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WO2008006105A2 - Marqueurs génétiques dans le gène du récepteur de la tachykinine nk1 (tacr1) correspondant à des troubles asthmatiques - Google Patents

Marqueurs génétiques dans le gène du récepteur de la tachykinine nk1 (tacr1) correspondant à des troubles asthmatiques Download PDF

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WO2008006105A2
WO2008006105A2 PCT/US2007/073066 US2007073066W WO2008006105A2 WO 2008006105 A2 WO2008006105 A2 WO 2008006105A2 US 2007073066 W US2007073066 W US 2007073066W WO 2008006105 A2 WO2008006105 A2 WO 2008006105A2
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marker
asthma
nucleic acid
tacrl
haplotype
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PCT/US2007/073066
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WO2008006105A9 (fr
WO2008006105A3 (fr
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Eva Halapi
Hakon Hakonarson
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Decode Genetics Ehf.
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Publication of WO2008006105A3 publication Critical patent/WO2008006105A3/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
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • Asthma (Morbidity Number (MIM) 600807], the most common chronic disease affecting children and young adults, is a complex genetic disorder with several overlapping phenotypes (Cookson and Moffatt, Hum. MoL Genet, 9:2359-64, 2000; Weiss, Ann. Allergy Asthma Immunol, 87 (Suppl l):5-8, 2001). Asthma attacks all age groups but often starts in childhood. It is a disease characterized by recurrent attacks of breathlessness and wheezing, which vary in severity and frequency from person to person.
  • the condition is due to inflammation of the air passages in the lungs and affects the sensitivity of the nerve endings in the airways so they become easily irritated.
  • the lining of the passages swell, causing the airways to narrow and reducing the flow of air in and out of the lungs.
  • the airways constrict, become inflamed, and become lined with mucous, often in response to one or more "triggers,” such as exposure to an allergen, cold air, exercise, or emotional stress.
  • asthma is caused by multiple interacting genes, some having a protective effect and others contributing to the disease pathogenesis, with each gene having its own tendency to be influenced by the environment (Koppelman et al, supra;; Cookson, 1999, supra; Holloway et al, 1999, supra).
  • the complex nature of the asthma phenotype, together with substantial locus heterogeneity and environmental influence, has made it difficult to uncover genetic factors that underlie asthma. [0004]
  • the human and economic burden associated with this condition is severe.
  • Asthma is not just a public health problem for developed countries. In developing countries, however, the incidence of the disease varies greatly. India has an estimated 15-20 million asthmatics. In Brazil, Costa Rica, Panama, Peru and Brazil, prevalence of asthma symptoms in children varies from 20% to 30%. Asthma is a chronic disease for which there is currently no cure. Although some of the underlying mechanisms have been elucidated in recent years, much more needs to be done to gain a clearer understanding of the many genetic and environmental factors that contribute to the development of various forms of asthma.
  • the present invention provides materials and methods that address one or more of the needs identified above.
  • the invention relates to methods of diagnosing an increased susceptibility to bronchial asthma, by evaluating certain markers or haplotypes relating to the TACRl gene (tachykinin receptor 1, also referred to as: NKlR, NKIR, SPR, NK 1 ).
  • the methods comprise detecting a genetic marker associated with the exon 2 LD block of TACRl gene.
  • the methods comprise detecting a genetic marker outside of the exon 2 LD block of the TACRl gene.
  • markers located outside of the exon 2 LD block have been identified that are in linkage disequilibrium with markers in the exon 2 LD block.
  • Aspects of the invention described with respect to exon 2LD block markers can be practiced with those markers that are outside of the block yet in linkage disequilbrium. All such variations are intended as aspects of the invention.
  • One aspect of the invention is a medical procedure comprising: analyzing nucleic acid from a human individual to measure a marker or haplotype in a tachykinin NK 1 receptor (TACRl) gene; and determining a status of a genetic indicator of an asthma disorder in the individual from the measurement of the marker or haplotype.
  • TACRl tachykinin NK 1 receptor
  • medical procedure is not intended to be limiting and describes a method or process that provides useful medical information to a clinician, and that optionally may include additional steps (e.g., selection of a therapeutic regimen and/or administration of a therapy).
