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WO1999050449A1 - Polymorphisme ii: liaison de l'asthme avec un locus du chromosome 4 - Google Patents

Polymorphisme ii: liaison de l'asthme avec un locus du chromosome 4 Download PDF

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Publication number
WO1999050449A1
WO1999050449A1 PCT/GB1999/000966 GB9900966W WO9950449A1 WO 1999050449 A1 WO1999050449 A1 WO 1999050449A1 GB 9900966 W GB9900966 W GB 9900966W WO 9950449 A1 WO9950449 A1 WO 9950449A1
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WO
WIPO (PCT)
Prior art keywords
allele
asthma
seq
locus
pair
Prior art date
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Ceased
Application number
PCT/GB1999/000966
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English (en)
Inventor
William Osmond Charles Michael Cookson
Sumit Bhattacharyya
Nicholas Leaves
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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.)
Filing date
Publication date
Application filed by Oxford University Innovation Ltd filed Critical Oxford University Innovation Ltd
Priority to AU31585/99A priority Critical patent/AU3158599A/en
Priority to EP99913467A priority patent/EP1066412A1/fr
Publication of WO1999050449A1 publication Critical patent/WO1999050449A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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 is concerned with methods for the diagnosis of asthma and with materials and methods relating thereto.
  • Asthma is a disease which is becoming more prevalent and is the most common disease of childhood (1). Most asthma in children and young adults is initiated by IgE mediated allergy (atopy) to inhaled allergens such as house dust mite and cat dander. However, not all asthmatics are atopic, and most atopic individuals do not have asthma, so that factors in addition to atopy are necessary to induce the disease (2,3). Asthma is strongly familial, and is due to the interaction between genetic and environmental factors. The genetic factors are thought to be variants of normal genes ("polymorphisms”) which alter their function to predispose to asthma.
  • Asthma may be identified by recurrent wheeze and intermittent air flow limitation.
  • An asthmatic tendency may be quantified by the measurement of bronchial hyper-responsiveness in which an individual's dose-response curve to a broncho-constrictor such as histamine or methacholine is constructed.
  • the curve is commonly summarised by the dose which results in a 20% fall in air flow (PD20) or the slope of the curve between the initial air flow measurement and the last dose given (slope).
  • PD20 20% fall in air flow
  • slope slope of the curve between the initial air flow measurement and the last dose given (slope).
  • Asthma is accompanied by blood eosinophilia, and eosinophils are prominent in asthmatic airways.
  • IgE In the atopic response, IgE is produced by B-cells in response to allergen stimulation. These antibodies coat mast cells by binding to the high affinity receptor for IgE (Fc ⁇ RI). When a multivalent allergen binds to an IgE- coated mast cell, the cross-linking of adjacent IgEs by allergen initiates a series of cellular events leading to the destabilisation of the cell membrane and release of inflammatory mediators. This results in mucosal inflammation, wheezing, coughing, sneezing and nasal blockage.
  • Fc ⁇ RI high affinity receptor for IgE
  • Atopy can be diagnosed by (i) a positive skin prick test in response to a common allergen; (ii) detecting the presence of specific serum IgE for allergen; or (iii) by detecting elevation of total serum IgE.
  • Genetic factors underlying a disease may be identified through localisation to particular chromosomal regions by genetic linkage. Genetic linkage is established by the study of families. It relies on matching the inheritance of disease with genetic polymorphisms of known localisation (known as "genetic markers"). In a complex disease such as asthma, genetic linkage will typically localise genes to within 10 - 20 Megabases (Mb) of DNA. A region of this size may contain 350 - 700 genes, and will be too large to permit immediate identification of the disease-causing gene.
  • Mb Megabases
  • Linkage disequilibrium occurs over 50-500 Kilobases (Kb) of DNA. Linkage disequilibrium may be detected by the study of individuals as well as by the study of families.
  • Disease-causing alleles will be in linkage disequilibrium with non-functional polymorphisms from the same chromosomal segment. It is therefore possible to detect allelic association with disease from particular chromosomal segments, without identifying the exact polymorphism and gene underlying the disease state.
  • allelic association may therefore give information as to disease susceptibility in a particular individual. Furthermore, allelic association is indicative of a disease-causing gene being present within 500 Kb of DNA in either direction from the allele (i.e. 1 Mb in total). Such a region may contain only 30 genes, within which the identification of the disease-causing gene is possible. The presence of linkage disequilibrium also means that other polymorphisms may be anticipated to associate with disease, and that these additional polymorphisms will also be diagnostic of disease susceptibility in particular individuals.
  • WO 95/05481 discloses that variants of the gene encoding the ⁇ -subunit of the high-affinity receptor for IgE (Fc ⁇ Rl ⁇ ) are associated with atopy. It teaches a method for diagnosing atopy which is based upon the demonstration of the presence or absence of one of two variants in a specific portion of the DNA sequence of the gene encoding Fc ⁇ Rl ⁇ , located near the commencement of exon 6 of the Fc ⁇ Rl ⁇ gene on chromosome 11. A further variant has also been found in which the unusual variant sequence is in the coding sequence for the C-terminal cytoplasmic tail of Fc ⁇ Rl ⁇ (4).
  • Tumour Necrosis Factor is a pro-inflammatory cytokine that is found in increased concentration in asthmatic airways (5). We have previously shown that polymorphisms within the TNF gene are associated with an increased risk of asthma (6).
  • the known polymorphisms do not account for all of the genetic factors which predispose to asthma.
  • asthma is not necessarily an atopic disease.
  • Identification of further genetic polymorphisms linked to asthma will allow the identification of children at risk of asthma before the disease, has developed (for example immediately after birth), with the potential for prevention of disease.
