WO2001004270A1 - Isogenes de ciblage de medicaments: polymorphismes dans le gene alpha du recepteur de l'interleukine 4 - Google Patents

Isogenes de ciblage de medicaments: polymorphismes dans le gene alpha du recepteur de l'interleukine 4 Download PDF

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Publication number
WO2001004270A1
WO2001004270A1 PCT/US2000/019094 US0019094W WO0104270A1 WO 2001004270 A1 WO2001004270 A1 WO 2001004270A1 US 0019094 W US0019094 W US 0019094W WO 0104270 A1 WO0104270 A1 WO 0104270A1
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seq
thymine
il4rα
nucleotide
cytosine
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PCT/US2000/019094
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English (en)
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Anne Chew
R. Rex Denton
Amy Duda
Krishnan Nandabalan
Joel Claiborne Stephens
Andreas K. Windemuth
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2000263446A priority Critical patent/AU2000263446A1/en
Publication of WO2001004270A1 publication Critical patent/WO2001004270A1/fr
Priority to US10/010,802 priority patent/US20030078220A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates to variation in genes that encode pharmaceutically important proteins.
  • this invention provides genetic variants of the human Interleukin 4 Receptor Alpha (IL4R ⁇ ) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • IL4R ⁇ human Interleukin 4 Receptor Alpha
  • nucleotide sequence of a particular gene may vary tremendously among individuals. Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a target protein may be manifested as significant, variation in expression of or in the structure and/or function of the protein. Such alterations may explain the relatively high degree of uncertainty inherent in treatment of individuals with drugs whose design is based upon a single representative example of the target. For example, it is well-established that some classes of drugs frequently have lower efficacy in some individuals than others, which means such individuals and their physicians must weigh the possible benefit of a larger dosage against a greater risk of side effects. In addition, variable information on the biological function or effects of a particular protein may be due to different scientists unknowingly studying different isoforms of the gene encoding the protein. Thus, information on the type and frequency of genomic variation that exists for pharmaceutically important proteins would be useful.
  • haplotype The organization of single nucleotide variations (polymorphisms) in the primary sequence of a gene into one of the limited number of combinations that exist as units of inheritance is termed a haplotype. Each haplotype therefore contains significantly more information than individual unorganized polymorphisms. Haplotypes provide an accurate measurement of the genomic variation in the two chromosomes of an individual. It is well-established that many diseases are associated with specific variations in gene sequences. However while there are examples in which individual polymo ⁇ hisms act as genetic markers for a particular phenotype, in other cases an individual polymorphism may be found in a variety of genomic backgrounds and therefore shows no definitive coupling between the polymorphism and the causative site for the phenotype (Clark AG et al.
  • the marker may be predictive in some populations, but not in other populations (Clark AG et al. 1998 supra). In these instances, a haplotype will provide a superior genetic marker for the phenotype (Clark AG et al. 1998 supra; Ulbrecht M et al. 2000, supra; Rua ⁇ o G & Stephens JC Gen EngNews 19 (21), December 1999). Analysis of the association between each observed haplotype and a particular phenotype permits ranking of each haplotype by its statistical power of prediction for the phenotype.
  • Haplotypes found to be strongly associated with the phenotype can then have that positive association confirmed by alternative methods to minimize false positives. For a gene suspected to be associated with a particular phenotype, if no observed haplotypes for that gene show association with the phenotype of interest, then it may be inferred that variation in the gene has little, if any, involvement with that phenotype (Ruano & Stephens 1999, supra). Thus, information on the observed haplotypes and their frequency of occurrence in various population groups will be useful in a variety of research and clinical applications.
  • IL4R ⁇ Interleukin 4 Receptor Alpha
  • IL4R is a transmembrane complex composed of two different protein subunits, a 140-kDa high affinity binding subunit named interleukin-4 receptor ct (IL-4R ⁇ ; also known as CD 124 antigen) and either a gamma-c subunit, which is present in several cytokine receptors, or an interleukin- 13 receptor 1 (IL-13R1) subunit.
  • IL-4R ⁇ interleukin-4 receptor ct
  • IL-13R1 interleukin- 13 receptor 1
  • Both subunits of the IL-4R are required to bind interleukin-4 (IL-4) and to mediate its transcription-activating effects through the tyrosine kinases, Jakl and Jak3.
  • IL-4 interleukin-4
  • Jak 1 and Jak 3 phosphorylate the IL- 4R ⁇ subunit, creating binding sites in the cytoplasmic domain for many other proteins, including SOS, Stat-6, c-fes, and src homology phosphatase 1 (SHP-1).
  • Activated Jak proteins also phosphorylate Stat- proteins, which travel into the nucleus and function as transcription factors.
  • Other IL-4 signal pathways exist, but are less well characterized.
  • IL-4 one of the most important cytokines involved in the allergic response, is produced when cells from the immune system, in particular T cells, are activated in response to an allergen. Regulation of the immune response involves Helper T-cells that differentiate into two subtypes, Thl and Th2. Thl cells express interferon-gamma and interluekin-2 (IL-2), and mediate a cell-based immunity, where macrophages and neutrophils are prominently involved. Thl cells also direct the IgE-producing B cells, as well as mast cells, basophils and eosinophils. Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13.
  • IL-2 interferon-gamma and interluekin-2
  • Th cell subtype represses the other, so the immune system is forced into differentiation into either a Thl or Th2 response against an external allergic challenge. In many instances an aberration of this response can render a pathological state such as a Th2 response against ragweed.
  • IL-4 induces in B cells the synthesis of IgE type antibodies that recognize specific allergens. IgE binds to receptors on mast cells and basophils and mediates the early humoral (sub-chronic) response on the B-side of the immune system.
  • mast cell-attached IgE If an allergen binds mast cell-attached IgE, the mast cell releases mediators like histamine, and the eicosanoid leukotrienes and prostaglandins products, some of which cause the familiar symptoms of an acute allergic reaction: swelling, itching, mucous, and reddening of the skin. Later in this process eosinophils and other inflammatory cells migrate to the site of inflammation. This later phase is important in asthma, because the eosinophils may instigate a more chronic inflammation which can adversely scar lung tissue.
  • IL-4 is at least partly responsible for recruiting eosinophils, because it induces synthesis of specific adhesion molecules on the capillary endothelium, and stimulates expression of IL-5 and eotaxin.
  • IL-5 leads to the development of a large number of eosinophils from precursor cells in the bone marrow, and eotaxin stimulates their migration into the lung tissue.
  • the gene for IL-4R ⁇ has eleven exons encoding an 825 amino acid protein and spans over 24 kb of the short arm of chromosome 16 (16pl2.1) (Pritchard et al., Genomics 10:801, 1991; GenBank
  • IL4R- ⁇ gene A reference sequence for IL4R- ⁇ gene, which corresponds to the reverse complement of nucleotides 100020-71331 in the GenBank Accession No. AC004525, is shown in Fig. 1 (SEQ ID NO:l).
  • Reference sequences for IL-4R ⁇ mRNA (GenBank Accession No. NM_000418) and the encoded IL4R ⁇ precursor protein (GenBank Accession No. P24394) are shown in Figs. 2 and 3, respectively (SEQ ID NOS:2 and 3).
  • Significant features reported for the IL-4R ⁇ precursor include: a signal peptide located between a.a. 1 and 25; an extracellular domain between a.a. 26 and 232; disulfide bonds between a.a.
  • the IL-4R ⁇ allele with isoleucine at amino acid position 75 (Ile75) in the extracellular domain is more responsive to IL-4 than the allele with valine at that position (Val75) and is associated with atopic asthma but not with non-atopic asthma (Mitsuyasu et al., supra).
  • the allele with arginine at position 576 (Arg576) in the cytoplasmic domain exhibits higher receptor activity than the glutamine allele (Glu576) due to reduced binding by the Arg576 allele of a negative regulatory molecule, src homology phosphatase 1 (Imani et al., J. Biol Chem. 272:7927-7931, 1997).
