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WO2008006105A9 - 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

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Publication number
WO2008006105A9
WO2008006105A9 PCT/US2007/073066 US2007073066W WO2008006105A9 WO 2008006105 A9 WO2008006105 A9 WO 2008006105A9 US 2007073066 W US2007073066 W US 2007073066W WO 2008006105 A9 WO2008006105 A9 WO 2008006105A9
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WO
WIPO (PCT)
Prior art keywords
marker
asthma
nucleic acid
tacrl
haplotype
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Ceased
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PCT/US2007/073066
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English (en)
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WO2008006105A2 (fr
WO2008006105A3 (fr
Inventor
Eva Halapi
Hakon Hakonarson
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Decode Genetics ehf
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Decode Genetics ehf
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Publication of WO2008006105A2 publication Critical patent/WO2008006105A2/fr
Publication of WO2008006105A9 publication Critical patent/WO2008006105A9/fr
Publication of WO2008006105A3 publication Critical patent/WO2008006105A3/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
    • C12Q2600/106—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
  • 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.
  • 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.
  • 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 molecule that is used to practice the invention comprises the nucleotide sequence of SEQ ID NO: 48 (or its complement) or a fragment thereof effective for identification, in human nucleic acid, of a marker or haplotype outside of the exon 2 LD block of the TACRl gene (SEQ ID NO: 1).
  • the TACRl nucleic acid comprises a fragment of SEQ ID NO: 48 (or its complement) of less than 250 nucleotides that includes a marker or haplotype within SEQ ID NO: 48 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.
  • Procedures, methods, and uses are contemplated which use marker(s) or haplotype(s) that are outside of the exon 2 LD block but are in linkage disequlibrium with marker(s) or haplotype(s) disclosed herein.
  • the identified marker(s) or haplotype(s) have an r 2 value of at least 0.1, or at least 0.15, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, or at least 0.55, or at least 0.6, or at least 0.65, or at least 0.7, or at least 0.75, or at least 0.8, or at least 0.85, or at least 0.9, or at least 0.91, or at least 0.92, or at least 0.93, or at least 0.94, or at least 0.95, or at least 0.96, or at least 0.97, or at least 0.98, or at least 0.99 or 1.0.
  • the r 2 value is at least 0.2.
  • linkage disequilibrium as described herein refers to linkage disequilibrium characterized by values of ID' I of at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5, or at least 0.6, or at least 0.7, or at least 0.8, or at least 0.85, or at least 0.9, or at least 0.95, or at least 0.96, or at least 0.97, or at least 0.98, or at least 0.99.
  • markers that are in LD with the markers and/or haplotypes of the exon 2 LD block may be used as surrogate markers and/or may be used in combination with markers located within the exon 2 LD block of the TACRl gene.
  • Procedures, methods, and uses are contemplated in which a marker or markers that are analyzed correlate with greater or lesser incidences of asthma than in a comparative population.
  • the marker or haplotype correlates with a decreased prevalence of asthma disorders in humans with the marker or haplotype, compared to humans lacking the marker or haplotype.
  • the marker or haplotype may be associated with a relative risk of an asthma disorder of less than 0.9, or 0.8., or 0.7, or 0.6, or 0.5, or 0.4, or 0.3.
  • 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.
  • the marker or haplotype correlates with an increased prevalence of asthma disorders in humans with the marker or haplotype compared to humans lacking the marker or haplotype.
  • the analyzing or measuring step comprises screening for the presence in the individual of a TACRl haplotype or marker that is found with greater frequency in humans with an asthma disorder compared to the frequency with which it is found in humans free of asthma disorders.
  • the haplotype or marker may be associated with a relative risk of at least 1.1, or 1.2, or 1.3, or 1.4, or 1.5, or 1.6, or 1.7, or 1.8.
  • information in addition to genetic information is used to evaluate a genetic indicator.
  • 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 an increased prevalence of an asthma disorder in members of the subpopulation with the marker or haplotype compared to members lacking the marker or haplotype.
  • the determining step optionally comprises diagnosing an increased genetic susceptibility for an asthma disorder.
  • the determining may comprise identifying the individual as having a TACRl asthmatic disorder.
  • haplotypes or markers comprise at least one marker selected from the group consisting of the markers listed in Tables 1 and 2.
  • the haplotype or marker comprises at least one marker selected from the group consisting of the markers listed in Table 2, Table 6 and Table 7.
  • the marker or haplotype comprises at least one member selected from the group consisting of: rs3771827, rs735668, rs7599593, rs 12475818, rs3771834, rs 13031966 and rs3771820; wherein the presence of 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 rs 13031966, or a G allele in rs3771820 is indicative of increased susceptibility to asthma.
  • the marker or haplotype comprises allele 4 (T) at marker SG02S550 (SEQ ID NO: 1, position 40,163).
  • Exemplary asthma disorders include atopic asthma, non-atopic asthma, and bronchial hyperresponsiveness (BHR).
  • BHR bronchial hyperresponsiveness
  • 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
  • Still another, related aspect of the invention is a method of screening a human subject for susceptibility for an asthma disorder comprising: analyzing nucleic acid of a human subject for the presence and absence of the TACRl haplotype comprised of at least one marker selected from the markers listed in Table 6; and identifying the subject as having an increased susceptibility to an asthma disorder, wherein the presence of the TACRl haplotype correlates with an increased risk of an asthma disorder compared to humans lacking the haplotype.
  • the invention is a medical procedure comprising: analyzing a biological sample from a human individual to measure the expression of TACRl mRNA; and determining a status of an indicator of an asthma disorder in the individual from the measurement of mRNA.
  • the invention is a medical procedure comprising: analyzing a biological sample from a human individual to measure the expression or distribution of a polypeptide encoded by a TACRl nucleic acid; and determining a status of an indicator of an asthma disorder in the individual from the measurement of expression or distribution of the protein.
  • Still another variation of the invention is a method of screening an individual for risk of developing an asthma disorder, comprising: obtaining a biological sample that comprises nucleic acid from said individual, and screening the nucleic acid for an at-risk TACRl marker or haplotype that is more frequently present in humans with asthma disorders (affected), compared to the frequency of its presence in healthy humans free of asthma disorders (control), wherein the presence of the at-risk marker or haplotype is indicative of a susceptibility to an asthma disorder.
  • the marker or haplotype comprises at least one member selected from the group consisting of: rs3771827, rs735668, rs7599593, rsl2475818, rs3771834, rsl3031966 and rs3771820; wherein the presence of 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, or a G allele in rs3771820 is indicative of increased susceptibility to asthma.
  • the marker or haplotype comprises allele 4 (T) at marker SG02S550 (SEQ ID NO: 1, position 40,163).
  • 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 tachykinin inhibitor comprises an inhibitor of the expression or activity of tachykinin NK 1 (TACRl).
  • TACRl tachykinin NK 1
  • Exemplary classes of inhibitors described herein include polypeptide binding agents; TACRl nucleic acid binding agents; agents that inhibit TACRl nucleic acid expression; and agents that inhibit TACRl polypeptide signaling.
  • Exemplary biological inhibitors include antibody substances that bind TACRl; TACRl antisense molecules; TACRl interfering RNA molecules; and ribozymes.
  • Exemplary small molecule inhibitors of TACRl are identified in the Agent Tables below.
  • One preferred genus of small molecule inhibitors comprises a compound of Formula I, or a pharmaceutically acceptable salt, ester, or pro-drug thereof:
  • R 1 is a lower alkyl or aryl, optionally substituted by a lower alkyl, lower alkoxy, halogen or trifluoromethyl;
  • 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)-;
  • 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 at least one reagent for determining the identity in human nucleic acid of at least one marker in the exon 2 LD block of TACRl, wherein the at least one marker correlates with either an increased prevalence of asthma disorders in humans, or correlates with a decreased prevalence of asthma disorders in humans.
  • the kits include at least one marker is selected from the group consisting of D2S286, DG2S104, DG2S107, SG02S549, and SG02S550.
  • kits for assessing susceptibility to asthma comprising at least one oligonucleotide probe that is from 5-100 nucleotides in length and specifically hybridizes under stringent conditions to all or a portion of SEQ ID NO:1 (or SEQ ID NO: 48), wherein at least one of said at least one oligonucleotide probes comprises at least one marker selected from the group of markers disclosed herein and markers in linkage disequilibrium therewith, wherein the at least one marker correlates with either an increased prevalence of asthma disorders in humans, or correlates with a decreased prevalence of asthma disorders in humans.
  • the kit further comprises at least one oligonucleotide pair for amplifying a genomic fragment comprising at least one marker listed in Table 7, the segment being from 40-500 nucleotides in length.
  • the oligonucleotide probe optionally comprises a detectable label.
  • the probe comprises a detectable label at its 3' terminus and a quenching moiety attached to the probe at a location that is 5' relative to the label.
  • the kit comprises two oligonucleotide probes and wherein one of said probes comprises a detectable label, a quencher and a marker listed in Table 7.
  • the invention also provides oligonucleotide(s) useful for detecting a polymorphism.
  • oligonucleotide(s) comprise 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), with the proviso that the nucleotide sequence hybridizes to a segment that includes a SNP listed in Table IA or Table 7, and the oligonucleotide is complementary to either SEQ ID NO: 1 (or its complement) or is complementary to the sequence variant containing an allelic variant of the SNP (or its complement); a label attached at the 3' end of the oligonucleotide; and a quenching moiety attached at the 5' end of the oligonucleotide.
  • 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).
  • Kits comprising two detecting oligonucleotides are also contemplated.
  • the first and second detecting oligonucleotides hybridize to the same segment of SEQ ID NO: 1 (or its complement), and include the same SNP, with the proviso that the first and second detecting oligonucleotides are complementary and hybridize to different alleles of the SNP.
  • the kit optionally comprises an enhancing oligonucleotide, said enhancing oligonucleotide comprising 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 wherein 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).
  • an enhancing oligonucleotide comprising 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 wherein the detecting and enhancing oligonucleotides hybridize to near- adjacent
  • the at least one reagent in the kit comprises at least one contiguous nucleotide sequence that is fully complementary to a region of human nucleic acid that comprises the at least one marker.
  • the reagent comprises an allele- specific oligonucleotide that differentially hybridizes to human nucleic acid that contains one of the alleles of the marker.
  • the at least one oligonucleotide is/are attached to a solid support.
  • the oligonucleotides are isolated as individual oligonucleotides for hybridizing to a single marker, or are isolated as a mixture of two or more oligonucleotides, for hybridizing to two or more markers.
