WO2002016631A1 - Haplotypes du gene ifng - Google Patents

Haplotypes du gene ifng Download PDF

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Publication number
WO2002016631A1
WO2002016631A1 PCT/US2001/026678 US0126678W WO0216631A1 WO 2002016631 A1 WO2002016631 A1 WO 2002016631A1 US 0126678 W US0126678 W US 0126678W WO 0216631 A1 WO0216631 A1 WO 0216631A1
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Prior art keywords
ifng
haplotype
gene
seq
individual
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PCT/US2001/026678
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English (en)
Inventor
Anne Chew
R. Rex Denton
Kevin Finkel
Krishnan Nandabalan
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2001286807A priority Critical patent/AU2001286807A1/en
Publication of WO2002016631A1 publication Critical patent/WO2002016631A1/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Definitions

  • This invention relates to variation in genes that encode pharmaceutically-important proteins.
  • this invention provides genetic variants of the human interferon, gamma (IFNG) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • IFNG human interferon, gamma
  • haplotype is the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymorphisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses AD supra; Ulbrecht M et al. 2000 Am JRespir Crit Care Med 161: 469-74) and drug response (Wolfe CR et al.
  • IFNG interferon, gamma
  • IFNG interferon, gamma
  • IFNG is a multifunctional cytokine released from activated T lymphocytes in response to specific antigens or mitogens.
  • IFNG belongs to the type II (or gamma) interferon family of proteins and has several immunoregulatory functions, including its abilities to act as a potent antiproliferative effector on transformed cells, to activate macrophages, and to potentiate the antiviral and anti-tumor effects of type I (alpha or beta) interferons.
  • IFNG is essential for the development of T-helper (Th) 1 cells and exhibits antiviral activity towards a variety of intracellular pathogens including the human immunodeficiency virus (FHV-l). Mutations in IFNG have been associated with such diseases as type I diabetes, multiple sclerosis, asthma and other immunological disorders as well (Robomi et al. J Interferon Cytokine Res 2000 Feb;20(2): 187-90; Bream et al. Immunogenetics 2000 Jan;51(l):50-8; Goris et al. J Interferon Cytokine Res 1999 Sep; 19(9): 1037-46; Barnes et al.
  • CA-repeat intronic dinuclotide
  • IFNG single nucleotide polymorphisms in IFNG have been correlated with FfTV-1 (Bream et al., supra). Taken together, these data suggest that IFNG plays a potentially important role in the development of a wide variety of diseases.
  • the interferon, gamma gene is located on chromosome 12ql4 and contains 4 exons that encode a 166 amino acid protein.
  • a reference sequence for the IFNG gene is shown in the contiguous lines of Figure l(Genaissance Reference No. 2212693; SEQ ID NO: 1). Reference sequences for the coding sequence (GenBank Accession No. X13274) and protein are shown in Figures 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3), respectively.
  • polymorphic sites correspond to the following nucleotide positions in Figure 1 : 1936 (PS1), 4490 (PS2) and 4874 (PS3).
  • PS1 polymorphic sites
  • PS2 polymorphic sites
  • PS3 polymorphic sites
  • the polymorphisms at these sites are thymine or cytosine at PS1, guanine or thymine at PS2 and adenine or guanine at PS3.
  • the inventors have determined the identity of the alleles at these sites in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino.
  • each of these IFNG haplotypes constitutes a code that defines the variant nucleotides that exist in the human population at this set of polymorphic sites in the IFNG gene.
  • each IFNG haplotype also represents a na rally-occurring isoform (also referred to herein as an "isogene") of the IFNG gene.
  • the frequency of each haplotype and haplotype pair within the total reference population and within each of the four major population groups included in the reference population was also determined.
  • the invention provides a method, composition and kit for genotyping the IFNG gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PS1, PS2 and PS3 in both copies of the IFNG gene from the individual.
  • a genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel IFNG polymorphic sites.
  • a genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel IFNG polymorphic sites. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 4-below or has one of the haplotype pairs in Table 3 below.
  • the invention also provides a method for haplotyping the IFNG gene in an individual.