  • the Arg576 allele has a higher frequency in patients with allergic inflammatory disorders, including atopy (Khurana Hershey et al., New Eng. J. Med. 337: 1720-1725, 1997).
  • Hershey et al. (WO 00/34789) reported that the Arg576 allele is significantly associated with asthma. Studies showed that patients who were homozygous for this allele had about a 9- fold higher risk towards asthma and that two copies of Arg576 are associated in an increase in asthma prevalence and severity. Kanemitsu et al.
  • IL-4R ⁇ gene polymo ⁇ hisms leading to amino acid changes in the cytoplasmic domain of the protein product have been identified at codons 400 (E400A), 431 (C431R) and 786 (S786P) (Kruse et al., supra; Deichmann et al., Biochem. Biophys. Res. Commun. 231 : 696-697, 1997).
  • a polymo ⁇ hism of guanine or adenine at a position corresponding to nucleotide 55328 in Figure 1 has also been reported as well as a polymo ⁇ hism of cytosine or thymine at a position corresponding to nucleotide 55430 (Buetow et al., 1999, Nat Genet. 21:323-5).
  • polymo ⁇ hisms in the IL4R ⁇ gene Because of the potential for polymo ⁇ hisms in the IL4R ⁇ gene to affect the expression and function of the encoded protein, it would be useful to determine whether additional polymo ⁇ hisms exist in the IL4R ⁇ gene, as well as how such polymo ⁇ hisms are combined in different copies of the gene. Such information would be useful for studying the biological function of IL4R ⁇ as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function.
  • polymo ⁇ hic sites correspond to the following nucleotide positions in the reverse complement of the indicated GenBank Accession Number: 32884 (PS1), 32903 (PS2), 32961 (PS3), 33135 (PS4), 35763 (PS6), 35770 (PS7), 35817 (PS8), 35905 (PS9), 35944 (PS10), 35958 (PS11), 37330 (PS12), 37473 (PS13), 37586 (PS14), 37591 (PS15), 37604 (PS16), 37644 (PS17), 37678 (PS18), 43446 (PS19), 43703 (PS20), 53008 (PS21), 53099 (PS22), 53153 (PS23), 53456 (PS25), 53507 (PS27), 53513 (PS28), 53915 (PS30), 53949 (PS32), 54237 (PS33),
  • the polymo ⁇ hisms at these sites are adenine or guanine at PS1, cytosine or thymine at PS2, guanine or thymine at PS3, guanine or cytosine at PS4, cytosine or thymine at PS6, guanine or adenine at PS7, thymine or cytosine at PS8, cytosine or thymine at PS9, cytosine or thymine at PS 10, guanine or adenine at PS11, guanine or adenine at PS12, cytosine or thymine at PS13, cytosine or thymine at PS14, guanine or adenine at PS 15, adenine or thymine at PS 16, cytosine or adenine at PS 17, cytosine or thymine at PS 18, guanine or adenine at PS 19, thymine or cytosine at PS20, adenine or cyto
  • IL4R ⁇ -encoding polynucleotides containing one or more of the novel polymo ⁇ hic sites reported herein will be useful in studying the expression and biological function of IL4R ⁇ , as well as in developing drugs targeting this protein.
  • information on the combinations of polymo ⁇ hisms in the IL4R ⁇ gene may have diagnostic and forensic applications.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymo ⁇ hic variant of a reference sequence for the IL4R ⁇ gene or a fragment thereof.
  • the reference sequence comprises SEQ ID NO: 1 and the polymo ⁇ hic variant comprises at least one polymo ⁇ hism selected from the group consisting of guanine at PS1, thymine at PS2, thymine at PS3, cytosine at PS4, thymine at PS6, adenine at PS7, cytosine at PS8, thymine at PS9, thymine at PS10, adenine at PS11, adenine at PS12, thymine at PS13, thymine at PS14, adenine at PS15, thymine at PS 16, adenine at PS 17, thymine at PS 18, adenine at PS 19, cytosine at PS20, cytosine at PS21 , thymine at PS22, cytosine at
  • the polymo ⁇ hic variant comprises one or more additional polymo ⁇ hisms selected from the group consisting of guanine at PS5, cytosine at PS24, cytosine at PS26, cytosine at PS29, guanine at PS31 , adenine at PS42, and thymine at PS43.
  • a particularly preferred polymo ⁇ hic variant is a naturally-occurring isoform (also referred to herein as an "isogene") of the IL4R gene.
  • An IL4R ⁇ isogene of the invention comprises adenine or guanine at PS1, cytosine or thymine at PS2, guanine or thymine at PS3, guanine or cytosine at PS4, cytosine or thymine at PS6, guanine or adenine at PS7, thymine or cytosine at PS8, cytosine or thymine at PS9, cytosine or thymine at PS 10, guanine or adenine at PS11, guanine or adenine at PS 12, cytosine or thymine at PS 13, cytosine or thymine at PS 14, guanine or adenine at PS 15, adenine or thymine at PS 16, cytosine or adenine at PS 17, cytosine or thymine at PS 18, guanine or adenine at PS 19, thymine or cytosine at PS20, adenine or cytosine
  • An IL4R ⁇ isogene may be defined by the combination and order of these polymo ⁇ hisms in the isogene, which is referred to herein as an IL4R ⁇ haplotype.
  • the invention also provides data on the number of different IL4R haplotypes found in the above four population groups. This haplotype data is useful in methods for deriving an IL4R ⁇ haplotype from an individual's genotype for the IL4R ⁇ gene and for determining an association between an IL4R ⁇ haplotype and a particular trait.
  • the invention provides a polynucleotide comprising a polymo ⁇ hic variant of a reference sequence for an IL4R ⁇ cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig. 2) and the polymo ⁇ hic cDNA comprises at least one poiymo ⁇ hism selected from the group consisting of thymine at a position corresponding to nucleotide 237, adenine at a position corresponding to nucleotide 244, cytosine at a position corresponding to nucleotide 291, thymine at a position corresponding to nucleotide 501, adenine at a position corresponding to nucleotide 554, cvtosine at a position corresponding to nucleotide 939, thymine at a position corresponding to nucleotide 1242, thymine at a position corresponding to nucleotide 1293, cytosine at a position corresponding to nucle
  • the polymo ⁇ hic variant comprises one or more additional polymo ⁇ hisms selected from the group consisting of guanine at a position corresponding to 223, cytosine at a position corresponding to nucleotide 1199, cytosine at a position corresponding to 1291, cytosine at a position corresponding to nucleotide 1507 and guanine at a position corresponding to 1737.
  • Polynucleotides complementary to these IL4R ⁇ genomic and cDNA variants are also provided by the invention.
  • the invention provides a recombinant expression vector comprising one of the polymo ⁇ hic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector.
  • the recombinant vector and host cell may be used to express IL4R ⁇ for protein structure analysis and drug binding studies.
  • the invention provides a polypeptide comprising a polymo ⁇ hic variant of a reference amino acid sequence for the IL4R ⁇ protein.
  • the reference amino acid sequence comprises SEQ ID NO:3 (Fig.
  • the polymo ⁇ hic variant comprises at least one variant amino acid selected from the group consisting of threonine at a position corresponding to amino acid 82, histidine at a position corresponding to amino acid 185, isoleucine at a position corresponding to amino acid 579, serine at a position corresponding to amino acid 675, and alanine at a position corresponding to amino acid 752.
  • the polymo ⁇ hic variant also comprises at least one variant amino acid selected from the group consisting of valine at a position corresponding to amino acid 75, alanine at a position corresponding to amino acid 400, arginine at a position correpsonding to amino acid 431 , proline at a position corresponding to amino acid 503, and arginine at a position corresponding to amino acid 576.
  • a polymo ⁇ hic variant of IL4R ⁇ is useful in studying the effect of the variation on the biological activity of IL4R ⁇ as well as studying the binding affinity of candidate drugs targeting IL4R ⁇ for the treatment of allergies, asthma, and other immune responses.
