WO2001028539A2 - Agent for treating cephalic pain - Google Patents

Abstract

Use of an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway in the manufacture of a medicament for use in a method of preventing or treating cephalic pain.

Description

AGENT FOR TREATING CEPHALIC PAIN

Field of the mvention

The invention relates to a modulator and to a method of screenin tog for the modulator.

Background to the invention

Cephalic pain disorders are generally multifactorial disorder, many of which have an unknown etiology. Both environmental and genetic factors are thought to contribute to cephalic pain disorders. In the case of migraine familial aggregation is observed, although segregation analysis of the pattern of inheritance of migraine within families indicates multifactorial inheritance (not a simple Mendelian inheritance). This implies that many genes contribute to the genetic predisposition to migraine, making it difficult to identify individual genes in linkage studies.

Summary of the invention

The inventors have shown that polymorphisms in the insulin receptor gene contribute to susceptibility to cephalic pain. The insulin receptor is an important component in the regulation of the glucose and lipid metabolism pathways. The present finding allows the treatment of cephalic pain, and in particular migraine, by the manipulation of components of the glucose and lipid metabolism pathways, in particular by manipulation of the insulin receptor.

Accordingly the invention provides use of an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway in the manufacture of a medicament for use in a method of preventing or treating cephalic pain.

Description of the Figure

Figure 1 shows the principle of the Taqman (trade mark) allelic discrimination assay, adapted to detect a polymorphism according to the invention. Two allelic specific primers, G and A, differ in their sequence at the polymorphic site (either G or A) and in the fluorescent dye attached to their 5' end (either F or H). In the Figure, only the allele corresponding to probe G is present. Probe G can therefore anneal without mismatch to the template and, as Taq DNA polymerase extends from the non- specific primer upstream, the nucleotides containing the fluorescent dye F and quenching agent can be removed from the specific primer by the 5' to 3' endonuclease activity of Taq. Released from the quenching agent, the dye then fluoresces and this can be detected to determine that the allele corresponding to probe G is present in the sample.

Description of sequences in Sequence Listing SEQ ID NO's: 1 to 22 are the sequences of exons 1 to 22 of the insulin receptor gene;

SEQ ID NO: 23 is the complete coding sequence of the insulin receptor mRNA;

SEQ ID NO: 24 is the sequence of the mRNA for the insulin receptor precursor;

SEQ ID NO: 25 is the complete sequence from exons 14 to 17 of the insulin receptor gene, including introns, and

SEQ ID NO: 26 is the amino acid sequence of human PPAR gamma.

Detailed description of the invention

The present invention is concerned with the prevention or treatment of cephalic pain by the use of an agent that modulates, typically agonises, the insulin receptor or insulin receptor signalling pathway. The cephalic pain may be a cluster headache, chronic paroxysmal hemicrania, headache associated with vascular disorders, headache associated with substances or their withdrawal (for example drug withdrawal), tension headache and in particular migraine with aura or migraine without aura.

The agent may modulate the insulin receptor or the insulin receptor signalling pathway (indirectly) by acting on a component which is able to affect (act on) the receptor or pathway. Such a component is one whose natural activity is generally able to affect the receptor or pathway (i.e. it is operatively linked to the receptor or pathway). Any activity of the receptor or pathway may be affected by the component

The agent typically modulates the expression or the activity of the component The component is typically a carbohydrate, lipid, protem or polynucleotide (such as genomic DNA or unsphced or spliced mRNA) The component may be an enzyme such as an enzyme m the glucose or lipid metabolic pathways or a kmase The component may be intracellular or extracellular In one embodiment the component is present m a neuron or a cell m the neurovascuiar network which is cπtical to the generation ot cephalic pain, such as a cell the tπgemmovascular network and all nociceptive connections and afferent modulatory connections to which it is mono- or poly-synaptically linked

The component may mediate a metabolic or other effect of receptor signalling activity such as GLUT4 expression at the cell surface, stimulation ot glucose or 2- deoxyglucose or 3-O-methyl glucose uptake into cells, increased glycogen synthase pho phorylation, activation and glycogen synthesis, decreased polysis, increased fatty acid synthesis and incorporation into tπglyceπde, inhibition of gluconeogenesis m hepatocytes

The component may be part of or directly involved in the intracellular signalling pathway of the insulin receptor, l e the component may be downstream of the receptor A downstream component typically mediates or is part of the intracellular changes which occur due to signalling activity The component may be one which is modified (typically phosphorylated or de-phosphorylated), or whose location in the cell changes, duπng signalling activity The component may be one which is capable of binding the insulin receptor Typically the downstream component is msulm receptor substrate- 1, -2, -3, or -4, p85, Grb2, Gabl, phosphatidyl mositol 3 k ase, pp60, ppl20, son of sevenless (SOS), MAP kinase, seπne phosphatase, threomne phosphatase, tyrosme kinase, ras, raf, syp, she or a G protem

The agent may modulate components related to the glucose or lipid pathways, l e components which are upstream of the insulin receptor The component which the agent modulates may be the msulm receptor itself The agent may thus modulate any of the following activities of the receptor insulin binding, IGF-1 binding, kinase activity (e g tyrosme, threomne or serine kinase activity) autophosphorylation, internalisation, re-cycling, interactions with regulatory proteins, or interactions with signalling complexes. The agent may modulate the ability of the receptor to cause directly (or indirectly through another component) post-translational modifications, such as serine/threonine phosphorylation, dephosphorylation (via serine /threonine- or tyrosine phosphatases) or glycosylation.

The agent may modulate a product which regulates or is part of the expression pathway of the component. The product may be one which is specific to the expression pathway of that component. The agent may act upon the product in any of the ways described herein in which the agent acts upon the component. The product may be the gene from which any of the components is expressed, an RNA polymerase that can express mRNA from the gene, the unspliced mRNA which is transcribed from the gene, factors that aid splicing of the mRNA. the spliced mRNA, nuclear factors that bind to the mRNA and/or transport the mRNA from the nucleus to the cytoplasm, translation factors that contribute to translating the mRNA to protein.

Thus the agent may modulate transcription from the component gene or translation of the component mRNA. Preferably the agent is a specific inhibitor of transcription from the component gene, and does not inhibit transcription from other genes. The agent may bind to the component gene either (i) 5' to the coding sequence, and/or (ii) to the coding sequence, and/or (iii) 3' to the coding sequence. Thus the agent may bind to the promoter, and inhibit the initiation of transcription. As discussed above the agent may bind and inhibit the action of a protein which is required for transcription from the component gene.

The agent may bind to the untranslated or translated regions of the component mRNA. This could modulate the initiation of translation. The agent may modulate, in particular agonise, expression by modulating the rate at which the component is broken down. In particular in the case where the component is the insulin receptor the agent may modulate the expression of different variants of the receptor (e.g. variants produced by different splicing of the mRNA), tissue-specific expression, subcellular localisation or hybridisation with other receptors (e.g. the IGF-1 receptor).

The agent typically has an activity which directly or indirectly (e.g. mediated through any of the components discussed above) results an effect on the msulm receptor or msulm receptor pathway which is generally counter (opposite) to the effect of a polymorphism m the sulm receptor gene which causes susceptibility to migraine The polymorphism will generally cause a change m any of the characteristics of the receptor discussed herein, such as expression, activity, expression variant, cellular localisation or the pattern of expression in different tissues The polymorphism may have an agonist effect, but preferably has an antagonist effect on any of these characteristics of the receptor Generally this will lead to a consequent increase or decrease m particular parts of the activity of the pathway (particular polymorphisms may cause an mcrease m activity in one part of the pathway and also cause a decrease in activity another part of the pathway) The polymoφhism may be any of the following polymorphisms INSBa, INSCa, exon8 poll , exonl 1 poll, exonl7 pol2 (the form of these polymoφhisms will be allele 2 as defined in table 2) exonό poll, exon7 poll, exon7 po!2, exonδ pol2, exon9 ρol3, exonl 4 poll or INSR-c 4479C>T (the form of these polymoφhisms will be allele 1 or 2 which is in linkage disequilibrium with the associated polymoφhism) These polymoφhisms are defined in Table 2 below with reference to the sequence flanking the polymoφhism The polymoφhism may be a polymoφhism at the same location as any of these particular polymoφhisms (in the case of a SNP, it will be an A, T, C or G at any of the locations)

The polymoφhism may be in linkage disequilibrium any of these particular polymoφhisms mentioned above Polymoφhisms which are in linkage disequilibrium with each other m a population tend to be found together on the same chromosome Typically one is found at least 30% of the times, for example at least 40 °0o, 50%, 70% or 90%, of the time the other is found on a particular chromosome in individuals m the population Polymoφhisms which are in linkage disequilibrium with any of the polymoφhisms mentioned herein are typically with 500kb, preferably with 400kb, 200kb, 100 kb, 50kb, lOkb, 5kb or 1 kb of the polymoφhism The polymoφhism is typically an insertion, deletion or substitution with a length of at least 1 , 2, 5 or more base pairs or ammo acids In the case of a gene region polvmoφhism the polymoφhism is typically a substitution of 1 base pair, I e a single polynucleotide polymoφhism (SNP) The polymoφhism may be 5' to the coding region, in the coding region, m an tron or 3' to the coding region

The polymoφhism will have a sequence which is different from or the same as the corresponding region in any one of SEQ ID NO's 1 to 25 Thus the activity of the agent (which is counter to the effect of the polymoφhism) will generally lead to an agonist effect on the receptor or pathway As discussed above the agent may act on a component which is downstream of the msulm receptor Such an agent may or may not have an effect on the receptor but will act on a part of the signalling pathway (in a way which is counter to the effect of the mutation on the pathway)

In one embodiment the agent has a mixed antagonist/agonist effect, acting as an antagonist towards some of the characteπstics or effects of the receptor, whilst acting as an agonist towards other characteπstics or effects of the receptor

Some of the components which are discussed herein will have an agonist effect on the expression or activity of the receptor or pathway, whilst others will have an antagonist effect Thus the activity of some of the components will have an effect which is the same as a mutation that causes susceptibility to migraine and the activity of others will have an effect which is counter to the effect of the mutation Thus the agents which act directly on these components will act as agonists or antagonists (as appropnate) order to lead to an effect on the receptor or pathway which is counter to the effects of the mutation

Typically the activity of the agent will cause at least a 2, 5, 10, 20 or 50 fold mcrease in the expression or activity of (l) the component which it acts on or (n) on the msulm receptor, for example as measured in any suitable m vitro or m vivo assay mentioned herein and typically at any of the administration doses mentioned herein Agents may cause an increase of at least 10%, at least 25%, at least 50%, at least 100%, at least, 200%, at least 500% or at least 1000% in such expression or activity at a concentration of the agent of l g ml^"1, lOug ml ^l, lOOμg ml ^l, 500μg ml ^l, lmg ml ' lOmg ml ', lOOmg ml ' Typically the percentage increase represents the percentage mcrease m expression or activity m a compaπson of assays m the presence and absence of the agent Any combination of the above mentioned degrees of percentage increase and 1/28539

7 concentration of agent may be used to define the agent, with a greater percentage increase at a lower concentration being preferred

Typically the agent binds to 1 , 2 or more of the components under physiological ( vivo) conditions Generally the binding is specific The binding is reversible or irreversible An agent which binds irreversibly dissociates very slowly from the component because it would be very tightly bound, either covalently or non- covalently Reversible binding, m contrast with irreversible binding, is characteπsed by a rapid dissociation of the agent/component complex

Typically the agent will affect the binding of another substance to the component (such as a substance which naturally bind the component) The agent may bind the component at the same site as the substance binds The agent is typically able to compete for, or inhibit, the binding of the substance to the component In one embodiment the agent does not bind the component at a site that overlaps with the site at which the substance binds Typically such an agent does not compete with the substance for binding to component, but may still inhibit the binding of the agent to the component

The agent may or may not cause a change m the structure of the component In one embodiment the agent causes the component to change to a less active or nonfunctional form This change may be reversible or irreversible Typically the component only adopts such a changed form when bound to the agent However the change may be irreversible, for example, if the component is chemically modified or is broken down by the agent, for example by the breaking of peptide bonds

The agent may affect the sensitivity of the receptor to msulm, i e may increase or decrease any insulin binding-dependent activity of the receptor The agent is typically one which can be used to prevent or treat diabetes, such as non- msulm dependent diabetes Typically the agent causes hypoglycemia or antihyperglycemia, stimulates msulm release or reduces the clearance of msulm The agent typically lowers glucose levels by enhancing msulm action action, such as at hepatic sites and/or peπpheral sites Thus the agent will typically increase msul - dependent glucose disposal and/or mhbit hepatic glucose output (HGO)

The agent which activates the receptor may be an agonist or antagonist of a peroxisome proliferator-activated receptor (PPAR), tvpically PPAR alpha or delta, preferably PPAR gamma.

