranslated region of the DRD1 gene (Cichon et al., Hum. Mol. Genet. 3, 209-209 (1994)). In the current dataset (n 246 individuals), the DRD1 Bl allele frequency is 0.37 and the B2 allele frequency is 0.63.

These values are similar to the DRD1 B1 and B2 allele frequencies reported in Caucasians In the overall dataset, 16% of individuals have the B1/B1 genotype, 41% have the B1/B2 genotype and 43% display the B2/B2 genotype.

An association of MWA and the DRD1 B1 allele is apparent in an analysis of genotype distributions (Table No significant difference is observed in the genotypic distribution between the control group and individuals with MO. The B2/B2 genotype in the dataset is significantly less frequent in individuals with MWA than in control individuals and individuals with MO (Chi-square 6.28; p 0.006). MWA is observed in 14% of the B2/B2 individuals, 26% of the Bl/B2 individuals and 31% of the B1/B1 individuals (Figure 1A).

WO 98/07426 PCTIUS97/14830 TABLE 3 DOPAMINE D1 RECEPTOR POLYMORPHISM FREQUENCIES IN A SAMPLE OF 246 INDIVIDUALS SGenotypes Allele Frequencies 81181 I% 81182 82/82 81 Controls In= 115) 15 45 55 0.33 0.67 MO Subjects In=77) 12 30 35 0.35 0.65 MWA Subjects 64) 12 27 15 0.47 .53' TOTAL In=246) 39 102 141%) 105 0.37 0.63 Chi-square 6.71 (p 0.01) vs. control group *Chi-square 3.91 (p 0.02) vs. MO group Chi-square 6.75 (p 0.009) vs. the combined control and MO group The DRD1 B1 and B2 allele frequencies were also determined in each subgroup of subjects. Similar allele frequencies are observed in both the control group and individuals with MO. By contrast, individuals with MWA have a significantly greater frequency of the DRD2 B1 allele (0.47) than either the control group (Chi-square 6.71; p 0.01) or individuals with MO (Chi-square 3.91; p 0.02).

2. DRD2 NcoT Al and A2 Allele Frequencies A C to T polymorphism, resulting in a silent mutation at amino acid 313, has been described in the DRD2 gene (Sarkar et al., Genomics 11, 8-14 In the current dataset, the DRD2 NcoI Al allele frequency is 0.73 and the A2 allele frequency is 0.27. These values are similar to the DRD2 NcoI allele frequencies reported in the North American population In the overall dataset, 54% of individuals have the A1/A1 genotype, 37% have the A1/A2 genotype and 8% display the A2/A2 genotype.

An association of MWA and the DRD2 Al allele is apparent in an analysis of genotype distributions (Table No significant difference is observed in the genotypic distribution between the control group and individuals with MO. The A1/A1 genotype in the dataset is significantly more frequent in individuals with MWA than in control individuals and individuals with MO (Chi-square 5.50; p 0.01). MWA is observed in 5% of the A2/A2 individuals, 17% of the A1/A2 individuals and 28% of the A1/A1 individuals (Figure lA).

WO 98/07426 PCTfUS97/14830 -21- TABLE 4 DOPAMINE D2 RECEPTOR NcoI POLYMORPHISM FREQUENCIES IN A SAMPLE OF 246 INDIVIDUALS Genotypes Allele Fre 5 Al/Al AlIA2 A9/A lot A quencies ao Controls 115) 57 48 10 0.70 0.30 MO Subjects (n=77) 40 28 9 0.70 0.30 MWA Subjects 64) 37 16 (30% 1 0.83 TnTAI In= 9AR 4A t n. 1. 91G Ig% 20 1 0,73 0 27 Chi-square 6.45 (p 0.01) vs. control group **Chi-squere 5.99 (p 0.01) vs. MO group Chi-square 7.28 (p 0.007) vs. the combined control and MO group The DRD2 NcoI A allele frequencies were also determined in each subgroup of subjects (Table Similar allele frequencies were observed in both the control group and individuals with MO. By contrast, individuals with migraine with aura had a significantly greater frequency of the DRD2 Al allele (0.83) than either the control group or individuals with migraine without aura.

3. DRD3 Al and A2 Allele Frequencies A polymorphism resulting in a glycine to serine substitution at position 9 in the N-terminal part of the DRD3 receptor was analyzed (Lannfelt et al., Psychiatr.

Genet. 2, 249-256 (1992)). The polymorphism consists of a A to G substitution which is 25 bp downstream from the start codon, creating a restriction site for Ball. This polymorphism has been hypothesized to play a role in receptor insertion into the cell membrane (Rietschel et al., Psychiatr. Res. 46, 253-259 (1993).

In the overall dataset, the DRD3 Al allele frequency is 0.62 and the A2 allele frequency is 0.37. These values are similar to the DRD3 allele frequencies reported in previous studies In the overall dataset, 37% of individuals have the A1/A1 genotype, have the A1/A2 genotype and 12% display the A2/A2 genotype.

No significant difference was observed in the genotypic distribution between the control group and individuals with MO (Table However, the DRD3 A2/A2 genotype in the dataset is significantly more frequent in individuals with MWA than in control individuals and individuals with MO (Chi-square 4.32; p 0.02).