  • the nucleic acid to be analyzed can be from any biological sample (e.g., tissue or fluid) obtainable from a human subject, such as a blood sample or tissue biopsy, that contains DNA or RNA from the individual. Genomic DNA is preferred for measurements of markers or haplotypes that map to non-coding portions of the gene.
  • human individual is intended to include both living humans (adults, children, infants), humans in utero, human embryos that may exist ex vivo, and deceased humans. Testing of a sample from deceased humans may provide valuable genetic information for living relatives.
  • exemplary measurements include determining the identity of an allele for the marker, e.g., determining the specific nucleotide present on a chromosome at the location of a polymorphic site (a SNP), or determining the number of bases/repeats defining an allele of a micro satellite marker. Determining the identity on one chromosome or on both chromosomes is specifically contemplated.
  • a haplotype measurement is a determination of alleles of two or more markers that, together, are indicative of the presence or absence of a haplotype.
  • haplotypes can be present or absent in a human subject.
  • haplotypes, or alleles of certain individual markers correlate with an increased or decreased prevalence of asthma in population cohorts that have been studied.
  • Such haplotypes or markers thereby serve as a genetic indicator of asthma.
  • the status of such a genetic indicator in a human subject refers to whether the correlative marker is present or absent in DNA from the human subject, and optionally, to conclusions that may be drawn from its presence or absence.
  • the identification of a marker correlated with increased incidence of asthma in human subjects provides an indication that the subject may be at increased risk for developing asthma.
  • the identification of the same marker provides information about a genetic factor that may contribute to the asthma, which has relevance to selection of the most effective therapeutic or prophylactic treatment regimen.
  • RNA or DNA nucleic acid
  • exemplary techniques include for analyzing nucleic acid (RNA or DNA) comprise sequencing, amplification, and/or hybridization procedures in which it is helpful to have an oligonucleotide or polynucleotide that hybridizes to a portion of a gene that contains a marker, or to an adjacent portion.
  • the analyzing employs a TACRl nucleic acid molecule.
  • TACRl nucleic acid molecule is intended to comprise all or part of a TACRl genomic DNA sequence, or a cDNA/mRNA sequence.
  • the molecule may be single or double stranded, may be DNA or RNA and may even include synthetic bases or other modifications (e.g., labels) that are helpful in nucleic acid analytical techniques.
  • the invention includes the use of TACRl nucleic acid molecule for analyzing nucleic acid from a human individual, to determine of a status of a genetic indicator of an asthma disorder in the human individual by measuring a tachykinin NK 1 receptor (TACRl) gene marker or haplotype in nucleic acid from the human individual.
  • TACRl tachykinin NK 1 receptor
  • the marker or haplotype is located in an exon 2 LD block of the TACRl gene (SEQ ID NO: 1). Data for this region is provided, e.g., in Example 1. It is contemplated that markers in other portions of the TACRl gene that also are predictive of asthma risk may be used in the invention.
  • the marker or haplotype is located in TACRl nucleic acid molecule that comprises the nucleotide sequence of SEQ ID NO: 48 (or its complement).
  • the TACRl nucleic acid molecule that is used to practice the invention comprises the nucleotide sequence of SEQ ID NO: 1 (or its complement) or a fragment thereof effective for identification, in human nucleic acid, of a marker or haplotype in an exon 2 LD block of the TACRl gene (SEQ ID NO: 1).
  • the TACRl nucleic acid comprises a fragment of SEQ ID NO: 1 (or its complement) of less than 250 nucleotides that includes a marker or haplotype within SEQ ID NO: 1 that is indicative of increased or decreased risk of developing asthma. Any integer length fragment that can be used, e.g., in a hybridization or annealing assay, is contemplated.
  • the TACRl nucleic acid comprises a fragment of SEQ ID NO: 1 (or its complement) of less than 250 nucleotides that is adjacent, within 250 bases, of a marker or haplotype within SEQ ID NO: 1 that is indicative of increased or decreased risk of developing asthma.
  • Such nucleic acids are useful, e.g., for amplification (e.g., PCR or primer extension) and sequencing procedures.
  • the TACRl nucleic acid comprises a fragment of SEQ ID NO: 48 (or its complement) of less than 250 nucleotides that is adjacent, within 250 bases, of a marker or haplotype within SEQ ID NO: 48 that is indicative of increased or decreased risk of developing asthma.