The agent is typically a compound as described in WO 97/31907, ΨO 00/08002 or US-A-5,902,726. For example, the agent may be a compound of general formula (I) or a tautomeric form or a pharmaceutically acceptable salt or solvate thereof:

C0₂R

Alk (1)

O -

wherein A is selected from the group consisting of:

(i) phenyl, wherein said phenyl is optionally substituted by one or more of the following groups; halogen atoms, C,.₆alkyl, C ₃ alkoxy, C,.₃ fluoroalkoxy, nitrile, or -NR⁷R⁸ where R⁷ and R⁸ are independently hydrogen or C,.₃alkyl;

(ii) a 5- or 6- membered heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur; and

(iii) a fused bicyclic ring wherein C represents a heterocyclic

group as defined in point (ii) above, which bicyclic ring is attached to group B via a

B is selected from the group consisting of:

(iv) C,_₆ alkylene;

(v) -MC,.₆ alkylene or C,_₆ alkyleneMC,_.6 alkylene, wherein M is O, S, or -NR² wherein R² represents hydrogen or C_[.₃ alkyl;

(vi) a 5- or 6- membered heterocyclic group containing at least one nitrogen heteroatom and optionally at least one further heteroatom selected from oxygen, nitrogen and sulfur and optionally substituted by C0₃ alkyl; and (vii) Het-C|.₆ alkylene, wherein Het represents a heterocyclic group as defined in point (vi) above; Alk represents C,_.3 alkylene; R' represents hydrogen or C,_.3 alkyl;

Z is selected from the group consisting of:

(viii) -(C,_₃ alkylene) phenyl, which phenyl is optionally substituted by one or more halogen atoms; and (ix) -NR^JR⁴, wherein R³ represents hydrogen or C,_₃ alkyl, and R⁴ represents -Y- (CO)-T-R⁵ , or -Y-(CH(OH))-T-R⁵, wherein:

(a) Y represents a bond, C0₆ alkylene, C₂.₆ alkenylene, C₄.₆ cycloalkylene or cycloalkenylene, a heterocyclic group as defined in point (vi) above, or phenyl optionally substituted by one or more C₁ alkyl groups and/or one or more halogen atoms; (b) T represents a bond, C,_.3 alkyleneoxy, -0- or -N(R⁶)-, wherein R⁶ represents hydrogen or C0₃ alkyl; (c) R^D represents C,_₆ alkyl, C .₆ cycloalkyl or cycloalkenyl, phenyl (optionally substituted by one or more of the following groups; halogen atoms, C,., alkyl, C alkoxy groups, C₀.₃ alkyleneNR⁹R'° (where each R⁹ and R'°is independently hydrogen, C,.₃ alkyl, -SO₂C0₃ alkyl, or -C0₂C,.₃alkyl, -S0₂NHC,.₃alkyl), C₀.₃ alkyleneC0₂H, C₀.₃ alkyleneCO₂C0₃alkyl, or -0CH₂C(0)NH₂), a 5- or 6- membered heterocyclic group as defined in point (ii) above, a bicylic fused ring

wherein ring D represents a 5- or 6-membered

heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur and optionally substituted by (=0), which bicyclic ring is attached to T via a ring atom of ring D: or -C,^ alkyleneMR¹¹ wherein M is O, S, or -NR'² wherein R'² and R" are independently hydrogen or C,.₃ alkyl. 1/28539

10

Such compounds are disclosed in WO97/31907.

The terms C ₃ alkyl or alkylene and C,_₆ alkyl or alkylene as used herein respectively contain 1 to 3 or 1 to 6 carbon atoms and appropriately include straight chained and branched alkyl or alkylene groups, typically methyl, methylene, ethyl and ethylene groups, and straight chained and branched propyl, propylene, butyl and but lene groups. The term C₂.₆ alkenyl or alkenylene as used herein contains 2 to 6 carbon atoms and appropriately includes straight chained and branched alkenyl and alkenylene groups, in particular propenylene or the like. The term C,.₃ alkyleneoxy as used herein denotes -0-C,. alkylene-, wherein

C,_.3 alkylene is substantially as defined above, e.g. -0-CH₂- etc.

The terms C₄.₆ cycloalkyl, C₄.₆ cycloalkylene, C₄.₆ cycloalkenyl and C_4.6 cycloaikenylene include cyclic groups containing 4 to 6 carbon atoms, such as cyclopentane, cyclopentylene, cyclohexane, cyclohexylene, cyclohexene and cyclohexenylene.

The term halogen as used herein includes fluorine, chlorine, bromine and iodine.

The term 5- or 6-membered heterocyclic group as used herein includes 5- or 6- membered unsubstituted heterocycloalkyl groups and substituted or unsubstituted heteroaryl groups, e.g. substituted or unsubstituted imidazolidinyl, piperidyl, piperazinyl, pyrrolidinyl, moφholinyl. pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, imidazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, triazolyl or tetrazolyl.

By substituted heterocyclic group is meant a 5 or 6 membered heteroaryl group substituted by one or more of the following; halogen atoms, C,.₃ alkyl, C,_₃ alkoxy groups, C_0.3 alkylene N R⁹R¹⁰ (where each R⁹ and R¹⁰ is idependently hydrogen, C,_₃ alkyl, -SO₂C,.₃ alkyl or C0₂C,.₃ alkyl, -S0₂NHC,.₃ alkyl), C₀.₃ alkylene CO_:H, C_0.3 alkylene CO.C^ alkyl, -OCH₂C(0)NH₂, -C,.₃ fluoroalkyl, -CN or SC,_.6 alkyl. In formula (I) above, in the case where Y represents a bond, the nitrogen atom of -NR R⁴ is directly linked to -(C=O) or (CH(OH)) of R⁴, ie. Z represents -N(R³)- (C=0)-T-R⁵ or -N(R³)(CH(OH))-T-R⁵. Similarly, m the case where T represents a bond. -(CO) or (CH(OH)) of R⁴ is directly linked to R , le Z represents -N(R^J)-Y- (C=O)-R³ or -N(R^J)-Y-(CH(OH))-R³ It may be the case that both Y and T represent a bond, whereby Z represents -N(Rø-(C=0)-R^Λ or -N(R^J)-(CH(OH))-R³

Aptly A represents any of phenyl, heteroaryl (e g pyπdyl) or

wherein fused ring C represents a 5-membered heteroaryl group contaimng at least one nitrogen heteroatom and optionally a further heteroatom selected from nitrogen and oxygen (e g oxazolyl, imidazolyl) Particularly A represents any of phenyl, pyndyl, piperazmyl, or benzoxazolyl, any of which can optionally be substituted by one or more C, _, alkyl, especially phenyl, piperazmyl, or pyndyl

B suitably represents any of C,._, alkylene (e g methylene), -N(CH₃)C_j- 3alkylene (e g -N(CH₃)(CH₂)₂-) or Het-C, ₀ alkylene, wherein Het represents a 5- membered heterocyclic group containing at least one nitrogen heteroatom and optionally at least one further heteroatom selected from oxygen and sulfur (e g pyrrolid yl, oxazolyl and thiazolyl) and aptly substituted by C_{t 3} alkyl Particularly B represents -N(CH₃)(CH₂)₂, oxazolyl -Ci-6 alkylene, which oxazolyl is optionally substituted by C, ₃ alkyl, or thiazolyl which is optionally substituted by C, ₃ alkyl Appropnately Alk represents methylene

Appropriately R¹ represents hydrogen, methyl or ethyl, especially hydrogen Suitably Z may represent -(C, ₃ alkylene) phenyl substituted by one or more halogen atoms, such as optionally substituted benzyl Preferably Z represents -NR³R⁴ substantially as hereinbefore described Generally R^J represents hydrogen As hereinbefore described, R⁴ represents -Y-(C=0)-T-R\ or -Y-(CH(OH))-T-R³ especially -Y(C=0)-T-R⁵, and particular groupings represented by R⁴ include

Y represents phenyl (optionally substituted by one or more halogen atoms, or one or more C, ₃ alkyl e g methyl groups), T represents a bond or an oxygen atom, and R' represents C, ₃ alkyl or phenyl (optionally substituted by one or more halogen atoms or one or more C_{{ 3} alkyl groups),

Y represents a heterocyclic group substantially as hereinbefore descπbed (e g thienyl), T represents a bond and R³ represents phenyl (optionally substituted by one or more halogen atoms or one or more C_{{ 3} alkyl groups). Y represents C₂.₆ alkenylene- (e.g. propenylene), T represents a bond and R^-1 represents phenyl (optionally substituted by one or more halogen atoms);

Y represents C .₆ cycloalkenylene- (e.g. cyclohexenylene), T represents a bond and R³ represents phenyl; Y represents phenyl, T represents a bond and R³ represents a heterocyclic group substantially as hereinbefore described (e.g. piperidyl);

Y represents a bond, T represents a bond and R³ represents a bicyclic ring

substantially as hereinbefore described (e.g. D represents a

6-membered heterocyclic ring, in particular pyranyl substituted by (C=0));

Y represents phenyl, T represents C_1-3 alkyleneoxy (e.g. -O-CH2-) or N(R⁶)- (e.g. -NH-) and R³ represents phenyl.

Preferably when Z represents NR^JR⁴ R^J represents H and R⁴ represents Y- (C=0)-T-R³ the said NH and said (C-O) are positioned ortho to each other on Y (which is phenyl), T is a bond or -0-, R³ is C,.₆ alkyl, or phenyl (optionally substituted by one or more: halogen atoms, C_[.₃ alkyl, C,.₃ alkoxy groups, C_{0. 3}alkyleneNR⁹R'° where each R⁹andR^l0 is independently hydrogen, C,.₃ alkyl, -SO₂C_[. ₃alkyl, or -CO₂C₁.₃alkyl, - SO₂NHC,_₃ alkyl, C₀.₃ alkyleneCO_:H, C_w alkyleneCO i. ₃alkyl, or - OCH₂C(0)NH₂).

Particularly suitably Y represents phenyl, T represents a bond or -O- and R³ represents C,_₃ alkyl or phenyl e.g. R⁴ represents

o

R¹³ wherein R¹³ represents phenyl or OCH₃.

An appropriate subgroup of compounds according to the present invention can be represented by formula (la) 1/28539

C0₂H

A — B — 0 - NH (la)

Ar

0 -

wherein A and B are substantially as hereinbefore described, and Ar represents phenyl or a 5- or 6- membered heteroaryl group containing at least one heteroatom selected from oxygen, nitrogen and sulfur; and salts and solvates thereof.

Suitably in formula (la), A is selected from phenyl, pyridyl and benzoxazoyl. In particular, A in Formula (la) represents phenyl or pyridyl. Furthermore, B in Formula la) is suitably selected from -NR²C,.₆ alkylene substantially as hereinbefore described and Het-C,.₆ alkylene optionally substituted by C,.₃ alkyl substantially as hereinbefore described. In particular, B in Formula (la) represents -N(CH₃)(CH₂)₂- or oxazolyl-C,_₆ alkylene, which oxazolyl is optionally substituted by C_[.₃ alkyl, e.g. methyl.

A particular subgroup of the compounds of formula 1 are compounds of formula (I): wherein;

A is selected from the group consisting of:

(i) phenyl optionally substituted by one or more halogen atoms; (ii) a 5- or 6- membered heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur; and

(iii) a fused bicyclic ring wherein C represents a heterocyclic

group as defined in point (ii) above, which bicyclic ring is attached to group

B via a ring atom of ring C; /28539

14

B is selected from the group consisting of:

(iv) C,_₆ alkylene;

(v) -NR²C[_₆alkylene, wherein R² represents hydrogen or C,.₃ alkyl; (vi) a 5- or 6- membered heterocyclic group containing at least one nitrogen heteroatom and optionally at least one further heteroatom selected from oxygen, nitrogen and sulfur and optionally substituted by C,_₃ alkyl; and (vii) Het-C,.₆alkylene, wherein Het represents a heterocyclic group as defined in point (vi) above;

Alk represents C,.₃alkylene;

R' represents hydrogen or C,.₃ alkyl; Z is selected from the group consisting of: (viii) -(C,.₃aikylene) phenyl, which phenyl is optionally substituted by one or more halogen atoms; and (ix) -NR³R⁴, wherein R³ represents hydrogen or C,_₃ alkyl, and R⁴ represents -Y-

(C=0)-T-R⁵ , or -Y-(CH(OH))-T-R⁵, wherein:

(a) Y represents a bond, C,.₆ alkylene, C₂.₆ alkenylene, C₄.₆ cycloalkylene or cycloalkenylene, a heterocyclic group as defined in point (vi) above, or phenyl optionally substituted by one or more C,_₃ alkyl groups and/or one or more halogen atoms;

(b) T represents a bond, C,.₃ alkyleneoxy, -0- or -N(R⁶)-, wherein R⁶ represents hydrogen or C,.₃ alkyl; (c) R³ represents C,.₆ alkyl, C₄.₆ cycloalkyl or cycloalkenyl, phenyl optionally substituted by one or more halogen atoms or one or more C_μ3 alkyl groups, a 5- or 6- membered heterocyclic group as defined

in point (ii) above, or a bicyclic fused ring

wherein ring D represents a 5- or 6-membered heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur and 1/28539

15 optionally substituted by (=0), which bicyclic ring is attached to T via a ring atom of ring D; or a tautomeric form thereof, and/or a pharmaceutically acceptable salt or solvate thereof. Preferred examples of the compounds of formula (I) include (S)-(2-benzoyl- phenylamino)-3-{4-[2-(5-methyl-2-phenyl-oxazol-4-yl) efhoxy]-phenyl}propionic acid, and 2-(S)-(l-carboxy-2-{4-{2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]- phenyl}-ethylamino)-benzoic acid methyl ester.

Alternatively the agent may be a compound of general formula (II) or a tautomeric form, pharmaceutically acceptable salt or solvate thereof:

(II) wherein;

R^2"' is hydrogen or C,.₃alkyl;

R^2"2 is hydrogen, or C,.₈aikyl optionally substituted by one or more halogens;

R^2"3 is C,.₆alkyl, C₄.₇cycloalkyl or cycloalkenyl, -OC,.₆alkyl, -NR'R (where each R' is independently hydrogen or C_N3alkyl), a 5 or 6 membered heterocyclic group containing at least one oxygen, nitrogen, or sulfur ring atom (optionally- substituted by one or more halogen, C_halky 1 optionally substituted by one or more halogens, -OC,_.6alkyl optionally substituted by one or more halogens, -CN, or -N0₂), or phenyl (optionally substituted by one or more halogen, C,.₆alkyl optionally substituted by one or more halogens, -OC,.₆alkyl optionally substituted by one or more halogens, -CN, or -N0₂);

R^2"4 is a 5 or 6 membered heterocyclic group containing at least one oxygen, nitrogen, or sulfur ring atom (optionally substituted by one or more halogen, C,_.6alkyl optionally substituted by one or more halogens, -OC,.₆alkyl optionally substituted by one or more halogens, -CN, or -N0₂), or phenyl (optionally substituted by one or more halogen, C₁.₆alkyl optionally substituted by one or more halogens, -OC_u6alkyl optionally substituted by one or more halogens, -NR'R' (as defined above), -CN, or - N0₂); R²° is hydrogen, halogen, or C,.₃alkyl optionally substituted by one or more halogens;

R^2'6 is hydrogen or C,.₃alkyl; X is O or S; and n is 1 , 2, or 3. Such compounds are disclosed in WO 00/08002.

As used above, C,._s alkyl is preferably C_u6 alkyl, and C,.₆ is preferably C,_.3 alkyl. Typical C,.₆ alkyl and C,.₃ alkyl groups are as defined above. Typical C₄_₇ cycloalkyl, C₄.₇ cycloalkenyl and 5- or 6-membered heterocyclic groups and typical halogen atoms are as defined above. Preferably, R^2'1 is hydrogen or methyl. Most preferably, R^2"1 is hydrogen.