WO 98/07426 PCT/US97/14830 -22- MWA is observed in 22% of the Al/Al individuals, 19% of the A1/A2 individuals and 37% of the A2/A2 individuals (Figure IC). The DRD3 A2 allele frequencies is increased in MWA compared to both control and MO individuals.

However, this difference does not reach statistical significance.

TABLE DOPAMINE D3 RECEPTOR POLYMORPHISM FREQUENCIES IN A SAMPLE OF 246 INDIVIDUALS Genotypes Allele Frequencies A1/A1 A1/A2 l%l A2/A2 A1 A2 Controls (n=115) 45 58 12 0.64 0.36 MO Subjects (n=77) 27 43 7 0.63 0.37 MWA Subjects (n=54) 20 23 11 0.58 0.42 TOTAL ln= 246) 92 124 30 0.63 0.37 SChi-square 4.32 (p 0.02) vs. the combined control and MO group 4. DRD4 Allele Frequencies The DRD4 gene contains a 48-bp sequence in the third cytoplasmic loop of the receptor that ranges from 2- to 8-fold repeat units (Van Tol et al., Nature 358, 149-152 (1992). In the current dataset, genotypes were obtained on 238 of the 246 individuals. The observed allele frequency distribution is similar to the DRD4 allele frequencies reported in the North American population 2 repeats (n 38; 0.08 allele frequency), 3 repeats (n 26; 0.05), 4 repeats (n 310; 0.65), repeats (n 1; 0.01) and 7 repeats (n 101; 0.21).

In the current dataset, 106 individuals display the most common genotype 4/4) whereas 91 individuals have at least one 7 allele (Table 6).

TABLE 6 DOPAMINE D4 RECEPTOR POLYMORPHISM FREQUENCIES IN A SAMPLE OF 238 INDIVIDUALS Genotypes Allele Frequencies 7 allele 4. 7 7 allele present aenotype absent 4 allele 7 allele Controls 110) 38 30 72 0.70 0.18 MO Subjects (n=75) 34 20 41 0.57 0.27 MWA Subjects (n =53) 19 13 34 0.65 0.20 TOTAL 2381 91 63 147 162%1 0.65 0.21 No significant difference is observed in the WO 98/07426 PCT/US97/14830 -23genotypic distribution between the control group, individuals with MO and individuals with MWA (data on the most common genotypes are summarized in Table MWA is observed in 21% of individuals with a 7 allele and 23% of individuals without a 7 allele. Similar allele frequencies are observed in the control group, individuals with MO and individuals with MWA (Table 6).

DRD5 Al and A2 Allele Frequencies In the current dataset, the DRD5 Al allele frequency is 0.68 and the A2 allele frequency is 0.32.

These values are similar to the DRD5 allele frequencies reported in the North American population (Sommeret al., Hum. Genet. 92, 633-634 (1993)). In the overall dataset, 47% of individuals have the A1/A1 genotype, 43% have the A1/A2 genotype and 10% display the A2/A2 genotype.

No significant difference was observed in the genotypic distribution between the control group, individuals with MO and individuals with MWA (Table 7).

MWA is observed in 22% of the A1/A1 individuals, 23% of the A1/A2 individuals and 16% of the A2/A2 individuals.

Similar allele frequencies are observed in the control group, individuals with MO and individuals with MWA (Table 7).

TABLE 7 DOPAMINE D5 RECEPTOR POLYMORPHISM FREQUENCIES IN A SAMPLE OF 246 INDIVIDUALS Genotypes Allele Frequencies Al/A1 Al/A2 A2/A2 Al A2 Controls (n=115) 58 45 12 0.70 0.30 MO Subjects (n=77) 32 36 9 0.65 0.35 MWA Subjects(n=54) 26 24 4 0.70 0.30 TOTAL (n=2461 116 105 25 0.68 0.32 6. DAT 9 and 10 Allele Frequencies A polymorphic 40 bp repeat in the 3' untranslated region as been identified in the DAT gene (Vandenbergh et al., Genomics 14, 1104-1106 (1992)). In the overall dataset, the DAT 9 allele frequency is 0.22, the allele frequency is 0.77 and more rare alleles have a WO 98/07426 PCT/US97/14830 -24frequency of 0.01. These values are similar to the DAT allele frequencies reported in previous studies In the overall dataset, 35% of individuals have the 9/10 genotype and 59% display the 10/10 genotype. The less frequent DAT genotypes polymorphisms are as follows: 9/9 (n 11), 1/2 (n 2) and 2/8 Since only 14 individuals have these less frequent genotypes, these individuals were not analyzed independently in the present study.

No significant difference was observed in the genotypic distribution between the control group and individuals with MO. An association of MWA and the 10/10 genotypes is observed in the present study (Table 8).

MWA is observed in 13% of the 9/10 individuals and 25% of the 10/10 individuals (Figure 1D). The 10/10 genotype frequency is significantly higher in the MWA group (69%) in comparison to the 9/10 genotype frequency in the control subjects Chi-square 4.46; p 0.02) and to the MO group Chi-square 4.41; p 0.02). A higher level of statistical significance is observed when the 10/10 genotype frequency in the MWA group is compared to the 9/10 genotype in the combined control and MO groups Chi-square 6.46; p 0.006).

