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WO1999027130A1 - Procede de detection d'une predisposition genetique a des formes courantes de migraine - Google Patents

Procede de detection d'une predisposition genetique a des formes courantes de migraine Download PDF

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Publication number
WO1999027130A1
WO1999027130A1 PCT/AU1998/000973 AU9800973W WO9927130A1 WO 1999027130 A1 WO1999027130 A1 WO 1999027130A1 AU 9800973 W AU9800973 W AU 9800973W WO 9927130 A1 WO9927130 A1 WO 9927130A1
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WO
WIPO (PCT)
Prior art keywords
htr
allele
migraine
2cser
individual
Prior art date
Application number
PCT/AU1998/000973
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English (en)
Inventor
Lynette Robyn Griffiths
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Griffith University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Griffith University filed Critical Griffith University
Priority to AU13273/99A priority Critical patent/AU1327399A/en
Publication of WO1999027130A1 publication Critical patent/WO1999027130A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • 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

Definitions

  • THIS INVENTION relates to a method of detecting a genetic predisposition to common forms of migraine.
  • Migraine is a common complex disorder the aetiology of which remains unknown. However, the disease does show strong familial aggregation, such that up to 50% of first-degree relatives of sufferers are also affected with the disorder (Dalsgaard-Nielson & Ulrich, 1972,
  • Familial Hemiplegic Migraine has been mapped to chromosome 19q13 (Joutel et al., 1993, Nature Genet. 5 40). Subsequent studies have indicated heterogeneity of FHM, with about 50% of tested families showing genetic linkage to this region (Joutel et al., 1993, supra, Ophoff et al., 1994, Genomics, 22 21 , Joutel et al., 1994, Am. J. Hum. Genet. 55 1186; Ophoff et a/., 1996, Eur. J. Hum. Genet. 4 321 ).
  • 5-HT 5- hydroxytryptamine
  • This neurotransmitter molecule has been implicated in several neurological disorders such as depression, aggression, suicidal tendencies, alcoholism and impulsive disorders (Lappalainen et al., 1995, Genomics 27 294, which is herein incorporated by reference).
  • the receptors for 5-HT are designated 5-HTR 2A 5-HTR 2B and 5-HTR 2C and are expressed widely throughout the brain.
  • polymorphic variants of these receptors also exist, such as encoded by the 5-HTR 2CSER allele which incorporates a G to C transversion at codon 23, and thereby encodes a non-conservative amino acid substitution of Ser 23 for Cys 23 in the first transmembrane domain of the receptor protein (Lappalainen et al., 1995, supra).
  • 5-HTR 2A and 5-HTR 2C antagonists prevent migraine (Humphrey et al., 1990, Ann. NY. Acd. Sci. 600 587) and that the gene encoding 5-HTR 2C is located on the X-chromosome (Milatovitch et al., 1992, Hum. Mol. Gen.
  • the present inventors have realized that, to date, no genetic markers have been identified which are associated with common forms of migraine, and that as a result, there are no reliable methods for detecting a genetic predisposition to common forms of migraine. However, the present inventors have discovered that individuals possessing the 5- HTR 2CSER allele show a statistically-significant likelihood of displaying clinical migraine symptoms.
  • the present invention resides in a method for detecting whether an individual is predisposed to common forms of migraine, which method includes the steps of (i) obtaining a sample of nucleic acid from said individual; and (ii) determining whether a 5-HTR 2CSER allele is present in said sample to indicate whether said individual is predisposed to common forms of migraine.
  • the present inventors have used this surprising result to provide the method of the invention, which method identifies human individuals predisposed to common forms of migraine such as migraine with aura and migraine without aura.
  • predisposed means having an increased probability that said individual may display symptoms of common forms of migraine.
  • the increased probability is based on experimental data obtained by the present inventors showing a statistically significant correlation between suffering from migraine symptoms and possession of the 5-HTR 2CSER allelic polymorphism.
  • the method of the invention is well-suited to genetic screening and counseling applications, as well as in assisting diagnosis of migraine sufferers.
  • HTR 2CSER allele and migraine with aura (MA) in particular.
  • the method of the invention detects whether said individual is predisposed to migraine with aura (MA).
  • said individual is a human.
  • Said nucleic acid sample is obtained from any suitable cells or tissues of a human, such as blood leukocytes, amniotic fluid, skin or the like.
  • said cells are blood leukocytes.
  • said nucleic acid is genomic DNA, although RNA is contemplated.
  • cDNA is reverse transcribed from RNA by methods well known in the art.
  • Determination of whether said sample contains a 5- HTR 2CSER allele may be performed using any of a variety of techniques such as PCR-RFLP analysis, fluorescence-based melt curve analysis, Single Strand Conformational Polymorphism (SSCP) analysis, Denaturing Gradient Gel Electrophoresis (DGGE) or simply by nucleotide sequencing. Whichever method is used, the ability to detect the presence of the 5-HTR 2CSER allele is essential.
  • melt curve analysis can be performed using a fluorochrome-labeled 5-HTR 2CSER allele-specific probe which forms base- pair mismatches with DNA strands that do not encode 5-HTR 2CSER . These are detectable by virtue of their lower melting temperature (TJ compared to fully complementary sequences.
  • TJ melting temperature
  • samples obtained from 5- HTR 2CSER homozygotes, 5-HTR 2CSER/CYS heterozygotes and 5-HTR 2CCYS homozygotes can be distinguished on the basis of melt curve analysis.
  • a demonstration of allele-specific melt curve analysis can be found, for example, in International Publication No. WO97/46714 which is herein incorporated by reference.