  • the haplotyping method comprises determining, for one copy of the IFNG gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS1, PS2 and PS3.
  • the haplotyping method comprises determining whether one copy of the individual's IFNG gene is defined by one of the IFNG haplotypes shown in Table 4, below, or a sub-haplotype thereof.
  • the haplotyping method comprises determining whether both copies of the individual's IFNG gene are defined by one of the IFNG haplotype pairs shown in Table 3 below, or a sub-haplotype pair thereof.
  • the haplotyping method can be used by the pharmaceutical research scientist to validate IFNG as a candidate target for treating a specific condition or disease predicted to be associated with IFNG activity. Determining for a particular population the frequency of one or more of the individual IFNG haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue IFNG as a target for treating the specific disease of interest.
  • variable IFNG activity is associated with the disease
  • one or more IFNG haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
  • variable IFNG activity has little, if any, involvement with that disease.
  • the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any IFNG haplotype or haplotype pair, apply the information derived from detecting IFNG haplotypes in an individual to decide whether modulating IFNG activity would be useful in treating the disease. .
  • the claimed invention is also useful in screening for compounds targeting IFNG to treat a specific condition or disease predicted to be associated with IFNG activity. For example, detecting which of the IFNG haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each of the IFNG isoforms present in the disease population, or for only the most frequent IFNG isoforms present in the disease population.
  • the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.
  • Haplotyping the IFNG gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with IFNG activity.
  • determining which of the IFNG haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute IFNG haplotypes and/or haplotype pairs evenly to treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of an IFNG haplotype or haplotype pair that is associated with response to the drug being studied in the trial, even if this association was previously unknown.
  • the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any IFNG haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and an IFNG genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein.
  • the method comprises comparing the frequency of the IFNG genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the IFNG genotype or haplotype in a reference population.
  • a higher frequency of the IFNG genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the IFNG genotype, haplotype, or haplotype pair.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • the IFNG haplotype is selected from the haplotypes shown in Table 4, or a sub-haplotype thereof.
  • Such methods have applicability in developing diagnostic tests and therapeutic treatments for type I diabetes, multiple sclerosis, asthma, and immune-related disorders.
  • the invention provides an isolated polynucleotide comprising a ' nucleotide sequence which is a polymorphic variant of a reference sequence for the IFNG gene or a fragment thereof.
  • the reference sequence comprises the contiguous sequences shown in Figure 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of cytosine at PS1, thymine at PS2 and guanine at PS3.
  • a particularly preferred polymorphic variant is an isogene of the IFNG gene.
  • An IFNG isogene of the invention comprises thymine or cytosine at PS1, guanine or thymine at PS2 and adenine or guanine at PS3.
  • the invention also provides a collection of IFNG isogenes, referred to herein as an IFNG genome anthology. Polynucleotides complementary to these EFNG genomic DNA variants are also provided by the invention. It is believed that polymorphic variants of the IFNG gene will be useful in studying the expression and function of IFNG, and in expressing IFNG protein for use in screening for candidate drugs to treat diseases related to IFNG activity.
  • the invention provides a recombinant expression vector comprising one of the polymorphic genomic DNA 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 IFNG for protein structure analysis and drug binding studies.
  • the present invention also provides nonhuman transgenic animals comprising one or more of the IFNG polymorphic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the IFNG isogenes in vivo, for in vivo screening and testing of drugs targeted against IFNG protein, and for testing the efficacy of therapeutic agents and compounds for type I diabetes, multiple sclerosis, asthma, and immune-related disorders in a biological system.
  • the present invention also provides a computer system for storing and displaying polymorphism data dete ⁇ nined for the IFNG gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymorphism data.
  • the polymorphism data includes one or more of the following: the polymorphisms, the genotypes, the haplotypes, and the haplotype pairs identified for the IFNG gene in a reference population.
  • the computer system is capable of producing a display showing IFNG haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the IFNG gene (Genaissance Reference No. 2212693; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • SEQ ID NO: 19 is a modified version of SEQ ID NO: 1 that shows the context sequence of each polymorphic site, PS1-PS3, in a uniform format to facilitate electronic searching.
