WO2001029256A2 - Marqueurs de susceptibilite aux maux de tete - Google Patents

Marqueurs de susceptibilite aux maux de tete Download PDF

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Publication number
WO2001029256A2
WO2001029256A2 PCT/GB2000/004050 GB0004050W WO0129256A2 WO 2001029256 A2 WO2001029256 A2 WO 2001029256A2 GB 0004050 W GB0004050 W GB 0004050W WO 0129256 A2 WO0129256 A2 WO 0129256A2
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Prior art keywords
polymoφhism
polynucleotide
protein
cephalic pain
individual
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PCT/GB2000/004050
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English (en)
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WO2001029256A3 (fr
Inventor
Ian James Purvis
Linda Catherine Mccarthy
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Glaxo Group Limited
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Priority to AU79356/00A priority Critical patent/AU7935600A/en
Publication of WO2001029256A2 publication Critical patent/WO2001029256A2/fr
Publication of WO2001029256A3 publication Critical patent/WO2001029256A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/101—Taqman
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the diagnosis of susceptibility to cephalic pain and agents which can be used in the diagnosis.
  • Cephalic pain disorders are generally multifactorial disorders, many of which have an unknown etiology. No biochemical marker has been found for many of these disorders, and therefore diagnosis can only be done by clinical symptoms. Both environmental and genetic factors are thought to contribute to cephalic pain disorders. In the case of susceptibility to migraine familial aggregation is observed, and segregation analysis of the pattern of inheritance of migraine within families indicates a multifactorial inheritance (not a simple Mendelian inheritance). A multifactorial inheritance means that many genes contribute to the genetic predisposition to migraine, making it difficult to identify the individual susceptibility genes in linkage studies.
  • the inventors have shown that the insulin receptor is involved in the etiology of migraine. They have found that polymorphisms in the insulin receptor gene cause susceptibility to cephalic pain, and in particular to migraine.
  • the invention provides a method of diagnosing susceptibility to cephalic pain in an individual comprising typing in a sample from the individual the insulin receptor gene region or insulin receptor protein ofthe individual and thereby determining whether the individual is susceptible to cephalic pain.
  • the invention also provides a method of diagnosing susceptibility to cephalic pain in an individual, which method comprises typing in vivo the insulin receptor gene region or insulin receptor protein of the individual and determining thereby whether the individual is susceptible to cephalic pain.
  • Figure 1 shows the principle ofthe 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).
  • 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.
  • SEQ ID NO's: 1 to 22 are the sequences of exons 1 to 22 ofthe insulin receptor gene
  • SEQ ID NO: 23 is the complete coding sequence of the insulin receptor mRNA
  • SEQ ID NO: 24 is the sequence ofthe mRNA for the insulin receptor precursor.
  • SEQ ID NO: 25 is the complete sequence from exons 14 to 17 ofthe insulin receptor gene, including introns.
  • the present invention is concerned with the diagnosis of cephalic pain.
  • the insulin receptor gene region or insulin receptor protein of an individual is typed.
  • the individual's susceptibility to cephalic pain can thus be determined.
  • the cephalic pain is typically 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 typing ofthe insulin receptor gene region or insulin receptor protein may comprise the measurement of any suitable characteristic ofthe gene region or receptor to determine whether the individual is susceptible to cephalic pain.
  • the characteristic which is measured is one which can be influenced by a cephalic pain susceptibility polymorphism in the insulin receptor gene region or protein (e.g any such polymorphism mentioned herein).
  • the individual may or may not have a cephalic pain susceptibility polymorphism, but the gene region or receptor may have been affected by other factors (environmental or genetic) which have caused an effect which is similar to the effect ofthe susceptibility polymo ⁇ hism. Such an effect may be any ofthe effects ofthe polymorphisms discussed herein.
  • the typing comprises identifying whether the individual has a cephalic pain susceptibility polymo ⁇ hism, or a polymo ⁇ hism which is in linkage disequilibrium with such a polymo ⁇ hism, in (i) the insulin receptor gene region or (ii) the insulin receptor protein.