TABLE 8 DOPAMINE TRANSPORTER POLYMORPHISM FREQUENCIES IN A SAMPLE OF 246 INDIVIDUALS Genotypes Allele Frequencies S9/10 10/10 other 9 Controls (n=115) 44 65 6 0.22 0.78 MO Subjects (n=77) 31 44 2 0.22 0.77 MWA Subjects (n=54) 11 37 6 0.21 0.79 TOTAL (n=243) 86 146 14 0.22 0.78 Chi-square 4.46 (p 0.02) vs. control group Chi-square 4.41 (p 0.02) vs. MO group 0 Chi-square 6.46 (p 0.006) vs. combined control and MO group 7. Allelic "Risk Factor" Assessment Risk factor analysis has proven to be an important approach to the assessment and management of cardiovascular disease (Kannel, Hosp. Pract. 25, 119-130 (1990); Wilson, Am. J. Hypertens. 7, 7S-12S (1994); WO 98/07426 PCTILJS97/14830 Hancock, Scientific American Medicine (eds Dale, D. C. Federman, D. 1-12 (Scientific American, Inc., New York, 1995). Applying the principles of risk factor analysis to MWA, an analysis of the data was performed based on the allelic variants which showed independent associations with MWA. The goal of this analysis was to determine if additive and/or synergistic effects exist between the dopaminergic genes in terms of MWA.

Based on the data in the present study, 5 specific allelic "risk factors" were identified that were associated independently with an increased susceptibility to MWA compared to alternative genotypes at the same molecular location within the dopaminergic genes: the DRD1 B1/B1 or B1/B2 genotype, the DRD2 A1/Al genotype, the DRD2 A1/A2 genotype, the DRD3 A2/A2 genotype and the DAT 10/10 genotype. Although the strength of the associations varied amongst the different genes, an initial attempt to develop a "risk factor" profile for MWA assigned equal weight to each allelic "risk factor" with a single exception: the DRD2 Al allele. Individuals with the DRD2 A1/A1 genotype were assigned a risk factor of "2" since the frequency of MWA appeared to be related to the number DRD2 Al alleles present in an individual (Table 4).

The number of allelic "risk factors" were determined for each individual in the present study dopaminergic allelic "risk factors" per individual). The frequency of MWA was then determined in each allelic "risk factor" group. As summarized in Figure 2, MWA was present in 0% of individuals (n 6) with 0 "risk factors", 11% of individuals (n 19) with 1 "risk factor", 14% of individuals (n 69) with 2 "risk factors", 18% of individuals (n 92), with 3 "risk factors", 39% of individuals (n 56) with 4 "risk factors" and 75% of individuals (n 4) with all 5 dopaminergic allelic "risk factors".

Finally, MWA frequency was determined in individuals with 0, 1 or 2 allelic "risk factors" vs. individuals with 4 or 5 allelic "risk factors". Individuals with 4 or WO 98/07426 PCTIUS97/14830 -26allelic "risk factors" have a significantly greater (Chisquare 16.67; p 0.00002) incidence of MWA than individuals with 0,1 or 2 allelic "risk factors" II. Association Between Comorbid Migraine. Anxiety and Depression and DRD2 NcoI Alleles In epidemiological studies, a clinical diagnosis of migraine significantly increases the risk of comorbid anxiety and depression. However, because all of these diseases are probably multifactorial, the fact that a specific genetic locus may correlate with one of these symptoms does not necessarily imply that it correlates with others. This example tests whether variant forms of dopaminergic genes correlate with depression and anxiety as well as with migraine with aura.

A. METHODS The data described in this study are derived from a clinical genetic relational database that was developed initially for the genetic analysis of migraine Peroutka Howell, Towards Migraine 2000, (Amsterdam, Elsevier Science 1996), pp. 35-48. Potential subjects were identified by physician or self-referral. Subjects were evaluated using a semi-structured interview for migraine.

Migraine evaluations were conducted by a neurologist and/or trained interviewer. The lifetime presence or absence was determined for each of the criteria in the International Headache Society (IHS) definition of migraine with (MWA) (Cephalalgia 8, 1-96 (1988).

A semi-structured interview based on the Structured Clinical Interview for DSM-III-R (SCID) (Spitzer et al., Arch Gen Psychiatr 1992;49:624-629), modified to include the criteria of the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) (Diagnostic and Statistical Manual of Mental Disorders. Vol. 4. (Washington, DC, American Psychiatric Association, 1994), pp. pp. 317-391 and 393-444) was used to evaluate anxiety and depressive disorders in the same individuals interviewed for WO 98/07426 PCTIUS97/14830 -27migraine. The interview included questions that were appropriate to establish a DSM-IV-based diagnosis of generalized anxiety disorder (GAD), phobias, panic attacks, panic disorder, obsessive-compulsive disorder (OCD) and major depression. Interviews were performed by physicians or trained psychiatric nurses. Diagnoses required the concurrence of at least 3 physicians.

Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems, Research Triangle Park, North Carolina). Genotyping of the DRD2 NcoI polymorphism was performed using previously described primers (Sarkar et al., Genomics 11,8-14 (1991).