  • 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.
  • kits comprise oligonucleotides for amplification of at least two of said markers, and more preferably at least three, four, five, six, seven, eight, nine, ten, or more such markers. Oligonucleotides for amplification of each marker described herein, and kits containing them, are specifically contemplated.
  • the contiguous exon 2 LD block sequence is provided in SEQ ID NO: 1, with the understanding that its sequence (and its complement) can be used to make oligonucleotides for sequencing, amplifying, or hybridizing to any marker in this region.
  • Preferred markers include those set forth in Tables 1, IA, 2, 2A, 6 and 7. Other oligonucleotides can be made from the sequence in SEQ ID NO: 48.
  • 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 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 selected from the group consisting of D2S286 and rs3771827, wherein the presence of a non-2 allele in D2S286 or a T allele in rs3771827 is indicative of increased susceptibility to asthma or allergic rhinitis.
  • the marker is rs3771827, and wherein the presence of a T allele in rs3771827 is indicative of increased susceptibility to asthma.
  • the marker or haplotype is indicative of decreased susceptibility of asthma.
  • the decreased susceptibility is in one embodiment characterized by a relative risk of less than 0.8, or a relative risk of less than 0.7.
  • the marker is selected from the group consisting of D2S286, rs3771827, rs735668, rs7599593, rsl2475818, rs3771834, rsl3031966 and rs3771820, and wherein the presence of a 2 allele in D2S286, a C allele in SNP rs3771827; a C allele in rs735668; a C allele in rs7599593; a G allele in rsl2475818; a G allele in rs3771834, an T allele in rsl3031966; and an A allele in rs3771820 is indicative of a decreased susceptibility to asthma
  • 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 present invention relates to a kit for assaying a sample from an individual to detect a susceptibility to bronchial asthma, wherein the kit comprises one or more reagents for detecting one or more markers associated with the exon 2 LD block of TACRl.
  • the one or more reagents comprise at least one contiguous nucleotide sequence (preferably isolated) that is completely complementary to a region comprising at least one marker associated with the exon 2 LD block of TACRl.
  • the one or markers is selected from the group consisting of D2S286, rs3771827, rs735668, rs7599593, rsl2475818, rs3771834, rsl3031966 and rs3771820.
  • the one or more marker is D2S286 or rs3771827.
  • the marker is the T allele in rs3771827.
  • the present invention relates to a method of assessing an individual for probability of response to a TACRl therapeutic agent, comprising: detecting a marker associated with the exon 2 LD block of TACRl, wherein the presence of the marker is indicative of a probability of a positive response to TACRl therapeutic agent.
  • the marker is selected from the group consisting of D2S286, rs3771827, rs735668, rs7599593, rsl2475818, rs3771834, rsl3031966 and rs3771820,.
  • the marker is marker D2S286 or marker rs3771827, and wherein the presence of a non-2 allele in D2S286 or a T allele in rs3771827 is indicative of a probability of a positive response to a TACRl therapeutic agent.
  • TACRl therapeutic agent for the manufacture of a medicament for the treatment of asthma.
  • the TACRl therapeutic agent is an agent that alters activity in TACRl mediated signaling.
  • the TACRl therapeutic agent is an agent selected from the group set forth in the Agent Tables.
  • nucleic acid comprises a nucleotide sequence that of a TACRl gene or fragment thereo, and wherein the polymorphic site is selected from the marker set forther in any one of Tables 1, IA, 2, 2A, 6 and 7;
  • the detection oligonucleotide probe comprises a detectable label at the 3' terminus thereof and a quenching moiety located 5' relative to the detectable label; wherein the detection oligonucleotide probe comprises a sequence that is complementary to a first segment of the nucleotide sequence, wherein the first segment includes the polymorphic site, wherein the enhancer oligonucleotide probe is complementary to a second segment of the nucleotide sequence that is located 5' relative to the first segment, such that the enhancer oligonucleotide probe is located three prime relative to the detection oligonucleotide probe when both oligonucleotide probes are hybridized to the nucleic acid; and wherein a single base gap exists between the first segment and the second segment, whereby when the detection oligonucleotide probe comprises a detectable label at the 3' terminus thereof and a quenching moiety located 5' relative to the detectable label; wherein the detection oligonucleo
  • 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.
  • the selecting of a human subject and use of biological samples shall be construed to be restricted to selecting based on testing of a biological sample that has previously been removed from a human body and/or based on information obtained from a medical history, patient interview, or other activity that is not practiced on the human body; and (2) the administering of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self- administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.).
  • any technique e.g., orally, inhalation, topical application, injection, insertion, etc.
  • the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above.
  • aspects of the invention may have been described by reference to a genus or a range of values for brevity, it should be understood that each member of the genus and each value within the range is intended as an aspect of the invention.
  • various aspects and features of the invention can be combined, creating additional aspects which are intended to be within the scope of the invention.
  • FIG. 1 depicts the TACRl region of interest with respect to linkage disequilibrium
  • 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.
  • asthma disorders are meant to refer to a disorder of the respiratory system characterized by inflammation, narrowing of the airways, and/or increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with atopic or allergic symptoms. Further symptoms of asthma disorders include airway hyperresponsiveness to one or more environmental or other allergens, airway inflammation, airway obstruction, tissue and/or blood eosinophilia, and/or mucus hypersecretion.
  • Chemokines stimulate the recruitment of eosinophils, macrophages, neutrophils, and T lymphocytes. Once present, effector cells, such as eosinophils, may be prompted to release a collection of toxic granules. These granules may cause further, prolonged bronchoconstriction and damage epithelial layers. This damage, coupled with profibrotic cytokines also released by eosinophils and epithelial cells, can lay the groundwork for the process of airway remodeling to begin. Further, cytokines released at the time of mast cell degranulation can have more global effects.
  • cytokines such as IL-4 and IL-5, can have the effect of ensuring that this cycle of allergic inflammation persists.
  • an aspect of the invention includes screening for predictive markers in the tachykinin receptor together with screening for markers for any of these other genes, to provide a more comprehensive evaluation of a subject's genetic indicators for asthma than has been possible previously.
  • 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.
  • the mammalian tachykinins a family of peptides traditionally classified as neurotransmitters that includes substance P (SP) and neurokinins A (NKA) and B (NKB), have been implicated to have a wide variety of biological actions including smooth muscle contraction, vasodilatation, pain transmission, neurogenic inflammation, and the activation of the immune system. They all share a common C-terminal region: Phe-X-Glu-Leu-Met-NH2 where X is hydrophobic.
  • the N-terminal sequence is believed to convey receptor specificity to each of the three known mammalian tachykinin receptors, NK 1 , NK 2 , and NK 3 .
  • SP shows preference for NKj, NKA for NK 2 , and NKB for NK 3 .
  • the Substance P precursor was first determined to be encoded on two complementary cDNA sequences.
  • the second sequence contained a previously uncharacterized tachykinin, now known as Neurokinin A (NKA), produced by alternative RNA splicing of two distinct transcripts encoding Substance P alone ( ⁇ tachykinin precursor l, ⁇ TACl, SEQ ID NOs: 6 and 7) or with NKA ( ⁇ TACl, SEQ ID NOs: 8 and 9).
  • a third TACl transcript containing both Substance P and NKA but differing in its sequence organization was subsequently reported and named ⁇ TACl (SEQ ID NOs: 10 and 11).
  • TACl The total number of human TACl transcripts discovered is now four after the more recent discovery of ⁇ TACl (SEQ ID NOs: 12 and 13).
  • Neurokinin B (NKB) the third mammalian tachykinin, is encoded on a single TAC3 transcript (SEQ ID NOs 13-17).
  • 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).
  • Substance P and neurokinin A are potent vasodilators and contractors of smooth muscle (Severini et al., Pharmacol. Rev., 54:285-322, 2002). In studies on rodent airways, substance P and neurokinin A have been implicated as the neurotransmitters mediating the excitatory part of the nonadrenergic, noncholinergic (NANC) nervous system. (Barnes, P., Lancet, 1:242-245, 1986; Lundberg et al., Pharmacol. Rev., 48:113-178, 1996). Substance P and neurokinin A have various effects that could contribute to the changes observed in the airways of patients with asthma and chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • tachykinins The biological activity of tachykinins depends on their interaction with three specific receptors, the tachykinin NK 1 (Genbank Ace. Nos. NM_015727 and NM_001058; SEQ ID NOs: 2-5), NK 2 (Genbank Ace. No. BC096842; SEQ ID NOs: 32 and 33) and NK 3 receptor (Genbank Ace. No. NM_001059; SEQ ID NOs: 34 and 35) (Maggi et al., Eur. Respir. J., 6:735-742, 1993; Almeida et al., Curr. Med. Chem., 11:2045-2081, 2004, the disclosures of which are incorporated herein by reference).
  • NK 1 Genbank Ace. Nos. NM_015727 and NM_001058; SEQ ID NOs: 2-5
  • NK 2 Genbank Ace. No. BC096842; SEQ ID NOs: 32 and 33
  • the tachykinin receptor displaying highest affinity for substance P was termed the tachykinin NKl receptor, or tachykinin- 1 receptor (TACRl).
  • the receptor showing highest affinity for neurokinin A was termed the tachykinin NK2 receptor and the receptor with the highest affinity for neurokinin B was called the tachykinin NK3 receptor (Maggi et al., J. Auton. Pharmacol. ,13:23-93, 2000, the disclosure of which is incorporated herein by reference). All tachykinins can act as full agonists on the three different receptors, but with lower affinities for second or third receptors compared to the preferred receptor (Maggi et al., 1993, supra).
  • 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).
  • the NK 1 receptor has preferential affinities for substance P and hemokinin (Khawaja et al., Int J Biochem Cell Biol, 28, 721-738, 1996, Pattachini, Trends in Pharmacological Sciences, 25:, 2004) over neurokinin A and neurokinin B.
  • the NK 1 receptor is broadly expressed in airways; bronchial glands; bronchial vessels; bronchial smooth muscle; and also in a host of immune cells, including macrophages, lymphocytes, neutrophils, dendritic cells and mast cells. (De Swert et al., Eur J Pharmacol., 8:533(1- 3):171-81, 2006).
  • Tachykinins including substance P, elicit a broad spectrum of biological activities within the cardiovascular, gastrointestinal, urogenital, immune and nervous systems.
  • Substance P has been shown to induce broncho-constriction in different species, including humans; cough; secretion of mucus; and increased alveolar and epithelial permeability (De Swert et al., Eur J Pharmacol., 8;533(1-3):171-81, 2006 ).