  • the present invention also provides antibodies that recognize and bind to the above polymo ⁇ hic IL4R ⁇ protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods. In other embodiments, the invention provides methods, compositions, and kits for haplotyping and/or genotyping the IL4R ⁇ gene in an individual.
  • the methods involve identifying the nucleotide or nucleotide pair present at one or more polymo ⁇ hic sites selected from PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS25, PS27, PS28, PS30, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS44, and PS45 in one or both copies of the IL4R ⁇ gene from the individual.
  • compositions contain oligonucleotide probes and primers designed to specifically hybridize to one or more target regions containing, or that are adjacent to, a polymo ⁇ hic site.
  • the methods and compositions for establishing the genotype or haplotype of an individual at the novel polymo ⁇ hic sites described herein are useful for studying the effect of the polymo ⁇ hisms in the etiology of diseases affected by the expression and function of the IL4R ⁇ protein, studying the efficacy of drugs targeting IL4R ⁇ , predicting individual susceptibility to diseases affected by the expression and function of the IL4R ⁇ protein and predicting individual responsiveness to drugs targeting IL4Roc.
  • the invention provides a method for identifying an association between a genotype or haplotype and a trait.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • Such methods have applicability in developing diagnostic tests and therapeutic treatments for allergies, asthma, and other immune responses.
  • the present invention also provides transgenic animals comprising one of the IL4R ⁇ genomic polymo ⁇ hic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the IL4R ⁇ isogenes in vivo, for in vivo screening and testing of drugs targeted against IL4R ⁇ protein, and for testing the efficacy of therapeutic agents and compounds for allergies, asthma, and other immune responses in a biological system.
  • the present invention also provides a computer system for storing and displaying polymo ⁇ hism data determined for the IL4R ⁇ gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymo ⁇ hism data.
  • the polymo ⁇ hism data includes the polymo ⁇ hisms, the genotypes and the haplotypes identified for the IL4R ⁇ gene in a reference population.
  • the computer system is capable of producing a display showing IL4R ⁇ haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the IL4R ⁇ gene that is the reverse complement of part of Genbank Accession Number AC004525.1; contiguous lines; SEQ ID NO:l), with the underlines indicating the start and stop codons, shading indicating the reference coding sequence, and the bold nucleotides indicating the polymo ⁇ hic sites and polymo ⁇ hisms identified by Applicants in a reference population.
  • Figure 2 illustrates a reference sequence for the IL4R coding sequence (GenBank Accession
  • Figure 3 illustrates a reference sequence for the IL4R ⁇ protein (GenBank Accession Number CAA36672; contiguous lines; SEQ ID NO:3), with the bold amino acids indicating the amino acid variations caused by the polymo ⁇ hisms of Fig. 2.
  • the present invention is based on the discovery of novel variants of the IL4R ⁇ gene.
  • the inventors herein discovered 38 novel polymo ⁇ hic sites by characterizing the IL4R ⁇ gene found in genomic DNAs isolated from Index Repository IA that contains immortalized cell lines from one chimpanzee and 93 human individuals and Index Repository IB that contains 70 human individuals. Theses two repositories contain 51 individuals in common.
  • Index Repository IA included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (22 individuals), African descent (20 individuals) Asian (20 individuals) Hispanic/Latino (17 individuals). To the extent possible, the members of this reference population were organized into population subgroups by the self- identified ethnogeographic origin of their four grandparents as shown in Table 1 below. In addition, Index Repository IA contains three unrelated indigenous American Indians (one from each of North, Central, and South America), one three-generation Caucasian family (From the CEPH Utah cohort) and one two-generation African- American family. Table 1. Population Groups in Index Repository IA
  • Index Repository IB contains a reference population of 70 human individuals comprised of 4 three-generation families (from the CEPH Utah cohort) as well as unrelated African- American, Asian, and Caucasian individuals. A total of 38 individuals in this reference population are unrelated.
  • the inventors herein also determined the haplotypes found on each chromosome for most human members of this repository.
  • the IL4 ⁇ genotypes and haplotypes found in the Index Repositories include those shown in Tables 4 and 5, respectively.
  • the polymo ⁇ hism and haplotype data disclosed herein are useful for studying population diversity, anthropological lineage, the significance of diversity and lineage at the phenotypic level, paternity testing, forensic applications, and for identifying associations between the IL4R ⁇ genetic variation and a trait such as level of drug response or susceptibility to disease.
  • the following terms shall be defined as follows unless otherwise indicated:
  • Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.
  • Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.
  • Genotype An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at one or more polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full-genotype and/or a sub-genotype as described below.
  • Sub-genotype The unphased 5' to 3' sequence of nucleotides seen at a subset of the known polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in a single individual.
  • Genotyping A process for determining a genotype of an individual.
  • Haplotype - A 5' to 3' sequence of nucleotides found at one or more polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • haplotype includes a full-haplotype and or a sub-haplotype as described below.
  • Full-haplotype The 5' to 3' sequence of nucleotides found at all known polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • Sub-haplotype The 5' to 3' sequence of nucleotides seen at a subset of the known polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • Haplotype pair The two haplotypes found for a locus in a single individual.
  • Haplotyping A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • Haplotype data Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.
  • Isoform - A particular form of a gene, mRNA, cDNA or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
  • Isogene - One of the isoforms of a gene found in a population. An isogene contains all of the polymo ⁇ hisms present in the particular isoform of the gene.
  • Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term "isolated" is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.
  • Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
  • Naturally-occurring - A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.
  • Nucleotide pair The nucleotides found at a polymo ⁇ hic site on the two copies of a chromosome from an individual.
  • phased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, phased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is known.
  • Polymorphic site (PS) - A position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.
  • Polymorphism The sequence variation observed in an individual at a polymo ⁇ hic site.
  • Polymo ⁇ hisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • Polymorphism data Information concerning one or more of the following for a specific gene: location of polymo ⁇ hic sites; sequence variation at those sites; frequency of polymo ⁇ hisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.
  • Polymorphism Database A collection of polymo ⁇ hism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
  • Polynucleotide A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.
  • Population Group A group of individuals sharing a common ethnogeographic origin.
  • Reference Population A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population. Typically, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
  • Single Nucleotide Polymorphism (SNP) Typically, the specific pair of nucleotides observed at a single polymo ⁇ hic site. In rare cases, three or four nucleotides may be found.
  • Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
  • Treatment A stimulus administered internally or externally to a subject.
  • Unphased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is not known. The inventors herein have discovered 38 novel polymo ⁇ hic sites , and confirmed the existence of 7 other sites, in the IL4R ⁇ gene.
  • PS1- 45 The polymo ⁇ hic sites identified by the inventors are referred to as PS1- 45 to designate the order in which they are located in the gene (see Table 3 below), with the novel polymo ⁇ hic site referred to as PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS25, PS27, PS28, PS30, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS44, and PS45 and the previously reported polymo ⁇ hic sites referred to as PS5, PS24, PS26, PS29, PS31, PS42, and PS43.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the IL4R ⁇ gene or a fragment of the gene which contains at least one of the novel polymo ⁇ hic sites described herein.
  • the nucleotide sequence of a variant IL4R ⁇ gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymo ⁇ hic sites PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS ⁇ 8, PS19, PS20, PS21, PS22, PS23, PS25, PS27, PS28, PS30, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS44, and PS45,and may also comprise one or more additional polymo ⁇ hisms selected from the
  • nucleotide sequence of a variant fragment of the IL4R ⁇ gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymo ⁇ hic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence (or other reported IL4R ⁇ sequences) or to portions of the reference sequence (or other reported IL4R ⁇ sequences), except for genotyping oligonucleotides as described below.
  • the location of a polymo ⁇ hism in a variant gene or fragment is identified by aligning its sequence against SEQ ID NO: 1.