  • Such nucleic acids are useful, e.g., for amplification (e.g., PCR or primer extension) and sequencing procedures.
  • the procedure, method, or use of the invention further comprises determining if the human individual is a member of a human subpopulation, wherein the marker or haplotype correlates with a decreased prevalence of an asthma disorder in members of the subpopulation with the marker or haplotype compared to members lacking the marker or haplotype.
  • Exemplary "subpopulations" are any groupings that doctors or population genetics use or into which humans self-classify. A population can be divided into subpopulations based on age, sex, race, ethnicity, religion, nation/region of origin, weight, behavioral factors, and environmental factors, for example. Exemplary subpopulations are those of a specific sex, age group, race, ethnicity, height, weight, or body mass index, for example.
  • various embodiments of the invention involve analyzing human nucleic acid.
  • the procedure, method, or use of the invention further comprising isolating nucleic acid from the human individual (e.g., from a blood or tissue sample from the individual) for the analysis.
  • the nucleic acid is purified or isolated for analysis.
  • the procedure or method or use further comprises amplifying nucleic acid from a biological sample from the individual to provide amplified nucleic acid for the analysis.
  • the amplifying optionally comprises a polymerase chain reaction (PCR) using at least one primer that comprises a nucleotide sequence identical to or complementary to at least 14 nucleotides of SEQ ID NO: 1 (or SEQ ID NO: 48).
  • PCR polymerase chain reaction
  • a preferred uses at least two primers, one identical to and one complementary to (end portions of) a segment targeted for amplification.
  • Some embodiments of the procedure or method or use of the invention comprise detecting a polymorphism in a Tachykinin NK 1 receptor (TACRl) nucleic acid, wherein the presence of the polymorphism in the nucleic acid is indicative of an increased or decreased susceptibility to an asthma disorder.
  • TACRl Tachykinin NK 1 receptor
  • the invention involves analyzing TACRl alone in an individual. However, in other variations, the invention involves analyzing TACRl in combination with any other gene implicated in asthma.
  • the procedure or method or use of the invention further comprises analyzing nucleic acid from the human individual to measure a marker or haplotype in a MAP3K9 gene; and determining a status of a genetic indicator of an asthma disorder in the individual from the measurement of the TACRl marker or haplotype and from the measurement of the MAP3K9 marker or haplotype.
  • MAP3K9 analysis procedures, MAP3K9 association with asthma, and relevant treatment information is set forth in co-owned U.S. Patent Publication No. US 2006/0014165 Al, published January 19, 2006, incorporated herein by reference in its entirety.
  • the procedure, use, or method as already described further comprises an additional step of administering to the human individual a composition comprising a tachykinin inhibitor, in an amount that is therapeutically or prophylactically effective to treat or prevent the asthma disorder or a symptom of said disorder.
  • the invention also provides the use of a tachykinin inhibitor for the manufacture of a medicament for treatment of prophylaxis of an asthma disorder in a human individual, wherein the individual is identified, according to a procedure, use, or method described herein as having a genetic indicator of asthma in a tachykinin NK 1 receptor (TACRl) gene.
  • TACRl tachykinin NK 1 receptor
  • Exemplary classes of tachykinin inhibitors for use in the invention include:
  • One preferred genus of small molecule inhibitors comprises a compound of Formula I, or a pharmaceutically acceptable salt, ester, or pro-drug thereof:
  • R 2 and R 2 are independently from each other hydrogen, halogen, trifluoromethyl, lower alkoxy, halogen or cyano; or
  • R 3 and R 3 are independently from each other hydrogen, lower alkyl or forming a cycloalkyl group together with the carbon atom, to which they are attached;
  • R 5 is, independently from each other, hydrogen, lower alkyl, or benzyl, which is optionally substituted by lower alkyl;
  • R 6 is hydrogen, hydroxy, lower alkyl, -(CH 2 ) n 0(CH 2 ) n 0H, -CHO, or a 5- or 6- membered heterocyclic group, optionally bonded via an alkylene group;
  • X is -C(O)N(R 5 )C(O)-;
  • n 0-4.
  • a highly preferred species comprises a compound of Formula II, or a pharmaceutically acceptable salt, ester, or pro-drug thereof:
  • composition is administered in an amount effective to ameliorate the occurrence and severity of symptoms of asthma.