  • tachykinin NKl receptors are involved in the broncho-constriction and the proinflammatory changes induced by substance P and neurokinin A.
  • the present invention relates to identification of an asthma-associated linkage disequilibrium (LD) block within the gene encoding Tachykinin receptor 1, NK 1 ("exon 2 LD block of TACRl").
  • LD asthma-associated linkage disequilibrium
  • markers within the exon 2 LD block of TACRl including microsatellite D2S286 and SNP markers rs3771827, rs 3771823, rs7588326, rs7599593, rsl2475818, rs3771834, rsl3031966 and rs3771820 have been found to be associated with asthma (See, e.g., Figure 2).
  • nucleotide sequence of this exon 2 LD block is set forth in SEQ ID NO: 1, in which nucleotide position 1 corresponds to nucleotide position 75,296,139 in human reference sequence (NCBI Build 34, July 2003) produced by the International Human Genome Sequencing Consortium.
  • Molecular biology techniques are used to identify the presence of particular haplotypes or marker alleles in a human subject, including a 2 allele in marker D2S286 (associated with a decreased susceptibility to asthma); a non-2 allele (e.g., 6, 10,4,8, 12,18, 14,or 16, or other allele) in marker D2S286 (associated with increased susceptibility to asthma); a C allele in SNP rs3771827 (associated with a decreased susceptibility to asthma); a T allele in SNP rs3771827 (associated with increased incidence of asthma); a C allele in SNP rs735668 (associated with a decreased susceptibility to type asthma); an A allele in SNP rs735668 (associated with increased susceptibility to asthma); a C allele in SNP rs7599593 (associated with a decreased susceptibility to asthma); a T allele in SNP rs7599593 (associated with an increased susceptibility to asthma); a G allele
  • markers or SNP' s, or haplotypes comprising multiple markers or SNP' s, identified using the methods described herein can be used for diagnosis of a susceptibility to asthma, and also for diagnosis of a decreased susceptibility to asthma or for identification of an allele that is protective against asthma.
  • the diagnostic assays presented below can be used to identify the presence or absence of these particular alleles.
  • diagnosis of a susceptibility to asthma is made by detecting a polymorphism in a TACRl nucleic acid as described herein (e.g., the alleles in marker D2S286 or in SNP r rs3771827, rs735668 , rs7599593 , rs 12475818 , rs3771834, rsl3031966 and rs3771820).
  • the polymorphism can be a variation in a TACRl nucleic acid that causes a difference in the polypeptide encoded by a TACRl nucleic acid, such as:
  • More than one such change may be present in a single gene.
  • a polymorphism associated with a disease or condition or a susceptibility to a disease or condition associated with a TACRl nucleic acid can be a synonymous alteration in one or more nucleotides (i.e., an alteration that does not result in a change in the polypeptide encoded by a TACRl nucleic acid).
  • Such a polymorphism may alter splicing sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the gene.
  • a TACRl nucleic acid that has any of the changes or alterations described above is referred to herein as an "altered nucleic acid.”
  • a polymorphism associated with an increased or decreased prevalence of a disease or condition also may have no effect whatsoever on the encoded amino acid sequence or the mRNA, or transcription or translation.
  • polymorphisms and haplotypes exist that are in linkage disequilibrium with causative mutations, the identity of which may or may not be known.
  • the test sample can be from any source which contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or virtually any tissue sample (e.g., from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs).
  • a test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling.
  • the DNA, RNA, or cDNA sample is then examined to determine whether a polymorphism in a TACRl nucleic acid is present, and/or to determine which allele or splicing variant(s) encoded by the TACRl is present.
  • 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.
  • Specific hybridization if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and TACRl nucleic acid in the test sample, then the TACRl has the polymorphism or allele, or is the splicing variant, that is complementary to and detectable by the nucleic acid probe. More than one nucleic acid probe can be used concurrently in this method.
  • Specific hybridization of any one of the nucleic acid probes is indicative of a polymorphism in the TACRl nucleic acid, or of the presence of a particular allele or splicing variant encoding the TACRl nucleic acid and can be diagnostic for a susceptibility to asthma, or for a decreased susceptibility to asthma (or indicative of a protective allele against asthma).
  • Specific hybridization of a nucleic acid probe, as described above, to RNA from the individual is indicative of a polymorphism or allele in a TACRl nucleic acid, or of the presence of a particular splicing variant encoded by a TACRl nucleic acid, and is therefore diagnostic for the susceptibility to asthma or the decreased susceptibility to asthma (or indicative of a protective allele against asthma).
  • a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods of the invention.
  • PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl) glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P. E. et al, Bioconjugate Chemistry 5, American Chemical Society, p. 1 (1994).
  • the PNA probe can be designed to specifically hybridize to a TACRl nucleic acid. Hybridization of the PNA probe to a TACRl nucleic acid can be diagnostic for a susceptibility to asthma or decreased susceptibility to asthma (or indicative of a protective allele against asthma).
  • alteration analysis by restriction digestion can be used to detect an alteration in the gene, if the alteration (mutation) or polymorphism in the gene results in the creation or elimination of a restriction site (e.g., restriction fragment length polymorphism, or RFLP).
  • a restriction site e.g., restriction fragment length polymorphism, or RFLP.
  • a test sample containing genomic DNA is obtained from the individual.
  • Polymerase chain reaction (PCR) can be used to amplify a TACRl nucleic acid (and, if necessary, the flanking sequences) in the test sample of genomic DNA from the test individual.
  • RFLP analysis is conducted as described (see Current Protocols in Molecular Biology, supra).
  • the digestion pattern of the relevant DNA fragment indicates the presence or absence of the alteration or polymorphism in the TACRl nucleic acid, and therefore indicates the presence or absence a susceptibility to asthma or a decreased susceptibility to asthma (or indicative of a protective allele against asthma).
  • Sequence analysis can also be used to detect specific polymorphisms in a TACRl nucleic acid.
  • a test sample of DNA or RNA is obtained from the test individual.
  • PCR or other procedure is used to amplify the gene or nucleic acid, and/or its flanking sequences, if desired.
  • the sequence of a TACRl nucleic acid, or a fragment of the nucleic acid, or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA, is determined, using standard methods.
  • the sequence of the nucleic acid, nucleic acid fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of the gene or cDNA or mRNA, as appropriate.
  • the presence of a polymorphism in the TACRl indicates that the individual has a susceptibility to asthma or a decreased susceptibility to asthma (or indicative of a protective allele against asthma).
  • Allele- specific oligonucleotides can also be used to detect the presence of a polymorphism in a TACRl nucleic acid, through the use of dot-blot hybridization of amplified oligonucleotides with allele- specific oligonucleotide (ASO) probes (see, for example, Saiki, R. et al, Nature 324:163-166 (1986)).
  • ASO allele-specific oligonucleotide
  • an “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15-30 base pairs, that specifically hybridizes to a TACRl nucleic acid, and that contains a polymorphism associated with a susceptibility to asthma or a polymorphism associated with a decreased susceptibility to asthma (or indicative of a protective allele against asthma).
  • An allele-specific oligonucleotide probe that is specific for particular polymorphisms in a TACRl nucleic acid can be prepared, using standard methods (see Current Protocols in Molecular Biology, supra).
  • 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.
  • Hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of a polymorphism in the TACRl nucleic acid, and is therefore indicative of susceptibility to asthma or is indicative of decreased susceptibility to asthma (or indicative of a protective allele against asthma).
  • the invention further provides allele-specific oligonucleotides that hybridize to the reference or variant allele of a gene or nucleic acid comprising a single nucleotide polymorphism or to the complement thereof. These oligonucleotides can be probes or primers.
  • An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer, which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product, which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single -base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • LNAs locked nucleic acids
  • the size of primers and probes can be reduced to as few as 8 bases.
  • LNAs are a novel class of bicyclic DNA analogs in which the 2' and 4' positions in the furanose ring are joined via an O- methylene (oxy-LNA), S-methylene (thio-LNA), or amino methylene (amino-LNA) moiety.
  • oxy-LNA O- methylene
  • thio-LNA S-methylene
  • amino-LNA amino methylene
  • arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual can be used to identify polymorphisms in a TACRl nucleic acid.
  • an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as "GenechipsTM,” have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092.
  • arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al, Science 251:767-777 (1991), Pirrung et al, U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al, PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186, the entire teachings are incorporated by reference herein. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261; the entire teachings are incorporated by reference herein. In another example, linear arrays can be utilized.
  • a nucleic acid of interest is hybridized with the array and scanned for polymorphisms.
  • Hybridization and scanning are generally carried out by methods described herein and also in, e.g., published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings are incorporated by reference herein.
  • a target nucleic acid sequence that includes one or more previously identified polymorphic markers is amplified by well-known amplification techniques, e.g., PCR.
  • Asymmetric PCR techniques may also be used.
  • Amplified target generally incorporating a label, is then hybridized with the array under appropriate conditions.
  • the array is scanned to determine the position on the array to which the target sequence hybridizes.
  • the hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array.
  • arrays can include multiple detection blocks, and thus be capable of analyzing multiple, specific polymorphisms.
  • detection blocks may be grouped within a single array or in multiple, separate arrays so that varying, optimal conditions may be used during the hybridization of the target to the array. For example, it may often be desirable to provide for the detection of those polymorphisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows for the separate optimization of hybridization conditions for each situation.
  • oligonucleotide arrays for polymorphism detection can be found, for example, in U.S. Patents Nos. 5,858,659 and 5,837,832, the entire teachings of which are incorporated by reference herein.
  • Other methods of nucleic acid analysis can be used to detect polymorphisms in an asthma gene or variants encoded by an asthma gene. Representative methods include direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. ScL USA 81:1991-1995 (1988); Sanger, F. et al, Proc. Natl. Acad. ScL USA 74:5463- 5467 (1977); Beavis et al, U.S. Pat. No.
  • diagnosis of a susceptibility to asthma, or of a decreased susceptibility to asthma (or indicative of a protective allele against asthma) can also be made by expression analysis by quantitative PCR (kinetic thermal cycling).
  • This technique utilizing TaqMan ® assays, can assess the presence of an alteration in the expression or composition of the polypeptide encoded by a TACRl nucleic acid or splicing variants encoded by a TACRl nucleic acid.
  • TaqMan ® probes can also be used to allow the identification of polymorphisms and whether a patient is homozygous or heterozygous. Further, the expression of the variants can be quantified as physically or functionally different.