  • SEQ ID NO: 19 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymorphic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein.
  • Figure 2 illustrates the coding sequence for IFNG (contiguous lines; SEQ ID NO:2).
  • Figure 3 illustrates the amino acid sequence of the IFNG protein (contiguous lines; SEQ ID NO:3).
  • the present invention is based on the discovery of novel variants of the IFNG gene.
  • the inventors herein discovered 4 isogenes of the IFNG gene by characterizing the IFNG gene found in genomic DNAs isolated from an Index Repository that contains immortalized cell lines from one chimpanzee and 93 human individuals.
  • the human individuals included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (21 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic/Latino (18 individuals). To the extent possible, the members of this reference population were organized into population subgroups by their self-identified ethnogeographic origin as shown in Table 1 below.
  • the Index Repository contains three unrelated indigenous American Indians (one from each of North, Central and South America), one three-generation Caucasian family (from the GEPH Utah cohort) and one two-generation African-American family.
  • the IFNG isogenes present in the human reference population are defined by haplotypes for 3 polymorphic sites in the IFNG gene, all of which are believed to be novel.
  • the novel IFNG polymorphic sites identified by the inventors are referred to as PS1-PS3 to designate the order in which they are located in the -gene (see Table 2 below).
  • the inventors herein also determined the pair of haplotypes for the IFNG gene present in individual human members of this repository.
  • the human genotypes and haplotypes found in the repository for the IFNG gene include those shown in Tables 3 and 4, respectively.
  • the polymorphism and haplotype data disclosed herein are useful for validating whether IFNG is a suitable target for drugs to treat type I diabetes, multiple sclerosis, asthma, and immune-related disorders, screening for such drugs and reducing bias in clinical trials of such drugs.
  • 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 polymorphic 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.
  • Full-genotype The unphased 5 ' to 3 ' sequence of nucleotide pairs found at all polymorphic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.
  • Sub-genotype The unphased 5 ' to 3 ' sequence of nucleotides seen at a subset of the polymorphic sites examined herein 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 polymorphic sites in a
  • 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 polymorphic sites examined herein 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 polymorphic sites examined herein 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 (e.g., alleles) of a gene found in a population.
  • An isogene (or allele) contains all of the polymorphisms 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, where physical features include polymorphic sites.
  • 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 polymorphic site on the two copies of a chromosome from an individual.
  • phased As applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, phased means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is known.
  • PS Polymorphic site
  • Polymorphism The sequence variation observed in an individual at a polymorphic site. Polymorphisms 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 polymorphic sites; sequence variation at those sites; frequency of polymorphisms 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 polymorphism 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.
  • Reference Population 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.
  • 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%.
  • SNP Single Nucleotide Polymorphism
  • 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 polymorphic sites in a locus, unphased means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is not known. As discussed above, information on the identity of genotypes and haplotypes for the IFNG gene of any particular individual as well as the frequency of such genotypes and haplotypes in any particular population of individuals is useful for a variety of drug discovery and development applications.
  • compositions and methods for detecting the novel IFNG polymorphisms, haplotypes and haplotype pairs identified herein comprise at least one oligonucleotide for detecting the variant nucleotide or nucleotide pair located at a novel IFNG polymorphic site in one copy or two copies of the IFNG gene.
  • oligonucleotides are referred to herein as IFNG haplotyping oligonucleotides or genotyping oligonucleotides, respectively, and collectively as IFNG oligonucleotides.
  • an IFNG haplotyping or genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, one of the novel polymorphic 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 exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
  • 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 Publishers, Inc. (1995), pages 617-620).
  • 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.
  • Haplotyping or genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of an IFNG polynucleotide.
  • the target region is located in an IFNG 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 another region in the IFNG polynucleotide or with a non-
  • oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • the skilled artisan can readily design and test oligonucleotide probes and primers suitable for detecting polymorphisms in the IFNG gene using the polymorphism information provided herein in conjunction with the known sequence information for the IFNG gene and routine techniques.
  • a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect” or "complete” complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule.