  • Polymo ⁇ hisms which are in linkage disequilibrium with each other in a population tend to be found together on the same chromosome. Typically one is found at least 30% ofthe times, for example at least 40 %, 50%, 70% or 90%, of the time the other is found on a particular chromosome in individuals in the population. Thus polymo ⁇ hisms which are not functional susceptibility polymo ⁇ hisms, but are in linkage disequilibrium with the functional polymorphisms, may act as a marker indicating the presence ofthe polymo ⁇ hism.
  • Polymo ⁇ hisms which are in linkage disequilibrium with any of the polymo ⁇ hisms mentioned herein are typically within 500kb, preferably within 400kb, 200kb, 100 kb, 50kb, lOkb, 5kb or 1 kb ofthe polymo ⁇ hism.
  • the term "insulin receptor gene region" generally encompasses any of these distances from 5' to the transcription start site and 3' to the transcription termination site.
  • polymo ⁇ hism which is typed may be in the insulin receptor gene region or protein.
  • the polymo ⁇ hism is typically an insertion, deletion or substitution with a length of at least 1, 2, 5 or more base pairs or amino acids.
  • the polymo ⁇ hism is typically a substitution of 1 base pair, i.e. a single polynucleotide polymo ⁇ hism (SNP).
  • SNP single polynucleotide polymo ⁇ hism
  • the polymo ⁇ hism may be 5' to the coding region, in the coding region, in an intron or 3' to the coding region.
  • the polymo ⁇ hism which is detected is typically the functional mutation which contributes to cephalic pain, but may be a polymo ⁇ hism which is in linkage disequilibrium with the functional mutation.
  • the polymo ⁇ hism will be associated with cephalic pain, for example as can be determined in a case/control study (e.g. as mentioned below).
  • the polymo ⁇ hism will generally cause a change in any ofthe characteristics ofthe receptor discussed herein, such as expression, activity, expression variant, cellular localisation or the pattern of expression in different tissues.
  • the agent may modulate any ofthe following activities ofthe insulin receptor: insulin binding, IGF-1 binding, kinase activity (e.g. tyrosine, threonine or serine kinase activity), autophosphorylation, internalisation, re-cycling, interactions with regulatory proteins, or interactions with signalling complexes.
  • the polymo ⁇ hism may modulate the ability ofthe 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 polymo ⁇ hism typically has an agonist or antagonist effect on any of these characteristics of the receptor. Generally this will lead to a consequent increase or decrease in the activity ofthe pathway.
  • the polymo ⁇ hism may be any ofthe following polymo ⁇ hisms: INSBa,
  • exon ⁇ .poll, exon7.poll , exon7.pol2, exon8.pol2, exon9.pol3, exonl4.poll and INSR-c.4479C>T the form of the polymo ⁇ hism is allele 1 or 2 as defined in Table2.
  • Each of exon ⁇ .poll, exon7.poll, exon7.pol2, exon8.pol2, exon9.pol3, exonl4.poll and INSR-c.4479C>T is in linkage disequilibrium with one of the associated polymo ⁇ hisms, i.e. with one of INSBa, INSCa, exon8.poll, exonl l .poll and exonl7.pol2.
  • the polymo ⁇ hism may be a polymo ⁇ hism at the same location as any of these particular polymo ⁇ hisms (in the case of a SNP, it will be an A, T, C or G at any ofthe locations).
  • the polymo ⁇ hism may be in linkage disequilibrium with any of these particular polymo ⁇ hisms.
  • the polymo ⁇ hism will have a sequence which is different from or the same as the corresponding region in any one of SEQ ID NOS: 1 to 25.
  • a polymo ⁇ hism which can be typed to determine susceptibility to cephalic pain may be identified by a method comprising determining whether a candidate polymo ⁇ hism in the insulin receptor gene region or insulin receptor protein is (i) associated with cephalic pain or (ii) is in linkage disequilibrium with a polymo ⁇ hism which is associated with cephalic pain, and thereby determining whether the polymo ⁇ hism can be typed to determine susceptibility to cephalic pain.
  • the polymo ⁇ hism is typically detected by directly determining the presence of the polymo ⁇ hism sequence in a polynucleotide or protein of the individual.
  • a polynucleotide is typically genomic DNA or mRNA, or a polynucleotide derived from these polynucleotides, such as in a library made using polynucleotide from the individual (e.g. a cDNA library).
  • a library made using polynucleotide from the individual e.g. a cDNA library.
  • the presence ofthe polymo ⁇ hism is determined in a method that comprises contacting a polynucleotide or protein of the individual with a specific binding agent for the polymo ⁇ hism and determining whether the agent binds to a polymo ⁇ hism in the polynucleotide or protein, the binding of the agent to the polymo ⁇ hism indicating that the individual is susceptible to migraine.