  • diagnosis of a susceptibility to asthma or of a decreased susceptibility to asthma can be made by examining expression and/or composition of a TACRl polypeptide, by a variety of methods, including enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • ELISAs enzyme linked immunosorbent assays
  • a test sample from an individual is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a TACRl nucleic acid, or for the presence of a particular variant encoded by a TACRl nucleic acid.
  • An alteration in expression of a polypeptide encoded by a TACRl nucleic acid can be, for example, an alteration in the quantitative polypeptide expression ⁇ i.e., the amount of polypeptide produced); an alteration in the composition of a polypeptide encoded by a TACRl nucleic acid is an alteration in the qualitative polypeptide expression ⁇ e.g., expression of an altered TACRl polypeptide or of a different splicing variant).
  • diagnosis of a susceptibility to asthma or of a decreased susceptibility to asthma can be made by detecting a particular splicing variant encoded by that TACRl nucleic acid, or a particular pattern of splicing variants.
  • alteration in the polypeptide expression or composition, as used herein, refers to an alteration in expression or composition in a test sample, as compared with the expression or composition of polypeptide by a TACRl nucleic acid in a control sample.
  • a control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from an individual who is not affected by a susceptibility to asthma (negative control) or an individual affected by asthma (positive control). Comparison to a statistical reference value, obtained from samples of multiple individuals, also can be performed.
  • an alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, is indicative of a susceptibility to asthma.
  • the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample is indicative of a susceptibility to asthma.
  • Various means of examining expression or composition of the polypeptide encoded by a TACRl nucleic acid can be used, including: spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al, U.S. Pat.
  • an antibody capable of binding to the polypeptide e.g., as described above, preferably an antibody with a detectable label, is used.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • the presence of a polypeptide encoded by a polymorphic or altered nucleic acid, or the absence of a polypeptide encoded by a non-polymorphic or non-altered nucleic acid, is diagnostic for a susceptibility to asthma, as is the presence (or absence) of particular splicing variants encoded by the TACRl nucleic acid.
  • the level or amount of polypeptide encoded by a TACRl nucleic acid in a test sample is compared with the level or amount of the polypeptide encoded by the TACRl in a control sample.
  • a level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant is indicative of an alteration in the expression of the polypeptide encoded by the TACRl nucleic acid, and is diagnostic for a susceptibility to asthma.
  • a difference from the control is indicative of a decreased susceptibility to asthma, and/or is indicative of a protective allele against asthma.
  • 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/).
  • haplotype refers to a segment of a genomic DNA strand that is characterized by a specific combination of genetic markers ("alleles") arranged along the segment, and inherited as a block.
  • haplotype also is used to refer to the set of alleles within a segment of genomic DNA that is inherited as a block.
  • the haplotype comprises one or more alleles, two or more alleles, three or more alleles, four or more alleles, or five or more alleles.
  • the genetic markers are particular "alleles” at "polymorphic sites” associated with the exon 2 LD block of TACRl.
  • exon 2 LD block of TACRl refers to the LD block on Chromosome 2p within which association of variants to asthma is observed.
  • NCBI Build 34 position of this LD block is from 75,296,139 - 75,355,488 base pairs.
  • a reference sequence containing these nucleotides is reproduced herein as SEQ ID NO: 1, wherein position 1 of SEQ ID NO: 1 corresponds to position 75,296,139 of the build.
  • a reference population such as the general human population or a recognized subset into which an individual is classified as a member.
  • particular markers and/or haplotypes of the invention may be characteristic of increased susceptibility to asthma, as characterized by a relative risk of greater than one. Markers and/or haplotypes that confer increased susceptibility to asthma are furthermore considered to be "at-risk", as they confer an increased risk of disease.
  • the markers and/or haplotypes of the invention are characteristic of decreased susceptibility to asthma, as characterized by a relative risk of less than one.
  • a nucleotide position at which more than one sequence is possible in a population is referred to herein as a "polymorphic site".
  • a polymorphic site is a single nucleotide in length, the site is referred to as a single nucleotide polymorphism ("SNP").
  • SNP single nucleotide polymorphism
  • Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed.
  • the person skilled in the art will realize that an allele also can be identified by assaying or reading the opposite strand of a double stranded nucleic acid, such as genomic DNA.
  • the assay employed may either measure the percentage or ratio of the two bases possible, i.e., A and G.
  • the percentage or ratio of the complementary bases T/C can be measured. Quantitatively (for example, in terms of relative risk), identical results would be obtained from measurement of either DNA strand (+ strand or - strand).
  • Polymorphic sites can allow for differences in sequences based on substitutions, insertions or deletions.
  • a polymorphic micro satellite has multiple small repeats of bases (such as CA repeats) at a particular site in which the number of repeat lengths varies in the general population.
  • Each version of the sequence with respect to the polymorphic site is referred to herein as an "allele" of the polymorphic site.
  • the SNP allows for both an adenine allele and a thymine allele.
  • SNPs and micro satellite markers located within the exon 2 LD block of TACRl found to be associated with asthma are described in Tables 1, IA or 2. able 1
  • Table IA contains markers identified in Table 1, plus additional markers that were identified by pool sequencing of the affected and control patients described in Example 1. Table IA also contains additional information about particular markers, such as updated location on Build 34 (superseding the location numbers provided in Table 1) and correlation information (r 2 ) of the identified markers to rs3771827, based on HapMap data for the CEU population (see worldwide web at www.hapmap.org).
  • Amplimer (SEQ ID NO: 44) : tgaagttgtgtgcctcctcataatatttattgactttgtggattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgaagcccatgttcaaaacctttgc tcattcttttattggatttcctgtctttttAATAGTGCTTAATTTAATTGAT
  • polypeptide encoded by the reference nucleotide sequence is the "reference” polypeptide with a particular reference amino acid sequence
  • polypeptides encoded by variant alleles are referred to as "variant" polypeptides with variant amino acid sequences.
  • a polymorphic micro satellite has multiple small repeats of bases that are 2-8 nucleotides in length (such as CA repeats) at a particular site, in which the number of repeat lengths varies in the general population.
  • Haplotypes are a combination of various genetic markers, e.g., SNPs and microsatellites, having particular alleles at polymorphic sites. Therefore, detecting haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques (Chen, X. et al., Genome Res. 9(5): 492-98 (1999)), PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. These markers and SNPs can be identified in at- risk haplotypes. Certain methods of identifying relevant markers and SNPs include the use of linkage disequilibrium (LD) and/or LOD scores.
  • LD linkage disequilibrium
  • the SNP genotyping method utilizes specially labeled probes and the specific cleavage by Endonuclease IV enzyme to differentiate alleles (Kutyavin et al., Nuc. Acids Res., 34:el28, published online September 27, 2006, the disclosure of which is incorporated herein by reference in its entirety). Briefly, basic PCR is performed, e.g., for 50 cycles, to amplify a segment of DNA (the amplicon) containing a SNP, after which Endonuclease IV, and ehancer oligo and two allele specific detection probes are added. The reaction is incubated for 50° C for one hour and end-point signal read on an ABI 7900 instrument.
  • an individual who is at-risk for asthma is an individual in whom an at-risk marker or haplotype is identified.
  • the at-risk marker or haplotype is one that confers a significant increased risk (or susceptibility) of asthma.
  • Risk associated with a marker or haplotype can be expressed as a relative risk, or as a percentage.
  • an increased risk may be expressed as a relative risk of at least about 1.1, including but not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0, or greater, depending on the data collected.
  • increase in risk may be at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or greater.
  • the risk is statistically significant in the general human population or in a defined subpopulation (defined by one or more factors such as race, age, ethnicity, national origin, sex, or genetic markers at another locus) in which a subject falls.
  • methods of the invention relating to measuring or predicting a genetic indicator of risk refer to risk that can be shown to be statistically significant in a human population or subpopulation that is studied. Statistical significance can be measured by the Fisher exact test, which is described elsewhere herein.
  • the marker or haplotype confers decreased risk (decreased susceptibility) of asthma.
  • significant decreased risk is measured as a relative risk at less than 0.9, including but not limited to 0.9, 0.8, 0.7, 0.6, 0.5, and 0.4.
  • significant relative risk is less than 0.7.
  • the decreased in risk (or susceptibility) is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%.
  • a significant decrease in risk is at least about 30%.
  • the term “susceptibility to asthma” indicates either an increased risk or susceptibility or a decreased risk or susceptibility of asthma, by an amount that is significant, when a certain allele, marker, SNP or haplotype is present; significance is measured as indicated above.
  • the terms “decreased risk”, “decreased susceptibility” and “protection against,” as used herein, indicate that the relative risk is decreased accordingly when a certain other allele, marker, SNP, and/or a certain other haplotype, is present. It is understood however, that identifying whether an increased or decreased risk is medically significant may also depend on a variety of factors, including the specific disease, the marker or haplotype, and often, environmental factors.
  • An at-risk marker or haplotype in, or comprising portions of, the TACRl gene is one where the marker or haplotype is more frequently present in individuals diagnosed with asthma (affected), compared to the frequency of its presence in healthy individuals (control).
  • a simple test for correlation would be a Fisher-exact test on a two by two table. Given a cohort of chromosomes, the two by two table is constructed out of the number of chromosomes that include both of the markers or haplotypes, one of the markers or haplotypes but not the other, and neither of the markers or haplotypes.
  • At-risk marker or haplotype is an at-risk marker or haplotype within or near TACRl that significantly correlates with asthma.
  • an at-risk marker or haplotype comprises an at-risk marker or haplotype within or near TACRl that significantly correlates with susceptibility to asthma.
  • the marker or haplotype is associated with the exon 2 LD block of TACRl as described herein.
  • the method comprises assessing in an individual the presence or frequency of SNPs and/or microsatellites in, comprising portions of, the TACRl gene, wherein the presence of the SNPs and/or microsatellites is indicative that the individual is susceptible to asthma.
  • SNPs and markers can form haplotypes that can be used as screening tools. These markers and SNPs can be identified in at-risk haploptypes.
  • an at-risk haplotype can include microsatellite markers and/or SNPs such as marker D2S286 and/or SNP rs3771827, rs735668, rs7577326, rs3771823, or rs735668.
  • the presence of an at-risk haplotype is indicative of increased susceptibility to asthma, and therefore is indicative of an individual who falls within a target population for the prophylaxis or treatment methods described herein.
  • the frequencies of haplotypes in the patient and the 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.
  • the patients and the controls are assumed to have identical frequencies.
  • 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.