  • the polymo ⁇ hism is selected from the group consisting of guanine at PS1, thymine at PS2, thymine at PS3, cytosine at PS4, thymine at PS6, adenine at PS7, cytosine at PS8, thymine at PS9, thymine at PS10, adenine at PS11, adenine at PS12, thymine at PS13, thymine at PS14, adenine at PS15, thymine at PS16, adenine at PS17, thymine at PS18, adenine at PS19, cytosine at PS20, cytosine at PS21, thymine at PS22, cytosine at PS23, thymine at PS25, thymine at PS27, cytosine at PS28, thymine at PS30, aden
  • Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the IL4R ⁇ gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the polymo ⁇ hic sites described herein. Any particular variant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
  • IL4R ⁇ isogenes may be isolated using any method that allows separation of the two "copies" of the IL4R ⁇ gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles. Separation methods include targeted in vivo cloning (TIVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and copending U.S. application Serial No.
  • 08/987,966 Another method, which is described in copending U.S. Application Serial No. 08/987,966, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets. Yet other methods are single molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruano et al., 17 Nucleic Acids. Res. 8392, 1989; Ruano et al., 19 Nucleic Acids Res. 6877-6882, 1991; Michalatos-Beloin et al., 24 Nucleic Acids Res. 4841-4843, 1996).
  • SMD single molecule dilution
  • the invention also provides IL4R ⁇ genome anthologies, which are collections of IL4R ⁇ isogenes found in a given population.
  • the population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same sex population.
  • An IL4R ⁇ genome anthology may comprise individual IL4R ⁇ isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the IL4R ⁇ isogenes in the anthology may be stored in separate containers.
  • a preferred IL4R ⁇ genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded IL4R ⁇ protein in a prokaryotic or a eukaryotic host cell.
  • expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SV40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence in a given host cell.
  • the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
  • Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
  • Host cells which may be used to express the variant IL4R ⁇ sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
  • the recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microi ⁇ jection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York).
  • eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used.
  • Non- limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, he ⁇ es virus vectors, and baculovirus transfer vectors.
  • Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282:1 145-1147).
  • Particularly preferred host cells are mammalian cells.
  • polymo ⁇ hic variants of the IL4R gene will produce IL4R ⁇ mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules.
  • mRNAs can be used for the preparation of an IL4R ⁇ cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the IL4R ⁇ reference coding sequence shown in Figure 2.
  • the invention also provides IL4R ⁇ mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
  • RNA sequence except for having one or more polymo ⁇ hisms selected from the group consisting of thymine at a position corresponding to nucleotide 237, adenine at a position corresponding to nucleotide 244, cytosine at a position corresponding to nucleotide 291 , thymine at a position corresponding to nucleotide 501, adenine at a position corresponding to nucleotide 554, cytosine at a position corresponding to nucleotide 939, thymine at a position corresponding to nucleotide 1242, thymine at a position corresponding to nucleotide 1293, cvtosine at a position corresponding to nucleotide 1299, thymine at a position corresponding to nucleotide 1701, adenine at a position corresponding to nucleotide 1735, thymine at a position corresponding to nucleot
  • Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain the novel polymo ⁇ hisms described herein.
  • the invention specifically excludes polynucleotides identical to previously identified and characterized IL4R ⁇ cDNAs and fragments thereof.
  • Polynucleotides comprising a variant RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
  • Genomic and cDNA fragments of the invention comprise at least one novel polymo ⁇ hic site identified herein and have a length of at least 10 nucleotides and may range up to the full I p no+h nf tli p gene.
  • a fragment accordmg to the present invention is between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
  • nucleic acid molecules containing the IL4R ⁇ gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand.
  • reference may be made to the same polymo ⁇ hic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymo ⁇ hic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the IL4R ⁇ genomic variants described herein.
  • Polynucleotides comprising a polymo ⁇ hic gene variant or fragment may be useful for therapeutic pu ⁇ oses.
  • an expression vector encoding the isoform may be administered to the patient.
  • the patient may be one who lacks the IL4R ⁇ isogene encoding that isoform or may already have at least one copy of that isogene.
  • IL4R ⁇ isogene expression of an IL4R ⁇ isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA for the isogene.
  • oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3 ' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred.
  • inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • Antisense oligonucleotides may also be designed to block translation of IL4R ⁇ mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of IL4R ⁇ mRNA transcribed from a particular isogene.
  • the oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo.
  • the oligonucleotides may be formulated as a pharmaceutical composition for administration to the patient.
  • Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life.
  • the invention also provides an isolated polypeptide comprising a polymo ⁇ hic variant of the reference IL4R ⁇ amino acid sequence shown in 3. The location of a variant amino acid in an IL4R ⁇ polypeptide or fragment of the invention is identified by aligning its sequence against Fig. 3.
  • An IL4R ⁇ protein variant of the invention comprises an amino acid sequence identical to SEQ ID NO: 3 except for having one or more variant amino acids selected from the group consisting of threonine at a position corresponding to amino acid 82, histidine at a position corresponding to amino acid 185, isoleucine at a position corresponding to amino acid 579, serine at a position corresponding to amino acid 675, and alanine at a position corresponding to amino acid 752, and may also comprise one or more additional variant amino acids selected from the group consisting of valine at a position corresponding to amino acid 75, alanine at a position corresponding to amino acid 400, arginine at a position correpsonding to amino acid 431, proline at a position corresponding to amino acid 503, and arginine at a position corresponding to amino acid 576.
  • IL4R ⁇ protein variants included within the invention comprise all amino acid sequences based on SEQ ID NO: 3 and having the combination of amino acid variations described in Table 2 below.
  • an IL4R ⁇ protein variant of the invention is encoded by an isogene defined by one of the observed haplotypes shown in Table 5.
  • the invention also includes IL4R peptide variants, which are any fragments of an IL4R ⁇ protein variant that contains one or more of the amino acid variations shown in Table 2.
  • An IL4R ⁇ peptide variant is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long.
  • Such IL4R ⁇ peptide variants may be useful as antigens to generate antibodies specific for one of the above IL4R ⁇ isoforms.
  • the IL4R ⁇ peptide variants may be useful in drug screening assays.
  • an IL4R ⁇ variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing one of the variant IL4R ⁇ genomic and cDNA sequences as described above.
  • the IL4R ⁇ protein variant may be isolated from a biological sample of an individual havine an IL4R isogene which encodes the variant protein.
  • a particular IL4R isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular IL4R ⁇ isoform but does not bind to the other IL4R ⁇ isoform.
  • the expressed or isolated IL4R ⁇ protein may be detected by methods known in the art, including
  • IL4R ⁇ variant proteins can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Current Protocols in Molecular Biology John Wiley and Sons, New York, New York). In the case of immunoaffinity chromatography, antibodies specific for a particular polymo ⁇ hic variant may be used.
  • a polymo ⁇ hic variant IL4R ⁇ gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric IL4R ⁇ protein.
  • the non-IL4R ⁇ portion of the chimeric protein may be recognized by a commercially available antibody.
  • the chimeric protein may also be engineered to contain a cleavage site located between the IL4R ⁇ and non-IL4R ⁇ portions so that the IL4R ⁇ protein may be cleaved and purified away from the non-IL4R ⁇ portion.
  • An additional embodiment of the invention relates to using a novel IL4R ⁇ protein isoform in any of a variety of drug screening assays.
  • Such screening assays may be performed to identify agents that bind specifically to all known IL4R ⁇ protein isoforms or to only a subset of one or more of these isoforms.
  • the agents may be from chemical compound libraries, peptide libraries and the like.
  • the IL4R ⁇ protein or peptide variant may be free in solution or affixed to a solid support.
  • high throughput screening of compounds for binding to an IL4R ⁇ variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the IL4R ⁇ protein(s) of interest and then washed. Bound IL4R ⁇ protein(s) are then detected using methods well-known in the art.
  • a novel IL4R ⁇ protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the IL4R ⁇ protein.
  • the invention provides antibodies specific for and immunoreactive with one or more of the novel IL4R ⁇ variant proteins described herein.
  • the antibodies may be either monoclonal or polyclonal in origin.
  • the IL4R ⁇ protein or peptide variant used to generate the antibodies may be from natural or recombinant sources or produced by chemical synthesis using synthesis techniques known in the art. If the IL4R ⁇ protein variant is of insufficient size to be antigenic, it may be conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity of the peptide.