Preferably, R^2"2 is C,.₈alkyl optionally substituted by one or more halogens. Preferably, said halogen is fluorine. Most preferably, R^2"2 is straight-chain. Preferably, R^2"3 is pyridine, pyrazine, thiophene, furan, thiazole, or phenyl (any of which may be optionally substituted by one or more halogen, C^alkyl optionally substituted by one or more halogens, -OC,.₆alkyl optionally substituted by one or more halogens, -CN, or -N0₂), or C₄.₇cycloalkyl. Most preferably. R^2"3 is phenyl (optionally substituted by one or more halogen, C,.₆alkyl optionally substituted by one or more halogens, -OC^alkyl optionally substituted by one or more halogens, -CN, or -N0₂). Preferably R^2"4 is phenyl (optionally substituted by one or more halogen, C,.

₆alkyl optionally substituted by one or more halogens, or -OC,._όalkyl optionally substituted by one or more halogens). Preferably, said halogen is fluorine. Most preferably R^2"4 is phenyl either unsubstituted or substituted with 1 , 2, or 3 fluorine atoms. Preferably, R²° is hydrogen, halogen, or C ₃alkyl optionally substituted by one or more halogens. Most preferably R²° is hydrogen. Preferably R^2"6 is methyl or ethyl. 1/28539

17

Preferably n is 2.

Preferably, the carbon atom bonded to C0₂R^2"' is in the S configuration. In other words, preferably, the absolute configuration around that carbon is:

Preferred examples of the compounds of general formula (II) include (2S)-2- ([(Z)-l-methyl-3-oxo-3-phenyl-l-propenyl]amino}-3-{4-(5-methyl-2-phenyl-l,3- oxazol-4-yl) ethoxy]phenyl}propanoic acid and (2S)-3-{4-[2-(5-methyl-2-phenyl- l ,3-oxazol-4-yl)ethoxy]phenyl}-2-{[(Z)-3-oxo-3-phenyl-l -(trifluoromethyl)- 1 - propenyl] amino] propanoic acid.

The agent may be a sulfonylurea (e.g. l -butyl-3-sulfonylurea, tolbutamide, chloφropamide, tolazamide, acetohexamide, glyburide, glipizide or gliclazide), a guanide (guanide or chioroguanide), a biguanide (e.g. phenformin, metformin or buformin) or an -glucosidase inhibitor (e.g. acarbose).

The agent may be selected from thiazolidinediones, such as the compounds of formula (III)

wherein

R^J"' is selected from the group consisting of hydrogen, C,._galkyi, aminoC,_{. 8}alkyl, C^alkylaminoC^alkyl, heteroarylaminoC^alkyl, (heteroaryl)(C,. ₈alkyl)aminoC,.₆alkyl, (C₄_₈cycloalkyl)C_|_₈alkyl, C,.₈alkylheteroarylC|.₈alkyl, 9 or 10 membered heterobicycles which are partially aromatic or substituted 9 or 10 membered heterobicycles which are partially aromatic.

A dashed line ( ) is none or one double bond between the two carbon atoms. Preferably, the dashed line ( ) represents no double bond. 1/28539

Such compounds are described in US-A-5,902,726.

As used in formula (III), C_[_₈ alkyl is preferably C,.₆ alkyl, more preferably C,-₃ alkyl. Typical C,.₆ alkyl and C,_₃ alkyl groups are as defined above. Typical heteroaryl groups are 5- or 6-membered heterocyclic groups as defined above. C_4.8 cycloalkyl is preferably C .₇ cycloalkyl such as those defined above. Typical 9 or 10 membered heterobicyles which are partially aromatic include 10-membered rings containing one or more heteroatoms selected from N, O or S.

Preferred compounds of formula (III) are those wherein R^3"' is selected from (i), (ii) or (iii) below:

(i) a substituted 9 or 10 membered heterobicycle which is partially aromatic such as a group

wherein

R^3"2 and R^3"3 are the same or different and each represents a hydrogen atom or a C,-C₅ alkyl group; R^J_4 represents a hydrogen atom, a C,-C₆ aliphatic acyl group, an alicyclic acyl group, an aromatic acyl group, a heterocyclic acyl group, an araliphatic acyl group, a (C,-C₆ alkoxy)carbonyl group or an aralkyloxycarbonyl group; and

R^J and R^3"6 are the same or different and each represents a hydrogen atom, a C_[-C₅ alkyl group or a C C₅ alkoxy group, or R³° and R^J"6 together represent a C_rC alkylenedioxy group.

Preferably R^3'2, R³0 R^3'5 and R^3'6 are each methyl and R^3"4 is hydrogen. In this case the agent is troglitazone;

(ii) heteroarylaminoC,.₆ alkyl group such as a group

wherein:

R^J" is hydrogen or a C,.₆ alkyl group and n is 1 , 2, 3 or 4. Preferably R^3"7 is methyl and n is 1 , in which case the agent is rosiglitazone.

(iii) a C,._s alkylheteroaryl C ₈ alkyl group such as a group

wherein:

R^3"s is a C,_.6 alkyl group and n is 1, 2, 3 or 4. Preferably R^3"8 is ethyl and n is 1. More preferably the C,_.s alkylheteroaryl C,_.s alkyl group is

in which case the agent is pioglitazone.

In the above groups (i), (ii) and (iii), where R^3'2, R^3"3, R³0 R^3"6, R^3'7 or R^3"8 represents an alkyl group, this may be a straight or branched chain alkyl group for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl.

Where R^3"4 represents an aliphatic acyl group, this preferably has from 1 to 6 carbon atoms and may include one or more carbon-carbon double or triple bonds. Examples of such groups include formyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, hexanoyl, acryloyl, methacryloyl and crotonoyl. Where R^3'4 represents an alicyclic acyl group, it is preferably a cyclopentanecarbonyl, cyclohexanecarbonyl or cycloheptanecarbonyl group. Where R^3"1 represents an aromatic acyl group, the aromatic moiety thereof may optionally have one or more substituents (for example nitro, amino, alkylamino, dialkylamino, alkoxy, halo, alkyl or hydroxy substituents); examples of such aromatic acyl groups include benzoyl, p-nitrobenzoyl, m- fluorobenzoyl. o-chlorobenzoyl, p-aminobenzoyl, m-(dimethylamino)benzoyl, o- methoxy benzoyl, 3.4-dichlorobenzoyl, 3,5-di-t-butyl-4-hydroxybenzoyl and 1- naphthoyl groups. Where R^3"4 represents a heterocyclic acyl group, the heterocyclic moiety thereof preferably has one or more, preferably one. oxygen, sulphur or nitrogen hetero atoms and has from 4 to 7 ring atoms. Where R^J"4 represents an araliphatic acyl group, the aliphatic moiety thereof may optionally have one or more carbon-carbon double or triple bonds and the aryl moiety thereof may optionally have one or more substituents (for example nitro, amino, alkylamino, dialkylamino, alkoxy, halo, alkyl or hydroxy substituents); examples of such araliphatic acyl groups include the phenylacetyl, p-chlorophenylacetyl, phenylpropionyl and cinnamoyl groups. Where R^3"4 represents a (C,-C₆ alkoxy)carbonyl group, the alkyl moiety thereof may be any one of those alkyl groups as defined above and the alkoxycarbonyl group represented by R^J0 Where R"⁴ represents an aralkyloxycarbonyl group, the aralkyl moiety thereof may be any one of those included within the araliphatic acyl group represented by R 0

Where R ° and R^J"6 represent alkoxy groups, these may be the same or different and may be straight or branched chain groups, preferably having from 1 to 4 carbon atoms. Examples include methoxy, ethoxy, propoxy, isopropoxy and butoxy. Alternatively, R³° and R^J"δ may together represent a C C₄ alkylenedioxy group, more preferably a methylenedioxy or ethylenedioxy group.

The agent may also be ciglitazone, darglitazone or englitazone or derivatives of any of the thiozoladinediones (e.g. derivatives referred to in WO 00/35437). Other agents include oxyzolidinediones, such as JTT 501 , and non-chiral acyclic agents, such as GW 262570, as well as substituted 4-hydroxyphenylalcanoic acid derivatives with agonist activity to PPAR gamma. The agent may be a thiazolidinedione as described in U.S. Pat Nos. 5,089,514, 4,342,771, 4,367,234, 4,340,605 or 5,306,726.

The agent may be a beta 3 agonist. The agent may antagonise atypical beta- adrenoceptors which occur in adipose tissue and the gastrointestinal tract. Such agonists have been found to be particularly useful as thermogenic anti-obesity agents and as anti-diabetic agents. These agonists are described for example in WO 97/21665, WO 97/21666, WO 98/43953, WO 99/65877, WO 95/33724, EP 0455006 and EP 0543662. The agent may be selected from non-thiazolidinedione insulin sensitizers such as those dislcosed in Buckle et al (1996) Bioorganic and Medicinal Chemistry Letters 6, 2121-6 and substituted 4-hydroxy-phenylalcanoιc acid drivatives, such as those described WO 97/31907, hypoglycemic alkaloids, such as qumdolme and cryptolepme, which may be obtained from extracts from Cryptolepsis sp as disclosed in US-A-5,629,31 , as well as tπteφenoid substances, such as those disclosed m US-A-5,691,386, and eremophilanohde sesquiteφenes, such as described in US-A-5,747,527

Other suitable agents include polymoφhic iorms of troghtazone, teφenoid- type quinones and C-substituted pentacycloazoles and N-alkyl substituted pentacycloazoles, for example as disclosed in US-A-5,700,820, US-A.-5,674,900, US-A-5, 641 ,796 The disclosure of all the US patents, WO publications and other publications mentioned herein is incoφorated herein by reference

Other agents include those that activate a RXR receptor that forms a heterodimer with PPAR, for example, ligand 100268, which is an RXR receptor ligand The agent may be an angiotensm II antagonist or angiotensm converting enzyme inhibitor In one embodiment the agent is a protem, polynucleotide, carbohydrate, lipid or small organic molecule

The invention may be carried out by admmistenng a substance which provides an agent with any of the above properties in vivo Such a substance is also included in the term "agent' Typically the substance is an inactive or precursor form of the agent which can be processed in vivo to provide the agent Thus the substance may comprise the agent associated, covalently or non-covalently, with a carrier The substance can typically be modified or broken down to provide the agent

The invention provides a method for screening for the agent comprising contacting a candidate substance with a product selected from (I) one or more components as defined above, (n) any part of the expression pathway for a component as defined in (l), or (m) a functional analogue of (l) or (n), and determining whether the candidate substance binds or modulates the product, typically in a manner which increases directly or indirectly the activity or expression of the receptor or pathway The method may be carried out in vitro (inside or outside a cell) or in vivo, l e the product may be provided in a form which is inside or outside a cell, which cell may be in vitro or in vivo In one embodiment the method is carried out on a cell, cell culture or cell extract which comprises the component The cell mav be any of the cells mentioned herein, and is preferably the cell is one in which the component or part is naturally expressed

The method may be earned out m an animal (such as any animal mentioned herein) whose msulm receptor gene comprises a polymoφhism which causes susceptibility to cephalic pain, such as any such polymoφhism mentioned herein Typically such an msulm receptor gene is a polynucleotide provided by the invention (as descnbed below) or comprises sequence from such a polynucleotide

In the case where the product is a functional analogue (m), this will have some or all of the relevant activity of (i) or (n) will have surface that mimics the surtace of (l) or (n) Typically the analogue is or comprises a fragment of (l) or (a) In the case where (l) or (a) is a polv nucleotide or polypeptide the analogue typically has homology with (I) or (u) The product (I), (a) or (in) may be a polynucleotide or protem of the invention as descnbed below Any suitable binding assay format can be used to determine whether the product binds the candidate substance, such as the formats discussed below

The term "modulate' includes any of the ways mentioned herein in which the agent of the invention is able to modulate a component Whether or not a candidate substance modulates the activity of (i) or (a) may be determined by providing the candidate substance to (i) or (a) under conditions that permit activity of (I) or (a), and determining whether the candidate substance ib able to modulate the activity of the component

The activity which is measured may be any of the activities which is mentioned herein, and may the measurement of a change in a component or an effect on a cell or an effect on an ammal in which the method is being carried out The effect may be one which is associated with cephalic pain, and in the case of an animal may be a symptom of cephalic pain, m particular migraine The symptom may be a behavioural change, vomiting, photophobia or phonophobia, or a electrophysiological or vasomodulatory change of the substance may be measured Typically the assay measure the effect of the candidate substance on the binding between the component and another substance (such as a ligand) Suitable assays in order to measure the changes in such interactions include fluorescence imaging plate reader assays, and radiohgand binding assays

In the case where the activity is transcription from a gene of a component the method may comprise measuring the ability of the candidate substance to modulate transcription, for example m a reporter gene assay Typically such an assay comprises

(a) providing a test construct comprising a first polynucleotide sequence with the promoter activity of the gene of the component operably linked to a second polynucleotide sequence to be expressed in the form of mRNA,

(b) contacting the candidate substance with the test construct under conditions that would permit the second polynucleotide sequence to be expressed in the form of mRNA in the absence of the substance, and

(c) determining whether the substance modulates expression from the construct

In a preferred embodiment the method tor screening for the agent determines whether the agent acts as an agonist or antagonist of a PPAR, preferably gamma (e g a PPAR which is the same or homologous to SEQ ID NO 26), in a manner that leads to activation/agonising of msulm receptor activity Such a method may be based on the methods described Willson et al (2000) J Medicinal Chemistry 43,527-550 In the embodiment the method determines whether the agent increases the expression or activity of an RXR ligand which has the desired effect on PPAR, l e an effect which leads to the activation of the insulin receptor

Suitable candidate substances which tested in the above screening methods include antibody agents (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies) Furthermore, combinatorial hbranes, defined chemical identities, peptide and peptide mimetics, oligonucleotides and natural agent libraries, such as display libraries (e g phage display libraries) may also be tested The candidate substances may be chemical compounds, which are typically derived from synthesis around small molecules which may have any of the properties of the agent mentioned herein Batches of the candidate substances may be used in an initial screen of, for example, ten substances per reaction, and the substances of batches which show inhibition tested individually The invention also provides an isolated polynucleotide or protein that comprises (i) a polymoφhism that causes susceptibility to cephalic pain, or (ii) a naturally occurring polymoφhism that is in linkage disequilibrium with (i). Such polymorphisms may be any of the polymoφhisms mentioned herein. The polymoφhism that causes susceptibility may be one which is or which is not found in nature.