  • DGGE also exploits T m differences, but uses differential electrophoretic migration through gradient gels as a means of distinguishing between polymorphic alleles.
  • An example of DGGE can be found in Folde & Loskoot, 1994, Hum. Mut. 3 83, which is herein incorporated by reference.
  • 5-HTR 2CSER allele is performed using PCR-RFLP analysis of human genomic DNA, essentially as described in Lappalainen et al., 1995, supra, and as will be described hereinafter.
  • the method of the invention includes the steps of:-
  • step (ii) to digestion with Hinfl restriction endonuclease to thereby generate one or more restriction fragments; and (iv) determining relative molecular sizes of said restriction fragments obtained in step (iii).
  • the presence of an 86 bp first said restriction fragment in step (iv) is indicative of the 5-HTR 2CSER allele being present in said sample.
  • the presence of said 86 bp fragment alone indicates that said individual is a homozygote
  • an additional 104 bp second said restriction fragment indicates that said individual is a heterozygote.
  • the 104 bp restriction fragment corresponds to the 5-HTR 2CCYS allele. Samples containing only the 104 bp restriction fragment are indicative of 5-HTR 2CCYS homozygotes.
  • the presence of an 18 bp third said restriction fragment, in addition to said 86 bp fragment, is indicative of he 5-HTR 2CSER allele being present in said sample.
  • the 86 bp, 104 bp and 18 bp relative molecular sizes attributed to said first, second and third restriction fragments respectively are approximate sizes and are subject to variation inherent in size determinations of macromolecules such as DNA.
  • said relative molecular sizes include such variations, and are not meant to be absolute size determinations.
  • the invention resides in a kit for detecting a genetic predisposition to common forms of migraine, said kit comprising:-
  • kits are applicable to PCR-based RFLP analysis.
  • the primers have the following nucleotide sequences:-
  • the molecular size markers are in the form of an 86 bp DNA molecule corresponding to a 5-HTR 2CSER allele, and a 104 bp DNA molecule corresponding to a 5-HTR 2CCYS allele.
  • An 18 bp DNA molecule may also be provided with the 86 bp size marker.
  • corresponding refers to the molecular size marker having approximately the same relative molecular size as Hinfl restriction fragments generated from 5- HTR 2C PCR amplification products.
  • Hinfl restriction endonuclease may also be included in said kit.
  • FIG. 1 5% Ultra High resolution gel electrophoresis of samples subjected to PCR-RFLP analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT MATERIALS AND METHODS 1.1 Families and Diagnoses Prior to commencement, all research was approved by
  • DNA was extracted from blood leukocyte samples by a standard SDS-Proteinase K method incorporating a salting out procedure (Nyholt et a/., 1996, supra).
  • PCR-RFLP analysis PCR was performed using 5-HTR 2C specific primers which do not discriminate between 5-HTR 2CCYS and 5-HTR 2CSER alleles (5-HTR 2C allele non-specific primers ) essentially as described in Lappalainen et al, 1995, supra.
  • the primer sequences used corresponded to SEQ ID NO: 1 and SEQ ID NO: 2. However, the following alternative thermal cycling conditions were used:
  • PCR fragments generated by this method were then digested with 12-16 Units Hinfl restriction endonuclease at 37°C for 16 hours. In some cases where it was difficult to obtain reproducible genotyping results, PCR fragments were repeatedly subjected to Hinfl digestion up to five times. Fragments were then subjected to 5% Ultra High resolution agarose gel electrophoresis, which is capable of resolving 40-130 base pair (bp) DNA fragments. This method was used in preference to 10% polyacrylamide (Lappalainen et al, 1995, supra) and standard 5 % agarose gel electrophoresis.
  • 5-HTR 2CCYS homozygote HH: 104 bp.
  • 5-HTR 2CSER homozygote hh: 86 bp and 18 bp;
  • 5-HTR 2CCYS/SER heterozygote Hh:104bp, 86b and 18bp.
  • FIG. 1 An example of an array of electrophoretically-separated bands is shown in FIG. 1.
  • the gel electrophoresis system did not allow resolution of the predicted 18 bp fragment.
  • genotypes could be distinguished by the presence of 86 bp and 104 bp (Hh) and 86 bp (hh) fragments respectively.
  • 5-HTR 2CSER and 5-HTR 2CCYS do not display significant differences in their responses to serotonin under normal physiological conditions (Lappalainen et al., 1996, supra). Although the functional significance of this discovery remains to be elucidated, it is clear that it is of great practical significance.
  • the method of the invention provides a means whereby predisposition to common forms of migraine can be determined by a genetic test. This test could be used:-
  • FIG. 1 A first figure.
  • restriction fragment is not visible.
  • HH 5-HTR 2CCYS homozygotes
  • Hh 5-HTR 2CCYS/SER heterozygotes
  • hh 5-HTR 2CSER homozygotes
  • d.f degrees of freedom
  • O observed frequency
  • H 5-HTR 2CCYS allele
  • H 5-HTR 2CCYS allele
  • h 5-HTR 2CSER allele
  • d.f 5-HTR 2CSER allele
  • H 5-HTR 2CCYS allele
  • h 5-HTR 2CSER allele
  • d.f 5-HTR 2CSER allele
  • HH 5-HTR 2CCYS homozygotes
  • Hh 5-HTR 2CCYS/SER heterozygotes
  • hh 5-HTR 2CSER homozygotes
  • d.f degrees of freedom.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détection permettant de déterminer si un individu est enclin à des formes courantes de migraine. Ce procédé consiste à déterminer si l'individu est porteur d'un allèle 5-HTR2CSER. La présence de cet allèle indique une prédisposition de l'individu à des formes courantes de migraine.
PCT/AU1998/000973 1997-11-24 1998-11-24 Procede de detection d'une predisposition genetique a des formes courantes de migraine WO1999027130A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13273/99A AU1327399A (en) 1997-11-24 1998-11-24 A method of detecting a genetic predisposition to common forms of migraine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPP0515 1997-11-24
AUPP0515A AUPP051597A0 (en) 1997-11-24 1997-11-24 A detection method