  • the agent will also bind to flanking nucleotides and amino acids on one or both sides of the polymo ⁇ hism, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side.
  • determination of the binding ofthe agent to the polymo ⁇ hism can be done by determining the binding ofthe agent to the polynucleotide or protein.
  • the agent is able to bind the corresponding wild-type sequence by binding the nucleotides or amino acids which flank the polymo ⁇ hism position, although the manner of binding will be different to the binding of a polynucleotide or protein containing the polymorphism, and this difference will generally be detectable in the method (for example this may occur in sequence specific PCR as discussed below).
  • the presence of the polymo ⁇ hism is being determined in a polynucleotide it may be detected in the double stranded form, but is typically detected in the single stranded form.
  • the agent may be a polynucleotide (single or double stranded) typically with a length of at least 10 nucleotides, for example at least 15, 20, 30 or more polynucleotides.
  • the agent may be molecule which is structurally related to polynucleotides that comprises units (such as purines or pyrimidines) able to participate in Watson-Crick base pairing.
  • the agent may be a protein, typically with a length of at least 10 amino acids, such as at least 20, 30, 50, 100 or more amino acids.
  • the agent may be an antibody (including a fragment of such an antibody which is capable of binding the polymo ⁇ hism).
  • a polynucleotide agent which is used in the method will generally bind to the polymo ⁇ hism, and flanking sequence, of the polynucleotide ofthe individual in a sequence specific manner (e.g. hybridise in accordance with Watson-Crick base pairing) and thus typically has a sequence which is fully or partially complementary to the sequence of the polymo ⁇ hism and flanking region.
  • the partially complementary sequence is homologous to the fully complementary sequence.
  • the agent is as a probe. This may be labelled or may be capable of being labelled indirectly. The detection of the label may be used to detect the presence ofthe probe on (and hence bound to) the polynucleotide or protein of the individual.
  • the binding ofthe probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein (and thus to separate it from one composition or solution).
  • the polynucleotide or protein of the individual is immobilised on a solid support and then contacted with the probe.
  • the presence of the probe immobilised to the solid support (via its binding to the polymo ⁇ hism) is then detected, either directly by detecting a label on the probe or indirectly by contacting the probe with a moiety that binds the probe.
  • the solid support is generally made of nitrocellulose or nylon.
  • the method may be based on an ELISA system.
  • the method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide which contains the polymo ⁇ hism, allowing (after binding) the two probes to be ligated together by an appropriate ligase enzyme. However the two probes will only bind (in a manner which allows ligation) to a polynucleotide that contains the polymo ⁇ hism, and therefore the detection of the ligated product may be used to determine the presence ofthe polymo ⁇ hism.
  • the probe is used in a heteroduplex analysis based system to detect polynucleotide polymo ⁇ hisms.
  • a heteroduplex analysis based system to detect polynucleotide polymo ⁇ hisms.
  • the probe when the probe is bound to polynucleotide sequence containing the polymo ⁇ hism it forms a heteroduplex at the site where the polymo ⁇ hism occurs (i.e. it does not form a double strand structure).
  • a heteroduplex structure can be detected by the use of an enzyme which single or double strand specific.
  • the probe is an RNA probe and the enzyme used is RNAse H which cleaves the heteroduplex region, thus allowing the polymo ⁇ hism to be detected by means of the detection of the cleavage products.
  • the method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3, 268-71 (1994) and Proc. Natl. Acad. Sci. 85, 4397-4401 (1998).
  • the polynucleotide agent is able to act as a primer for a
  • PCR reaction only if it binds a polynucleotide containing the polymo ⁇ hism (i.e. a sequence- or allele-specific PCR system).
  • a PCR product will only be produced if the polymo ⁇ hism is present in the polynucleotide of the individual.
  • the presence of the polymo ⁇ hism may be determined by the detection ofthe PCR product.
  • the region of the primer which is complementary to the polymo ⁇ hism is at or near the 3' end of the primer.