  • carrier molecules include, but are not limited to, albumins (e.p human bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).
  • an antibody specifically immunoreactive with one of the novel IL4R ⁇ protein isoforms described herein is administered to an individual to neutralize activity of the IL4R ⁇ isoform expressed by that individual.
  • the antibody may be formulated as a pharmaceutical composition which includes a pharmaceutically acceptable carrier.
  • Antibodies specific for and immunoreactive with one of the novel IL4R ⁇ protein isoform described herein may be used to immunoprecipitate the IL4R ⁇ protein variant from solution as well as react with IL4R ⁇ protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
  • the antibodies will detect IL4R ⁇ protein isoforms in paraffin or frozen tissue sections, or in cells which have been fixed or unfixed and prepared on slides, coverslips, or the like, for use in immunocytochemical, immunohistochemical, and immunofluorescence techniques.
  • an antibody specifically immunoreactive with one of the novel IL4R ⁇ protein variants described herein is used in immunoassays to detect this variant in biological samples.
  • an antibody of the present invention is contacted with a biological sample and the formation of a complex between the IL4R ⁇ protein variant and the antibody is detected.
  • suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J.
  • Neoman Stockton Press, New York, New York; Current Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York).
  • Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and Campbell et al., 1984, Methods in Immunology, W.A. Benjamin, Inc.).
  • Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g.,
  • Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Current Protocols in Molecular Biology, supra.
  • Exemplary antibody molecules for use in the detection and therapy methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site.
  • Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al. , Volume 13, Elsevier Science Publishers, Amsterdam).
  • the antibodies or antigen binding fragments thereof may also be produced by genetic engineering.
  • the technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication number WO 901443, WO 901443 and WO 9014424 and in Huse et al., 1989, Science, 246: 1275-1281.
  • the antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci. 86; 10029).
  • Effect(s) of the polymo ⁇ hisms identified herein on expression of IL4R ⁇ may be investigated by preparing recombinant cells and/or organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the IL4R ⁇ gene.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into IL4R protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the desired IL4R ⁇ isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the IL4R ⁇ isogene is introduced into a cell in such a way that it recombines with the endogenous IL4R ⁇ gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired IL4R ⁇ gene polymo ⁇ hism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
  • Examples of cells into which the IL4R ⁇ isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the IL4R ⁇ isogene. Such recombinant cells can be used to compare the biological activities of the different protein variants.
  • Recombinant organisms i.e., transgenic animals, expressing a variant IL4R ⁇ gene are prepared using standard procedures known in the art.
  • a construct comprising the variant gene is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage.
  • Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art.
  • One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053.
  • Another method involves directly injecting a transgene into the embryo.
  • a third method involves the use of embryonic stem cells. Examples of animals into which the IL4R ⁇ isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice" and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M.
  • Transgenic animals stably expressing a human IL4R ⁇ isogene and producing human IL4R ⁇ protein can be used as biological models for studying diseases related to abnormal IL4R ⁇ expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • compositions for treating disorders affected by expression or function of a novel IL4R ⁇ isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel IL4R ⁇ isogenes; an antisense oligonucleotide directed against one of the novel IL4R ⁇ isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel IL4R ⁇ isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • a pharmaceutically acceptable carrier examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
  • the pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound.
  • Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • the dose can be estimated initially either in cell culture assays or in animal models.
  • the animal model may also be used to determine the appropriate concentration range and route of administration.
  • Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • compositions and methods for detecting the novel IL4R polymo ⁇ hisms identified herein comprise at least one IL4R ⁇ genotyping oligonucleotide.
  • an IL4R ⁇ genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one of the novel polymo ⁇ hic sites described herein.
  • oligonucleotide refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a preferred oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length.
  • the oligonucleotide may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives.
  • oligonucleotides may have a phosphate- free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH
  • Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • the oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • Genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of an IL4R ⁇ polynucleotide, i.e., an IL4R ⁇ isogene.
  • specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with a non-target region or a non-IL4R ⁇ polynucleotide under the same hybridizing conditions.
  • the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect" or
  • nucleic acid molecule is "substantially complementary” to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions.
  • Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D. et al. in Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).
  • an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary to the target region.
  • non-complementary nucleotides may be interspersed into the oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • Preferred genotyping oligonucleotides of the invention are allele-specific oligonucleotides.
  • ASO allele-specific oligonucleotide
  • allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps.
  • Allele-specific oligonucleotide probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 th or 8 th position in a 15 mer, the 8 th or 9 th position in a 16mer, the 10 th or 11 th position in a 20 mer).
  • a preferred ASO probe for detecting IL4R ⁇ gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • TTGCACCACTGCACT SEQ ID NO: 5
  • TTGCACCGCTGCACT SEQ ID NO: 5
  • TTTTGTGCTATTCCC (SEQ ID NO: 6) and its complement
  • TTTTGTGTTATTCCC (SEQ ID NO: 7) and its complement
  • AGAACAACGGAGGCG (SEQ ID NO: 12) and its complement
  • AG7 ⁇ ACAATGGAGGCG (SEQ ID NO: 13) and its complement
  • GCCTGGGCTGAGGGT SEQ ID NO:20 and its complement
  • GCCTGGGTTGAGGGT SEQ ID NO:21 and its complement
  • TGGGGTGGGCAGGGG SEQ ID NO: 22 and its complement
  • TGGGGTGAGCAGGGG SEQ ID NO: 23 and its complement
  • TTCTCCCGCAGTGAA SEQ ID NO:24 and its complement
  • TTCTCCCACAGTGAA SEQ ID NO:25 and its complement
  • GTGAAAACGACCCGG (SEQ ID NO:2 ⁇ and its complement, GTGAAAATGACCCGG (SEQ ID NO:27 and its complement,
  • GGCAAGCCCTGGGGC SEQ ID NO: 28 and its complement
  • GGCAAGCTCTGGGGC SEQ ID NO: 29 and its complement
  • GCCCTGGGGCTGGAT [SEQ ID NO:30 and its complement
  • GCCCTGGAGCTGGAT [SEQ ID NO:31 and its complement
  • ATAGCAAATCCCAGG (SEQ ID NO: 32 and its complement, ATAGCAATTCCCAGG (SEQ ID NO: 33 and its complement,
  • GCTCTGCCCTAGGCA SEQ ID NO:34 and its complement
  • GCTCTGCACTAGGCA SEQ ID NO:35 and its complement
  • TCCCTCCGCATCGCA SEQ ID NO: 38 and its complement
  • TCCCTCCACATCGCA SEQ ID NO: 39 and its complement
  • ATGTCTGCAGTAGAC SEQ ID NO: 43 and its complement
  • TGGACCTGCTCGGAG SEQ ID NO: 48 and its complement
  • TGGACCTTCTCGGAG SEQ ID NO: 49 and its complement
  • AGTCATGCCTTCTTC (SEQ ID NO:50 and its complement, AGTCATGTCTTCTTC (SEQ ID NO:51 and its complement,
  • GCCTTCTTCCACCTT (SEQ ID NO: 52 and its complement, GCCTTCTCCCACCTT (SEQ ID NO: 53 and its complement, CAGCCCCCGTCTCGG (SEQ ID NO:54 and its complement, CAGCCCCTGTCTCGG (SEQ ID NO:55 and its complement,
  • GGAGTTTGTACATGC SEQ ID NO: 56 and its complement
  • GGAGTTTATACATGC SEQ ID NO:5 " 7 and its complement
  • CAGCTCCCCAGAGCA SEQ ID NO:58 and its complement
  • CAGCTCCTCAGAGCA SEQ ID NO:59 and its complement
  • AGACAGGTCCTCGCC SEQ ID NO: 60 and its complement
  • AGACAGGGCCTCGCC SEQ ID NO: 61 and its complement
  • AGGTGCATGTCCTCT (SEQ ID NO: 64 and its complement, AGGTGCACGTCCTCT (SEQ ID NO: 65 and its complement,
  • GTGCATGTCCTCTTG (SEQ ID NO: 66 and its complement, GTGCATGCCCTCTTG (SEQ ID NO: 67 and its complement,
  • GGCTTATCCATGCCT SEQ ID NO: 68 and its complement
  • GGCTTATTCATGCCT SEQ ID NO: 69 and its complement
  • AGCCAGGCTGGCAGA SEQ ID NO:70 and its complement
  • AGCCAGGGTGGCAGA SEQ ID NO:71 and its complement
  • GGCCCACATGGAGGC SEQ ID NO:72 and its complement
  • GGCCCACGTGGAGGC SEQ ID NO:73 and its complement
  • T7AACACAGCCATCAA SEQ I D NO : 74 and its complement
  • TAACAC7 ⁇ ACCATCAA SEQ I D NO : 75 and its complement
  • TAATGCTCGTCTGTG SEQ ID NO:76 and its complement
  • TAATGCTTGTCTGTG SEQ ID NO:77 and its complement
  • ACTTGCCGTCTGGGT SEQ ID NO:78 and its complement
  • ACTTGCCATCTGGGT SEQ ID NO: 79 and its complement.