Briefly, 40 ng of genomic DNA was amplified in 10 uL of a solution containing Ix Perkin Elmer PCR amplification buffer, 400 uM each dNTP, 0.5 U TaqGold polymerase (Perkin Elmer, Foster City, CA) and 1 uM primers DRD2.35 (ATCCTGCAGCCATGG) and DRD2.38 (ATTGTCCGGCTTTACC). The enzyme was activated with an initial incubation at 94 0

C

for 10 minutes, followed by 14 cycles of amplification with denaturation at 94 0 C for 20 seconds, annealing at 63 0 C for 1 minute, elongation at 72 0 C for 30 seconds with a decrease of 0.5 0 C and 3 seconds for each annealing step, and an additional 40 cycles of denaturation at 94 0 C for seconds, annealing at 56 0 C for 30 seconds and elongation at 72 0 C for 1 minute. After amplification, 10 uL of a solution containing 2x NEB4 buffer and 2 U NcoI (New England Biolabs, Beverly, MA) were added directly to the amplification reaction and incubated at 37 0 C for greater than 4 hours. The digested products were separated on a 1.2% agarose SFR gel (Amresco, Solon, OH). Analysis of the genotype was performed by two individuals blinded to the clinical status.

B. RESULTS 1. Clinical characteristics of individuals in the present study Direct diagnostic interviews were completed on all individuals in the present study (n 242). A diagnosis was made only if the individual met DSM or IHS criteria WO 98/07426 PCT/US97/14830 -28for the disorders listed in Table 9. If a clear clinical diagnosis could not be made, the subject was not included in any further statistical analyses for that particular disorder. For each of the conditions analyzed, a diagnosis was made in at least 98% of the individuals.

In the overall dataset, 55% (134/242) of individuals were diagnosed with at least one of the clinical disorders analyzed. As shown in Table 9, anxiety disorders are the most common diagnosis, being present in 46% (122/242) of the current dataset. Major depression is the single most common diagnosis 38%) amongst the group of analyzed disorders in the present study. The incidences of panic attacks and phobia are similar. MWA is present in 21% of the individuals. Panic disorder, GAD and OCD are present in less than 20% of the current study group.

TABLE 9 INCIDENCE OF MIGRAINE WITH AURA, ANXIETY DISORDERSAND MAJOR DEPRESSION IN THE INDIVIDUALS (n=242) IN THE PRESENT STUDY The clinical diagnoses were based on DSM criteria for the anxiety disorders and major depression and on IHS criteria for MWA. If a clear diagnosis could not be made, the individual was "not diagnosed" and was not included in further statistical analyses for the disorder.

Affected Unaffected Not diagnosed MWA, anxiety or depression 55% 45% 0% Any anxiety disorder 46% 54% 0% Major Depression 38% 61% 1% Panic Attacks 31% 68% 1% Phobia 29% 69% 2% Migraine with Aura 21% 79% 0% Panic Disorder 19% 81% 1% Generalized Anxiety Disorder 17% 81% 2% Obsessive Comouleive Disorder 14% 86% 0% 2. Freauency of neuropsychiatric disorders based on DRD2 Nco I genotypes The incidences of the various clinical diagnoses based on DRD2 NcoI genotypes is provided in Table 10. A present or past history of MWA, anxiety disorders or major depression is present in 69% of the A1/Al individuals, 53% of the A1/A2 individuals and 22% of the A2/A2 individuals. The incidence of any of these neuropsychiatric diagnoses is significantly higher in the WO 98/07426 PCT/US97/14830 -29- Al/Al individuals when compared to either the A1/A2 individuals (Chi-square=6.53; p 0.005), A2/A2 individuals (Chi-square=15.29; p 0.00005), or the combined A2/any group of individuals (Chi-square=12.72; p 0.0002).

TABLE INCIDENCE OF MIGRAINE WITH AURA, ANXIETY DISORDERS AND MAJOR DEPRESSION IN THE CURRENT DATABASE BASED ON DRD2 NcoI GENOTYPES A1/A1 A1/A2 A2/A2 Chi-square analysis (n=1311 (n=931) n=18) Al/Al vs. A2/anvy value MWA, anxiety or depression 69% 53% 22% 12.72 0.0002 Any anxiety disorder 64% 41% 17% 7.20 0.004 Generalized Anxiety Disorder 23% 11% 11% 6.11 0.007 Major Dep. 45% 33% 17% 5.18 0.01 Panic Attacks 38% 26% 17% 4.96 0.01 Migraine with Aura 26% 17% 6% 4.09 0.02 Phobia 34% 27% 11% 2.60 0.05 Panic Disorder 22% 16% 11% 1.45 n.s.

Obsessive Comoulsive Disorder 14% 16% 0% 0.01 n.s.

The presence of an anxiety disorder is significantly more frequent in the A1/A1 individuals than in either the A1/A2 individuals (Chi-square=3.87; p 0.02), A2/A2 individuals (Chi-square=8.92; p 0.001), or the combined A2/any group of individuals (Chi-square=7.20; p 0.004).

A similar pattern in seen with GAD. Major depression, panic attacks, MWA and phobia are also all increased significantly in the A1/A1 vs. A2/any individuals (Table 10). Although both panic disorder and OCD are more frequent in the A1/A1 vs. A2/any individuals, the difference does nor reach statistical significance.

However, OCD is more frequent in the A1/A1 individuals than in the A2/A2 individuals (Chi-square=2.84; p 0.05).

3. DRD2 Nco I allele frequencies in neuropsychiatric disorders in the current study DRD2 NcoI allele frequencies were determined in individuals based on the presence or absence of the neuropsychiatric disorders analyzed in the present study.