The polynucleotide or protein may comprise human or animal sequence (or be homologous to such sequence). Such an animal is typically a mammal, such as a rodent (e.g a mouse, rat or hamster) or a primate. Such a polynucleotide or protein may comprise any of the human polymorphisms mentioned herein at the equivalent positions in the animal polynucleotide or protein sequence.

The polynucleotide or protein typically comprises the insulin receptor gene region sequence or the insulin receptor protein sequence, or is homologous to such sequences; or is part of (a fragment of) such sequences (as discussed below such sequences may be of a human or animal). In particular the part of the sequence may correspond to any of the sequences given herein in or parts of such sequences. The polynucleotide is typically at least 5, 10, 15, 20, 30, 50, 100, 200, 500, bases long, such as at least lkb, lOkb, lOOkb, 1000 kb or more in length. The polynucleotide of the invention is generally capable of hybridising selectively with a polynucleotide comprising all or part of the insulin receptor gene region sequence, including sequence 5' to the coding sequence, coding sequence, intron sequence or sequence 3' to the coding sequence. Thus it may be capable of selectively hybridising with all or part of the sequence shown in any one of SEQ ID NOS: 1 to 25 (including sequence complementary to that sequence).

Selective hybridisation means that generally the polynucleotide can hybridize to the gene region sequence at a level significantly above background. The signal level generated by the interaction between a polynucleotide of the invention and the gene region sequence is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the gene region sequence. The intensity of interaction may be measured, for example, by radiolabelling the polynucleotide, e.g. with ³²P. Selective hybridisation is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and either 0.003 or 0.03M sodium citrate at from about 50°C to about 60°C).

Polynucleotides of the invention may comprise DNA or RNA. The polynucleotides may be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or poly ly sine chains at the 3' and/or 5' ends of the molecule. For the puφoses of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art.

The protein of the invention can be encoded by a polynucleotide of the invention. The protein may comprise all or part of a polypeptide sequence encoded by any of the polynucleotides represented by SEQ ID NO's: l to 25, or be a homologue of all or part of such a sequence. The protein may have one or more of the activities of the insulin receptor, such as being able to bind insulin and/or signalling activity. The protein is typically at least 10 amino acids long, such as at least 20, 50, 100, 300 or 500 amino acids long.

The protein may be used to produce antibodies specific to the polymoφhism, such as those mentioned herein. This may be done for example by using the protein as an immunogen which is administered to a mammal (such as any of those mentioned herein), extracting B cells from the animal, selecting a B cell from the extracted cells based on the ability of the B cell to produce the antibody mentioned above, optionally immortalising the B cell and then obtaining the antibody from the selected B cell.

Polynucleotides or proteins of the invention may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ^J3S, fluorescent labels, enzyme labels or other protein labels such as biotin.

Polynucleotides of the invention can be incoφorated into a vector. Typically such a vector is a polynucleotide in which the sequence of the polynucleotide of the invention is present. The vector may be recombinant replicable vector, which may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.

The vector may be an expression vector. In such a vector the polynucleotide of the invention in the vector is typically operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Such vectors may be transformed into a suitable host cell as described above to provide for expression of the protein of the invention. Thus, in a further aspect the invention provides a process for preparing the protein of the invention, which process comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression of the protein, and optionally recovering the expressed protein.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes. Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.

The invention also provides an animal which is transgenic for a polymoφhism as mentioned above. The animal may be any of the animals mentioned herein. Typically the genome of all or some of the cells of the animal comprises a polynucleotide of the invention. Generally the animal expresses a protein of the invention. Typically the animal suffers from cephalic pain, such as The binding assay generally comprises contacting the candidate substance with the product and determining whether the binding occurs between the candidate substance and the product. The binding may be determined by measuring a characteristic of the product which changes upon binding, such as spectroscopic changes.

The assay format may be a "band shift' system, for example based on determining whether the candidate substance advances or retards the product during gel electrophoresis. The assay may be a competitive binding assay . This determines whether the candidate substance is able to inhibit the binding of the product to an agent which is known to bind to the product, such as an antibody specific for the product.

The agent, polynucleotide, protein of the invention or any of the cells mentioned herein may be present in a substantially isolated form. They may be mixed with carriers or diluents and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case it will generally comprise at least 90%, e.g. at least 95%, 98% or 99% of the dry mass of the preparation.

Homologues of polynucleotide or protein sequences are referred to herein. Such homologues typically have at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99% homology, for example over a region of at least 15, 20, 30, 100 more contiguous nucleotides or amino acids. The homology may calculated on the basis of amino acid identity (sometimes referred to as "hard homology").

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the

National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 1 1 , the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915- 10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001. The homologous sequence typically differ by at least 1 , 2, 5, 10, 20 or more mutations (which may be substitutions, deletions or insertions of nucleotide or amino acids). These mutation may be measured across any of the regions mentioned above in relation to calculating homology. In the case of proteins the substitutions are preferably conservative substitutions. These are defined according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

The formulation of the agent for use in preventing or treating cephalic pain will depend upon factors such as the nature of the substance and the condition to be treated. The agent may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The inhibitors may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The modulators may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.

Typically the agent is formulated for use with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymefhylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes. Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate. pectin, methylcellulose, carboxymefhylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. Solutions for intravenous or infusions may contain as earner, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

A therapeutically effective amount of agent is administered to a patient. The dose of modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1 mg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration.

Preferably, daily dosage levels are from 5 mg to 2 g.

The dose of agent may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A suitable dose may however be from 0.1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. The following Example illustrates the invention

EXAMPLE

Clinical criteria for identifying individuals with migraine

The following criteria were used to identify individuals with specific types of migraine

Migraine without aura

HA (head ache) lasting 4-72 hrs if unsuccessfully treated, HA with at least 2 of the following unilateral pain, pulsating quality, moderate to severe intensity, aggravation by physical activity, - HA with nausea, or vomiting, or photophobia, or phonophobia (at least 1)

Migraine with aura

Aura lasting 4-60 minutes,

FIA defined as above, with onset accompanying or following aura withm 60 minutes

Familial hemiplegic migrame

HA fulfills migrame with aura characteristics, aura includes hemiparesis that may be prolonged (> 60 minutes) at least 1 first-degree relative with similar HAs

Genotyping of individuals for SNPs

Samples were obtained from the saidy group and genomic DNA extracted using a standard kit and a slating out technique (Cambπdge Molecular) The genotypes of the migrameurs with aura and control individuals for individual SNPs within the insulin receptor gene were then determined from the DNA samples obtained using the Taqman allelic discrimination assay For each polymoφhic site the allelic discrimination assay used two allele specific primers labeled with a different fluorescent dye at their 5 ' ends but with a common quenching agent at their 3' ends. Both primers had a 3' phosphate group so that Taq polymerase could not add nucleotides to them. The allele specific primers comprised the sequence encompassing the polymoφhic site and differed only in the sequence at this site. The allele specific primers were only capable of hybridizing without mismatches to the appropriate allele.

The allele specific primers were used in typing PCRs in conjunction with a third primer, which hybridized to the template 5^" of the two specific primers. If the allele corresponding to one of the specific primers was present the specific primer would hybridize perfectly to the template. The Taq polymerase, extending the 5' primer, would then remove the nucleotides from the specific probe releasing both the fluorescent dye and the quenching agent. This resulted in an increase in the fluorescence from the dye no longer in close proximity to the quenching agent. If the allele specific primer hybridized to the other allele the mismatch at the polymorphic site would inhibit the 5' to 3^' endonuclease activity of Taq and hence prevent release of the fluorescent dye.

The ABI7700 sequence detection system was used to measure the increase in fluorescence from each specific dye during the thermal cycling PCR directly in PCR reaction tubes. The information from the reactions was then analyzed. If an individual was homozygous for a particular allele only fluorescence corresponding to the dye from that specific primer would be released, if the individual was heterozygous both dyes would fluoresce. Table 1 shows the P values for the co-inheritance of the associated SNPs with migraine. Table 2 shows the SNPs typed in the sample group to determine association of the SNP with migraine. The polymoφhic site typed is given together with the flanking sequence 5' and 3'. Table 1

en c m ω

C H m en x m rπ

H

C r m rυ

Claims

1. Use of an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway in the manufacture of a medicament for use in a method of preventing or treating cephalic pain.

2. Use according to claim 1 wherein the agent is an agonist.

3. Use according to claim 1 wherein the agent directly agonises any one of the following components: the insulin receptor, insulin receptor substrate- 1, -2, -3, or -4, p85, Grb2, Gabl, phosphatidyl inositol 3 kinase, pp60, ppl20, son of sevenless (SOS), MAP kinase, serine phosphatase, threonine phosphatase, tyrosine kinase, ras, raf, syp, she or a G protein.

4. Use according to claim 3, wherein the component is the insulin receptor.

5. Use according to any one of the preceding claims wherein the agent agonises insulin sensitivity.

6. Use according to any one of the preceding claims wherein the agent agonises the effect of a cephalic pain susceptibility polymoφhism in the insulin receptor gene.

7. Use according to claim 6 wherein the agent agonises the effect of a polymoφhism which is selected from INSBa, INSCa, exon8.pol 1, exonl l.pol 1 , exonl7.pol2, exonό.pol l, exon7.pol l, exon7.pol2, exon8.pol2, exon9.pol3, exonl4.pol l and INSR-C.4479C.T or which is in linkage disequilibrium therewith.

8. A method of screening for an agent as defined in claim 1 comprising contacting a candidate substance with (i) one or more components which affect the insulin receptor or the insulin signalling pathway, (ii) any part of the expression pathway for a component as defined in (i); or (iii) a functional analogue of (i) or (ii), and determining whether the candidate substance binds or modulates (i) ,(ii) or (iii).

9. A method according to claim 8 wherein at least one of the components is the insulin receptor protein, a peroxisome proliferator-activated receptor or an RXR ligand.

10. A method according to claim 8 comprising administering the candidate substance to a mammal whose insulin receptor gene comprises a polymoφhism which is selected from INSBa, INSCa, exonδ.pol 1, exonl l.pol 1, exonl7.pol2, exonό.pol 1, exon7.pol 1, exon7.pol2, exon8.pol2, exon9.pol3, exonl4.pol 1 and INSR-C.4479C.T or which is in linkage disequilibrium therewith; and determining whether the substance counteracts the effect of the polymoφhism.

11. A method according to claim 10 wherein the effect is a symptom of migraine.

12. An isolated polynucleotide or protein that comprises (i) a polymoφhism that causes susceptibility to cephalic pain or (ii) a naturally occurring polymoφhism that is in linkage disequilibrium with (i).

13. A polynuclotide or protein according to claim 12 which comprises insulin receptor gene region sequence or insulin receptor protein sequence.

14. A polynucleotide or protein according to claim 13 wherein the polymoφhism is as defined in claim 7.

15. A polynucleotide or protein according to any one of claims 12 to 14 which has a length of at least 15 nucleotides or at least 15 amino acids.

16. A vector incoφorating a polynucleotide as defined in any one of claims 12 to 15.

17. A process for the preparation of a protein that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway, which process comprises:

(a) culturing a host cell transformed or transfected with a vector as defined in claim 16 under conditions to provide for expression of the protein, and optionally

(b) recovering the expressed protein.

18. A non-human animal which is transgenic for a polymoφhism which is selected from INSBa, INSCa, exonδ.pol 1, exonl l .pol 1 , exonl7.pol2, exonό.pol l, exon7.pol l , exon7.pol2, exon8.pol2, exon9.pol3, exonl4.pol l and INSR- C.4479C.T or which is in linkage disequilibrium therewith.

19. A method of treating cephalic pain, which comprise adminstering to a subject in need thereof an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway.

SEQUENCE LISTING

<210> 1 <211> 2085

<212> DNA

<213> Homo sapiens

<400> I agatctggcc attgcactcc agcctgggca acagagaaaa actccatcta aaaaaaaaaa 60 aaaaaaaaaa aaaaaacaga gagagagaga gagagagaga gaaggaaacg gaactggggg 120 gaggatttgc aaaaatatgg ttagggatgg cacttcagag atgaagccat cctggagtgt 180 tacgggcaag ggaaatgctg gggcaaagcc ccagaggcag gaataggttt ggcctgttgc 240 atgaacagtg ggtccagctc ctagcaaact gtttattgaa tgaaagaaga atgaatgcct 300 tgggtctagg gttgtgctgg gcgctttctt aagttttctt tcccgggtac ctccccagaa 360 ctggcatgca ggtattatta aacccattac acaagtgaaa ctggcccaga gacagaaaag 420 tccctggtcc aagaccacac aggagtgagg ggtggaggaa ccctcctccc attgagttct 480 ggctttccta tactgaaagc cccttcctct cctgcagtaa ggtaggtgga accgctgtcc 540 cgccttgttg gtgaatgtcg ttgctagact tcagacacat acaggctggt ctgctgaaaa 600 tcagagatgt ccacctgcgc cctattcgag gtctccggcg tcttctttgg cgtcgtcttt 660 gccctttcag aagcgtctgc acatttttcc aggtgtcatt tctccaactt gaacacaggg 720 agcgcactgg gcacgcgggc acgtggctgt ccccaggggc ctggcttggg tctcgcccct 780 gggccggggc gcacgcgcgg gcgggacatc tgggggcgcc cacgcgctct gggacgagtg 840 tcgctggcca ggcccggact gaggaaaggc gagtgagaca ctactcgcct ggggtgcaaa 900 atttaaggga gtgaaaaaaa aaaaaaaaga aagaaaccaa aaccacctcg agtcaccaaa 960 ataaacattt taatgcagta ttttttaaaa aatcaacagg aatcctccaa agcccactat 1020 gaacaaaata gcaaaatggt agagaaagga tctgtgccgc tgcgtcgggc ctgtggggcg 1080 cctccggggg tctgaaactg gaggagactc ggggctgtag ggcgcgcgga tctggggcgc 1140 gccctcggtc ccggcgcgcc cagggcctcc cgcgcggggc ccggcacagg gaggcgggga 1200 ggcgggcggg gcggggcggg accgggcggc acctccctcc cctgcaagct ttccctccct 1260 ctcctgggcc tctcccgggc gcagagtccc ttcctaggcc agatccgcgc cgccttttcc 1320 cgcggcccgc acggggccca gctgacgggc cgcgttgttt acgggccgga gcagccctct 1380 ctcccgccgc ccgcccgcca cccgccagcc caggtgcccg cccgccagtc agctagtccg 1440 tcggtccgcg cgtccctctg tcccggagcc cgcagatcgc gacccagagc gcgcggggcc 1500 gagagccgag agacagtccc gggcgcagcg cggagctccg ggccccgaga tcctgggacg 1560 gggcccgggc cgcagcggcc ggggggtcgg ggccaccacc gcaagggcct ccgctcagta 1620 tttgtagctg gcgaagccgc gcgcgccctt cccggggctg cctctgggcc ctccccggca 1680 ggggggctgc ggcccgcggg tcgcgggcgt ggaagagaag gacgcgcggc ccccagcgcc 1740 2 tcttgggtgg ccgcctcgga gcatgacccc cgcgggccag cgccgcgcgc tctgatccga 1800 ggagaccccg cgctcccgca gccatgggca ccgggggccg gcggggagcg gcggccgcgc 1860 cgctgctggt ggcggtggcc gcgctgctac tgggcgccgc gggccacctg taccccggag 1920 agggtgagtc tgggggcgcg ggcgtgggcg gggagcgccg cgatggggag aggaccccac 1980 ccaagccaaa atcgatcccc cgcttgtgga ctgagaaccc tccccagggg cggggggcgg 2040 tggccaggac ggtagctcct gcatcgcgta gggggagcgg gaagc 2085