  • An allele-specific oligonucleotide primer of the invention has a 3' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present. Allele-specific oligonucleotide primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a preferred ASO primer for detecting IL4R ⁇ gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • CTGAGATTGCACCAC (SEQ ID NO:80); GGCTGGAGTGCAGTG (SEQ ID NO:81); CTGAGATTGCACCGC (SEQ ID NO: 82); GGCTGGAGTGCAGCG (SEQ ID NO: 83); CTGTGCTTTTGTGCT (SEQ ID NO:84); ACCAAGGGG7AATAGC (SEQ ID NO:85); CTGTGCTTTTGTGTT (SEQ ID NO:86); ACCAAGGGGAATAAC (SEQ ID NO:87); GAGTTCCTGGGCCGC SEQ ID NO 88); GGAGCAGCCTGAGCG (SEQ ID NO: 89); GAGTTCCTGGGCCTC SEQ ID NO 90); GGAGCAGCCTGAGAG (SEQ ID NO: 91);
  • TCCGAGTAAGCCTGC SEQ ID NO 92 TCCAGCTCCAGCGCA (SEQ ID NO:93); TCCGAGTAAGCCTCC SEQ ID NO 94); TCCAGCTCCAGCGGA (SEQ ID NO:95);
  • TGGTCAGTGCGGATA SEQ ID NO :104 CCAGTGTATAGTTAT SEQ ID NO: 105) ; TGGTCAGTGCGGACA SEQ ID NO :106 ; CCAGTGTATAGTTGT SEQ ID NO: 107) ;
  • GGCTGGATAGCAAAT SEQ ID NO :136 CTAGCTCCTGGGATT SEQ ID NO:137) ; GGCTGGATAGCAATT SEQ ID NO :138 ; CTAGCTCCTGGGAAT SEQ ID NO: 139) ; CACCTGGCTCTGCCC SEQ ID NO :140 ; GGGACTTGCCTAGGG SEQ ID NO: 141) ; CACCTGGCTCTGCAC SEQ ID NO :142 ; GGGACTTGCCTAGTG SEQ ID NO: 143) ;
  • GAACCCTCCCTCCGC SEQ ID NO :148 ; GCTGGCTGCGATGCG SEQ ID NO: 149) ; GAACCCTCCCTCCAC SEQ ID NO :150 ; GCTGGCTGCGATGTG SEQ ID NO: 151) ;
  • TAGATACACCTGCTG SEQ ID NO :152 GCAGATACACCACAG SEQ ID NO: 153) ; TAGATACACCTGCCG SEQ ID NO :154 ; GCAGATACACCACGG SEQ ID NO: 155) ;
  • GAAGGCATGTCTGAA SEQ ID NO :156 ; ATGGCTGTCTACTTC SEQ ID NO:157) ; GAAGGCATGTCTGCA SEQ ID NO :158 ; ATGGCTGTCTACTGC SEQ ID NO: 159) ; GAACCCTGACCAACC SEQ ID NO :160 ; TGC7 ⁇ AAAGCAAAGGA SEQ ID NO: 161) ; G7AACCCTGACC7 ⁇ ATC SEQ ID NO :162 ; TGCAAAAGCAAAGAA SEQ ID NO: 163) ; TCTTGCCCTGTTTTC SEQ ID NO: 164 TGTTGTGCTCCAGAA SEQ ID NO: 165 TCTTGCCCTGTTTCC SEQ ID NO:166 TGTTGTGCTCCAGGA SEQ ID NO: 167
  • TGTTCCTGGACCTGC SEQ ID NO: 168 TCTCCTCTCCGAGCA SEQ ID NO: 169 TGTTCCTGGACCTTC SEQ ID NO: 170 TCTCCTCTCCGAGAA SEQ ID NO: 171
  • TGGGGGAGTCATGCC SEQ ID NO: 172 AAGGTGGAAGAAGGC SEQ ID NO: 173 TGGGGGAGTCATGTC SEQ ID NO: 174 AAGGTGGAAGAAGAC SEQ ID NO: 175 AGTCATGCCTTCTTC SEQ ID NO: 176 TTCCCGAAGGTGGAA SEQ ID NO: 177 AGTCATGCCTTCTCC SEQ ID NO: 178 TTCCCGAAGGTGGGA SEQ ID NO: 179
  • CCCAAGCAGCTCCCC SEQ ID NO:188 CCCAGGTGCTCTGGG SEQ ID, NO: 189 CCCAAGCAGCTCCTC SEQ ID NO: 190 CCCAGGTGCTCTGAG SEQ ID NO: 191
  • TCTTAGGTGCATGTC SEQ ID NO:204 CAGCAACAAGAGGAC SEQ ID NO: 205
  • TCTTAGGTGCATGCC SEQ ID NO:206 CAGCAACAAGAGGGC SEQ ID NO: 207
  • GACTAGGGCTTATCC SEQ ID NO:208 TTTCCCAGGCATGGA SEQ ID NO:209
  • GACTAGGGCTTATTC SEQ ID NO:210 TTTCCCAGGCATGAA SEQ ID NO: 211
  • G7 ⁇ AGGCAGCCAGGCT SEQ ID NO:212 TGGAAATCTGCCAGC SEQ ID NO:213 GAAGGCAGCCAGGGT SEQ ID NO:214 TGGAAATCTGCCACC SEQ ID NO:215 GATCATGGCCCACAT SEQ ID NO: 216 AGGTGGGCCTCCATG SEQ ID NO:217 GATCATGGCCCACGT SEQ ID NO: 218 AGGTGGGCCTCCACG SEQ ID NO:219
  • AGA7 ⁇ ACTAACACAGC SEQ ID NO: 220 ATTCCCTTGATGGCT SEQ ID NO:221
  • AGA7 ⁇ ACTAACACAAC SEQ ID NO:222 ATTCCCTTGATGGTT SEQ ID NO:223
  • TAAGA7AACTTGCCGT SEQ ID NO:228 ACCCA7AACCCAGACG (SEQ ID NO: 229) TAAGAAACTTGCCAT SEQ ID NO:230 and ACCCAAACCCAGATG (SEQ ID NO: 231 ]
  • genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymo ⁇ hic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymo ⁇ hisms described herein and therefore such genotyping oligonucleotides are referred to herein as "primer-extension oligonucleotides”.
  • the 3 '-terminus of a primer- extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymo ⁇ hic site.
  • a particularly preferred oligonucleotide primer for detecting IL4R ⁇ gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • TAGGGCTTAT (SEQ ID NO: 296)
  • CCCAGGCATG SEQ ID NO : 297 )
  • AACTAACACA (SEQ ID NO: 302) CCCTTGATGG ( SEQ ID NO : 303 )
  • GAGTAATGCT SEQ ID NO: 304
  • ACACACAGAC SEQ ID NO : 305
  • GAAACTTGCC SEQ ID NO: 306
  • CAAACCCAGA SEQ ID NO : 307
  • a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymo ⁇ hic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymo ⁇ hic site.