In individuals with MWA, anxiety disorders and/or major depression, the Al allele frequency is 0.80 and the A2 allele frequency is 0.20. In individuals who have none of these neuropsychiatric disorders, the Al allele WO 98/07426 PCTfUS97/14830 frequency is 0.63 and the A2 allele frequency is 0.37.

The difference in the DRD2 NcoI Al allele frequencies between these two groups of individuals is highly significant (Chi-square=17.13; p 0.00002).

Table 11 DRD2 NcoI ALLELE FREQUENCIES IN INDIVIDUALS WITH OR WITHOUT MIGRAINE WITH AURA, ANXIETY DISORDERS OR MAJOR DEPRESSION IN THE PRESENT STUDY Al A2 Chi-spuare D value MWA, anxiety and/or depression 0.80 0.20 17.13 0.00002 No MWA, anxiety or depression 0.63 0.37 In conclusion, the present data indicate that MWA, anxiety disorders and major depression are comorbidly associated with allelic variations in the DRD2 gene. As a result, some manifestations of these diseases may constitute a distinct clinical syndrome resulting from a single underlying genetic variation. The clinical recognition that all three disorders are associated with the same genetic variant has significant diagnostic and therapeutic implications.

The foregoing description of the preferred embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teaching.

All publications and patent applications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually so denoted.

TABLE 1 SPECTRA BIOMEDICAL, INC.

Development DRUG BRAND INDICATIONS DOSE DRD2 OTHER MAJOR SIDE COMIPANY NAME AFFINITY RECEPTORS EFFECTS: CONTACT PERSON.

SED/HYPOTENSION

OTHER

Clinically Effective DRD2 Anti-Migzraine Dru~s haloperidol Haldol psychosis 2-20 mg 4 alpha =14 McNeil Tourette's ADHD P0 IM 5HIT2 =45 domperidone Motilium 10-30 mg 6 alpha =74 Janssen (Europe) (Europe) PO prochlorperazine Compazine psychosis 10 mg 7 alpha 200 generic anxiety P0 IM antiemnetic IV PR chlorpromnazine Thorazine psychosis 200-800 mg 25 alpha 4 .1 generic anxiety P0 IM 5HT2 19

(SKB)

antiemetic IV PR

MDI

hiccups flunarizine Sibelium 10-20 mg 110 Janssen (Europe) (Europe) P0 metoclopramide Rglan GE refiux 10 mg 160 alpha 10,000 generic (Robins) antiemetic P0 I! stasis IVIIIII 0

'C

00 TABLE 1 SPECTRA BIOMEDICAL, INC.

~DRD2_Development Program DRUG BRAND INDICATIONS DOSE DRD2 OTHER MAJOR SIDE COMPANY NAME AFFINITY RECEPTORS EFFECTS: CONTACT PERSON

SEDIHYPOTENSION

OTHER

Commercially Available But Untested DRD2 Anta onists risperidone Risperdal psychosis 2-8 mg/day 1 alpha I 10 Janssen 5HT2 Dr. Jim Gibb perphenazine Trilafon psychosis antiemetic 8-32 mg I Schering PO IM

IV

fluphenazine Prolixin psychosis 2-20 mng 2 alpha =8 Apothecon PO IM 5HT2=25....

IV

droperidol Inapsine antiemnetic 2.5-10 mg 2 alpha =I Janssen pre-anes IM 5HT7

IV

trifluoperazine Stelazine psychosis 5-20 mg 3 alpha 68 SKB anxiety P0 IM...

IV

pimozide Orap Tourette's 2-6 mg 3 alpha 78 Gate Pharmaceuticals PO 5HT2 (half life thiothixene Navane psychosis 5-30 mg 3 alpha I I1 Roerig PO IM 5HT2 36...

C,

C=

M

-4

QD

TABLE 1 SPECTRA BIOMEDICAL, INC.

DRD2 Development Program DRUG BRAND INDICATIONS DOSE DRD2 OTHER MAJOR SIDE COMPANY NAME AFFINITY RECEPTORS EFFECTS: CONTACT PERSON

SED/HYPOTENSION

EPS OTHER chiorprothixene Taractin psychosis 50400 mg 8 Roche PO TM thioridazine Meilaril psychosis 150-600 mg 63 alpha =7 Sandoz ___________anxiety/depression PO 5HIT2 =63 loxapine Loxitane psychosis 60-100 mg 100 Lederle P0 TM

___IV

molindone Moban psychosis 5-250 mg 150 Gate Pharmaceuticals PO prometliazine Phenergan antiemetic 25 mg 240 generic (Wyeth P0 TM Ayerst) clozapine Clozaril psychosis 150-450 mg 380 alpha =20 Sandoz PO 5HT2 29 0 agranulocytosis seizures trimetho- Tigan antiemetic 200-250 mg 640 generic (SKB) benzamide mesoridazine Serentil psychosis EtOH 75-300 mg Boehringer Ingelheim abuse P0 EM "Psychoneurotic IV manifestations" TABLE 1 SPECTRA BIOMEDICAL, INC.