<210> 2 <211> 928 <212> DNA <213> Homo sapiens

<400> 2 tactttacag agaaagctac tcatcccggc tggctgcaga gtttacaggg cccgggatga 60 aaacacaggg cccaggtttc ctgtccatga agccggctct gcccctgatc cttctgatgc 120 atccaccgtg cgtctgctca cctgtcttgc tttctgttca ttttctcttg tagtgtgtcc 180 cggcatggat atccggaaca acctcactag gttgcatgag ctggagaatt gctctgtcat 240 cgaaggacac ttgcagatac tcttgatgtt caaaacgagg cccgaagatt tccgagacct 300 cagtttcccc aaactcatca tgatcactga ttacttgctg ctcttccggg tctatgggct 360 cgagagcctg aaggacctgt tccccaacct cacggtcatc cggggatcac gactgttctt 420 taactacgcg ctggtcatct tcgagatggt tcacctcaag gaactcggcc tctacaacct 480 gatgaacatc acccggggtt ctgtccgcat cgagaagaac aatgagctct gttacttggc 540 cactatcgac tggtcccgta tcctggattc cgtggaggat aattacatcg tgttgaacaa 600 agatgacaac gaggagtgtg gagacatctg tccgggtacc gcgaagggca agaccaactg 660 ccccgccacc gtcatcaacg ggcagtttgt cgaacgatgt tggactcata gtcactgcca 720 gaaaggtacg ccggggatac agggttctaa gcagtgtctc gtgccttgtt ctagaaagct 780 taaaatgttt tatggcttaa aaatgttaaa tggtcattag gtaggggccg gggaatagtg 840 ggtggtggca ttcactagcc cagggagtgg cagacatttt ctgtaaagac tcagatagta 900 gatacttcag attttgcagg ccatatgg 928

<210> 3 <211> 639 <212> DNA <213> Homo sapiens

400> 3 gatccagaat tgctgcatat gcagacagga attggacaaa gccatttatt tatttattta 60 tttatttatt tatttattta tttattτccc tctctctctc tctcτctctc cagtttgccc 120 gaccatctgt aagtcacacg gctgcaccgc cgaaggcctc tgttgccaca gcgagtgcct 180 gggcaactgt tctcagcccg acgaccccac caagtgcgtg gcctgccgca acttctacct 240 ggacggcagg tgtgtggaga cctgcccgcc cccgtactac cacttccagg actggcgctg 300 tgtgaacttc agcttctgcc aggacctgca ccacaaatgc aagaactcgc ggaggcaggg 360 ctgccaccaa tacgtcattc acaacaacaa gtgcatccct gagtgtccct ccgggtacac 420 gatgaattcc agcaagtgag ttctggatgt gggtctgggg ggcagccgag aggagaagga 480 acgtggggtt ggttgtgacg atgccgcttg ttaaaactgt gtgcaaaccc agggttaatt 540 ggctatgagt gaggtctctg ctctcagatg ctacttttgc accctgtttt ggtcctgggc 600 ttgggagtgg gagttgacta cctttttctc taaaggacc 639

<210> 4 <211> 663 <212> DNA

<213> Homo sapiens

<400> 4 ccaacatggt aaccccgtct ctactcaaaa atacaaaaat tagccaggca cggtggcggg 60 cacctataat cccagctact gtggaggctg aggcaggaga atctcttgaa cccagaaggc 120 agaggttgca gtgagctgag atcgcaccac tgcactccag cctgggcaac agagcgagac 180 tctgtcacac aaacacacac acacacacaa agaaatacca tatcaggcag aaagatgcct 240 gagatgtctg aaggaccttg gataccgtga cacccccctc ccctttctct ttctctctct 300 ctctgctccg tccttagctt gctgtgcacc ccatgcctgg gtccctgtcc caaggtgtgc 360 cacctcctag aaggcgagaa gaccatcgac tcggtgacgt ctgcccagga gctccgagga 420 tgcaccgtca tcaacgggag tctgatcatc aacattcgag gaggcagtga gtgtctctgt 480 gtgggcgtcg ggggtgcctg ttgggctcca tgtccctctg agctgtgagc ggggaagaaa 540 agcagtgcag accctgctgc gtgctcctac agcactttta ggatggtcgt tcagtggctc 600 ccccatggat agaaccatgc tgggagtctg cctcaaaacc tgaaatgaac agctcagtct 660 tec 663

<210> 5 <211> 410 <212> DNA <213> Homo sapiens <400> 5 gggcagaagt atgcttgacc catttaagga atgctaagga cttcagattg tgttctaagc 60 atgatgagtt ttgagctggg tatgtccagt catttgcagc ctgagggtta tcttctcacc 120 atggagaatc atgagaagat tgaaatatgt ctatagaaac ccactggata ttctctcctt 180 tccttagaca atctggcagc tgagctagaa gccaacctcg gcctcattga agaaatttca 240 gggtatctaa aaatccgccg atcctacgct ctggtgtcac tttccttctt ccggaagtta 300 cgtctgattc gaggagagac cttggaaatt gggtacgtgg gcctgattgt gtgtatggcc 360 tgagtgctaa ctaggaagtt cgtgtattag aacaacttaa ggattttttt 410

<210> 6 <211> 554 <212> DNA <213> Homo sapiens

<400> 6 ggccatgaaa acttcctcaa cttcctctgt tatccacatt caacaaatat gtgttgagta 60 tgtgccaagc aagtggagag gattaggcac gtagcactga acaagatcaa ctccgagcat 120 ggccacacca tcttggagtt gtagaagacc agccgttgaa tgactagatg tgtgtgtttt 180 ttccatagga actactcctt ctatgccttg gacaaccaga acctaaggca gctctgggac 240 tggagcaaac acaacctcac catcactcag gggaaactct tcttccacta taaccccaaa 300 ctctgcttgt cagaaatcca caagatggaa gaagtttcag gaaccaaggg gcgccaggag 360 agaaacgaca ttgccctgaa gaccaatggg gaccaggcat cctgtaagtc actggtcccc 420 aacctttttg gcacgaggga ccggtttagt ggaagatggt ttttccatgg actggtggtg 480 ggtggggatg gtttcagcat gattcaagtg cattacattt actatgcact ttattcctat 540 tatgattaca ttgt 554

<210> 7

<211> 592

<212> DNA

<213> Homo sapiens

<400> 7 ttgcgcgggt acagactgcg cttattcagt tgactgtctg gctgagtcaa gtcattggct 60 tacgtgagtg tgagtggcca agttgcaaaa ctggctctta cctttgaatc ttcccccatt 120 catactcagc caggcacatg gggaggagac ccttaaggga atagcagcat cacctctgcc 180 ttctcacggt ccctccagga agtgtggggg tcccaggctt tggtcτgaaa ctacactgaa 240 atagctcatt tttgcctttt gttttaactt ttccaggtga aaatgagtta cttaaatttt 300 cttacattcg gacatctttt gacaagatct tgctgagatg ggagccgtac tggccccccg 360 acttccgaga cctcttgggg ttcatgctgt tctacaaaga ggcgtaagta gaagagttag 420 agagacgctg aggaggcgag ggctggctgg ctctgtgctt gctacgtttg tgctccaatc 480 tgcccctctt gggttcctgt ctatctccct cctcctcctg gaataaatat cttaggttcc 540 tttttacaat ctcaccagtc gatggcatgc aaagtcaata gtgtctgctt tt 592

<210> 8 <211> 401 <212> DNA <213> Homo sapiens

<400> 8 cattagattg ttgggtgagt aacatgtgac cctatgggat gtaacttccc aggcctcatc 60 tgcacggcac tcagtgtgac ggtcttgtaa gggtaactgc cttctgctgt tttgtcttga 120 aagcccttat cagaatgtga cggagttcga tgggcaggat gcgtgtggtt ccaacagttg 180 gacggtggta gacattgacc cacccctgag gtccaacgac cccaaatcac agaaccaccc 240 agggtggctg atgcggggtc tcaagccctg gacccagtat gccatctttg tgaagaccct 300 ggtcaccttt tcggatgaac gccggaccta tggggccaag agtgacatca tttatgtcca 360 gacagatgcc accagtgagt gtgtcttggg aatgtgaatt c 401

<210> 9 <211> 420 <212> DNA

<213> Homo sapiens

<400> 9 ggtgccctca tgatgtcttt aacttgtgtg tcccccgcca tcctcccacc agctttcttt 60 gcacactgtt tctcatgatg gacccgtttc ctttctccct ggcagacccc tctgtgcccc 120 tggatccaat ctcagtgtct aactcatcat cccagattat tctgaagtgg aaaccaccct 180 ccgaccccaa tggcaacatc acccactacc tggttttctg ggagaggcag gcggaagaca 240 gtgagctgtt cgagctggat tattgcctca aaggtgagtg caggcagctg tgctaggatc 300 ggtggggttt gcacacgtgt gtctgatgca ctttgcttca cctctaggga agcagctatc 360 tcttcctgtg tctcagtgtc ggaaggcaca cacacacact ccattctatc tcatatgaaa 420 <210> 10

<211> 517

<212> DNA <213> Homo sapiens

<400> 10 tttgtggtgt gtgtatgtgt ggtgtgttgt gtgatgtgtg tggtgtgtgt gtgggggggt 60 gtgtggtgtg tgtatgtgtg gtgtgtgtgg tgtgtgtgtg tggtgtgtgt gtgtgggggg 120 ggtgtgtgtg tgtatgtgtg ttcagccgca gagacttgag cccccctttt ctgtttcttt 180 ctccagggct gaagctgccc tcgaggacct ggtctccacc attcgagtct gaagattctc 240 agaagcacaa ccagagtgag tatgaggatt cggccggcga atgctgctcc tgtccaaaga 300 cagactctca gatcctgaag gagctggagg agtcctcgtt taggaagacg tttgaggatt 360 acctgcacaa cgtggttttc gtccccaggt caggacttgg cgctgggctc tcttagtggg 420 tgccaattgg cttggtgttg gtggaaggtc attacttagg gaccgagagg tagtgggagg 480 gagagacggc agaaccctgg gtggagtctg aatggag 517

<210> 11 <211> 343 <212> DNA <213> Homo sapiens

<400> 11 tggtccaggg tcaaagccag ggtgccctta ctcggacaca tgtggcctcc aagtgtcaga 60 gcccagtggt ctgtctaatg aagttccctc tgtcctcaaa ggcgttggtt ttgtttccac 120 agaaaaacct cttcaggcac tggtgccgag gaccctaggt atgactcacc tgtgcgaccc 180 ctggtgcctg ctccgcgcag ggccggcggc gtgccaggca gatgcctcgg agaacccagg 240 ggtttctctg gctttttgca tgcggcgggc agctgtgctg gagagcagat gcttcaccaa 300 ttcagaaatc caatgccttc actctgaaat gaaatctggg cat 343

<210> 12

<211> 719 <212> DNA

<213> Homo sapiens

<400> 12 ggtcattcct ggcagtctgt attgtaatcc atgttcccca ttgctgcacc ctcctgcgct 60 7 ctgatctttc ttcttaatca agccttttat tctccagtgt cactttttta aaaaaaatga 120 tggtgatggt gtcatcatac atgtcctact gtcgttccag gccatctcgg aaacgcaggt 180 cccttggcga tgttgggaat gtgacggtgg ccgtgcccac ggtggcagct ttccccaaca 240 cttcctcgac cagcgtgccc acgagtccgg aggagcacag gccttttgag aaggtggtga 300 acaaggagtc gctggtcatc tccggcttgc gacacttcac gggctatcgc atcgagctgc 360 aggcttgcaa ccaggacacc cctgaggaac ggtgcagtgt ggcagcctac gtcagtgcga 420 ggaccatgcc tgaaggtagg gctgctggtc cggggtccga gtgtcatggg tgggacatca 480 aggctgactt tttgtttgag acggagcctt gctctgtcgc ccaggctgga gtacagtggt 540 gcgacctcag ctcactccag cctctgccac ctatgtcaag tgattccctg cttcagcctc 600 ccaagtagct gggactacag gtgtctgcca ccacgcccag ctaatttttg tatttttagt 660 agagatgggg tttcaccata ttgcccaggc tggtcttgaa ctcctgggct caagtgatc 719

<210> 13 <211> 439 <212> DNA <213> Homo sapiens

<400> 13 gtcaccagcc caaggttgca ccatggacag gtggcagaag tgggatctca tccaagagtt 60 acatccctgc ctctcacttc ctctccttac agccaaggct gatgacattg ttggccctgt 120 gacgcatgaa atctttgaga acaacgtcgt ccacttgatg tggcaggagc cgaaggagcc 180 caatggtctg atcgtgctgt atgaagtgag ttatcggcga tatggtgatg aggtaaggcc 240 cttgactctt gggcatgccc ctgcaccact tcagcatgcc ccttcagagt tgcacttggt 300 acctccttcc tctgctgaaa ttttgattcc agtgcttctc tcatcaggta ctgtgctatt 360 agtacttaaa gccttgatac ctgacttcgc aggaagatgg gtcagaaatg ccaatctacc 420 agcttgttac ttttcttag 439