  • the polynucleotide agent will bind to the wild-type sequence but will not act as a primer for a PCR reaction.
  • the method may be an RFLP based system. This can be used if the presence of the polymo ⁇ hism in the polynucleotide creates or destroys a restriction site which is recognised by a restriction enzyme. Thus treatment of a polynucleotide with such a polymo ⁇ hism will lead to different products being produced compared to the corresponding wild-type sequence. Thus the detection of the presence of particular restriction digest products can be used to determine the presence of the polymo ⁇ hism.
  • the presence ofthe polymo ⁇ hism may be determined based on the change which the presence ofthe polymo ⁇ hism makes to the mobility of the polynucleotide or protein during gel electrophoresis.
  • SSCP polynucleotide single-stranded conformation polymo ⁇ hism
  • Denaturing gradient gel electrophoresis is a similar system where the polynucleotide is electrophoresed through a gel with a denaturing gradient, a difference in mobility compared to the corresponding wild-type polynucleotide indicating the presence of the polymo ⁇ hism.
  • the presence ofthe polymo ⁇ hism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system. This is illustrated in Figure 1.
  • this assay uses an allele specific primer comprising the sequence around, and including, the polymo ⁇ hism.
  • the specific primer is labelled with a fluorescent dye at its 5' end , a quenching agent at its 3' end and a 3' phosphate group preventing the addition of nucleotides to it.
  • the allele specific primer is used in conjunction with a second primer capable of hybridising to either allele 5' ofthe polymo ⁇ hism.
  • Taq DNA polymerase adds nucleotides to the nonspecific primer until it reaches the specific primer. It then releases polynucleotides, the fluorescent dye and quenching agent from the specific primer through its endonuclease activity. The fluorescent dye is therefore no longer in proximity to the quenching agent and fluoresces.
  • the mismatch between the specific primer and template inhibits the endonuclease activity of Taq and the fluorescent dye is not release from the quenching agent. Therefore by measuring the fluorescence emitted the presence or absence of the polymo ⁇ hism can be determined.
  • a polynucleotide comprising the polymo ⁇ hic region is sequenced across the region which contains the polymo ⁇ hism to determine the presence ofthe polymo ⁇ hism.
  • the presence of the polymo ⁇ hism may be determined indirectly, for example by measuring an effect which the polymo ⁇ hism causes. This effect may be in the expression or activity ofthe insulin receptor.
  • the presence of the polymo ⁇ hism may be determined by measuring the activity or level ofthe expression of the insulin receptor in the individual.
  • the expression ofthe insulin receptor may be determined by directly measuring the level of the receptor in the cell or indirectly by measuring the level of any other suitable component in the cell, such as measuring mRNA levels (e.g. using quantitative PCR, such as by a Taqman based method).
  • the method is carried out in vivo, however typically it is carried out in vitro on a sample from the individual.
  • the sample typically comprises a body fluid ofthe individual and may for example be obtained using a swab, such as a mouth swab.
  • the sample may be a blood, urine, saliva, cheek cell or hair root sample.
  • the sample is typically processed before the method is carried out, for example DNA extraction may be carried out.
  • the polynucleotide or protein in the sample may be cleaved either physically or chemically (e.g. using a suitable enzyme).
  • the part of polynucleotide in the sample is copied (or amplified), e.g. by cloning or using a PCR based method. Polynucleotide produced in such a procedure is understood to be covered by the term polynucleotide of the individual" herein.
  • the invention also provides a diagnostic kit that comprises a probe, primer, antibody (including an antibody fragment) or agent as defined herein.
  • the kit may additionally comprise one or more other reagents or instruments (such as mentioned herein) which enable any of the embodiments of the method mentioned above to be carried out.
  • Such reagents or instruments include one or more of the following: a means to detect the binding ofthe agent to the polymo ⁇ hism, a detectable label (such as a fluorescent label), an enzyme able to act on a polynucleotide (typically a polymerase, restriction enzyme, ligase, RNAse H or an enzyme which can attach a label to a polynucleotide), suitable buffer(s) (aqueous solutions) for enzyme reagents, PCR primers which bind to regions flanking the polymo ⁇ hism, a positive and/or negative control, a gel electrophoresis apparatus, a means to isolate DNA from sample, a means to obtain a sample from the individual (such as swab or an instrument comprising a needle) or a support comprising wells on which detection reactions can be done.