  • the presence of particular polymorphisms may predict the clinical course of disease (e.g. severe as opposed to mild) or the response to particular treatments. This diagnostic information will be of use to the health care, pharmaceutical and insurance industries.
  • D4S3032 * 5 a genetic polymorphism known as D4S3032 * 5 on chromosome 4 and a nearby polymorphism known as D4S2921 * 13 are associated with asthmatic traits. Specifically, D4S3032 * 5 is associated with bronchial hyper-responsiveness and D4S2921 * 13 is associated with peripheral eosinophilia, both of these being traits which underlie asthma. The two polymorphisms can therefore be used as diagnostic tools.
  • the invention therefore provides a method for diagnosing an individual as being asthmatic, or as having a predisposition to asthma, which method comprises demonstrating in the individual the presence or absence of one or more alleles which are associated with asthma, wherein the one or more alleles are situated at a locus in a region of chromosome 4 of up to 1 megabase in length, which region contains the locus D4S3032 and/or D4S2921.
  • the 1 Mb region of chromosome 4 referred to flanks the
  • D4S3032 and D4S2921 loci may be the subject of identification in the method according to the invention.
  • Equally two or more such alleles may be the subject of identification, including in particular the combination of D4S3032 * 5 and D4S2921*13.
  • Current diagnostic methods involving detection at the nucleic acid level normally comprise the steps of: (i) obtaining a suitable tissue sample from the individual; (ii) preparing from the tissue sample a nucleic acid sample; (iii) analysing the nucleic acid sample for the presence or absence of the relevant nucleic acid sequence, such as a specific allele.
  • an amplification step is performed prior to the analysis, such that the locus at which the allele is situated is amplified.
  • a preferred amplification technique is the PCR, although any suitable method of nucleic acid amplification may be employed.
  • the invention provides a pair of oligonucleotide primers for amplification of an allele which is associated with asthma, which allele is situated at a locus in a region of chromosome 4 of up to 1 megabase in length, which region contains the locus D4S3032 and/or D4S2921; and an assay kit comprising the pair of oligonucleotide primers.
  • the specific allele for identification may take the form of microsatellite repeats, which are nucleotide sequences containing short, repeated nucleotide motifs, usually a dinucleotide or a trinucleotide motif.
  • a pair of primers which hybridize under suitably stringent conditions, to sequences at a position on either side of the microsatellite repeats, may be used to amplify the microsatellite repeats by PCR. Differences in the number of repeats are recognised by size differences in the PCR products. An allele which has a specified number of repeats and therefore a known size can thus be identified.
  • D4S3032 * 5 and D4S2921 * 13 are examples of such alleles.
  • the primers employed in the method comprise nucleic acid sequences which are complementary to, or substantially complementary to unique sequences either side of the microsatellite repeats, such that only the relevant polymorphic region of the genome is amplified.
  • the conditions under which the amplification is performed are gauged such that specific hybridization of the primers to the flanking sequences occurs and non-specific hybridization is avoided.
  • the hybridization conditions are suitably stringent for that purpose. Standard techniques can be used to identify an appropriate set of reaction conditions.
  • the PCR products are detected by means of a detectable label attached to one of the PCR primers.
  • another form of labeling may be used such as a labeled sequence specific probe which hybridizes to the amplified sequences.
  • the label may be a fluorescent or other label.
  • the PCR products are subjected to size determination, typically involving size-separation for example by gel electrophoresis, and the presence or absence of the allele of interest is determined.
  • the allele for identification may be an allele other than D4S3032 * 5 or D4S2921 * 13 which is in linkage disequilibrium with D4S3032 * 5 or D4S2921 * 13 and is associated with asthma.
  • Functional polymorphisms include polymorphisms within genes, usually within coding sequences of genes.
  • Non-functional polymorphisms are polymorphisms which do not themselves cause the disease.
  • Panel A consisted of 80 nuclear families sub-selected from an
  • Bronchial responsiveness to methacholine was measured as previously described (8): the maximum dose administered was 12 ⁇ mol.
  • the slope of the dose-response curve was calculated as (pre-dose forced expiratory volume in one second (FEV1) - last FEV1) ⁇ the cumulative dose of methacholine).
  • FEV1 pre-dose forced expiratory volume in one second
  • FEV1 last FEV1
  • a constant of 0.01 was added to each measurement, to allow loge transformation when Slope was ⁇ 0.
  • Eosinophils in peripheral blood were Coulter-counted and the values log e transformed before analysis.
  • microsatellite markers D4S3032 and D4S2921 were typed by semi-automated fluorescent methods, as described previously (8). These markers are in close proximity at the telomeric region of the long arm of chromosome 4.
  • the polymerase chain reaction primer sequences for the markers were as follows: D4S3032 5' TGA AAT TCT ATT GAC CAA TGA TGT G (SEQ ID NO: 1) UD4S3032 5' TAG CAC CTG GAT TTA CCA TGA C (SEQ ID NO: 2) D4S2921 5' TCC TTC AGG AAC TGG TG (SEQ ID NO: 3) UD4S2921 5' TTA AAA ATC TAC AGA CAA GGG C (SEQ ID NO: 4)
  • the polymerase chain reaction conditions were as follows: The reaction volumes were 10 ⁇ l, containing 50ng of genomic DNA, 200mM dNTPs, 1 x NH4+ buffer, 50ng oligonucleotide primers (forward labelled fluorescently), 0.5 to 3.0mM MgCI 2 and 0.2U Taq polymerase.
  • PCR products were mixed with a size standard (GS350 TAM) in loading buffer (80% (v/v) formamide, 20% (vlv) 50mM EDTA, 0.1 % (w/v) blue dextran). Samples were denatured at 95°C for 4 min immediately prior to loading onto a 6% polyacrylamide gel and were electrophoresed at 800v for 6h on an Applied Biosystems (ABI) 313 DNA sequencer. Allele sizes were assigned using the ABI GENESCAN and ABI GENOTYPER software.
  • the recombination fraction between D4S3032 and D4S2921 was 3%, indicating in this telomeric region that the distance between the markers is of the order of 0.5 to 1 megabase.