<210> 14

<211> 386

<212> DNA

<213> Homo sapiens

<400> 14 tggctgtgag ctccctgcga ggggtggaca ctcccagatg tgcaaagctc agccaccctc 60 cttctcctcc tctcttcctc ccaggagctg catctctgcg tctcccgcaa gcacttcgct 120 ctggaacggg gctgcaggct gcgtgggctg tcaccgggga actacagcgt gcgaatccgg 180 gccacctccc ttgcgggcaa cggctcttgg acggaaccca cctatttcta cgtgacagac 240 tattgtaagt ctccatggca gcctcagctg actggggctg tgcttagcac tgagcatggt 300 gggacattgc aggggatgac ttggagaggc cgcagtgctg gccctggcct tgactctcag 360 gcctatcagc tgctgcggtg cttgcc 386

<210> 15 <211> 429 <212> DNA

<213> Homo sapiens

<400> 15 cccacccatt ccaggagtgg atgtgatttt tgatgtgaac tttgttggaa acacattgat 60 atgaaacata tattttctta ttctatttca gtagacgtcc cgtcaaatat tgcaaaaatt 120 atcatcggcc ccctcatctt tgtctttctc ttcagtgttg tgattggaag tatttatcta 180 ttcctgagaa agaggtgagt tcagtgagtt cagtggtgtg ctgggaacag ttggttctct 240 gggggaaaac atgccttgat ataggtatag gcatatttaa gtttattatg aattttgctg 300 atataggatg tgtaacatgc aatttacaga taattgtcat aatatgatat acacaactct 360 ttattgtaaa ttccctctag acagttgatt ctcacagaat gtttttattg attttttttt 420 ttgcccaaa 429

<210> 16 <211> 480 <212> DNA

<213> Homo sapiens

<400> 16 aaaaacaaaa acaaaaacaa aacaaaaaaa aaaccaccca gggagggatg agtgctccca 60 tgttgatgca cttacatacc tgtctgatgg gcttccattc aaaacataaa ggtcccccat 120 ccctgcccta gactgcatct aggattatgg ggattctgct ggtaagggct gccatttgcc 180 ttggggagtc ttgtatgaaa cacctttctg cagagtccca tgagaatctc aagctaacgt 240 gcctcgtttt cctcctccag gcagccagat gggccgctgg gaccgcttta cgcttcttca 300 aaccctgagt atctcagtgc cagtgatggt gagtaccatc ccttccctgt gggtggccag 360 aaccctactc atcagcttcc tttgccttca ccattgagtg agagtgaagg atgggttccc 420 cagggaggcc aagaaaagcc ctcttattca tttgagcttg ccaaactgcc cttgctgcag 480 <210> 17 <211> 485 <212 DNA <213> Homo sapiens

<400> 17 cccggcatgg gtcctggatc acagaactca tttcatgagt gttttcgagg gggtttgggt 60 gagggcttgg gtggaaggtg gctgcagacc cccaagggat cctccaagga tgctgtgtag 120 ataagtaaga agtagtgttt ccatgctctg tgtacgtgcc ggacgagtgg gaggtgtctc 180 gagagaagat caccctcctt cgagagctgg ggcagggctc cttcggcatg gtgtatgagg 240 gcaatgccag ggacatcatc aagggtgagg cagagacccg cgtggcggtg aagacggtca 300 acgagtcagc cagtctccga gagcggattg agttcctcaa tgaggcctcg gtcatgaagg 360 gcttcacctg ccatcacgtg gtgagtccag tgggggtggg acatgggctg gctttcctga 420 cccttccctt tctctgcctc ctcctcctgc acagagcgac agaggacaca gggtgtatcc 480 tccta 485

<210> 18 <211> 287 <212> DNA <213> Homo sapiens

<400> 18 acgctgcatc caggccacag ggtgctgtgt gtgacataga caccagggag ggaggagaac 60 cctggtgagt cgaatcacgg accctcctcc aagaaccctg gttgcttgct ctgcaggtgc 120 gcctcctggg agtggtgtcc aagggccagc ccacgctggt ggtgatggag ctgatggctc 180 acggagacct gaagagctac ctccgttctc tgcggccaga ggctgaggta agctgcttcg 240 ggggacccag cggggtactc ggtggagcac ccgctcctgg cctcctc 287

<210> 19

<211> 322

<212> DNA <213> Homo sapiens

<400> 19 gatcccagtg ctgctgaaac accaaccccg tgtttctgtt ttagaataat cctggccgcc 60 ctccccctac ccttcaagag atgattcaga tggcggcaga gattgctgac gggatggcct 120 10 acctgaacgc caagaagttt gtgcatcggg acctggcagc gagaaactgc atggtcgccc 180 atgattttac tgtcaaaatt ggaggttcgt ctggctttct gctttgaaaa cataacgacc 240 caggccaggt ttgatttcag aaggaagttg tctataatga gccgttaagt cttttctgat 300

5 aatataaagg ggcaagtact tc 322

<210> 20 <211 288 10 <212> DNA

<213> Homo sapiens

<400> 20 gacgtgggcc aggtgaaccc ctcttagggc tctgtgagag gtggggcagt caaggtggca 60 15 gatgctagga ccaaggctga aggttaagag cgtgtgaacc ttttgtgttg tcagactttg 120 gaatgaccag agacatctat gaaacggatt actaccggaa agggggcaag ggtctgctcc 180 ctgtacggtg gatggcaccg gagtccctga aggatggggt cttcaccact tcttctgaca 240 tgtggtgagt tgtgtgtgga tgggtggatg gacgctgggc ttgaattc 288

20

<210> 21

<211> 407

<212> DNA

<213> Homo sapiens

<400> 21 ttgcgtgtgt gtgtgcgttt gcgtgtgtgt gtttgcgcgc gcgcgtgtgt gtgtgtgtct 60 aaatggcttc tttgttacta ctatcaactg tcatcggcag gtcctttggc gtggtccttt 120 gggaaatcac cagcttggca gaacagcctt accaaggcct gtctaatgaa caggtgttga 180 aatttgtcat ggatggaggg tatctggatc aacccgacaa ctgtccagag agagtgtaag 240 tgtagaaagg gtttaaggtg tgtgaggtgt tcgttgaaag ggtattgccc tttacacgtg 300 tgcttggttt tgcctttcct atgtctacac gctcaccgtg tttgcatgct gtatgttaca 360 ggtgtgtttg tgtttgcata gcttgtcttt acatgcatgc ttgcatt 407

jj

<210> 22

<211> 873

<212> DNA

<213> Homo sapiens 1 1

<400> 22 ctgcagggac aagagtgggg gtttgggagg atgcgtggca gggcccccag actcacccag 60 gacgtgtcct tctgccccgc agcactgacc tcatgcgcat gtgctggcaa ttcaacccca 120 agatgaggcc aaccttcctg gagattgtca acctgctcaa ggacgacctg caccccagct 180 ttccagaggt gtcgttcttc cacagcgagg agaacaaggc tcccgagagt gaggagctgg 240 agatggagtt tgaggacatg gagaatgtgc ccctggaccg ttcctcgcac tgtcagaggg 300 aggaggcggg gggccgggat ggagggtcct cgctgggttt caagcggagc tacgaggaac 360 acatccctta cacacacatg aacggaggca agaaaaacgg gcggattctg accttgcctc 420 ggtccaatcc ttcctaacag tgcctaccgt ggcgggggcg ggcaggggtt cccattttcg 480 ctttcctctg gtttgaaagc ctctggaaaa ctcaggattc tcacgactct accatgtcca 540 gtggagttca gagatcgttc ctatacattt ctgttcatct taaggtggac tcgtttggtt 600 accaatttaa ctagtcctgc agaggattta actgtgaacc tggagggcaa ggggtttcca 660 cagttgctgc tcctttgggg caacgacggt ttcaaaccag gattttgtgt tttttcgttc 720 cccccacccg cccccagcag atggaaagaa agcacctgtt tttacaaatt cttttttttt 780 tttttttttt tttttttttg ctggtgtctg agcttcagta taaaagacaa aacttcctgt 840 ttgtggaaca aaatttcgaa agaaaaaacc aaa 873

<210> 23 <211> 4723 <212> DNA <213> Homo sapiens

<400> 23 ggggggctgc gcggccgggt cggtgcgcac acgagaagga cgcgcggccc ccagcgctct 60 tgggggccgc ctcggagcat gacccccgcg ggccagcgcc gcgcgcctga tccgaggaga 120 ccccgcgctc ccgcagccat gggcaccggg ggccggcggg gggcggcggc cgcgccgctg 180 ctggtggcgg tggccgcgct gctactgggc gccgcgggcc acctgtaccc cggagaggtg 240 tgtcccggca tggatatccg gaacaacctc actaggttgc atgagctgga gaattgctct 300 gtcatcgaag gacacttgca gatactcttg atgttcaaaa cgaggcccga agatttccga 360 gacctcagtt tccccaaact catcatgatc actgattact tgctgctctt ccgggtctat 420 gggctcgaga gcctgaagga cctgttcccc aacctcacgg tcatccgggg atcacgactg 480 ttctttaact acgcgctggt catcttcgag atggttcacc tcaaggaact cggcctctac 540 aacctgatga acatcacccg gggttctgtc cgcatcgaga agaacaatga gctctgttac 600 ttggccacta tcgactggtc ccgtatcctg gattccgtgg aggataatca catcgtgttg 660 aacaaagatg acaacgagga gtgtggagac atctgtccgg gtaccgcgaa gggcaagacc 720 aactgccccg ccaccgtcat caacgggcag tttgtcgaac gatgttggac tcatagtcac 780 tgccagaaag tttgcccgac catctgtaag tcacacggct gcaccgccga aggcctctgt 840 12 tgccacagcg agtgcctggg caactgttct cagcccgacg accccaccaa gtgcgtggcc 900 tgccgcaact tctacctgga cggcaggtgt gtggagacct gcccgccccc gtactaccac 960 ttccaggact ggcgctgtgt gaacttcagc ttctgccagg acctgcacca caaatgcaag 1020 aactcgcgga ggcagggctg ccaccaatac gtcattcaca acaacaagtg catccctgag 1080 tgtccctccg ggtacacgat gaattccagc aacttgctgt gcaccccatg- cctgggtccc 1140 tgtcccaagg tgtgccacct cctagaaggc gagaagacca tcgactcggt gacgtctgcc 1200 caggagctcc gaggatgcac cgtcatcaac gggagtctga tcatcaacat tcgaggaggc 1260 aacaatctgg cagctgagct agaagccaac ctcggcctca ttgaagaaat ttcagggtat 1320 ctaaaaatcc gccgatccta cgctctggtg tcactttcct tcttccggaa gttacgtctg 1380 attcgaggag agaccttgga aattgggaac tactccttct atgccttgga caaccagaac 1440 ctaaggcagc tctgggactg gagcaaacac aacctcacca ccactcaggg gaaactcttc 1500 ttccactata accccaaact ctgcttgtca gaaatccaca agatggaaga agtttcagga 1560 accaaggggc gccaggagag aaacgacatt gccctgaaga ccaatgggga caaggcatcc 1620 tgtgaaaatg agttacttaa attttcttac attcggacat cttttgacaa gatcttgctg 1680 agatgggagc cgtactggcc ccccgacttc cgagacctct tggggttcat gctgttctac 1740 aaagaggccc cttatcagaa tgtgacggag ttcgatgggc aggatgcgtg tggttccaac 1800 agttggacgg tggtagacat tgacccaccc ctgaggtcca acgaccccaa atcacagaac 1860 cacccagggt ggctgatgcg gggtctcaag ccctggaccc agtatgccat ctttgtgaag 1920 accctggtca ccttttcgga tgaacgccgg acctatgggg ccaagagtga catcatttat 1980 gtccagacag atgccaccaa cccctctgtg cccctggatc caatctcagt gtctaactca 2040 tcatcccaga ttattctgaa gtggaaacca ccctccgacc ccaatggcaa catcacccac 2100 tacctggttt tctgggagag gcaggcggaa gacagtgagc tgttcgagct ggattattgc 2160 ctcaaagggc tgaagctgcc ctcgaggacc tggtctccac cattcgagtc tgaagattct 2220 cagaagcaca accagagtga gtatgaggat tcggccggcg aatgctgctc ctgtccaaag 2280 acagactctc agatcctgaa ggagctggag gagtcctcgt ttaggaagac gtttgaggat 2340 tacctgcaca acgtggtttt cgtccccaga aaaacctctt caggcactgg tgccgaggac 2400 cctaggccat ctcggaaacg caggtccctt ggcgatgttg ggaatgtgac ggtggccgtg 2460 cccacggtgg cagctttccc caacacttcc tcgaccagcg tgcccacgag tccggaggag 2520 cacaggcctt ttgagaaggt ggtgaacaag gagtcgctgg tcatctccgg cttgcgacac 2580 ttcacgggct atcgcatcga gctgcaggct tgcaaccagg acacccctga ggaacggtgc 2640 agtgtggcag cctacgtcag tgcgaggacc atgcctgaag ccaaggctga tgacattgtt 2700 ggccctgtga cgcatgaaat ctttgagaac aacgtcgtcc acttgatgtg gcaggagccg 2760 aaggagccca atggtctgat cgtgctgtat gaagtgagtt atcggcgata tggtgatgag 2820 gagctgcatc tctgcgtctc ccgcaagcac ttcgctctgg aacggggctg caggctgcgt 2880 gggctgtcac cggggaacta cagcgtgcga atccgggcca cctcccttgc gggcaacggc 2940 tcttggacgg aacccaccta tttctacgtg acagactatt tagacgtccc gtcaaatatt 3000 gcaaaaatta tcatcggccc cctcatcttt gtctttctct tcagtgttgt gattggaagt 3060 atttatctat tcctgagaaa gaggcagcca gatgggccgc tgggaccgct ttacgcttct 3120 13 tcaaaccctg agtatctcag tgccagtgat gtgtttccat gctctgtgta cgtgccggac 3180 gagtgggagg tgtctcgaga gaagatcacc ctccttcgag agctggggca gggctccttc 3240 ggcatggtgt atgagggcaa tgccagggac atcatcaagg gtgaggcaga gacccgcgtg 3300 gcggtgaaga cggtcaacga gtcagccagt ctccgagagc ggattgagtt cctcaatgag 3360 gcctcggtca tgaagggctt cacctgccat cacgtggtgc gcctcctggg agtggtgtcc 3420 aagggccagc ccacgctggt ggtgatggag ctgatggctc acggagacct gaagagctac 3480 ctccgttctc tgcggccaga ggctgagaat aatcctggcc gccctccccc tacccttcaa 3540 gagatgattc agatggcggc agagattgct gacgggatgg cctacctgaa cgccaagaag 3600 tttgtgcatc gggacctggc agcgagaaac tgcatggtcg cccatgattt tactgtcaaa 3660 attggagact ttggaatgac cagagacatc tatgaaacgg attactaccg gaaagggggc 3720 aagggtctgc tccctgtacg gtggatggca ccggagtccc tgaaggatgg ggtcttcacc 3780 acttcttctg acatgtggtc ctttggcgtg gtcctttggg aaatcaccag cttggcagaa 3840 cagccttacc aaggcctgtc taatgaacag gtgttgaaat ttgtcatgga tggagggtat 3900 ctggatcaac ccgacaactg tccagagaga gtcactgacc tcatgcgcat gtgctggcaa 3960 ttcaacccca agatgaggcc aaccttcctg gagattgtca acctgctcaa ggacgacctg 4020 caccccagct ttccagaggt gtcgttcttc cacagcgagg agaacaaggc tcccgagagt 4080 gaggagctgg agatggagtt tgaggacatg gagaatgtgc ccctggaccg ttcctcgcac 4140 tgtcagaggg aggaggcggg gggccgggat ggagggtcct cgctgggttt caagcggagc 4200 tacgaggaac acatccctta cacacacatg aacggaggca agaaaaacgg gcggattctg 4260 accttgcctc ggtccaatcc ttcctaacag tgcctaccgt ggcgggggcg ggcaggggtt 4320 cccattttcg ctttcctctg gtttgaaagc ctctggaaaa ctcaggattc tcacgactct 4380 accatgtcca gtggagttca gagatcgttc ctatacattt ctgttcatct taaggtggac 4440 tcgtttggtt accaatttaa ctagtcctgc agaggattta actgtgaacc tggagggcaa 4500 ggggtttcca cagttgctgc tcctttgggg caacgacggt ttcaaaccag gattttgtgt 4560 tttttcgttc cccccacccg cccccagcag atggaaagaa agcacctgtt tttacaaatt 4620 cttttttttt tttttttttt tttttttttg ctggtgtctg agcttcagta taaaagacaa 4680 aacttcctgt ttgtggaaca aaatttcgaa agaaaaaacc aaa 4723