DRD2_DevelopmentProgramI DRUG BRAND INDICATIONS DOSE DRD2 OTHER MAJOR SIDE COMEPANY NAME AFFINITY RECEPTORS EFFECTS: CONTACT PERSON

SED/HYPOTENSION

E P S O T H E R thiethyperazine Torecan antiemnetic 10-30 mg .Roxane Labs PR I DRD2 Antaizonists (In Development) amnisulpiride launched psychosis 25-50 mg (half Synthelabo life 5 hrs) nemnonapride launched psychosis 9-36 mg/day (Japan) sultopride launched psychosis Synthelabo zoeielaunched psychosis anxiety 5-HT ant Fuzisawa zuclopenthixol launched psychosis Lundbeck (except Us) sertindole Serdolect psychosis anxiety 12-20 mg 5HT2 ant Abbott and Lundbeck (approved PO alpha 1 ant rzole 7/96)

DI

olanzepine Zyprex (pre- psychosis 2.5-15 mg 5-HT2 ant Lilly LY 170053) registration)

PO

quetiapine Seroquel psychosis 5-11T2 ant Zeneca TABLE 1 SPECTRA BIOMEDICAL, INC.

DRD2 Development DRUG BRAND INDICATIONS DOSE DRD2 OTHER MAJOR SIDE COMPANY NAME AFFINIT RECEPTORS EFFECTS: CONTACT PERSON

SED/HYPOTENSION

OTHER

ziprasidone Phase III psychosis 40-120 mg 5-HT2A ant (l0x Pfizer (CP-88059) depression PO D2) (half-life =5-HT2C ant 4 hours) 5-HTID ant 5-HT1A agonist perospirone Phase III psychosis ant Sumitomo iloperidone Phase III psychosis 110 alpha =0.4 HMR (HP-873) 5-HT2A =6 5-HT2c =43 5-HT6=31 5-HT7=22 setoperone Phase II psychosis 5-HT2 ant RW Johnson AD-5423 Phase 1[ psychosis 15 5HT2 =8 Dainippon mnazepertifie Phase 11 psychosis 3 5-HT1A agonist RW Johnson <3 alphalI ant 3 D3 ant bromerguride Phase HI psychosis 5-HTlA agonist Schering AG ___Lynn Botheil 1 192U90 Phase H psychosis 5-HT2 ant Glaxo Wellcome 5-H1TIA agonist carvotroline Phase 1 psychosis 200 mng 5-H12 ant Wyeth Ayerst Jim Barrett raciopride preclinical 3 Plough TABLE 1 SPECTRA BIOMEDICAL, INC.

DRD2_Development Program DRUG BRAND INDICATIONS DOSE DRD2 OTHER MAJOR SIDE COMPANY NAME AFFINITY RECEPTORS EFFECTS: CONTACT PERSON

SED/HYPOTENSION

OTHER

zalospirone preclinical American Home Products Jim Barrett remoxipride preclinical 110 Astra cyclic benzamides; preclinical psychosis 5-HT7 ant Glaxo 5-HTIA agonist Wellcome P 706-A preclinical psychosis 5-1112 ant Hoechst A.G.

p-9662 preclinical psychosis 5-HT2 ant Hoechst-Roussell ocaperidone preclinical psychosis antiemetic 1-2 mg/day 5-HT2 ant Janssen LEK-882 preclinical psychosis 5-HT2 ant LEK Pharmaceuticals 5-HT1A ant agonist EMD-56551 preclinical anxiety 5-HTla agonist Merck KGaA EMD-67478 preclinical anxiety 5-HTla agonist Merck KGaA EMD-77697 preclinical psychosis anxiety 5-HTla agonist Merck KGaA NNC-22-0031 preclinical psychosis 5-HT3 ant Novo Nordisk A/S ant ORG-10490 preclinical psychosis ant Organon ORG-20223 preclinical psychosis 5-HTla agonist 0 0

-J

TABLE 1 SPECTRA BIOMEDICAL, INC.

DRD2 Development Program DRUG BRAND INDICATIONS DOSE .DRD2 OTHER MAJOR SIDE COMPANY NAME AFFINITY RECEPTORS EFFECTS: CONTACT PERSON

SED/HYPOTENSION

EPS OTHER OPC-14597 preclinical S-16924 preclinical psychosis 5-HT1A ant Servier 5-HT2 ant D4 ant DU-29894 preclinical psychosis 5-HTIA agonist Solvay Duphar umespirone preclinical psychosis agonist Solvay Duphar SM-13496 preclinical psychosis 5-HT2 ant Sumitomo SM-9018 preclinical psychosis _________Sumitomo SDZ-MAR-327 preclinical psychosis 5-HTIA agonist Tsububa 5-HT2 ant DI ZD-3638 preclinical psychosis ant Zeneca alentemol ?psychosis 2 ant Parmacia Upjohn ORG-5222 suspended psychosis 5-11T ant Organon acetophenazine Tindal 40-120 mg not in Us) PO amperoxide inactive psychosis Pharmacia Upjohn/Sandoz HDC-912 preclinical psychosis Sandoz for licensing TABLE 1 SPECTRA BIOMEDICAL, INC.

~DRD2 DevelopmentProgram__________ DRUG BRAND INDICATIONS DOSE DRD2 OTHER MAJOR SIDE COMPANY NAME AFFINITY RECEPTFORS EFFECTS: CONTACT PERSON

SED/HYPOTENSION

EPS OTHER

Claims (45)

1. A method of diagnosing a patient for susceptibility to a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms of anxiety, or a combination of these symptoms, comprising detecting a variant allele of two or more dopaminergic genes in the patient, wherein risk factors associated with the presence of each variant allele are combined to indicate susceptibility to the syndrome.