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

Abstract

L'invention concerne une méthode de diagnostic de l'asthme ou d'une prédisposition à l'asthme chez un individu, la méthode consistant à démontrer chez l'individu la présence ou l'absence d'un ou de plusieurs allèles associés à l'asthme, le ou les allèles étant situés dans un locus d'une région de chromosome 4 allant jusqu'à 1 mégabase en longueur, la région contenant le locus D4S3032 et/ou D4S2921.
PCT/GB1999/000966 1998-03-27 1999-03-26 Polymorphisme ii: liaison de l'asthme avec un locus du chromosome 4 Ceased WO1999050449A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU31585/99A AU3158599A (en) 1998-03-27 1999-03-26 Polymorphism ii: linkage of asthma to a locus on chromosome
EP99913467A EP1066412A1 (fr) 1998-03-27 1999-03-26 Polymorphisme ii: liaison de l'asthme avec un locus du chromosome 4

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9806653.3A GB9806653D0 (en) 1998-03-27 1998-03-27 Polymorphism II
GB9806653.3 1998-03-27

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WO1999050449A1 true WO1999050449A1 (fr) 1999-10-07

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PCT/GB1999/000966 Ceased WO1999050449A1 (fr) 1998-03-27 1999-03-26 Polymorphisme ii: liaison de l'asthme avec un locus du chromosome 4

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EP (1) EP1066412A1 (fr)
AU (1) AU3158599A (fr)
GB (1) GB9806653D0 (fr)
WO (1) WO1999050449A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005481A1 (fr) * 1993-08-18 1995-02-23 Isis Innovation Limited Procede de diagnostic et therapie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005481A1 (fr) * 1993-08-18 1995-02-23 Isis Innovation Limited Procede de diagnostic et therapie

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ABRAMSON M ET AL: "The new asthma genetics and its implications for public health", PUBLIC HEALTH REVIEW, vol. 26, no. 2, February 1998 (1998-02-01), pages 127 - 144, XP002110731 *
DANIELS S ET AL: "A genome-wide search for quantitative trait loci underlying asthma", NATURE, vol. 383, 1996, pages 247 - 50, XP002110730 *
DIB C ET AL: "A comprehensive genetic map of the human genome based on 5264 microsatellites", NATURE, vol. 380, 14 March 1996 (1996-03-14), pages 152 - 154, XP002110732 *
MARONE G: "Asthma: recent advances", TRENDS IMMUNOLOGY TODAY, vol. 19, no. 1, January 1998 (1998-01-01), pages 1 - 5, XP004101456 *

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EP1066412A1 (fr) 2001-01-10
GB9806653D0 (en) 1998-05-27
AU3158599A (en) 1999-10-18

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