  • composition administered to target TACRl can be administered alone; administered with another composition targeting the same protein or pathway; or administered with other asthma therapeutic agents.
  • the procedure, use, or method of the invention optionally further comprises administering to the individual a composition that contains a compound selected from the group consisting of inhaled glucocortecoids, oral glucocortecoids, B 2 antagonists (long or short acting), and leukotriene antagonists.
  • Still another aspect of the invention is a method of assessing a human individual for probability of response to a tachykinin pathway antagonist therapeutic agent for prophylaxis or therapy, the method comprising: analyzing nucleic acid from a human individual for a marker or haplotype associated with an exon 2 LD block of the tachykinin NK 1 receptor (TACRl); and determining a probability of response to the agent from the analyzing, wherein the presence of a marker or haplotype that correlates with an elevated incidence of asthma in humans identifies a human with a greater probability for positive response to the therapeutic agent.
  • TACRl tachykinin NK 1 receptor
  • Still another aspect of the invention is articles of manufacture that are useful for practicing methods of the invention.
  • the invention is an apparatus for determining a genetic indicator for asthma in a human individual, comprising:
  • the apparatus further comprises a routine stored on the computer readable memory, adapted to be executed on a processor, to analyze genetic sequencing data or hybridization data for a human individual with respect to the human TACRl gene and generate an output comprising an allele or a haplotype for at least one marker shown in any of Tables 1, 2, and 6.
  • An exemplary status output comprises a determination of risk for an asthma disorder in the individual, or a determination of whether the individual, known already to have asthma, has a TACRl allele that contributes to the asthma.
  • the invention is a system comprising an apparatus as already described, and further comprising a display operably coupled to the processor to display the output.
  • the system further comprises a database operatively connected to the processor and adapted to store information about human individuals, said information including identity of one or more markers or haplotypes for a tachykinin NK 1 receptor (TACRl) gene.
  • TACRl tachykinin NK 1 receptor
  • the database further includes the diagnosis status of the individual with respect to asthma or other diseases in which TACRl may be implicated, including arthritic conditions or anxiety.
  • Kits comprising a detecting oligonucleotide and an enhancing oligonucleotide are also contemplated.
  • the enhancing oligonucleotide comprises a nucleotide sequence of 10-50 nucleotides, wherein the nucleotide sequence is complementary to and hybridizes to a segment of SEQ ID NO: 1 (or its complement), and the detecting and enhancing oligonucleotides hybridize to near-adjacent segments of SEQ ID NO: 1 (or its complement), such that a one abasic gap exists between the detecting and the enhancing oligonucleotides when both oligonucleotides are hybridized to SEQ ID NO: 1 (or its complement).
  • the reagent comprises at least one oligonucleotide for amplification of human nucleic acid that includes the at least one marker.
  • the reagent comprises at least one oligonucleotide primer pair for amplification of human nucleic acid that includes the at least one marker.
  • the oligonucleotides are isolated as individual oligonucleotides, or are isolated as a mixtures of one or more pairs of oligonucleotides, for amplifying one or more markers.
  • One aspect of the invention relates to a method of diagnosing a susceptibility to asthma or in an individual, comprising analyzing a nucleic acid sample obtained from the individual for a marker or haplotype associated with the exon 2 LD block of TACRl, wherein the presence of the marker or haplotype is indicative of a susceptibility to asthma or allergic rhinitis.
  • the marker or haplotype comprises at least one marker selected from the markers listed in Tables 1 and 2.
  • the marker or haplotype is a marker listed in Table 6.
  • the marker or haplotype is indicative of increased susceptibility of asthma or allergic rhinitis.
  • the increased susceptibility is in one embodiment characterized by a relative risk of at least 1.1, or at least 1.2, at least 1.3, or at least 1.4.
  • the marker is selected from the group consisting of D2S286, rs3771827, rs735668, rs7599593, rsl2475818, rs3771834, rsl3031966 and rs3771820 and wherein the presence of a non-2 allele (e.g., 6, 10, 4, 8, 12, 18, 14, 16, or other non-2 allele) in D2S286, a T allele in rs3771827; an A allele in rs735668; a T allele in rs7599593; a T allele in rsl2475818, an A allele in rs3771834, an G allele in rsl3031966, and a G allele in rs3771820, is indicative of increased susceptibility to bronchial asthma.