2. The method of claim 1, wherein the two or more dopaminergic genes are selected from a group consisting of DRDl, DRD2, DRD3 and DAT.

3. The method of claim 1, wherein the variant allele is DRD2 NcoI Al, and the to presence of homozygous Al alleles indicates increased susceptibility to the syndrome relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence of homozygous A2/A2 alleles.

4. The method of claim 1, wherein the variant allele is DRD1 B1 and the presence of homozygous or heterozygous B1 alleles indicates increased susceptibility to the syndrome relative to homozygous B2 alleles. The method of claim 1, wherein the variant allele is DRD3 A2 and the presence of homozygous A2 alleles indicates increased susceptibility to the syndrome relative to homozygous Al alleles. S 20 6. The method of claim 1, wherein the variant allele is DAT 10, and the presence of homozygous D10 alleles indicates increased susceptibility to the syndrome relative to heterozygous D10/D9 alleles.

7. The method of claim 2, wherein variant alleles are detected in each of the DRD1, DRD2, DRD3 and DAT genes and risk factors associated with the presence of each variant allele detected are combined to indicate susceptibility to the syndrome.

8. A method of treating a patient suffering from a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety, comprising administering to the patient a therapeutically effective amount of an agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT.

9. An agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT when used in treating a patient suffering from the syndrome characterised by symptoms of migraine with aura, depression and/or anxiety. The use of an agent that antagonises binding of dopamine to DRD1, DRD2, 'IRL^i^ DRD3 and/or DAT for the manufacture of a medicament for treating a patient suffering [I:\DayLib\LIBA]03616.doc:nss from the syndrome characterised by symptoms of migraine with aura, depression and/or anxiety.

11. The method of claim 8, the agent when used according to claim 9 or the use of claim 10, wherein the agent lacks specific binding to DRD4 and/or

12. The method of claim 8, the agent when used according to claim 9 or the use of claim 10, wherein the agent is any one of the agents shown in Table 1.

13. The method of claim 8, the agent when used according to claim 9 or the use of claim 10, wherein the blood-brain barrier of the patient is impermeable to the agent.

14. The method of claim 8, the agent when used according to claim 9 or the use of to claim 10, wherein the agent is administered intravenously, orally or intramuscularly. The method of claim 8, the agent when used according to claim 9 or the use of claim 10, wherein the agent is administered prophylactically.

16. The method of claim 8, the agent when used according to claim 9 or the use of claim 10, wherein the patient has a variant allele of one or more dopaminergic genes.

17. The method, the agent when used or the use according to claim 16, wherein the one or more dopaminergic genes are selected from DRD1, DRD2, DRD3 and DAT.

18. The method, the agent when used, or the use according to claim 16 or claim 17, wherein the variant allele is DRD2 NcoI Al and the presence of homozygous Al alleles indicates increased susceptibility to the syndrome relative to the presence of 20 heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence of homozygous A2/A2 alleles. S: 19. The method, the agent when used or the use according to any one of claims 16 to 18, wherein the syndrome is characterised by symptoms of migraine with aura.

20. The method, the agent when used or the use according to any one of claims 16 25 to 19, wherein the agent is any one of the agents shown in Table 1.

21. A method of treating a patient suffering from migraine with aura and having homozygous DRD2 NcoI Al alleles comprising administering to the patient a therapeutically effective amount of an agent that antagonises binding of dopamine to DRD2.

22. An agent that antagonises binding of dopamine to DRD2 when used in treating a patient suffering from migraine with aura and having homozygous DRD2 NcoI Al alleles.

23. The use of an agent that antagonises binding of dopamine to DRD2 for the manufacture of a medicament for treating a patient suffering from migraine with aura and v 4 having homozygous DRD2 Ncol Al alleles. [I:\DayLib\LIBA]03616.doc:nss

24. The method of claim 21, the agent when used according to claim 22 or use of claim 23, further comprising determining that the patient has homozygous NcoI Al alleles. A method of screening for a drug effective to treat the syndrome of claim 1, comprising determining the capacity of the drug to antagonise binding of dopamine to DRD1, DRD2, DRD3 and/or DAT.

26. The method of claim 25, wherein the drug lacks specific binding to DRD4

27. A method for determining the suitability of a patient suffering from a 0t syndrome characterised by symptoms of migraine with aura, depression and/or anxiety for treatment with an agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT comprising the steps of detecting a variant allele of one or more dopaminergic genes in the patient.

28. The method according to claim 27, wherein the one or more dopaminergic genes are selected from DRD1, DRD2, DRD3 and DAT.

29. The method according to claim 27 or claim 28, wherein the variant allele is DRD2 Ncol Al and the presence of homozygous Al alleles indicates increased suitability relative to the presence of heterozygous Al/A2 alleles, and the presence of heterozygous A/A2 alleles indicates increased susceptibility relative to the presence of homozygous 20o A1/A2 alleles.