  • a detectable label such as a fluorescent label
  • an enzyme able to act on a polynucleotide typically a polymerase, restriction enzyme
  • the invention further provides an isolated polynucleotide or protein that comprises (i) a polymo ⁇ hism that causes susceptibility to cephalic pain or (ii) a naturally occurring polymo ⁇ hism that is in linkage disequilibrium with (i).
  • a polymo ⁇ hism that causes susceptibility may be any of the polymo ⁇ hisms mentioned herein.
  • the polymo ⁇ hism 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.
  • rodent e.g a mouse, rat or hamster
  • a primate e.g. a primate.
  • Such a polynucleotide or protein may comprise any of the human polymo ⁇ hisms 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.
  • sequences may be of a human or animal.
  • the part ofthe 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 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:l 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 ofthe 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 32 P.
  • Selective hybridisation is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and either 0.003M 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 polylysine chains at the 3' and/or 5' ends of the molecule.
  • methylphosphonate and phosphorothioate backbones addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • 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 ofthe polynucleotides represented by SEQ ID NOS: 1 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 polymo ⁇ hism, 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 ofthe 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. Labels are also mentioned above in relation to the method of the invention. Suitable labels include radioisotopes such as 32 P or 35 S, fluorescent labels, enzyme labels or other protein labels such as biotin.
  • Polynucleotides ofthe invention can be inco ⁇ orated into a vector.
  • 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.
  • 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.
  • 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.
  • 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 ofthe 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. 5
  • the proteins and polynucleotides ofthe invention may be present in a substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use 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 10 preparation.
  • homologues of polynucleotide or protein sequences are referred to herein. Such homologues typically have at least 70% homology, preferably at least 80, 90%,
  • 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").
  • the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) 0 (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. 5 Software for performing BLAST analyses is publicly available through the
  • HSPs high scoring sequence pair
  • T some positive-valued threshold score
  • 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 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.
  • P(N) the smallest sum probability
  • 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 invention also provides a non-human animal transgenic for a polymorphism as mentioned above.
  • the animal may be any suitable mammal such as a rodent (e.g. a mouse, rat or hamster) or primate.
  • a rodent e.g. a mouse, rat or hamster
  • the genome of all or some ofthe cells of the animal comprises a polynucleotide of the invention.
  • the animal expresses a protein ofthe invention.
  • the animal suffers from cephalic pain and can be therefore used in a method to assess the efficacy of agents in relieving anti-cephalic pain.
  • the transgenic model can further be used to assess the ability of agents to modulate insulin receptor signalling activity.
  • the invention provides a method for treating a patient who has been diagnosed as being susceptible to cephalic pain by a method ofthe invention, comprising administering an effective amount of an anti-cephalic pain agent to the patient.
  • the anti-cephalic pain agent may therefore be administered to a patient to prevent the onset of such pain or to combat an episode of cephalic pain.
  • the invention also provides: use of an anti-cephalic pain agent in the manufacture of a medicament for use in treating a patient who has been diagnosed as being susceptible to cephalic pain by a method of the invention; and a pharmaceutical pack comprising an anti-cephalic pain agent and instructions for administering of the agent to humans diagnosed by the method of the invention.