  • a non-2 allele e.g., 6, 10, 4, 8, 12, 18, 14, 16, or other non-2 allele
  • the marker is D2S286, and wherein the presence of a 2 allele in D2S286 is indicative of decreased susceptibility to asthma.
  • the marker is rs3771827, and wherein the presence of a C allele in rs3771827 is indicative of decreased susceptibility to asthma.
  • the contacting step optionally comprises contacting the nucleic acid with a first detection oligonucleotide probe comprising a label that is a Gig Harbor Green and a second detection oligonucleotide probe comprising a label that is Yakima Yellow.
  • the contacting step and the treating step are performed simultaneously.
  • LD HapMap project Build 19.
  • the 148 kb gene spans seven LD blocks as outlined schematically (based on NCBI RefSeq). Exons 1-5 are indicated.
  • Microsatellite marker D2S286 is located at 75.3 Mb on chromosome 2 (NCBI Build 34) in intron 2 of the TACRl gene, within a 39.1 kb block that incorporates part of intron 1, the whole of exon 2 and part of intron 2 (herein referred to as the "exon 2 LD block of TACRl").
  • Rs 3771827 and rs 735668 are located in intron 1 of the exon 2 LD block of TACRl.
  • the markers are plotted equidistantly, rather than according to their physical positions.
  • the figure shows two measures of LD, i.e., D' (upper left part of figure) and r (lower right part).
  • FIG. 2 is a graph showing the association results between asthma and various SNP' s located in the exon 2 LD block of TACRl, based on data from an Icelandic cohort of human subjects.
  • Tachykinins are a family of structurally related neuropeptides sharing the C- terminal sequence Phe-X-Gly-Leu-Met-NH 2 . In mice, they are encoded by two genes, Tacl and Tac2.
  • HK-I hemokinin 1
  • TAC4 TAC4
  • SEQ ID NOs: 18 and 19 TAC4
  • This tachykinin is uniquely expressed outside of neuronal tissue in immune tissues.
  • HK-I has subsequently been found to be a full agonist at the NKi, NK 2 , and NK 3 receptors, with highest selectivity for NKi (Bellucci et al., Br. J. Pharmacol. 135, 266-274, 2002).
  • the TACRl gene product is a 7 transmembrane G-protein coupled receptor protein. Ligand binding to Tachykinin receptor NK 1 has been shown to mediate cellular activation via inisitol-3-phosphate (IP3) and adenyl cyclase (AC) pathways (Khawaja 1996). This receptor is known to be expressed in at least two different isoforms, for which cDNA and deduced amino acid sequences are provided in SEQ ID NOs: 2-5).
  • hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to a TACRl nucleic acid.
  • Specific hybridization indicates exact hybridization (e.g., with no mismatches).
  • Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above. In a particularly preferred aspect, the hybridization conditions for specific hybridization are high stringency.
  • a test sample of DNA is obtained from the individual.
  • PCR can be used to amplify all or a fragment of a TACRl nucleic acid and its flanking sequences.
  • the DNA containing the amplified TACRl nucleic acid (or fragment of the gene or nucleic acid) is dot-blotted, using standard methods (see Current Protocols in Molecular Biology, supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified TACRl nucleic acid is then detected.
  • composition of the polypeptide encoded by a TACRl nucleic acid in a test sample is compared with the composition of the polypeptide encoded by the TACRl nucleic acid in a control sample (e.g., the presence of different splicing variants).
  • a difference in the composition of the polypeptide in the test sample, as compared with the composition of the polypeptide in the control sample, is diagnostic for a susceptibility to asthma.
  • both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample.
  • a difference from the control is indicative of a decreased susceptibility to asthma, and/or is indicative of a protective allele against asthma.
  • a "marker”, as described herein, refers to a genomic sequence characteristic of a particular variant allele (i.e., polymorphic site).
  • the marker can comprise any allele of any variant type found in the genome, including SNPs, microsatellites, insertions, deletions, duplications and translocations. Some markers may be truncated as part of mRNA and/or identifiable in cDNA, whereas other markers may not be.
  • SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI) (http://www.ncbi.nlm.nih.gov/SNP/).