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WO1999027130A1 true WO1999027130A1 (fr) 1999-06-03

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996031621A2 (fr) * 1995-04-07 1996-10-10 Smithkline Beecham Plc Procede pour determiner la sensibilite de sujets a des agents modulateurs des recepteurs 5-ht¿2?
WO1997011175A1 (fr) * 1995-09-23 1997-03-27 Medical Research Council Depistage de troubles lies au dysfonctionnement serotoninergique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996031621A2 (fr) * 1995-04-07 1996-10-10 Smithkline Beecham Plc Procede pour determiner la sensibilite de sujets a des agents modulateurs des recepteurs 5-ht¿2?
WO1997011175A1 (fr) * 1995-09-23 1997-03-27 Medical Research Council Depistage de troubles lies au dysfonctionnement serotoninergique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CEPHALAGIA, Volume 16, No. 7, November 1996, NYHOLT D.R. et al., "Migraine Association and Linkage Analysis of the Human 5-Hydroxytryptamine (5-HT2A) Receptor Gene", pages 463-467. *
HEADACHE, Volume 36, No. 4, April 1996, BUCHWALDER A. et al., "Exclusion of 5-HT2A and 5-HT2C Receptor Genes as Candidate Genes for Migraine", pages 254-258. *
NEUROREPORT, Volume 8, No. 12, 18 August 1997, BURNET P.W. et al., "Allelic Variation in the Serotonin 5-HT2C Receptor Gene and Migraine", pages 2651-2653. *
NEUROSCIENCE LETTERS, Volume 212, No. 1, 5 July 1996, GUTIERREZ B. et al., "Allelic Association Analysis of the 5-HT2C Receptor Gene in Bipolar Affective Disorder", pages 65-67. *

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