  • One general approach to haplotype analysis involves using likelihood-based inference applied to NEsted MOdels (Gretarsdottir S., et al, Nat. Genet. 55:131-38 (2003)).
  • the method is implemented in the program NEMO, which allows for many polymorphic markers, SNPs and microsatellites.
  • the method and software are specifically designed for case-control studies where the purpose is to identify haplotype groups that confer different risks. It is also a tool for studying LD structures.
  • maximum likelihood estimates, likelihood ratios and p-values are calculated directly, with the aid of the EM algorithm, for the observed data treating it as a missing-data problem.
  • the Fisher exact test can be used to calculate two-sided p-values for each individual allele. All p-values are presented unadjusted for multiple comparisons unless specifically indicated. The presented frequencies (for microsatellites, SNPs and haplotypes) are allelic frequencies as opposed to carrier frequencies. To minimize any bias due the relatedness of the patients who were recruited as families for the linkage analysis, first and second-degree relatives can be eliminated from the patient list. Furthermore, the test can be repeated for association correcting for any remaining relatedness among the patients, by extending a variance adjustment procedure described in Risch, N. & Teng, J.
  • relative risk and the population attributable risk (PAR) can be calculated assuming a multiplicative model (haplotype relative risk model) (Terwilliger, J.D. & Ott, J., Hum. Hered. 42:331-46 (1992) and FaIk, CT. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt 3):221-33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply.
  • a multiplicative model haplotype relative risk model
  • haplotypes are independent, i.e., in Hardy- Weinberg equilibrium, within the affected population as well as within the control population.
  • haplotype counts of the affecteds and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis.
  • h r and h p risk(/zO/risk(/z ; ) (fjp ⁇ /if j /p j ), where/ and p denote, respectively, frequencies in the affected population and in the control population. While there is some power loss if the true model is not multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p-values are always valid since they are computed with respect to null hypothesis.
  • Linkage Disequilibrium refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrance of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements.
  • a particular genetic element e.g., an allele of a polymorphic marker, or a haplotype
  • Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurence of each allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles for each genetic element (e.g., a marker, haplotype or gene).
  • ID' I indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause ID' I to be ⁇ 1, but for single nucleotide polymorphisms (SNPs) this is usually regarded as being less likely than recombination).
  • the measure r 2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present.
  • the r 2 measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r 2 and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model). Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics.
  • a significant linkage disequilibrium is defined as r 2 > 0.1 and ID' I >0.8. In another embodiment, a significant linkage disequilibrium is defined as r > 0.2 and ID' I >0.9. Other combinations and permutations of values of r 2 and ID' lfor determining linkage disequilibrium are also possible, and within the scope of the invention.
  • Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population. In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations (Caucasian, african, Japanese, Chinese), as defined (http://www.hapmap.org).
  • Multipoint, affected-only allele- sharing methods can be used in the analyses to assess evidence for linkage.
  • Results both the LOD-score and the non-parametric linkage (NPL) score, can be obtained using the program Allegro (Gudbjartsson et al., Nat. Genet. 25:12-3 (2000)).
  • Our baseline linkage analysis uses the S pa irs scoring function (Whittemore, A.S., Halpern, J. Biometrics 50:118-27 (1994); Kruglyak L. et al., Am. J. Hum. Genet. 55:1347-63 (1996)), the exponential allele- sharing model (Kong, A. and Cox, NJ. , Am. J. Hum. Genet.
  • blocks can be defined as regions of DNA that have limited haplotype diversity (see, e.g., Daly, M. et al, Nature Genet. 29:229-232 (2001); Patil, N. et al, Science 294:1719-1723 (2001); Dawson, E. et al, Nature 4iS:544-548 (2002); Zhang, K. et al, Proc. Natl. Acad. ScL USA 99:7335-7339 (2002)), or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium (see, e.g., Gabriel, S.B.
  • the map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD.
  • the map can therefore be used to define haplotype blocks/LD blocks as regions flanked by recombination hotspots.
  • haplotype block or "LD block” includes blocks defined by either characteristic, or other alternative methods used by the person skilled in the art to define such regions.
  • Haplotype blocks can be used readily to map associations between phenotype and haplotype status.
  • the main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified.
  • These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. If desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.
  • markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such are useful for use in the methods and kits of the present invention.
  • One or more causative (functional) variants or mutations may reside within the region found to be associating to the disease or trait.
  • 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 and haplotypes comprising such markers are found to be useful for determination of susceptibility to asthma - i.e., they are found to be useful for diagnosing a susceptibility to asthma.
  • Particular markers and haplotypes are found more frequently in individuals with asthma than in individuals without asthma. Therefore, these markers and haplotypes have predictive value for detecting asthma, or a susceptibility to asthma, in an individual.
  • Haplotype blocks i.e., the exon 2 LD block of TACRl
  • TACRl the exon 2 LD block of TACRl
  • these "at-risk” tagging markers within the haplotype block also have predictive value for detecting asthma, or a susceptibility to asthma, in an individual.
  • "At-risk” tagging markers within the haplotype or LD blocks can also include other markers that distinguish among the haplotypes, as these similarly have predictive value for detecting asthma or a susceptibility to asthma.
  • a large number of markers or other variants and/or haplotypes comprising such markers or variants in association with the haplotype block (LD block) may be found to be associated with a certain trait and/or phenotype.
  • markers and/or haplotypes residing within the exon 2 LD block of TACRl as defined herein or in strong LD (characterized by r 2 greater than 0.2) with the exon 2 LD block of TACRl are associated with asthma (i.e., they confer increased or decreased susceptibility of asthma).
  • the identification of such additional variants can be achieved by methods well known to those skilled in the art, for example by DNA sequencing of the LD block, followed by statistical analysis in affected versus unaffected cohorts as described herein.
  • the present invention also encompasses such additional variants.
  • the at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant.
  • the present invention encompasses the assessment of such surrogate markers for the markers as disclosed herein. Such markers are annotated, mapped and listed in public databases or can alternatively be readily identified by sequencing the region (or a part of the region) identified by the markers of the present invention in a group of individuals, and identifying polymorphisms in the resulting group of sequences. As a consequence, the teachings herein permit scientists in the field of the invention to genotype surrogate markers that are in linkage disequilibrium with the markers and/or haplotypes as described herein.
  • the tagging or surrogate markers in LD with the at-risk variants detected also have predictive value for detecting association to asthma, or a susceptibility to asthma, in an individual.
  • These tagging or surrogate markers that are in LD with the markers of the present invention can also include other markers that distinguish among haplotypes, as these similarly have predictive value for detecting susceptibility to asthma.
  • markers within the exon 2 LD block of TACRl are found in decreased frequency in individuals with asthma, and haplotypes comprising two or more of those markers are also found to be present at decreased frequency in individuals with asthma. These markers and haplotypes are thus correlative of protection against asthma. These markers are postulated to identify one or more TACRl gene alleles that confer a decreased risk of developing asthma.
  • haplotypes and markers described herein are, in some cases, a combination of various genetic markers, e.g., SNPs and microsatellites. Therefore, detecting haplotypes can be accomplished by methods known in the art and/or described herein for detecting sequences at polymorphic sites. Furthermore, correlation between certain haplotypes or sets of markers and disease phenotype can be verified using standard techniques. A representative example of a simple test for correlation would be a Fisher-exact test on a two by two table.
  • a marker or haplotype associated with the exon 2 LD block of TACRl is one in which the marker or haplotype is more frequently present in individuals diagnosed with asthma (affected), compared to the frequency of its presence in healthy individuals (control), in which case the presence of the marker or haplotype is deemed indicative of asthma or a 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 variants described herein in general do not, by themselves, provide an absolute identification of individuals who will develop a particular disease.
  • the variants described herein do, however, indicate increased and/or decreased likelihood that individuals carrying the at-risk or protective variants of the invention will develop symptoms associated with asthma.
  • This information is extremely valuable in itself, as outlined in more detail in the below, as it can be used to, for example, initiate preventive measures at an early stage, perform regular physical and/or mental exams to monitor the progress and/or appearance of symptoms, or to schedule exams at a regular interval to identify the condition in question, so as to be able to apply treatment at an early stage.
  • the knowledge about a genetic variant that confers a risk of developing asthma offers the opportunity to apply a genetic-test to distinguish between individuals with increased risk of developing the disease (i.e., carriers of the at-risk variant), relative to controls, and also to identify those with decreased risk of developing the disease (i.e., carriers of the protective variant).
  • Such genetic testing confers the ability to diagnose the disease at an early stage, and to provide information to the clinician about prognosis/aggressiveness of the disease in order to be able to apply the most appropriate treatment.
  • the application of a genetic test for asthma can provide an opportunity for the detection of the disease at an earlier stage which may lead to the application of therapeutic measures at an earlier stage, and thus can minimize the deleterious effects of the symptoms and serious health consequences conferred by asthma.
  • Genetic testing can categorize people into categories of increased risk for developing a disease, permitting administration of prophylactic pharmaceutical agents and/or lifestyle management choices to minimize the likelihood of developing a disease or to lessen its symptoms or severity.
  • methods can be employed for the treatment of asthma and/or allergic rhinitis.
  • treatment refers not only to ameliorating symptoms associated with the disease or condition, but also preventing or delaying the onset of the disease or condition; preventing or delaying the occurrence of a another episode of the disease or condition; lessening the severity or frequency of symptoms of the disease or condition; and/or also lessening the need for concomitant therapy with other drugs that ameliorate symptoms associated with the disease or condition, e.g., corticosteroids.
  • Methods are additionally available for assessing an individual's risk for developing asthma and/or other respiratory diseases.
  • the individual to be treated is an individual who is identified according to the invention as having increased susceptibility (an increased risk) for asthma.
  • exemplary individuals for treatment include an individual having 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 rs 13031966, and a G allele in rs3771820.
  • a non-2 allele e.g., 6, 10, 4, 8, 12, 18, 14, 16, or other non-2 allele
  • methods can be employed for the treatment of other diseases or conditions associated with TACRl.
  • a TACRl therapeutic agent can be used both in methods of treatment of asthma, as well as in methods of treatment of other diseases or conditions associated with TACRl.
  • TACRl therapeutic agents can inhibit TACRl polypeptide activity or nucleic acid expression by a variety of mechanisms, such as, for example, interfering with ligand binding to TACRl polypeptide; interfering with TACRl signaling following ligand binding; inhibiting transcription or translation of the TACRl nucleic acid; by altering posttranslational processing of the TACRl polypeptide; by altering transcription of TACRl splicing variants; or by inhibiting expression of TACRl ligands.