  • the anti-cephalic pain agent is typically an anti-migraine agent.
  • Suitable anti-migraine agents are a steroid (e.g. hydrocortisone or dexamethasone, a NSAIDs (non-steroidal anti-inflammatory drug)(e.g. ibuprofen), a 5HT1D agonist, lidocaine (e.g. in the form of a nasal spray), an opioid (e.g. codeine or mo ⁇ hine), an Ergot preparation (e.g. ergotamine or dihydroergotamine), a triptan (e.g.
  • An effective amount of such an agent may be given to a human patient in need thereof.
  • 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 formulation of the agent will depend upon factors such as the nature of the substance and the condition to be treated.
  • the agent is formulated for use with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent For example it may be formulated for oral, parenteral, intravenous, intramuscular or subcutaneous administration. A physician will be able to determine the required route of administration for each particular patient.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • the effectiveness of particular anti-cephalic agents may be affected by or dependent on whether the individual has particular polymo ⁇ hisms in the insulin receptor gene region or insulin receptor.
  • the invention can allow the determination of whether an individual will respond to a particular anti-cephalic pain agent by determining whether the individual has a polymo ⁇ hism which affects the effectiveness of that agent.
  • the invention includes a method of treating a patient who has been identified as being able to respond to the agent comprising administering the agent to the patient.
  • certain anti-cephalic agents may produce side effects in individuals with particular polymo ⁇ hisms in the insulin gene region or protein.
  • the invention can also allow the identification of a patient who is at increased risk of suffering side effects due to such an anti-cephalic agent by identifying whether an individual has such a polymo ⁇ hism.
  • Individuals who carry a particular polymo ⁇ hism in the insulin receptor gene may exhibit differences in their ability to regulate metabolic pathways under different physiological conditions and will display altered reactions to different diseases.
  • differences in metabolic regulation arising as a result of the polymo ⁇ hism may have a direct effect on the response of an individual to gene therapy.
  • the polymo ⁇ hism may therefore have the greatest effect on the efficacy of drugs designed to modulate the activity ofthe insulin receptor or other components in its signalling pathway.
  • the polymo ⁇ hisms may also affect the response to agents acting on other biochemical pathways regulated by the insulin receptor.
  • the invention may therefore be useful both to predict the clinical response to such agents and to determine therapeutic dose.
  • the invention can be used to assess the predisposition and /or susceptibility of an individual to diseases mediated by the insulin receptor.
  • Polymo ⁇ hisms may be particularly relevant to the development of such diseases.
  • the present invention may be used to recognise individuals who are particularly at risk from developing these conditions.
  • the invention may further be used in the development of new drug therapies which selectively target one or more allelic variants of the insulin receptor gene (i.e. which have different polymo ⁇ hisms). Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy may have a significant impact on the design of new drugs. Drugs may be designed to regulate the biological activity ofthe variants implicated in the disease process while minimising effects on other variants.
  • the following Example illustrates the invention:
  • Migraine without aura - HA (head ache) lasting 4-72 firs if unsuccessfully treated;
  • HA with at least 2 ofthe 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).
  • HA defined as above, with onset accompanying or following aura within 60 minutes.
  • HA fulfills migraine with aura characteristics; aura includes hemiparesis that may be prolonged (> 60 minutes): at least 1 first-degree relative with similar HAs.
  • Samples were obtained from the study group and genomic DNA extracted using a standard kit and a slating out technique (Cambridge Molecular). The genotypes ofthe migraineurs 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.
  • 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 polymo ⁇ hic 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' ofthe 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.
  • the allele specific primer hybridized to the other allele 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 polymo ⁇ hic site typed is given together with the flanking sequence 5' and 3'.