  • Amplimer (SEQ ID NO: 44) : tgaagttgtgtgcctcctcataatatttattgactttgtggattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgaagcccatgttcaaaacctttgc tcattcttttattggatttcctgtctttttAATAGTGCTTAATTTAATTGAT
  • SNPs and microsatellite markers located outside of the exon 2 LD block of TACRl found to be associated with asthma are described in Table 2A.
  • LD between pairs of markers can be calculated using the standard definition of D' and R 2 (Lewontin, R., Genetics 49:49-67 (1964); Hill, W.G. & Robertson, A. Theor. Appl. Genet. 22:226-231 (1968)).
  • D' and R 2 Lewontin, R., Genetics 49:49-67 (1964); Hill, W.G. & Robertson, A. Theor. Appl. Genet. 22:226-231 (1968).
  • NEMO frequencies of the two marker allele combinations are estimated by maximum likelihood and deviation from linkage equilibrium is evaluated by a likelihood ratio test.
  • the definitions of D' and R 2 are extended to include microsatellites by averaging over the values for all possible allele combination of the two markers weighted by the marginal allele probabilities.
  • Such variants may confer a higher relative risk (RR) or odds ratio (OR) than observed for the tagging markers used to detect the association.
  • the present invention thus refers to the markers used for detecting association to the disease, as described herein, as well as markers in linkage disequilibrium with the markers.
  • markers that are in LD with the markers and/or haplotypes of the invention, as described herein may be used as surrogate markers.
  • the surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to be associating with the disease, as described herein.
  • the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein.
  • An example of such an embodiment would be a rare, or relatively rare ( ⁇ 10% allelic population frequency) variant in LD with a more common variant (> 10% population frequency) initially found to be associating with the disease, such as the variants described herein. Identifying and using such markers for detecting the association with asthma described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention.
  • markers found to be associated with asthma are markers comprising one or more allele that is more frequently present in an individual at risk for asthma, compared to the frequency of their presence in a healthy individual (control), wherein the presence of the markers is indicative of increased susceptibility to asthma.
  • the strength of the association of a marker or haplotype to increased risk for an asthma condition is measured by relative risk (RR).
  • RR is the ratio of the incidence of the condition among subjects who carry one copy of the marker or haplotype to the incidence of the condition among subjects who do not carry the marker or haplotype. This ratio is equivalent to the ratio of the incidence of the condition among subjects who carry two copies of the marker or haplotype to the incidence of the condition among subjects who carry one copy of the marker or haplotype.
  • the marker or haplotype has a relative risk of at least 1.2. In other embodiments, the marker or haplotype has a relative risk of at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 5.0.
  • Relative risk also can be used to express the strength of the association of a marker or haplotype with an apparent protective effect.
  • the relative risk (RR) is less than unity.
  • the marker or haplotype has a relative risk of less than 0.9.
  • the marker or haplotype has a relative risk of less than 0.8, less than 0.7, less than 0.6, less than 0.5 or less than 0.4.
  • the frequencies of haplotypes in patient and control groups can be estimated using an expectation-maximization algorithm (Dempster A. et al, J. R. Stat. Soc. B, 39:1-38 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-risk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately under both hypotheses and a corresponding 1- df likelihood ratio statistic is used to evaluate the statistical significance.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art and described above.
  • a “positive response" to a TACRl therapeutic agent is a physiological response that indicates treatment of asthma.
  • treatment refers not only to ameliorating symptoms associated with asthma, but also preventing or delaying the onset of asthma lessening the severity or frequency of symptoms of asthma; and/or also lessening the need for concomitant therapy with other drugs that ameliorate symptoms associated with asthma.
  • the current invention also pertains to methods of monitoring the response of an individual, such as an individual having an asthma disorder, to treatment with a tachykinin antagonist.
  • the test agent when expression of mRNA or polypeptide is greater (statistically significantly greater) in the presence of the test agent than in its absence, the test agent is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • the test agent when expression of the mRNA or polypeptide is less (statistically significantly less) in the presence of the test agent than in its absence, the test agent is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of mRNA or polypeptide expression in the cells can be determined by methods described herein for detecting mRNA or polypeptide.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in the methods of treatment described herein.