  • the TACRl therapeutic is a tachykinin antagonist, such as those described in European Patent Nos. EP 1 035 115, EP 1 103 545, EP 1 349 541, EP 1 103 546 and EP 1 368 295, the disclosure of which are incorporated herein by reference in their entireties.
  • the antagonist comprises the compound set forth in formula I or a pharmaceutically acceptable salt, ester, or pro-drug thereof:
  • R 1 is a lower alkyl or aryl, optionally substituted by a lower alkyl, lower alkoxy, halogen or trifluoromethyl;
  • 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 O(CH 2 ) n OH, -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.
  • the TACRl antagonist comprises the compound set forth in formula II or a pharmaceutically acceptable salt, ester, or pro-drug thereof:
  • lower alkyl denotes a straight- or branched-chain alkyl group containing from 1-7 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, n- butyl, i-butyl, t-butyl and the like
  • Preferred lower alkyl groups are alkyl groups with 1-4 carbon atoms.
  • lower alkoxy denotes a group wherein the alkyl residues are as defined above, and which is attached via an oxygen atom
  • halogen denotes chlorine, iodine, fluorine and bromine.
  • cycloalkyl denotes a saturated carbocyclic group containing 3-6 carbon atoms.
  • cyclic tertiary amine denotes, for example, pyyrol-1-yl, imidazol-1-yl, piperidin-1-yl, piperazin-1-ul, morpholin-4-yl, thiomorpholin-4-yl, l-oxo-thionorpholin-4-yl or l,l-dioxo-thiomorpholin-4-yl.
  • 5 or 6-membered heterocyclic group denotes, for example, pyridinyl, pyrimidinyl, oxadiazolyl, triazolyl, tetrazolyl, thienyl, furyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, piperazinyl or piperidyl.
  • the TACRl therapeutic agent is an agent that interferes with the activity of TACRl, such as, for example, an agent that interferes with TACRl signaling or interaction of TACRl with members of the tachykinin family (e.g., substance P, hemokinin, neurokinin A, etc.).
  • an agent that interferes with TACRl signaling or interaction of TACRl with members of the tachykinin family e.g., substance P, hemokinin, neurokinin A, etc.
  • the TACRl therapeutic agent(s) are administered in a therapeutically effective amount (i.e., an amount that is sufficient for "treatment," as described above).
  • a therapeutically effective amount i.e., an amount that is sufficient for "treatment," as described above.
  • the amount which will be therapeutically effective in the treatment of a particular individual's disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a nucleic acid of the invention in another embodiment, can be used in "antisense" therapy, in which a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the mRNA and/or genomic DNA of a asthma gene is administered or generated in situ.
  • the antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA inhibits expression of the TACRl polypeptide, e.g., by inhibiting translation and/or transcription. Binding of the antisense nucleic acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.
  • An antisense construct of the present invention can be delivered, for example, as an expression plasmid as described above. When the plasmid is transcribed in the cell, it produces RNA that is complementary to a portion of the mRNA and/or DNA which encodes the TACRl polypeptide.
  • the antisense construct can be an oligonucleotide probe that is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and/or genomic DNA of the polypeptide.
  • the oligonucleotide probes are modified oligonucleotides, which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, thereby rendering them stable in vivo.
  • exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).
  • oligodeoxyribonucleotides derived from the translation initiation site are preferred.
  • oligonucleotides are designed that are complementary to mRNA encoding the TACRl gene.
  • the antisense oligonucleotides bind to TACRl mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA, as referred to herein, indicates that a sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double- stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.
  • the oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotides can include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad.
  • the oligonucleotide may be conjugated to another molecule (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent).
  • another molecule e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent.
  • the antisense molecules are delivered to cells that express TACRl in vivo.
  • a number of methods can be used for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
  • a recombinant DNA construct is utilized in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g., pol III or pol II).
  • 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 plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.
  • viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
  • RNA interference small double- stranded interfering RNA
  • RNAI is a post-transcription process, in which double- stranded RNA is introduced, and sequence-specific gene silencing results, though catalytic degradation of the targeted mRNA. See, e.g., Elbashir, S. M. et al., Nature 411:494-498 (2001); Lee, N. S., Nature Biotech. 19:500-505 (2002); Lee, S-K. et al., Nature Medicine 8(7):681-686 (2002) the entire teachings of these references are incorporated herein by reference.
  • RNAi is used routinely to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases (Chi et al., PNAS,100 (ll):6343-6346 (2003)).
  • RNA-induced silencing complex protein complex RISC (RNA-induced silencing complex) with dual function helicase.
  • RISC protein complex RISC
  • the helicase has RNAas activity and is able to unwind the RNA.
  • the unwound siRNA allows an antisense strand to bind to a target. This results in sequence dependent degradation of cognate mRNA. Aside from endogenous RNAi, exogenous RNAi, chemically synthesized or recombinantly produced can also be used.
  • Endogenous TACRl polypeptide expression can also be reduced by inactivating or "knocking out” the gene, nucleic acid or its promoter using targeted homologous recombination (e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al, Cell 5:313-321 (1989)).
  • targeted homologous recombination e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al, Cell 5:313-321 (1989)).
  • an altered, non-functional gene or nucleic acid flanked by DNA homologous to the endogenous gene or nucleic acid (either the coding regions or regulatory regions of the nucleic acid) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the gene or nucleic acid in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the gene or nucleic acid.
  • the recombinant DNA constructs can be directly administered or targeted to the required site in vivo using appropriate vectors, as described above.
  • endogenous TACRl nucleic acid expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of a TACRl nucleic acid (i.e., the TACRl promoter and/or enhancers) to form triple helical structures that prevent transcription of the TACRl nucleic acid in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of a TACRl nucleic acid i.e., the TACRl promoter and/or enhancers
  • the antisense constructs described herein by antagonizing the normal biological activity of one of the TACRl proteins, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures.
  • the anti- sense techniques e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a asthma gene mRNA or gene sequence
  • Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
  • TACRl therapeutic agents as described herein can also be used in the treatment of asthma gene.
  • the therapeutic agents can be delivered in a composition, as described above, or by themselves. They can be administered systemically, or can be targeted to a particular tissue.
  • the therapeutic agents can be produced by a variety of means, including chemical synthesis; recombinant production; in vivo production (e.g., a transgenic animal, such as U.S. Pat. NO: 4,873,316 to Meade et al.), for example, and can be isolated using standard means such as those described herein.
  • a combination of any of the above methods of treatment e.g., administration of non-altered polypeptide in conjunction with antisense therapy targeting altered mRNA of TACRl; administration of a first splicing variant encoded by a TACRl nucleic acid in conjunction with antisense therapy targeting a second splicing encoded by a TACRl nucleic acid
  • administration of non-altered polypeptide in conjunction with antisense therapy targeting altered mRNA of TACRl e.g., administration of non-altered polypeptide in conjunction with antisense therapy targeting altered mRNA of TACRl; administration of a first splicing variant encoded by a TACRl nucleic acid in conjunction with antisense therapy targeting a second splicing encoded by a TACRl nucleic acid
  • the present invention additionally pertains to methods of assessing an individual's probability of response to a TACRl therapeutic agent.
  • markers or haplotypes relating to the TACRl gene are assessed, as described above in relation to assessing an individual for susceptibility to asthma.
  • the presence of an allele, marker, SNP or haplotype associated with an increased frequency of asthma is indicative of a greater probability of a positive response to a TACRl therapeutic agent.
  • "Greater probability of a positive response" indicates that the individual is more likely to have a positive response to a TACRl therapeutic agent than an individual with asthma that does not have such an allele, marker, SNP or haplotype.
  • 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 invention includes a method of assessing a probability of response to a TACRl therapeutic agent wherein a human subject with asthma is screened for the presence of an allele, marker, SNP, or haplotype that correlates with decreased incidence of asthma, where the presence of such allele, marker, SNP, or haplotype is indicative of a reduced probability of a positive response to a TACRl therapeutic agent.
  • a therapeutic that targets another pathway involved in asthma is predicted to be a more suitable regimen.
  • 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.
  • an assessment of the level of inflammatory markers of the individual both before, and during, treatment with the tachykinin antagonist may indicate whether the treatment has successfully decreased production of tachykinins in the airway wall (such as in ASM cells) or in bone-marrow derived inflammatory cells (such as peripheral blood mononuclear (PBM cells).
  • an individual having an asthma disorder can be assessed for response to treatment with a tachykinin antagonist, by examining the individuals TACRl levels in different cells and body fluids.
  • a level that is "significantly lower”, as used herein, is a level that is less than the amount that is typically found in control individual(s), or is less in a comparison of disease risk in a population associated with the other bands of measurement (e.g., the mean or median, the highest quartile or the highest quintile) compared to lower bands of measurement (e.g., the mean or median, the other quartiles; the other quintiles).
  • the level of a TACRl is assessed in an individual before treatment with a tachykinin antagonist; and during or after treatment with the tachykinin antagonist, and the levels are compared.
  • production of a TACRl is analyzed in a first test sample from the individual, and is also determined in a second test sample from the individual, during or after treatment with the tachykinin antagonist, and the level of production in the first test sample is compared with the level of production of the TACRl in the second test sample.
  • a level of the TACRl in the second test sample that is significantly lower than the level of the TACRl in the first test sample is indicative of efficacy of treatment with the tachykinin antagonist.
  • the efficacy of treatment is indicated by a decrease in the level of the inflammatory marker, that is, a level of the inflammatory marker during or after treatment that is significantly different (e.g., significantly lower), than the level of inflammatory marker before treatment, is indicative of efficacy.
  • Representative inflammatory markers include plasma IL-2, IL-6, IL- l ⁇ and TNF- ⁇ levels and exhaled nitric oxide (NO).
  • the present invention also pertains to pharmaceutical compositions comprising agents that alter TACRl activity or the activity of members of the G protein coupled receptor kinase family.
  • the pharmaceutical compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the carrier and composition preferably are sterile. The formulation should suit the mode of administration.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • Nebulized formulation for inhalation can include sodium chloride, sodium saccharine or sorbitani trioleas
  • inhalation via compressed carbonated formulation in a puffer can include 1,1,1,2-tetrafluoroethanum, monofluorotrichloromethanum tetrafluorodichloroaethanum or diflurodichloromethanum.
  • Methods of introduction of these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal.
  • Other suitable methods of introduction can also include gene therapy (as described below), rechargeable or biodegradable devices, particle acceleration devises ("gene guns") and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents (See, e.g., Agent Table II).