<210> 24 <211> 5180 <212> DNA <213> Homo sapiens

<400> 24 accgggagcg cgcgctctga tccgaggaga ccccgcgctc ccgcagccat gggcaccggg 60 ggccggcggg gggcggcggc cgcgccgctg ctggtggcgg tggccgcgct gctactgggc 120 gccgcgggcc acctgtaccc cggagaggtg tgtcccggca tggatatccg gaacaacctc 180 14 actaggttgc atgagctgga gaattgctct gtcatcgaag gacacttgca gatactcttg 240 atgttcaaaa cgaggcccga agatttccga gacctcagtt tccccaaact catcatgatc 300 actgattact tgctgctctt ccgggtctat gggctcgaga gcctgaagga cctgttcccc 360 aacctcacgg tcatccgggg atcacgactg ttctttaact acgcgctggt catcttcgag 420 atggttcacc tcaaggaact cggcctctac aacctgatga acatcacccg gggttctgtc 480 cgcatcgaga agaacaatga gctctgttac ttggccacta tcgactggtc ccgtatcctg 540 gattccgtgg aggataatta catcgtgttg aacaaagatg acaacgagga gtgtggagac 600 atctgtccgg gtaccgcgaa gggcaagacc aactgccccg ccaccgtcat caacgggcag 660 tttgtcgaac gatgttggac tcatagtcac tgccagaaag tttgcccgac catctgtaag 720 tcacacggct gcaccgccga aggcctctgt tgccacagcg agtgcctggg caactgttct 780 cagcccgacg accccaccaa gtgcgtggcc tgccgcaact tctacctgga cggcaggtgt 840 gtggagacct gcccgccccc gtactaccac ttccaggact ggcgctgtgt gaacttcagc 900 ttctgccagg acctgcacca caaatgcaag aactcgcgga ggcagggctg ccaccagtac 960 gtcattcaca acaacaagtg catccctgag tgtccctccg ggtacacgat gaattccagc 1020 aacttgctgt gcaccccatg cctgggtccc tgtcccaagg tgtgccacct cctagaaggc 1080 gagaagacca tcgactcggt gacgtctgcc caggagctcc gaggatgcac cgtcatcaac 1140 gggagtctga tcatcaacat tcgaggaggc aacaatctgg cagctgagct agaagccaac 1200 ctcggcctca ttgaagaaat ttcagggtat ctaaaaatcc gccgatccta cgctctggtg 1260 tcactttcct tcttccggaa gttacgtctg attcgaggag agaccttgga aattgggaac 1320 tactccttct atgccttgga caaccagaac ctaaggcagc tctgggactg gagcaaacac 1380 aacctcacca tcactcaggg gaaactcttc ttccactata accccaaact ctgcttgtca 1440 gaaatccaca agatggaaga agtttcagga accaaggggc gccaggagag aaacgacatt 1500 gccctgaaga ccaatgggga ccaggcatcc tgtgaaaatg agttacttaa attttcttac 1560 attcggacat cttttgacaa gatcttgctg agatgggagc cgtactggcc ccccgacttc 1620 cgagacctct tggggttcat gctgttctac aaagaggccc cttatcagaa tgtgacggag 1680 ttcgacgggc aggatgcatg tggttccaac agttggacgg tggtagacat tgacccaccc 1740 ctgaggtcca acgaccccaa atcacagaac cacccagggt ggctgatgcg gggtctcaag 1800 ccctggaccc agtatgccat ctttgtgaag accctggtca ccttttcgga tgaacgccgg 1860 acctatgggg ccaagagtga catcatttat gtccagacag atgccaccaa cccctctgtg 1920 cccctggatc caatctcagt gtctaactca tcatcccaga ttattctgaa gtggaaacca 1980 ccctccgacc ccaatggcaa catcacccac tacctggttt tctgggagag gcaggcggaa 2040 gacagtgagc tgttcgagct ggattattgc ctcaaagggc tgaagctgcc ctcgaggacc 2100 tggtctccac cattcgagtc tgaagattct cagaagcaca accagagtga gtatgaggat 2160 tcggccggcg aatgctgctc ctgtccaaag acagactctc agatcctgaa ggagctggag 2220 gagtcctcgt ttaggaagac gtttgaggat tacctgcaca acgtggtttt cgtccccagg 2280 ccatctcgga aacgcaggtc ccttggcgat gttgggaatg tgacggtggc cgtgcccacg 2340 gtggcagctt tccccaacac ttcctcgacc agcgtgccca cgagtccgga ggagcacagg 2400 ccttttgaga aggtggtgaa caaggagtcg ctggtcatct ccggcttgcg acacttcacg 2460 15 ggctatcgca tcgagctgca ggcttgcaac caggacaccc ctgaggaacg gtgcagtgtg 2520 gcagcctacg tcagtgcgag gaccatgcct gaagccaagg ctgatgacat tgttggccct 2580 gtgacgcatg aaatctttga gaacaacgtc gtccacttga tgtggcagga gccgaaggag 2640 cccaatggtc tgatcgtgct gtatgaagtg agttatcggc gatatggtga tgaggagctg 2700 catctctgcg tctcccgcaa gcacttcgct ctggaacggg gctgcaggct gcgtgggctg 2760 tcaccgggga actacagcgt gcgaatccgg gccacctccc ttgcgggcaa cggctcttgg 2820 acggaaccca cctatttcta cgtgacagac tatttagacg tcccgtcaaa tattgcaaaa 2880 attatcatcg gccccctcat ctttgtcttt ctcttcagtg ttgtgattgg aagtatttat 2940 ctattcctga gaaagaggca gccagatggg ccgctgggac cgctttacgc ttcttcaaac 3000 cctgagtatc tcagtgccag tgatgtgttt ccatgctctg tgtacgtgcc ggacgagtgg 3060 gaggtgtctc gagagaagat caccctcctt cgagagctgg ggcagggctc cttcggcatg 3120 gtgtatgagg gcaatgccag ggacatcatc aagggtgagg cagagacccg cgtggcggtg 3180 aagacggtca acgagtcagc cagtctccga gagcggattg agttcctcaa tgaggcctcg 3240 gtcatgaagg gcttcacctg ccatcacgtg gtgcgcctcc tgggagtggt gtccaagggc 3300 cagcccacgc tggtggtgat ggagctgatg gctcacggag acctgaagag ctacctccgt 3360 tctctgcggc cagaggctga gaataatcct ggccgccctc cccctaccct tcaagagatg 3420 attcagatgg cggcagagat tgctgacggg atggcctacc tgaacgccaa gaagtttgtg 3480 catcgggacc tggcagcgag aaactgcatg gtcgcccatg attttactgt caaaattgga 3540 gactttggaa tgaccagaga catctatgaa acggattact accggaaagg gggcaagggt 3600 ctgctccctg tacggtggat ggcaccggag tccctgaagg atggggtctt caccacttct 3660 tctgacatgt ggtcctttgg cgtggtcctt tgggaaatca ccagcttggc agaacagcct 3720 taccaaggcc tgtctaatga acaggtgttg aaatttgtca tggatggagg gtatctggat 3780 caacccgaca actgtccaga gagagtcact gacctcatgc gcatgtgctg gcaattcaac 3840 cccaacatga ggccaacctt cctggagatt gtcaacctgc tcaaggacga cctgcacccc 3900 agctttccag aggtgtcgtt cttccacagc gaggagaaca aggctcccga gagtgaggag 3960 ctggagatgg agtttgagga catggagaat gtgcccctgg accgttcctc gcactgtcag 4020 agggaggagg cggggggccg ggatggaggg tcctcgctgg gtttcaagcg gagctacgag 4080 gaacacatcc cttacacaca catgaacgga ggcaagaaaa acgggcggat tctgaccttg 4140 cctcggtcca atccttccta acagtgccta ccgtggcggg ggcgggcagg ggttcccatt 4200 ttcgctttcc tctggtttga aagcctctgg aaaactcagg attctcacga ctctaccatg 4260 tccaatggag ttcagagatc gttcctatac atttctgttc atcttaaggt ggactcgttt 4320 ggttaccaat ttaactagtc ctgcagagga tttaactgtg aacctggagg gcaaggggtt 4380 tccacagttg ctgctccttt ggggcaacga cggtttcaaa ccaggatttt gtgttttttc 4440 gttcccccca cccgccccca gcagatggaa agaaagcacc tgtttttaca aattcttttt 4500 tttttttttt ttttttgctg gtgtctgagc ttcagtataa aagacaaaac ttcctgtttg 4560 tggaacaaaa gttcgaaaga aaaaacaaaa caaaaacacc cagccctgtt ccaggagaat 4620 ttcaagtttt acaggttgag cttcaagatg gtttttttgg tttttttttt ttctctcatc 4680 caggctgaag gatttttttt ttctttacaa aatgagttcc tcaaattgac caatagctgc 4740 16 tgctttcata ttttggataa gggtctgtgg tcccggcgtg tgctcacgtg tgtatgcacg 4800 tgtgtgtgtc cattagacac ggctgacgtg tgtgcaaagt atccatgcgg agttgatgct 4860 ttgggaattg gctcatgaag gttcttctca agggtgcgag ctcatccccc tctctccttc 4920 cttcttattg actgggagac tgtgctctcg acagattctt cttgtgtcag aagtctagcc 4980 tcaggtttct accctccctt cacattggtg gccaagggag gagcatttca tttggagtga 5040 ttatgaatct tttcaagacc aaaccaagct aggacattaa aaaaaaaaaa aagaaaaaga 5100 aagaaaaaac aaaatggaaa aaggaaaaaa aaaaagaact gagatgacag agttttgaga 5160 atatatttgt accatattta 5180