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Abstract

La présente invention concerne un procédé permettant de diagnostiquer la susceptibilité aux maux de tête d"un individu, ledit procédé comprenant le typage dans un échantillon de l"individu, de la portion de gène de récepteur à insuline ou de la protéine récepteur à insuline de l"individu, et la détermination de la susceptibilité ou non de l"individu aux maux de tête.
PCT/GB2000/004050 1999-10-19 2000-10-19 Marqueurs de susceptibilite aux maux de tete WO2001029256A2 (fr)

Priority Applications (1)

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AU79356/00A AU7935600A (en) 1999-10-19 2000-10-19 Cephalic pain susceptibility marker

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GB9924717.3 1999-10-19
GBGB9924717.3A GB9924717D0 (en) 1999-10-19 1999-10-19 Diagnostic

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PCT/GB2000/004050 WO2001029256A2 (fr) 1999-10-19 2000-10-19 Marqueurs de susceptibilite aux maux de tete

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001028540A2 (fr) * 1999-10-19 2001-04-26 Glaxo Group Limited Therapie de la douleur cephalique
WO2001028539A2 (fr) * 1999-10-19 2001-04-26 Glaxo Group Limited Agent pour le traitement de la douleur cephalique

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7107882B2 (ja) * 2019-04-22 2022-07-27 ジェネシスヘルスケア株式会社 偏頭痛のリスクを判定する方法

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US4761371A (en) * 1985-02-12 1988-08-02 Genentech, Inc. Insulin receptor
EP0503440A1 (fr) * 1991-03-08 1992-09-16 Glaxo Group Limited Compositions à base de sumatriptan
US5677279A (en) * 1996-12-16 1997-10-14 Amylin Pharmaceuticals, Inc. Methods and compositions for treating pain with amylin or agonists thereof
EP0803570A1 (fr) * 1994-05-12 1997-10-29 Otsuka Pharmaceutical Co., Ltd. Adn mutant recepteur de l'insuline humaine

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US4761371A (en) * 1985-02-12 1988-08-02 Genentech, Inc. Insulin receptor
EP0503440A1 (fr) * 1991-03-08 1992-09-16 Glaxo Group Limited Compositions à base de sumatriptan
EP0803570A1 (fr) * 1994-05-12 1997-10-29 Otsuka Pharmaceutical Co., Ltd. Adn mutant recepteur de l'insuline humaine
US5677279A (en) * 1996-12-16 1997-10-14 Amylin Pharmaceuticals, Inc. Methods and compositions for treating pain with amylin or agonists thereof

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BARBETTI F ET AL: "DETECTION OF MUTATIONS IN INSULIN RECEPTOR GENE BY DENATURING GRADIENT GEL ELECTROPHORESIS" DIABETES, NEW YORK, NY, US, vol. 41, no. 4, 1 April 1992 (1992-04-01), pages 408-415, XP000615530 ISSN: 0012-1797 *
KROOK A ET AL: "RAPID AND SIMULTANEOUS DETECTION OF MULTIPLE MUTATIONS BY POOLED AND MULTIPLEX SINGLE NUCLEOTIDE PRIMER EXTENSION: APPLICATION TO THE STUDY OF INSULIN-RESPONSIVE GLUCOSE TRANSPORTER AND INSULIN RECEPTOR MUTATIONS IN NON-INSULIN-DEPENDENT DIABETES" HUMAN MOLECULAR GENETICS, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 1, no. 6, 1992, pages 391-395, XP000939000 ISSN: 0964-6906 *
MORRIS B.: "Insulin receptor gene in hypertension" CLINICAL AND EXPERIMENTAL HYPERTENSION, vol. 19, no. (5 & 6), 1997, pages 551-65, XP001053711 *
NYHOLT D ET AL: "Familial typical migraine" NEUROLOGY, vol. 50, no. 5, May 1998 (1998-05), pages 1428-32, XP001053727 *
SPLIT W ET AL: "HEADACHES IN NON INSULIN-DEPENDENT DIABETES MELLITUS" FUNCTIONAL NEUROLOGY, CIC EDIZIONI INTERNATIONALI, ROME, IT, vol. 12, no. 6, November 1997 (1997-11), pages 327-332, XP001028137 ISSN: 0393-5294 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001028540A2 (fr) * 1999-10-19 2001-04-26 Glaxo Group Limited Therapie de la douleur cephalique
WO2001028539A2 (fr) * 1999-10-19 2001-04-26 Glaxo Group Limited Agent pour le traitement de la douleur cephalique
WO2001028540A3 (fr) * 1999-10-19 2002-07-11 Glaxo Group Ltd Therapie de la douleur cephalique
WO2001028539A3 (fr) * 1999-10-19 2002-09-12 Glaxo Group Ltd Agent pour le traitement de la douleur cephalique

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WO2001029255A3 (fr) 2002-05-16
WO2001029255A9 (fr) 2002-02-07
WO2001029255A2 (fr) 2001-04-26
JP2003512062A (ja) 2003-04-02
WO2001029256A3 (fr) 2002-06-13
GB9924717D0 (en) 1999-12-22
EP1226276A2 (fr) 2002-07-31
AU1152001A (en) 2001-04-30

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