  • agents known to be useful in the inhalation treatment of asthma include cromolyn sodium; anticholinergic agents (e.g., atropine and ipratropium bromide); ⁇ 2 -agonists (e.g., adrenaline, isoproterenol, ephedrine, salbutamol, terbutaline, orciprenaline, fenoterol, and isoetharine), methylxanthines (e.g., theophylline); calcium-channel blockers (e.g., verapamil); and glucocorticoids (e.g, prednisone, prednisolone, dexamethasone, beclomethasone dipropionate, and beclomethasone valerate), as described in Ch.
  • anticholinergic agents e.g., atropine and ipratropium bromide
  • ⁇ 2 -agonists e.g., adrenaline, isoproterenol, ephedrin
  • Criteria for selecting a therapeutically-useful NO-donor compound will include its stability in storage prior to inhalation and its ability to decompose to release NO at a therapeutically beneficial rate upon deposition in the appropriate part of the respiratory tract.
  • S-nitroso-N-acetylpenicillamine (“SNAP”) has been shown to be stable in its solid form, but under physiological conditions (such as in the film of physiological fluid on the surface of the bronchiolar or alveolar lumen), the compound readily decomposes to release NO (Ignarro, Circ. Res., 1989).
  • the nitric-oxide-releasing compound could be provided in powder form, or it could be dissolved or suspended in a biologically-compatible liquid carrier.
  • compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion
  • it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Administration by inhalation includes a mixture of the active drug and the above mentioned ingredients.
  • nonsprayable forms viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • the agent may be incorporated into a cosmetic formulation.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
  • a pressurized volatile, normally gaseous propellant e.g., pressurized air.
  • Agents described herein can be formulated as neutral or salt forms.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), the disclosure of which is incorporated herein by reference.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • prodrugs or “pharmaceutically acceptable prodrugs” as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present invention.
  • Prodrug as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of Formula I.
  • prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). "Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1- 38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq.
  • the agents are administered in a therapeutically effective amount.
  • the amount of agents which will be therapeutically effective depends in part on the nature of the disorder and/or extent of symptoms, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration.
  • the pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like.
  • the pack or kit may also include means for reminding the patient to take the therapy.
  • the pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages.
  • the agents can be separated, mixed together in any combination, present in a single vial or tablet.
  • Agents assembled in a blister pack or other dispensing means is preferred.
  • unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
  • the invention also provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) which alter (e.g., increase or decrease) the activity of the TACRl, which otherwise interact with TACRl or with one or more members of the G protein-coupled receptor kinase pathway.
  • agents e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes
  • Warabi et al ( FEBS Letters, 521:140-144, 2002) report that the membrane bound human substance P receptor (NK 1 receptor, TACRl) is phosphorylated by G protein-coupled receptor kinase 5 (GRK5) and that both the rate and extent of phosphorylation increase dramatically in the presence of substance P in vitro. Accordingly, it is contemplated that agents that directly modulate GRK5 phosphorylating activity would be useful as inhibitors of TACRl activity.
  • GRK5 G protein-coupled receptor kinase 5
  • such agents can be agents which bind to TACRl; which have a stimulatory or inhibitory effect on, for example, activity of TACRl; or which change (e.g., enhance or inhibit) the ability of TACRl to interact with other members of the tachykinin peptide family or with one or more members of the G protein-coupled receptor kinase family, or which alter posttranslational processing of TACRl.
  • such agents can be agents which alter activity or function of one or more members of the G protein-coupled receptor kinase family, such as GRK5.
  • the invention provides assays for screening candidate or test agents that bind to or modulate the activity of TACRl protein (or biologically active portion(s) thereof), as well as agents identifiable by the assays.
  • Test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, KS., Anticancer Drug Des. 12:145 (1997)).
  • a cell, cell lysate, or solution containing or expressing TACRl, or a fragment or derivative thereof can be contacted with an agent to be tested; alternatively, the protein can be contacted directly with the agent to be tested.
  • the level (amount) of TACRl activity is assessed (e.g., the level (amount) of TACRl activity is measured, either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity of the TACRl protein or active fragment or derivative thereof in the absence of the agent to be tested).
  • the agent is an agent that alters the activity of TACRl.
  • An increase in the level of activity relative to a control indicates that the agent is an agent that enhances (is an agonist of) activity.
  • a decrease in the level of activity relative to a control indicates that the agent is an agent that inhibits (is an antagonist of) activity.
  • the level of activity of TACRl or a derivative or fragment thereof in the presence of the agent to be tested is compared with a control level that has previously been established. A level of the activity in the presence of the agent that differs from the control level by an amount that is statistically significant indicates that the agent alters TACRl activity.
  • the present invention also relates to an assay for identifying agents which alter the expression of the TACRl gene (e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) which alter (e.g., increase or decrease) expression (e.g., transcription or translation) of the gene or which otherwise interact with TACRl, as well as agents identifiable by the assays.
  • agents which alter the expression of the TACRl gene e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes
  • alter e.g., increase or decrease expression (e.g., transcription or translation) of the gene or which otherwise interact with TACRl, as well as agents identifiable by the assays.
  • the solution can comprise, for example, cells containing the nucleic acid or cell lysate containing the nucleic acid; alternatively, the solution can be another solution that comprises elements necessary for transcription/translation of the nucleic acid. Cells not suspended in solution can also be employed, if desired.
  • the level and/or pattern of TACRl expression e.g., the level and/or pattern of mRNA or of protein expressed, such as the level and/or pattern of different splicing variants
  • a control i.e., the level and/or pattern of the TACRl Expression in the absence of the agent to be tested.
  • the agent is an agent that alters the expression of a asthma gene.
  • Enhancement of TACRl expression indicates that the agent is an agonist of TACRl activity.
  • inhibition of TACRl expression indicates that the agent is an antagonist of TACRl activity.
  • the level and/or pattern of TACRl polypeptide(s) e.g., different splicing variants
  • the level and/or pattern of TACRl polypeptide(s) is compared with a control level and/or pattern that have previously been established. A level and/or pattern in the presence of the agent that differs from the control level and/or pattern by an amount or in a manner that is statistically significant indicates that the agent alters TACRl expression.
  • agents which alter the expression of TACRl or which otherwise interact with TACRl or one or more members of the G protein- coupled receptor kinase pfamily can be identified using a cell, cell lysate, or solution containing a nucleic acid encoding the promoter region of the TACRl gene or nucleic acid operably linked to a reporter gene.
  • the level of expression of the reporter gene e.g. , the level of mRNA or of protein expressed
  • a control i.e., the level of the expression of the reporter gene in the absence of the agent to be tested.
  • the agent is an agent that alters the expression of TACRl, as indicated by its ability to alter expression of a gene that is operably linked to the TACRl gene promoter. Enhancement of the expression of the reporter indicates that the agent is an agonist of TACRl activity. Similarly, inhibition of the expression of the reporter indicates that the agent is an antagonist of TACRl activity.
  • the level of expression of the reporter in the presence of the agent to be tested is compared with a control level that has previously been established. A level in the presence of the agent that differs from the control level by an amount or in a manner that is statistically significant indicates that the agent alters expression.
  • Agents which alter the amounts of different splicing variants encoded by TACRl e.g., an agent which enhances activity of a first splicing variant, and which inhibits activity of a second splicing variant
  • agents which are agonists of activity of a first splicing variant and antagonists of activity of a second splicing variant can also be identified using variations of the methods described above.
  • assays can be used to assess the impact of a test agent on the activity of a polypeptide in relation to a TACRl binding agent.
  • a cell that expresses a TACRl polypeptide is contacted with TACRl ligand in the presence (and preferably also in the absence) of a test agent, and the ability of the test agent to alter the interaction between the TACRl and the TACRl ligand is determined.
  • a cell fraction such as cell membranes that contain the TACRl polypeptide, can be used. Determining the ability of the test agent to bind to TACRl or a TACRl ligand can be accomplished, for example, by coupling the test agent with a radioisotope or enzymatic label.
  • modulators of expression of nucleic acid molecules of the invention are identified in a method wherein a cell that expresses TACRl is contacted with a test agent and the expression of TACRl mRNA or polypeptide is determined. The level of expression of appropriate mRNA or polypeptide(s) in the presence of the test agent is compared to the level of expression of mRNA or polypeptide(s) in the absence of the test agent. The test agent can then be identified as a modulator of expression based on this comparison.
  • 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 isolated nucleic acid molecules comprising all or a portion of a human TACRl gene sequence.
  • the TACRl nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA or genomic DNA.
  • DNA or RNA molecules can be double- stranded or single- stranded, and can be DNA- RNA duplexes; single stranded RNA or DNA can be the coding, or sense, strand or the non- coding, or antisense strand.
  • the nucleic acid molecule can include all or a portion of the coding sequence of the gene and can further comprise additional non-coding sequences such as introns and non-coding 3' and 5' sequences (including regulatory sequences, for example).
  • the nucleic acid can be a fragment containing only intronic, promoter, or other non-coding sequences.
  • nucleic acid molecules of the invention can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of an encoded polypeptide.
  • a marker sequence for example, a sequence that encodes a polypeptide to assist in isolation or purification of an encoded polypeptide.
  • sequences include, but are not limited to, those that encode a glutathione-S-transferase (GST) fusion protein and those that encode a hemagglutinin A (HA) polypeptide marker from influenza.
  • an "isolated" nucleic acid molecule is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
  • an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • an isolated nucleic acid molecule comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.
  • genomic DNA the term “isolated” also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • the isolated nucleic acid molecule can contain less than about 5 kb but not limited to 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
  • the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution.
  • isolated nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.
  • An isolated nucleic acid molecule can include a nucleic acid molecule or nucleic acid sequence that is synthesized chemically or by recombinant means.
  • isolated DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleic acid molecules include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution.
  • RNA transcripts of the DNA molecules of the present invention are also encompassed by “isolated” nucleic acid sequences.
  • Such isolated nucleic acid molecules are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern or Southern blot analysis.
  • homologous sequences e.g., from other mammalian species
  • gene mapping e.g., by in situ hybridization with chromosomes
  • tissue e.g., human tissue
  • 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.
  • the invention also encompasses nucleic acid molecules encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of a TACRl polypeptide.
  • variants can be naturally occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes.
  • Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions.
  • nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of a TACRl polypeptide.
  • nucleic acid sequences are fragments that comprise one or more polymorphic microsatellite markers.
  • nucleotide sequences are fragments that comprise one or more single nucleotide polymorphisms in a TACRl gene.
  • nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).
  • synthetic molecules that mimic nucleic acid molecules in the ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide).
  • the invention includes variants described herein that hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence encoding an amino acid sequence or a polymorphic variant thereof.
  • the variant that hybridizes under high stringency hybridizations has an activity of a TACRl polypeptide.
  • nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high stringency conditions).
  • Specific hybridization refers to the ability of a first nucleic acid to hybridize to a second nucleic acid in a manner such that the first nucleic acid does not hybridize to any nucleic acid other than to the second nucleic acid (e.g., when the first nucleic acid has a higher similarity to the second nucleic acid than to any other nucleic acid in a sample wherein the hybridization is to be performed).
  • nucleic acid or amino acid “homology” is equivalent to nucleic acid or amino acid "identity".
  • the length of a sequence aligned for comparison purposes is at least 30%, for example, at least 40%, in certain aspects at least 60%, and in other aspects at least 70%, 80%, 90% or 95% of the length of the reference sequence.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. ScL USA 90:5873-5877 (1993).
  • NBLAST nucleic Acids Res. 25:389-3402
  • Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 4(1): 11-17 (1988). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package (Accelrys, Cambridge, UK). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson and Lipman, Proc. Natl. Acad. ScL USA 85:2444-8 (1988).
  • the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package using either a BLOSUM63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package using a gap weight of 50 and a length weight of 3.
  • the present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence of TACRl, or the complement of such a sequence, and also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence encoding an amino acid sequence or polymorphic variant thereof.
  • the nucleic acid fragments of the invention are at least about 15, preferably at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic polypeptides described herein, are particularly useful, such as for the generation of antibodies as described below.
  • 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).
  • a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, for example about 20-25, and in certain aspects about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a contiguous nucleotide sequence of TACRl or polymorphic variant thereof.
  • Preferred primer or probe sequences hybridize to all or a portion of the exon2 LD block of TACRl, whose sequence is set forth in SEQ ID NO: 1.
  • a probe or primer comprises any integer number of nucleotides from SEQ ID NO: 1, preferably any integer number of 100 or fewer nucleotides. Exemplary ranges include from 6 to 50 nucleotides, or from 12 to 30 nucleotides.
  • the probe or primer is preferably at least 70% identical to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence, and more preferably for example at least 80% identical, or at least 90% identical, and in other aspects at least 95%, 96%, 97%, 98%, 99% identical.
  • the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Probes or primers that overlap a polymorphic or marker region of the TACRl sequence are useful for techniques described herein to identify the presence or absence of a specific allele in a sample.
  • 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.
  • a preferred probe or primer comprises a nucleotide sequence that overlaps a polymorphic site (any polymorphic site described herein) such that, when the probe/primer hybridizes to a TACRl reference/target sequence taught herein (or its complement), one of the nucleotides to which it hybridizes is a polymorphic site.
  • This preferred probe has a 3' label (e.g., a fluorescent label), and a quenching moiety attached to it, with the quenching moiety preferably located at the 5' end.
  • the polymorphism is no closer than six bases from the labeled 3' end of the probe/primer, and the quenching moiety is sufficiently close to the label to quench the signal given by the label.
  • the sequence of the probe is perfectly complementary to either the reference sequence or the reference sequence allelic variant defined by the polymorphism, or the complements of such sequences.
  • the probe described in the preceding paragraph is packaged with a second "enhancer" probe that hybridizes to the target sequence almost adjacent to the 3' end of where the first probe hybridizes, such that a one-base gap exists between the two probes when they are hybridized to the target sequence.
  • nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided herein.
  • nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on the sequence of TACRl or the complement of such a sequence, or designed based on nucleotides based on sequences encoding one or more of the amino acid sequences provided herein. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • 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 antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).
  • the nucleic acid sequences can also be used as probes or tools to compare with endogenous DNA sequences in patients to identify one or more of the markers/SNPs/microsatellites described herein. They can also be used as probes to hybridize and discover related DNA sequences or to subtract out known sequences from a sample.
  • the nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses.
  • Portions or fragments of the nucleotide sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways, such as polynucleotide reagents.
  • these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample.
  • the nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization or therapeutic use, or as markers for tissues in which the corresponding polypeptide is expressed, either constitutively, during tissue differentiation, or in diseased states.
  • nucleic acid sequences can additionally be used as reagents in the screening and/or diagnostic assays described herein, and can also be included as components of kits (e.g., reagent kits) for use in the screening and/or diagnostic assays described herein.
  • kits e.g., reagent kits
  • Kits useful in the methods of diagnosis comprise components useful in any of the methods described herein, including for example, hybridization probes or primers as described herein (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele- specific oligonucleotides, antibodies which bind to altered or to non-altered (native) TACRl polypeptide, means for amplification of nucleic acids comprising a TACRl nucleic acid or for a portion of TACRl, or reagents for analyzing the nucleic acid sequence of a TACRl nucleic acid or for analyzing the amino acid sequence of a TACRl polypeptide as described herein, etc.
  • hybridization probes or primers as described herein e.g., labeled probes or primers
  • reagents for detection of labeled molecules e.g., restriction enzymes (e.g., for
  • the kit for diagnosing a susceptibility to asthma can comprise primers for nucleic acid amplification of a region in the TACRl nucleic acid comprising one or more of the markers or SNPs described herein.
  • the primers can be designed using portions of the nucleic acids flanking SNPs that are indicative of asthma.
  • a machine- accessible medium includes recordable/non- recordable media (e.g., read only memory (ROM); random access memory (RAM); magnetic disk storage media; optical storage media; flash memory devices; etc.), as well as electrical, optical, acoustical or other form of propagated signals (e.g., carrier waves, infrared signals, digital signals, etc.); etc.
  • recordable/non- recordable media e.g., read only memory (ROM); random access memory (RAM); magnetic disk storage media; optical storage media; flash memory devices; etc.
  • electrical, optical, acoustical or other form of propagated signals e.g., carrier waves, infrared signals, digital signals, etc.
  • Routines e.g., software for practicing methods of the invention, hardware, firmware, machine accessible media, and programmed machines for practicing all or part of the invention described herein are all aspects of the invention.
  • Vectors and Host Cells are all aspects of the invention.
  • nucleic acid constructs containing a nucleic acid molecules described herein and the complements thereof (or a portion thereof).
  • the constructs comprise a vector (e.g., an expression vector) into which a sequence of the invention has been inserted in a sense or antisense orientation.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • Expression vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
  • recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell.
  • the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • 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.
  • Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells e.g., E. coli
  • insect cells e.g., insect cells
  • yeast or mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al, (supra), and other laboratory manuals.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture can be used to produce (i.e., express) a polypeptide of the invention.
  • the invention further provides methods for producing a polypeptide using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • Antibodies are also provided which bind a portion of either the variant or the reference gene product that contains the polymorphic site or sites.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that specifically bind an antigen.
  • a molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or a fragment thereof.
  • a desired immunogen e.g., polypeptide of the invention or a fragment thereof.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985, Inc., pp. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985, Inc., pp. 77-96) or trioma techniques
  • hybridomas The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al, (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
  • lymphocytes typically splenocytes
  • a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al, Bio/Technology 9: 1370-1372 (1991); Hay et al, Hum. Antibod. Hybridomas 3:81-85 (1992); Huse et al, Science 246: 1275-1281 (1989); and Griffiths et al, EMBO J. 12:725-734 (1993).
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • antibodies of the invention can be used to isolate a polypeptide of the invention by standard techniques, such as affinity chromatography or immunoprecipitation.
  • a polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells.
  • an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • the antibody can be coupled to a detectable substance to facilitate its detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 1, 35 S or 3 H.
  • TACRl tachykinin receptor 1
  • New sequence repeats i.e., dinucleotide, trinucleotide, and tetronucleotide repeats
  • Tandem repeats finder software Bosset, Nucleic Acids Res 27, 573, 1999
  • the size in basepairs of the lower allele of the CEPH sample 1347-02 (CEPH genomics repository) was subtracted from the size of the micro satellite amplicon and used as a reference.
  • SNP genotyping was carried using direct DNA sequencing (Applied BioSystems) or the Centaurus platform (Nanogen).
  • asthma and atopy measures have also been obtained on these patients, including pulmonary function tests, methacholine challenge tests, IgE levels, skin test results, use of anti-asthma drugs and response to therapy as well as information collected via comprehensive questionnaire addressing asthma severity, history of atopic disease (atopic dermatitis, allergic rhinitis), smoking history, environmental/occupational exposures and lifestyle measures. All patients have been "re-phenotyped" with respect to the above measures by their primary asthma specialist.
  • COPSAC Copenhagen Prospective Study on Asthma in Childhood
  • the allele 2 of D2S286 was associated with a significantly decreased risk (Relative risk (RR) 0.72; P 8.6x10-4) while allele 6 was associated with a significantly increased risk for allergic asthma with airway hyper reactivity.
  • the allele 2 of DS2286 was also associated with decreased risk (0.7) in the UK, although the difference was not statistically significant.
  • DG2S104 Two additional microsattelites located within the exon 2 LD block, DG2S104 and DG2S106 were typed on Icelandic patients and controls.
  • DG2S104 is a biallelic marker, allele 8 was associated with increased risk ( RR 1.44) while the allele 0 was associated with decreased risk (RR 0.70).
  • the micro satellite DG2S106 have 4 alleles with population frequencies greater than 1 %, , the allele 6 is associated with an increased risk ( 1.57) whereas allele 0 is associated with decreased risk in Icelandic patients.
  • the LD block was sequenced. Pools containing equal volume of a 15ng/ul DNA solutions were created from 105 patients carrying 2 copies of the rs3771827 T allele (affected homozygote risk carrier), 94 patients homozygous for C allele ( affected non carriers), 176 control individuals carrying two copies of the rs3771827 T allele ( controls homozygote risk allele carrier) and 220 controls individuals homozygous for rs3771827 C allele (control non carriers). Each pool was sequenced in duplicate using a total of 188 primer pairs. The sequences obtained were assembled and aligned to the reference sequence. Two previously undiscovered polymorphisms, TACLD.33_430 and TACLD.117_335 were selected on the basis of showing different levels in patients and controls pools. TACLD.33_430 and
  • TACLD.33_430 A and TACLD.117_335 T allele was significantly increased in affected patients compared to controls ( 7.6% versus 2.9% and 7.5 vs 3% respectively).

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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 Ceased WO2008006105A2 (fr)

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