  • an agent identified as described herein can be used to alter activity of a protein encoded by a TACRl gene, or to alter expression of TACRl by contacting the protein or the nucleic acid (or contacting a cell comprising the polypeptide or the nucleic acid) with the agent identified as described herein.
  • the present invention also pertains to nucleic acid molecules which are not necessarily found in nature but which encode a TACRl polypeptide, or another splicing variant of a TACRl polypeptide or polymorphic variant thereof.
  • the invention pertains to DNA molecules comprising a sequence that is different from the naturally occurring nucleotide sequence but which, due to the degeneracy of the genetic code, encode a TACRl polypeptide of the present invention.
  • nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein.
  • Probes or “primers” are oligonucleotides that hybridize in a base- specific manner to a complementary strand of nucleic acid molecules.
  • probes and primers include polypeptide nucleic acids, as described in Nielsen et al, Science 254:1497-1500 (1991).
  • Probes or primer that are adjacent to a polymorphic or marker region are useful, e.g., for amplifying or sequencing the region that includes the polymorphic site.
  • the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequence of TACRl and/or the complement or a portion, and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid molecule e.g., an antisense oligonucleotide
  • an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • COPSAC Copenhagen Prospective Study on Asthma in Childhood

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Abstract

Les polymorphismes du gène TACR1 faisant l'objet de la présente invention se révèlent, par analyse associative, analogues à un gène de sensibilité à l'asthme. L'invention concerne également des procédés permettant de diagnostiquer une sensibilité à l'asthme, une sensibilité réduite à l'asthme et une protection contre l'asthme, de même que des procédés de traitement de l'asthme.
PCT/US2007/073066 2006-07-07 2007-07-09 Marqueurs génétiques dans le gène du récepteur de la tachykinine nk1 (tacr1) correspondant à des troubles asthmatiques WO2008006105A2 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016399A2 (fr) * 2000-08-25 2002-02-28 Genaissance Pharmaceuticals, Inc. Haplotypes du gene tacr1
EP1262565A2 (fr) * 2001-05-25 2002-12-04 Pfizer Products Inc. Polymorphismes génétiques dans le gène humain du récepteur de la neurokinine 1 et leurs usages dans diagnostic et traitement de maladies
JP2003259875A (ja) * 2002-03-08 2003-09-16 Japan Science & Technology Corp ヒト遺伝子の一塩基多型（４）
WO2005100986A1 (fr) * 2004-04-15 2005-10-27 Bayer Healthcare Ag Diagnostics et methodes therapeutiques pour des maladies associees au recepteur de la tachykinine (tacr1)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016399A2 (fr) * 2000-08-25 2002-02-28 Genaissance Pharmaceuticals, Inc. Haplotypes du gene tacr1
EP1262565A2 (fr) * 2001-05-25 2002-12-04 Pfizer Products Inc. Polymorphismes génétiques dans le gène humain du récepteur de la neurokinine 1 et leurs usages dans diagnostic et traitement de maladies
JP2003259875A (ja) * 2002-03-08 2003-09-16 Japan Science & Technology Corp ヒト遺伝子の一塩基多型（４）
WO2005100986A1 (fr) * 2004-04-15 2005-10-27 Bayer Healthcare Ag Diagnostics et methodes therapeutiques pour des maladies associees au recepteur de la tachykinine (tacr1)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ADCOCK I M ET AL: "Increased tachykinin receptor gene expression in asthmatic lung and its modulation by steroids." JOURNAL OF MOLECULAR ENDOCRINOLOGY AUG 1993, vol. 11, no. 1, August 1993 (1993-08), pages 1-7, XP009092353 ISSN: 0952-5041 *
ALMEIDA T A ET AL: "Tachykinins and tachykinin receptors: structure and activity relationships." CURRENT MEDICINAL CHEMISTRY AUG 2004, vol. 11, no. 15, August 2004 (2004-08), pages 2045-2081, XP002459773 ISSN: 0929-8673 *
DATABASE EMBL [Online] ebi; SNP 6 May 2004 (2004-05-06), "human SNP" XP002479802 retrieved from EMBL Database accession no. ADK91051 *
DATABASE SNP [Online] nih; SNP in TACR1 17 February 2004 (2004-02-17), XP002459774 retrieved from NCBI.NLM.NIH.GOV Database accession no. ss16811250 *

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