  • IL4R ⁇ genotyping oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019).
  • Such immobilized genotyping oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized IL4R ⁇ genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymo ⁇ hisms in multiple genes at the same time.
  • the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
  • IL4R ⁇ genotype and “IL4R ⁇ haplotype” mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymo ⁇ hic sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymo ⁇ hic sites in the IL4R ⁇ gene.
  • the additional polymo ⁇ hic sites may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
  • One embodiment of the genotyping method involves isolating from the individual a nucleic acid mixture comprising the two copies of the IL4R ⁇ gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more of the polymo ⁇ hic sites selected from PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16,
  • the two "copies" of a gene in an individual may be the same allele or may be different alleles.
  • the genotyping method comprises determining the identity of the nucleotide pair at each of PS 1-45.
  • the nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample.
  • tissue samples include whole blood, semen saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the nucleic acid mixture may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from an organ in which the IL4R ⁇ gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in introns or in 5' and 3' nontranscribed regions. If an IL4R ⁇ gene fragment is isolated, it must contain the polymo ⁇ hic site(s) to be genotyped.
  • One embodiment of the haplotyping method comprises isolating from the individual a nucleic acid molecule containing only one of the two copies of the IL4R ⁇ gene, or a fragment thereof, that is present in the individual and determining in that copy the identity of the nucleotide at one or more of the polymo ⁇ hic sites PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS25, PS27, PS28, PS30, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS44, and PS45 in that copy to assign an IL4R ⁇ haplotype to the individual.
  • the nucleic acid may be isolated using any method capable of separating the two copies of the IL4R ⁇ gene or fragment such as one of the methods described above for preparing IL4R ⁇ isogenes, with targeted in vivo cloning being the preferred approach.
  • any individual clone will only provide haplotype information on one of the two IL4Roc gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional IL4R ⁇ clones will need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the IL4R ⁇ gene in an individual.
  • the haplotyping method also comprises identifying the nucleotide atone or more of the polymo ⁇ hic sites PS5, PS24, PS26, PS29, PS31 , PS42, and PS43. In a particularly preferred embodiment, the nucleotide at each of PS 1-45 is identified.
  • an IL4R ⁇ haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more of the polymo ⁇ hic sites selected from PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS25, PS27, PS28, PS30, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS44, and PS45 in each copy of the IL4R ⁇ gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS 1-45 in each copy of the IL4R ⁇ gene.
  • the identifying step is preferably performed with each copy of the gene being placed in separate containers.
  • the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be possible in some cases to perform the method in the same container.
  • first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymo ⁇ hic site(s), then detecting a combination of the first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination of the second and third dyes would identify the polymo ⁇ hism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic site(s) may be determined by amplifying a target region(s) containing the polymo ⁇ hic site(s) directly from one or both copies of the IL4R ⁇ gene, or fragment thereof, and the sequence of the amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled- artisan that only one nucleotide will be detected at a polymo ⁇ hic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymo ⁇ hism may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).
  • the identity of the allele(s) present at any of the novel polymo ⁇ hic sites described herein may be indirectly determined by genotyping a polymo ⁇ hic site not disclosed herein that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest. Two sites are said to be in linkage disequilibrium if the presence of a particular variant at one site enhances the predictability of another variant at the second site (Stevens, JC 1999, Mol. Diag. 4: 309-17). Polymo ⁇ hic sites in linkage disequilibrium with the presently disclosed polymo ⁇ hic sites may be located in regions of the gene or in other genomic regions not examined herein.
  • Genotyping of a polymo ⁇ hic site in linkage disequilibrium with the novel polymo ⁇ hic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymo ⁇ hic site.
  • the target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241: 1077-1080, 1988). Oligonucleotides useful as primers or probes in such methods should specifically hybridize to a region of the nucleic acid tfiat contains or is adjacent to the polymo ⁇ hic site.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length. Most preferably, the oligonucleotides are 20 to 25 nucleotides long. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
  • Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392- 396, 1992).
  • a polymo ⁇ hism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art.
  • allele-specific oligonucleotides are utilized in performing such methods.
  • the allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymo ⁇ hic site may be detected at once using a set of allele-specific oligonucleotides or oligonucleotide pairs.
  • the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymo ⁇ hic sites being detected.
  • Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
  • Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads.
  • the solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the IL4R ⁇ gene of an individual may also be determined by hybridization of a nucleic sample containing one or both copies of the gene to nucleic acid arrays and subarrays such as described in WO 95/11995.
  • the arrays would contain a battery of allele-specific oligonucleotides representing each of the polymo ⁇ hic sites to be included in the genotype or haplotype.
  • polymo ⁇ hisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • riboprobes Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985
  • proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • variant alleles can be identified by single strand conformation polymo ⁇ hism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232- 236, 1989).
  • SSCP single strand conformation polymo ⁇ hism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymo ⁇ hism(s).
  • multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).
  • an individual's IL4R ⁇ haplotype pair is predicted from its IL4R ⁇ genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying an IL4R ⁇ genotype for the individual at two or more polymo ⁇ hic sites selected from PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS25, PS27, PS28, PS30, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS44, and PS45, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing IL4R ⁇ haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data.
  • the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world.
  • a preferred reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each of the four population groups named above.
  • a particularly preferred reference population includes a 3-generation family representing one or more of the four population groups to serve as controls for checking quality of haplotypine Drocedures.
  • the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L.
  • the assigning step involves performing the following analysis.
  • each of the possible haplotype pairs is compared to the haplotype pairs in the reference population.
  • the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual.
  • only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
  • either no haplotypes in the reference population are consistent with the possible haplotype pairs, or alternatively, multiple reference haplotype pairs are consistent with the possible haplotype pairs.
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
  • a direct molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
  • the invention also provides a method for determining the frequency of an IL4R ⁇ genotype or IL4R ⁇ haplotype in a population.
  • the method comprises determining the genotype or the haplotype pair for the IL4R ⁇ gene that is present in each member of the population, wherein the genotype or haplotype comprises the nucleotide pair or nucleotide detected at one or more of the polymo ⁇ hic sites PS1, PS2, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS25, PS27, PS28, PS30, PS32, PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS44, and PS45 in the IL4Rct gene; and calculating the frequency any particular genotype or haplotype is found in the population.
  • the population may be a reference population,
  • frequency data for IL4R ⁇ genotypes and/or haplotypes found in a reference population are used in a method for identifying an association between a trait and an IL4R ⁇ genotype or an IL4R ⁇ haplotype.
  • the trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency of the genotype(s) or haplotype(s) of interest in a reference population as well as in a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above.
  • the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written or electronic form.
  • the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies of the genotype(s) or haplotype(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes and/or haplotypes observed in the populations are compared.
  • the trait is predicted to be associated with that IL4R ⁇ genotype or haplotype.
  • the IL4R ⁇ genotype or haplotype being compared in the trait and reference populations is selected from the full-genotypes and full- haplotypes shown in Tables 4 and 5, respectively, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.
  • the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting IL4R ⁇ or response to a therapeutic treatment for a medical condition.
  • medical condition includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders.
  • clinical response means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects).
  • clinical population In order to deduce a correlation between clinical response to a treatment and an IL4R ⁇ genotype or haplotype, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population".
  • This clinical data may be obtained by analyzing the results of a clinical trial that has already been run and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials.
  • the term "clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects. It is preferred that the individuals included in the clinical population have been graded for the existence of the medical condition of interest.
  • the therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses.
  • the IL4R ⁇ gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
  • correlations between individual response and IL4R ⁇ genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their IL4R ⁇ genotype or haplotype (or haplotype pair) (also referred to as a polymo ⁇ hism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated.
  • the most-common and least common nucleotides at the polymo ⁇ hic site are first defined. Then, for each individual in the trial population, one calculates a "dose" as the number of least- common nucleotides the individual has at the polymo ⁇ hic site of interest. This value can be 0 (homozygous for the least-common nucleotide), 1 (heterozygous), or 2 (homozygous for the most common nucleotide).