<210> 25

<211> 7240

<212> DNA

<213> Homo sapiens

<400> 25 gagctccctg cgaggggtgg acactcccag atgtgcaaag ctcagccacc ctccttctcc 60 tcctctcttc ctcccaggag ctgcatctct gcgtctcccg caagcacttc gctctggaac 120 ggggctgcag gctgcgtggg ctgtcaccgg ggaactacag cgtgcgaatc cgggccacct 180 cccttgcggg caacggctct tggacggaac ccacctattt ctacgtgaca gactattgta 240 agtctccatg gcagcctcag ctgactgggg ctgtgcttag cactgagcat ggtgggacat 300 tgcaggggat gacttggaga ggccacaggt gctggccctg gccttgactc tcaggcctat 360 cagctgctgc ggtgcttgcc ctctttgatc ctgcactttt tttttttttg agatggaggc 420 ttgctttgga gtgcactggc acaatctcag ctcactgtag cctccgcctc ccgggttcaa 480 gtgattctcc cacctcagcc tcacagtagc tgggactaca ggtgcccacc accacgcccg 540 gctaattctt gtatttttag tagagatggc atttcaccat gttggccagg ctggtctcaa 600 actcctgacc tcaagtgatc cgcccacctc ggcctcccaa agtgctggaa ttacaggcat 660 gagccaccat gcctggcctg atcctgcact taaaaaaaaa aaaaaaaaaa gtttcagagg 720 tactcgtgca gttcattata taagtaaatt gtggctgggc acggtggctc acacctgtaa 780 tcccagcact ttgggaggcc gaggcgggca gatcacaagg tcaggagatc gagaccatcc 840 tggccaacat ggtgaaaccc catctctact aaaaatacaa aaataaatta gccaggcatg 900 gtggcgggcg cctgtagtcc cagctactca ggaggctgag gcaggagcct caggaacccg 960 ggaagcagag cttgcagtga accgagatcg tgccactgca ctccagcctg ggcaacacag 1020 tgagactcct tctcaaaaaa taaaataaaa taagtaaatt gggtgttgtt gggggtttgc 1080 tgtacagata attttgtcac ccatgtaatc agcatagtac ctgataggtc gttttttgat 1140 cctttccctc ttctcaccca ccactctcaa gtaggcacct gtgttagtct gtacttacac 1200 tgcaataaag aaatacctgg ccgggcacag tggctcacac ctgtaatccc agcactttgg 1260 gaggccgagg tgggcggatc acttgaggtc aggagttcga gaccagcctg accaacatgg 1320 tgaaacccca tctctaccca aaaatacaaa aattagctgg gcatagtggt gtgcacccgc 1380 17 agtcccagct actcaggagg ctgaggcagg agaatcactt gaacccggga ggcggaggtt 1440 gcagtgagcc gacatcacgc cactgcactc cagcctgggt gacagagtga gactctgtct 1500 caaaaaataa aaaagaaaga aagagtgaaa gagagagaga agaaaaagaa aaagaaagaa 1560 aaggaaagaa agagagaaaa agagagataa aagaaagaaa aagaaagaag agaagagaga 1620 ggaagaggaa gaggtatgcg actgggactc agtaatttat aaagaaaaga ggtttaattg 1680 gctcccagat ctgcaggcag tacaggaacc atgatgctgg catctgctca gcttctgagg 1740 aagcctcaag aaactttcaa tcatggtgga aggtgaagtg ggagcaaggt gttaagacgg 1800 ggagatggtg ctacacactt cttaaacaac cagatcccat gagaacccac ttattataca 1860 gtacccagta gggatgttgc taacccatta gaaaccgcct ccatgatcca atcacctccc 1920 acccggccac tcctccaaca ttggggatta catttcaaca agagctttgg gtggggacac 1980 agatccaaac catagcagtc ccggtgtcta ctgttcactt ctttctgtcc atgtgtggtc 2040 agtgtctcac tctcacttat gggtgagaac atgaggtagt tggttttctg tccctgtgtt 2100 aattcaatta ggataatcat ctccagctcc attcatgttg ctgcaaagaa catgatctca 2160 ttctttttca tggctgtgta gtattccatg gtgtgtatgt ataacatttt ctttatctgg 2220 caatcctgca cttcctcatc tgtacatgga gataataaca gaaccacttc aggaggtgga 2280 gggacatttt aatgacacaa atgttaagtg cctggcacct gttgctagtg tctccatctt 2340 tgttactaga gttttttggc tagatgaggt ggctcacacc tgtaatccca gaactttggg 2400 aggctgaggc aacaggattg cttgaggaca ggcattagag accagcctga gcaacatagc 2460 gagactctgt ctccacaaaa aaatacaaaa attagccagc tatggtggtg catgcttgta 2520 atcccaggta cttgggaggc tgagacaggg ggatcacttg ggcccaggaa tttgaggttg 2580 cggtgaactg tgattgtgcc actatactcc agcctgggtg acagagtaag accctgtctc 2640 taaaaaataa aaattaaaag aagtttacat ctgtcaaaag tcatgctggg atcgggacta 2700 gctatgtaat ttgcaagacc cagtgaacaa tgaaaatgca gaactccttg ctttaaaatt 2760 attaagaatt tggccgggca cgttggctca cgcctgtcat cccagtactt tcggaggctg 2820 aggcgggagg atcacctgag gtcaggagtt tgaggccagc ctggccagca aggtgaaacc 2880 ctgtctctat taaaaataca aaaattagcc gggtgtggtg gtgcatgcct gtagtcccag 2940 ctacttggga ggctgaggca ggagaatcac ttgaacccga aaggaggagg ttccagtgag 3000 ccgagatcgt gccactgaac tctagcctgg gtgtcagagc aagactctgt cacaaaaata 3060 agtaaataaa taaaaattaa aataaaatga ataagcattt cagaggggca acagcagagc 3120 attaaactga cagaaaaggg tcctgcatcc actgcctgag atgtgggagg gatggaaatg 3180 agcagtgatt tggggcaggg gtggggaaga gtgtgcttcc agaatactga cctctgagcc 3240 cactgcctgg tcccactgca cctacgggac tgtttcggga ctgctggaaa atcaggatgt 3300 ggaagagcag cagagaggtt catggacaag ggagggaagg aacagggtgg cccacccatt 3360 ccaggagtgg atgtgatttt tgatgtgaac tttgttggaa acacattgat atgaaacata 3420 tattttctta ttctatttca gtagacgtcc cgtcaaatat tgcaaaaatt atcatcggcc 3480 ccctcatctt tgtctttctc ttcagtgttg tgattggaag tatttatcta ttcctgagaa 3540 agaggtgagt tcagtgagtt cagtggtgtg ctgggaacag ttggttctct gggggaaaac 3600 atgccttgat ataggtatag gcatatttaa gtttattatg aattttgctg atataggatg 3660 18 tgtaacatgc aatttacaga taattgtcat aatatgatat acacaactct ttattgtaaa 3720 ttccctctag acagttgatt ctcacagaat gtttttattg attttttttt ttgcccaaac 3780 ctttatatcc gaagctaacc tattattgca attgataaac aagtaaagct ccaatgtgaa 3840 tgttgattaa tttttcaaaa tttacattaa ggagtaggac ttgactgggc acagtggctc 3900 acacctgtaa tgctagcact ttgggaggcc aaggcgggtg aatcacctga ggtcaggagt 3960 ttgagaccag cctggccaac atggtgaaac ctcgtctcta ctaaaaatac taaaaaatta 4020 accgggcatg gtggtgggcg cctgtaatcc cagctactca ggaggctgag gcaggagaat 4080 tactgaaccc tggtggcgga ggttgcagtg agctgaaatc gcaccattgc attctagcct 4140 gggcgacaga gggagactcc gtctcgggaa aaaaaaaaaa agtaggacaa aactgaaata 4200 agacatatat gttcatcagt gatatgagtg acgtctttgc tgagtcagat ggtaattttt 4260 aaatatcaga agaacatttt gtgccacatg caacatcaca gttgcagaca tgacacgctt 4320 ttaagtttaa tctacatgat taaacatttt tctcagctgg gcacggtggc tcacacctgt 4380 aatcccaaca ctttgggagg ccgaggcggg cggatcatga ggtcaggagt tcgagaccag 4440 cctgaccaac atggtgaaac cccgtctcta ctaaaaatac aaaaattagc aaggtgtggt 4500 gatgtgcgcc tgtaatccca gcttctcagg aggctgaggc aggagaatca cttgaaccca 4560 ggaggcagag gttgcagtga gccgagatcg caccattgca ctccagcctg ggcaacagag 4620 tgagactttg tctcaaaaac aaaacaaaac aaaaaaatat ttttctcatc actttctcaa 4680 gcctggacaa acaacagaac aacaaatcca gtcctgagtt atagcatttg ccagtttctg 4740 taatgtaaat attcccagga tgtctaaatt caagctgtag acataatatt actgagtgca 4800 gtgttagaaa gagatacata atagctcccc attgaatcca ccctatggat acaatatggt 4860 gtataaatga tataatgtaa ataacctcaa ctgcattgat catatttaaa tgtagtatga 4920 gagttaggaa gtgatgagtt ttgaacatgt attgtctttg cttttaggat aatttattta 4980 attgtaagcc tctataattt atattttttg ttctatttgg aaggcattgt aaaatttaat 5040 ctttaatgat gcttgtattt aacaactggc tcactagttt cctgaaaatt taataattgt 5100 ttctcatcag tcgggatgag ctcgctctag aacagtactg ggtgagtggc ttttaagtgt 5160 tacatggatg gccataaatt atttaaaaag ccagccagag ccctgcatgg tcgtgcatat 5220 ctgtagtccc agccgctcgg gaggatgagg caggaggatc acttgagacc aagagttcaa 5280 gaccagcctg ggcaacatag tgagaccctg tctctatgaa attttacaaa ttagccaggt 5340 gtggtggtga gcacctgtat tcccagctat tcagaaactg aagtgggagg atctctggag 5400 cccagaaggt taagactgca gtgagctatg attataccac tgccctccag ccacaacaga 5460 gcaagactgc aactctgaaa tgtaaaaaca aaaacaaaaa caaaacaaaa aaaaaaccac 5520 ccagggaggg atgagtgctc ccatgttgat gcacttacat acctgtctga tgggcttcca 5580 ttcaaaacat aaaggtcccc catccctgcc ctagactgca tctaggatta tggggattct 5640 gctggtaagg gctgccattt gccttgggga gtcttgtatg aaacaccttt ctgcagagtc 5700 ccatgagaat ctcaagctaa cgtgcctcgt tttcctcctc caggcagcca gatgggccgc 5760 tgggaccgct ttacgcttct tcaaaccctg agtatctcag tgccagtgat ggtgagtacc 5820 atcccttccc tgtgggtggc cagaacccta ctcatcagct tcctttgcct tcaccattga 5880 gtgagagtga aggatgggtt ccccagggag gccaagaaaa gccctcttat tcatttgagc 5940 19 ttgccaaact gcccttgctg cagaaacctc attactgtgt gcatctggac acatggtatt 6000 tggcacctgc ctgaatgggc tcatctagcc ggtctgggac ccttgggcag ggtcgaccac 6060 ttgggctggg ctcagctggg cggttcttct ggtcttgcct ggcttcaccc atgtagctac 6120 attttgctgg tgtgtcagct agcactcggt agtcttagat gatttcactc acatgtctgg 6180 tggtcagcag gctgggtggt cccaaggtgg gggccctaag ctgggggagg ctgagcctca 6240 ctctgtccat ctagcctctt aggctccagc aggctggctc aggcttcatt ccatggtcct 6300 ctgttggttc ctagtagcaa gctccagggc aagctccagg gcaacagtcc attccaaatc 6360 tctgcttgga caattcttgt tgattcccat tgaccaaaat aactcacaag gccatgccca 6420 gggccaaggg gtggtgagat agactccacc ttttcatggg aagagctcca agtatcctgg 6480 caaaaaaaaa ccccaaccta ttacaacctg tcttccatcc ccttggcact ttgcagaaac 6540 agtagtctca ggtgggaagt agcatcattc catagcaagg gtctgaaatc agacaagaag 6600 gatggggatg caggtttgcc tcaggacata ttggccagga tcttggacca gttgtggctc 6660 cttccttgag tctctgccat gccctctcca tgggtgcaga tgcctgtcct gttctcggcc 6720 atatgcccag tgcccggcat gggtcctgga tcacagaact catttcatga gtgttttcga 6780 gggggtttgg gtgagggctt gggtggaagg tggctgcaga cccccaagga tcctccaagg 6840 atgctgtgta gataagtaag aagtagtgtt tccatgctct gtgtacgtgc cggacgagtg 6900 ggaggtgtct cgagagaaga tcaccctcct tcgagagctg gggcagggct ccttcggcat 6960 ggtgtatgag ggcaatgcca gggacatcat caagggtgag gcagagaccc gcgtggcggt 7020 gaagacggtc aacgagtcag ccagtctccg agagcggatt gagttcctca atgaggcctc 7080 ggtcatgaag ggcttcacct gccatcatgt ggtgagtcca gtgggggtgg gacacgggct 7140 ggctttcctg acccttccct ttctctgcct cctcctcctg cacagagcga cagaggacac 7200 agggtgtaac ctcctaccca cccctcactc cactaagctt 7240

<210> 26 <211> 478 <212> PRT

<213> Homo sapiens

<400> 26

Met Thr Met Val Asp Thr Glu He Ala Phe Trp Pro Thr Asn Phe Gly

1 5 10 15 lie Ser Ser Val Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe

20 25 30

Asp He Lys Pro Phe Thr Thr Val Asp Phe Ser Ser He Ser Thr Pro 35 40 45

His Tyr Glu Asp He Pro Phe Thr Arg Thr Asp Pro Val Val Ala Asp

50 55 60

Tyr Lys Tyr Asp Leu Lys Leu Gin Glu Tyr Gin Ser Ala He Lys Val

65 70 75 80

Glu Pro Ala Ser Pro Pro Tyr Tyr Ser Glu Lys Thr Gin Leu Tyr Asn 85 90 95

Lys Pro His Glu Glu Pro Ser Asn Ser Leu Met Ala He Glu Cys Arg

100 105 110

Val Cys Gly Asp Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys 20

115 120 125

Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr He Arg Leu Lys Leu He

130 135 140 Tyr Asp Arg Cys Asp Leu Asn Cys Arg He His Lys Lys Ser Arg Asn

145 150 155 160

Lys Cys Gin Tyr Cys Arg Phe Gin Lys Cys Leu Ala Val Gly Met Ser

165 170 175

His Asn Ala He Arg Phe Gly Arg He Ala Gin Ala Glu Lys Glu Lys 180 185 190

Leu Leu Ala Glu He Ser Ser Asp He Asp Gin Leu Asn Pro Glu Ser

195 200 205

Ala Asp Leu Arg Gin Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr He

210 215 220 Lys Ser Phe Pro Leu Thr Lys Ala Lys Ala Arg Ala He Leu Thr Gly

225 230 235 240

Lys Thr Thr Asp Lys Ser Pro Phe Val He Tyr Asp Met Asn Ser Leu

245 250 255

Met Met Gly Glu Asp Lys He Lys Phe Lys His He Thr Pro Leu Gin 260 265 270

Glu Gin Ser Lys Glu Val Ala He Arg He Phe Gin Gly Cys Gin Phe

275 280 285

Arg Ser Val Glu Ala Val Gin Glu He Thr Glu Tyr Ala Lys Ser He

290 295 300 Pro Gly Phe Val Asn Leu Asp Leu Asn Asp G n Val Thr Leu Leu Lys

305 310 315 320

Tyr Gly Val His Glu He He Tyr Thr Met Leu Ala Ser Leu Met Asn

325 330 335

Lys Asp Gly Val Leu He Ser Glu Gly Gin Gly Phe Met Thr Arg Glu 340 345 350

Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys

355 360 365

Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp

370 375 380 Leu Ala He Phe He Ala Val He He Leu Ser Gly Asp Arg Pro Gly

385 390 395 400

Leu Leu Asn Val Lys Pro He Glu Asp He Gin Asp Asn Leu Leu Gin

405 410 415

Ala Leu Glu Leu Gin Leu Lys Leu Asn His Pro Glu Ser Ser Gin Leu 420 425 430

Phe Ala Lys Leu Leu Gin Lys Met Thr Asp Leu Arg Gin He Val Thr

435 440 445

Glu His Val Gin Leu Leu Gin Val He Lys Lys Thr Glu Thr Asp Met 450 455 460 Ser Leu His Pro Leu Leu Gin Glu He Tyr Lys Asp Leu Tyr 465 470 475