  • a 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). While perfectly complementary oligonucleotides are preferred for detecting polymorphisms, departures from complete complementarity are contemplated where such departures do not prevent the molecule from specifically hybridizing to the target region.
  • 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 probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • Preferred haplotyping or genotyping oligonucleotides of the invention are allele-specific oligonucleotides.
  • allele-specific oligonucleotide means an oligonucleotide that is able, under sufficiently stringent conditions, to hybridize specifically to one allele of a gene, or other locus, at a target region containing a polymorphic site while not hybridizing to the corresponding region in another allele(s).
  • 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 oligonucleotides of the invention include ASO probes and ASO primers.
  • ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymorphic site in the target region (e.g., approximately the 7 th or 8 th position in a 15mer, the 8 th or 9 th position in a 16mer, and the 10 th or 11 th position in a 20mer).
  • An ASO 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.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a preferred ASO probe for detecting IFNG gene polymorphisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • CTGTTGCYTGCTTTC (SEQ ID NO: 4) and its complement
  • TAATACGKTCCATTT (SEQ ID NO: 5) and its complement
  • GAAAATGRATATCTA SEQ ID NO: 6
  • a preferred ASO primer for detecting IFNG gene polymorphisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of: ATTTTCCTGTTGCYT (SEQ ID NO:7); CCATCAGAAAGCARG (SEQ ID NO:8); TTAAGGTAATACGKT (SEQ ID NO: 9); TTTAAAAAATGGAMC (SEQ ID NO: 10); TGTAATGAAAATGRA (SEQ ID NO: 11); and TAT AATAGAT TYC (SEQ ID NO: 12).
  • oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymorphic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymorphisms described herein and therefore such 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 polymorphic site.
  • a particularly preferred oligonucleotide primer for detecting IFNG gene polymorphisms by primer extension terminates in a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • TTCCTGTTGC SEQ ID NO: 13
  • TCAGAAAGCA SEQ ID NO:,14
  • AGGTAATACG SEQ ID NO: 15
  • AAAAAATGGA SEQ ID.NO:16
  • a composition contains two or more differently labeled IFNG oligonucleotides for simultaneously probing the identity of nucleotides or nucleotide pairs at two or . more polymorphic 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 polymorphic site.
  • IFNG 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 oligonucleotides may be used in a variety of polymorphism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized IFNG oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymorphisms in multiple genes at the same time.
  • the invention provides a kit comprising at least two IFNG 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.
  • the additional polymorphic sites may be currently known polymorphic sites or sites that are subsequently discovered.
  • One embodiment of a genotyping method of the invention involves isolating from the individual a nucleic acid sample comprising the two copies of the IFNG gene, mRNA transcripts * thereof or cDNA copies thereof, or a fragment of any of the foregoing, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymorphic sites selected from the group consisting of PSl, PS2 and PS3 in the two copies to assign an IFNG genotype to the individual.
  • the two "copies" of a gene, mRNA or cDNA (or fragment of such IFNG molecules) in an individual may be the same allele or may be different alleles.
  • a genotyping method of the invention comprises determining the identity of the nucleotide pair at each of PS1-PS3.
  • the nucleic acid sample is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample. Suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the nucleic acid sample may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from a tissue in which the IFNG gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymorphisms located in introns or in 5 ' and 3 ' untranslated regions if not present in the mRNA or cDNA. If an IFNG gene fragment is isolated, it must contain the polymorphic site(s) to be genotyped.
  • One embodiment of a haplotyping method of the invention comprises isolating from the individual a nucleic acid sample containing only one of the two copies of the IFNG gene, mRNA or cDNA, or a fragment of such IFNG molecules, that is present in the individual and determining in that copy the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PSl, PS2 and PS3 in that copy to assign an IFNG haplotype to the individual.
  • the nucleic acid used in the above haplotyping methods of the invention may be isolated using any method capable of separating the two copies of the IFNG gene or fragment such as one of the methods described above for preparing IFNG isogenes, with targeted in vivo cloning being the preferred approach.
  • any individual clone will typically only provide haplotype information on one of the two IFNG gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional IFNG clones will usually 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 IFNG gene in an individual.