  • An individual's "response” is the value of the clinical measurement. Standard linear regression methods are then used to fit all the individuals' doses and responses to a single model (see e.g., L.D. Fisher and G.
  • the outputs of the regression calculation are the intercept r 0 , the slope S, and the variance (which measures how well the data tits this simple linear model).
  • the Students t-test value and the level of significance can then be calculated for each of the polymo ⁇ hic sites.
  • a second method for finding correlations between IL4R ⁇ haplotype content and clinical responses uses predictive models based on error-minimizing optimization algorithms.
  • One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Their Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997).
  • Simulated annealing Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K.
  • C is the measured clinical outcome, i goes over all polymo ⁇ hic sites, ⁇ over all candidate genes, C 0 , w j a and w i ' a are variable weight values, R j a is equal to 1 if site i in gene ⁇ in the first haplotype takes on the most common nucleotide and -1 if it takes on the less common nucleotide.
  • L s a is the same as R j a except for the second haplotype.
  • the constant term C 0 and the weights w j a and w i ' a are varied by the genetic algorithm during a search process that minimizes the error between the measured value of C and the value calculated from Equation 1.
  • Models other than the one given in Equation 1 can be readily inco ⁇ orated by those skilled in the art for analyzing the clinical and polymo ⁇ hism data.
  • the genetic algorithm is especially suited for searching not only over the space of weights in a particular model but also over the space of possible models (Judson, supra). Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymo ⁇ hic sites in the IL4R ⁇ gene.
  • ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10). These traits or variables are called the independent variables.
  • the independent variable(s) are measured and individuals are placed into groups based on their values for these variables.
  • the independent variable(s) refers to the combination of polymo ⁇ hisms present at a subset of the polymo ⁇ hic sites, and thus, each group contains those individuals with a given genotype or haplotype pair.
  • the variation in response within the groups and also the variation between groups is then measured. If the within-group response variation is large (people in a group have a wide range of responses) and the response variation between groups is small (the average responses for all groups are about the same) then it can be concluded that the independent variables used for the grouping are not causing or correlated with the response variable.
  • the calculated F-ratio is preferably compared with the critical F-distribution value at whatever level of significance is of interest.
  • the F- ratio is greater than the Critical F-distribution value, then one may be confident that the individual's genotype or haplotype pair for this particular subset of polymo ⁇ hic sites in the IL4R ⁇ gene is at least partially responsible for, or is at least strongly correlated with the clinical response.
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of IL4R ⁇ genotype or haplotype content.
  • the model is validated in one or more follow-up clinical trials designed to test the model.
  • the identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the IL4R ⁇ gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug.
  • the diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL4R ⁇ gene), a serological test, or a physical exam measurement.
  • a direct DNA test i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL4R ⁇ gene
  • serological test i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL4R ⁇ gene
  • a physical exam measurement i.e., a direct DNA test (i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL4R ⁇ gene)
  • serological test i.e., a serological test
  • a physical exam measurement i.e., a physical exam measurement.
  • this diagnostic method uses the predictive haplotyping method described above.
  • any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer.
  • the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the IL4R ⁇ gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymo ⁇ hism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
  • the IL4R ⁇ polymo ⁇ hism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files). These polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network.
  • a relational database e.g., an instance of an Oracle database or a set of ASCII flat files.
  • These polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD ROM or on one or more other storage devices accessible by the computer.
  • the data may be stored on one or more databases in communication with the computer via a network.
  • This example illustrates examination of various regions of the IL4R ⁇ gene for polymo ⁇ hic sites using DNA from Index Repository IA.
  • Target Regions The following target regions of the IL4R ⁇ gene were amplified using the PCR primer pairs listed below, with the sequences presented in the 5 ' to 3 ' direction and nucleotide positions shown for each region corresponding to the indicated GenBank Accession No.
  • Amplification profile 94°C - 2 min. 1 cycle
  • PCR products were purified by Solid Phase Reversible Immobilization using the protocol developed by the Whitehead Genome Center. A detailed protocol can be found at http://www.genome.wi.mit.edu/sequencing/protocols/pure/SPRI_pcr.html.
  • the purified PCR products were sequenced in both directions using the primer sets described previously or those listed, in the 5' to 3' direction, below.
  • This example illustrates examination of the IL4R ⁇ gene for polymo ⁇ hic sites in the following target regions: 1000 base pairs upstream ot the A1U start codon; each ot the exons, including approximately 100 base pairs on either side of the exon; and 500-1000 base paris downstream of the termination codon.
  • PCR primers which were designed based on the nearly complete IL4R genomic sequence reported in the GenBank database (Accession No: AC004525), are set forth below:
  • Amplification profile 94°C - 2 min. 1 cycle
  • PCR products were purified by Solid Phase Reversible Immobilization using the protocol developed by the Whitehead Genome Center. A detailed protocol can be found at http://www.genome.wi.mit.edu sequencing/protocols/pure/SPRI_pcr.html.
  • the purified PCR products were sequenced in both directions using the primer sets described previously or those listed, in the 5 ' to 3 ' direction, below.
  • Example 2 This example illustrates analysis of the IL4R ⁇ polymo ⁇ hisms identified in the Index Repositories for human genotypes and haplotypes for all polymo ⁇ hic sites except PSl, PS9, PSl 1, PS21, PS22, and PS23.
  • Table 4 A sampling of different genotypes containing these polymo ⁇ hisms that were observed in these reference populations are shown in Table 4 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
  • Table 4 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 4 can typically be inferred based on linkage disequilibrium and/or Mendelian inheritance.
  • haplotype pairs shown in Table 4 were estimated from the unphased genotypes using an extension of Clark's algorithm (Clark, A.G. (1990) Mol Bio Evol 7, 111-122), as described in U.S. Provisional Patent Application filed April 19, 2000 and entitled "A Method and System for Determining Haplotypes from a Collection of Polymo ⁇ hisms".
  • haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites. This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.

Abstract

L'invention porte sur des polynucléotides comprenant un ou plusieurs polymorphismes de 38 nouveaux nucléotides dans le gène humain Alpha du récepteur de l'Interleukine 4 (IL4Rα). L'invention porte également sur des compositions et sur des procédés de détection d'un ou plusieurs de ces polymorphismes, ainsi que sur divers génotypes et haplotypes pour le gène IL4Rα qui existent dans la population.
PCT/US2000/019094 1999-07-13 2000-07-13 Isogenes de ciblage de medicaments: polymorphismes dans le gene alpha du recepteur de l'interleukine 4 WO2001004270A1 (fr)

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WO2014207435A1 (fr) * 2013-06-25 2014-12-31 Ucl Business Plc Agents antimicrobiens et leurs utilisations

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Cited By (13)

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US11031098B2 (en) 2001-03-30 2021-06-08 Genetic Technologies Limited Computer systems and methods for genomic analysis
US6969589B2 (en) 2001-03-30 2005-11-29 Perlegen Sciences, Inc. Methods for genomic analysis
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WO2003057718A3 (fr) * 2002-01-07 2003-12-04 Perlegen Sciences Inc Systemes et procedes d'analyse genetique
US6897025B2 (en) * 2002-01-07 2005-05-24 Perlegen Sciences, Inc. Genetic analysis systems and methods
JP2006504392A (ja) * 2002-01-07 2006-02-09 パーレジェン サイエンス インク． 遺伝分析の系および方法
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US7427480B2 (en) 2002-03-26 2008-09-23 Perlegen Sciences, Inc. Life sciences business systems and methods
US6955883B2 (en) 2002-03-26 2005-10-18 Perlegen Sciences, Inc. Life sciences business systems and methods
US7335474B2 (en) 2003-09-12 2008-02-26 Perlegen Sciences, Inc. Methods and systems for identifying predisposition to the placebo effect
WO2014207435A1 (fr) * 2013-06-25 2014-12-31 Ucl Business Plc Agents antimicrobiens et leurs utilisations

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