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WO1999050450A1 - Polymorphisme iii: liaison de l'atopie avec un locus du chromosome 13 - Google Patents

Polymorphisme iii: liaison de l'atopie avec un locus du chromosome 13 Download PDF

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Publication number
WO1999050450A1
WO1999050450A1 PCT/GB1999/000967 GB9900967W WO9950450A1 WO 1999050450 A1 WO1999050450 A1 WO 1999050450A1 GB 9900967 W GB9900967 W GB 9900967W WO 9950450 A1 WO9950450 A1 WO 9950450A1
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WO
WIPO (PCT)
Prior art keywords
allele
locus
region
atopy
pair
Prior art date
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Ceased
Application number
PCT/GB1999/000967
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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 EP99913468A priority Critical patent/EP1066413A1/fr
Priority to AU31586/99A priority patent/AU3158699A/en
Priority to JP2000541337A priority patent/JP2003508006A/ja
Publication of WO1999050450A1 publication Critical patent/WO1999050450A1/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 atopic disease and with materials and methods relating thereto.
  • Atopy is a tendency to develop high levels of IgE and immediate hypersensitivity to allergens.
  • Atopic diseases include hay fever, infantile eczema and most forms of asthma.
  • 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).
  • 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).
  • 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
  • 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. Closer localisation of disease-causing genes may be accomplished by the detection of associations between particular alleles and the disease phenotype. Over short segments of DNA, distinctive alleles of the individual polymorphisms will show non-random association with alleles of neighbouring polymorphisms. This phenomenon, known as “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.
  • Kb Kilobases
  • 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.
  • 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 1 1. 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 atopy. Identification of further genetic polymorphisms linked to atopy will allow the identification of individuals with susceptibility to atopy, for example children at risk before atopic disease has developed, 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 healthcare, pharmaceutical and insurance industries. We have previously established linkage of atopy to chromosome
  • the invention therefore provides a method for diagnosing an individual as being atopic, or as having a predisposition to atopy, which method comprises demonstrating in the individual the presence or absence of an allele which is associated with atopy, wherein the allele is situated at a locus in a region of chromosome 13 of up to 1 megabase in length, which region contains the locus D13S273.
  • the specific allele D13S273*4, or other polymorphisms in the region which are associated with atopy 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.
  • obtaining a suitable tissue sample from the individual (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 13 of up to 1 megabase in length, which region contains the locus D13S273; 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. D13S273 is one such allele.
  • 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 D13S273 which is in linkage disequilibrium with D13S273*4 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 Australian population sample of 230 families (8).
  • the panel contained a total of 203 offspring forming 172 sib-pairs. Fifty-two % of the children were atopic.
  • Panel B consisted of 77 nuclear and extended families recruited from atopy and allergy clinics in the United Kingdom. These families contained 215 offspring forming 268 sib-pairs. Sixty-one % of the children were atopic.
  • STI Skin Test Index
  • microsatellite markers were typed by semi-automated fluorescent methods, as described previously (8).
  • the polymerase chain reaction primer sequences for the marker D13S273 were as follows:
  • 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. Cycling conditions were 1 min at 95°C, 1 min at 55°C and 45s at 72°C; 28 cycles were used. PCRs were performed on an Hybaid Omnigene thermal cycler.
  • Electrophoresis and allele scoring were as follows: PCR products were mixed with a size standard (GS350 TAM) in loading buffer (80% (vlv) formamide, 20% (v/v) 50mM EDTA, 0.1 % (w/v) blue dextran). Samples were denatured at 95°C for 4min immediately prior to loading onto a 6% polyacrylamide gel and were electrophoresed at 800v for 6h on an Applied Biosystems (ABI) 373 DNA sequencer. Allele sizes were assigned using the ABI GENESCAN and ABI GENOTYPER software.

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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)
  • Analytical Chemistry (AREA)
  • 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)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode de diagnostic d'atopie ou d'une prédisposition à l'atopie chez un individu, la méthode consistant à démontrer chez l'individu la présence ou l'absence d'un allèle associé à l'atopie, l'allèle étant situé dans un locus d'une région de chromosome 13 allant jusqu'à 1 mégabase en longueur, la région contenant le locus D13S273.
PCT/GB1999/000967 1998-03-27 1999-03-26 Polymorphisme iii: liaison de l'atopie avec un locus du chromosome 13 Ceased WO1999050450A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99913468A EP1066413A1 (fr) 1998-03-27 1999-03-26 Polymorphisme iii: liaison de l'atopie avec un locus du chromosome 13
AU31586/99A AU3158699A (en) 1998-03-27 1999-03-26 Polymorphism iii: linkage of atopy to a locus on chromosome 13
JP2000541337A JP2003508006A (ja) 1998-03-27 1999-03-26 多型ｉｉｉ：第１３染色体上の遺伝子座へのアトピーの連鎖

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9806656.6 1998-03-27
GBGB9806656.6A GB9806656D0 (en) 1998-03-27 1998-03-27 Polymorphism III

Publications (1)

Publication Number Publication Date
WO1999050450A1 true WO1999050450A1 (fr) 1999-10-07

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PCT/GB1999/000967 Ceased WO1999050450A1 (fr) 1998-03-27 1999-03-26 Polymorphisme iii: liaison de l'atopie avec un locus du chromosome 13

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EP (1) EP1066413A1 (fr)
JP (1) JP2003508006A (fr)
AU (1) AU3158699A (fr)
GB (1) GB9806656D0 (fr)
WO (1) WO1999050450A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817838A (zh) * 2021-08-31 2021-12-21 皖南医学院 一种粉尘螨微卫星标记、其引物及应用和引物的获取方法

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 (5)

* 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 *
WILTSHIRE S ET AL: "A genome scan for loci influencing total serum immunoglobulin levels: possible linkage of IgA to the chromosome 13 atopy locus", HUMAN MOLECULAR GENETICS, vol. 7, no. 1, January 1998 (1998-01-01), pages 27 - 31, XP002110729 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817838A (zh) * 2021-08-31 2021-12-21 皖南医学院 一种粉尘螨微卫星标记、其引物及应用和引物的获取方法

Also Published As

Publication number Publication date
GB9806656D0 (en) 1998-05-27
EP1066413A1 (fr) 2001-01-10
AU3158699A (en) 1999-10-18
JP2003508006A (ja) 2003-03-04

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