30. The method according to any one of claims 27 to 29, wherein the syndrome is migraine with aura.

31. The method according to any of claims 27 to 30, wherein the agent is any one of the agents shown in Table 1. 25 32. Use of an agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT for the manufacture of a medicament for use in therapy, the therapy comprising the step of: a) diagnosing a patient for susceptibility to a syndrome characterised by i symptoms of migraine with aura, depression and/or anxiety by a method comprising the step of detecting a variant allele of one or more dopaminergic genes in the patient; optionally further comprising the step of b) administering to the patient a therapeutically effective amount of an agent that antagonises binding of dopamine to DRD DRD2, DRD3 and/or DAT. c33. The use according to claim 32, wherein the one or more dopaminergic genes re selected from DRD1, DRD2, DRD3 and/or DAT. I:\DayLib\LIBA]03616.doc:nss 23. JUL 2001 15:06 SPRUSON AND FERGUSON 61292615486 NO. 0843 P. 7 42

34. The use according to claim 32 or 33, wherein the variant allele is DRD2 Ncol Al and the presence of homozygous Al alleles indicates increased susceptibility to the syndrome relative to the presence of heterozygous A1/A2 alleles and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence of s homozygous A2/A2 alleles. The use according to any of claims 32 to 34, wherein the syndrome is migraine with aura.

36. The use according to any of claims 32 to 35, wherein the agent is any one of the agents shown in Table 1.

37. A diagnostic agent or diagnostic agents for detecting a variant allele of two or more dopaminergic genes when used in therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms of anxiety, or a combination of these symptoms.

38. An agent when used according to claim 37, wherein the dopaminergic genes S I s are selected from DRD1, DRD2, DRD3 and DAT.

39. An agent when used according to claim 37 or 38, wherein the variant allele is DRD2 Ncol Al. 0 An agent for detecting a variant allele of one or more dopaminergic genes when used in therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms Yo@ 20 of migraine with aura.

41. Use of an agent or agents for detecting a variant allele of two or more S dopaminergic genes for the manufacture of a diagnostic for use in therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms of anxiety, or a combination of these symptoms.

42. Use according to claim 41, wherein the dopaminergic genes are selected from DRD1, DRD2, DRD3 and DAT.

43. Use according to claim 41 or claim 42, wherein the variant allele is DRD2 NcoI Al.

44. Use of an agent for detecting a variant allele of one or more dopaminergic genes for the manufacture of a diagnostic for use in therapy, prophylaxis or diagnosis of migraine with aura, wherein the syndrome is characterised by symptoms of migraine with aura. Use of an agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT for the manufacture of a medicament for use in the treatment of a (I:\DayUlA\uMBA]3616.Adcn 43 syndrome characterised by symptoms of migraine with aura, depression and/or anxiety associated with the presence of a variant allele of one or more dopaminergic genes.

46. The use according to claim 45, wherein the one or more dopaminergic genes are selected from DRD1, DRD2, DRD3 and DAT.

47. The use according to claim 45 or 46, wherein the variant allele is DRD2 NcoI Al and the presence of homozygous Al alleles indicates increased susceptibility to the syndrome relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence of homozygous A2/A2 alleles. 0t 48. The use according to any one of claims 45 to 47, wherein the variant allele is homozygous DRD2 NcoI Al.

49. The use according to any one of claims 45 to 48, wherein the syndrome is characterised by symptoms of migraine with aura. The use according to any one of claims 45 to 49, wherein the agent is any one of the agents shown in Table 1.

51. The use according to any one of claims 45 to 50, wherein the medicament is administered prophylactically.

52. A method of diagnosing a patient for susceptibility to migraine with aura or susceptibility thereto comprising detecting a variant allele of one or more dopaminergic 20 genes in the patient.

53. The method of claim 52, wherein the one or more dopaminergic genes are selected from a group consisting of DRD DRD2, DRD3 and DAT.

54. The method of claim 52, wherein the variant allele is DRD2 NcoI Al, and the presence of homozygous Al alleles indicates increased susceptibility to the syndrome 25 relative to the presence of heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles, and the presence of heterozygous A1/A2 alleles indicates increased susceptibility relative to the presence of homozygous A2/A2 alleles.

55. The method of claim 52, wherein the variant alleles are detected in two or more of the DRD1, DRD2, DRD3 and DAT genes, and risk factors associated with the presence of each variant allele detected are combined to indicate susceptibility to the syndrome.

56. A method of diagnosing a patient for susceptibility to a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms R Pa of anxiety, or a combination of these symptoms, comprising detecting a variant allele of wo or more dopaminergic genes in the patient, wherein risk factors associated with the [I:\DayLib\LIBA]03616.doc:nss 44 presence of each variant allele are combined to indicate susceptibility to the syndrome, substantially as hereinbefore described with reference to any one of the Examples I II.

57. A method for determining the suitability of a patient suffering from a syndrome characterised by symptoms of migraine with aura, depression and/or anxiety for treatment with an agent that antagonises binding of dopamine to DRD1, DRD2, DRD3 and/or DAT comprising the steps of detecting a variant allele of one or more dopaminergic genes in the patient, substantially as hereinbefore described with reference to any one of the Examples I II.

58. A diagnostic agent or diagnostic agents for detecting a variant allele of two or to more dopaminergic genes for use in therapy, prophylaxis or diagnosis of a syndrome characterised by symptoms of migraine with aura, symptoms of depression, or symptoms of anxiety, or a combination of these symptoms, substantially as hereinbefore described with reference to any one of the Examples I II. Dated 15 January, 2001 is Glaxo Group Ltd. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON I :\DayLib\LIBA)03616.doc:nss