  • the haplotype for the other allele may be inferred if the individual has a known genotype for the polymorphic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known.
  • the nucleotide at each of PS1-PS3 is identified.
  • the haplotyping method comprises determining whether an individual has one or more of the IFNG haplotypes shown in Table 4. This can be accomplished by identifying, for one or both copies of the individual's IFNG gene, the phased sequence of nucleotides present at each of PS1-PS3. This identifying step does not necessarily require that each of PS1-PS3 be directly examined. Typically only a subset of PS1-PS3 will need to be directly examined to assign to an individual one or more of the haplotypes shown in Table 4. This is because at least one polymorphic site in a gene is frequently in strong linkage disequilibrium with one or more other polymorphic sites in that gene (Drysdale, CM et al.
  • an IFNG haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymorphic sites selected from the group consisting of PSl, PS2 and PS3 in each copy of the IFNG gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS3 in each copy of the IFNG 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 polymorphic site(s), then detecting a combination of the first and third dyes would identify the polymorphism in the first gene copy while detecting a combination of the second and third dyes would identify the polymorphism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymorphic site(s) may be determined by amplifying a target region(s) containing the polymorphic site(s) directly from one or both copies of the IFNG gene, or a 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 polymorphic 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 polymorphism 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 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).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No.
  • a polymorphism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known iii 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 polymorphic 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 polymorphic 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 IFNG gene of an individual may also be determined by hybridization of. a nucleic acid sample containing one or both copies of the gene, mRNA, cDNA or fragment(s) thereof, 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 polymorphic sites to be included in the genotype or haplotype.
  • polymorphisms 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).
  • variant alleles can be identified by single strand conformation polymorphism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of.
  • SSCP single strand conformation polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymorphism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524. Related methods are disclosed in W091/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283. Extended primers containing a polymorphism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res.
  • multiple polymorphic 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. (W089/10414).
  • the identity of the allele(s) present at any of the novel polymorphic sites described herein may be indirectly determined by haplotyping or genotyping another polymorphic site that is in linkage disequilibrium with the polymorphic site that is of interest.
  • Polymorphic sites in linkage disequilibrium with the presently disclosed polymorphic sites may be located in regions of the gene or in other genomic regions not examined herein. Detection of the allele(s) present at a polymorphic site in linkage disequilibrium with the novel polymorphic 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 polymorphic site.
  • an individual's IFNG haplotype pair is predicted from its IFNG genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying a IFNG genotype for the individual at two or more IFNG polymorphic sites described herein, accessing data containing IFNG haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the genotype data.
  • the reference haplotype pairs include the IFNG haplotype pairs shown in Table 3.
  • the IFNG haplotype pair can be assigned by comparing the individual's genotype with the genotypes corresponding to the haplotype pairs known to exist in the general population or in a specific population group, and determining which haplotype pair is consistent with the genotype of the individual.
  • comparison of the genotype of the individual to the haplotype pairs identified in a reference population and determination of which haplotype pair is consistent with the genotype of the individual may be performed by visual inspection (for example, by consulting Table 3).
  • haplotype pair frequency data (such as that presented in Table 6) may be used to determine which of these haplotype pairs is most likely to be present in the individual.
  • This determination may also be performed in some embodiments by visual inspection upon consulting Table 6. If a particular IFNG haplotype pair consistent with the genotype of the individual is more frequent in the reference population than others consistent with the genotype, then that haplotype pair with the highest frequency is the most likely to be present in the individual, h other embodiments, the comparison may be made by a computer-implemented algorithm with the genotype of the individual and the reference haplotype data stored in computer-readable formats.
  • one computer-implemented algorithm to perform this comparison entails enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing IFNG haplotype pairs frequency data determined in a reference population to determine a probability that the individual has a possible haplotype pair, and analyzing the determined probabilities to assign a haplotype pair to the individual.
  • 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 haplotyping procedures.
  • 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.
  • haplotyping the individual using a direct haplotyping method such as, for example, CLASPER SystemTM technology (U.S. Patent No. 5,866,404), single molecule dilution, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).
  • CLASPER SystemTM technology U.S. Patent No. 5,866,404
  • single molecule dilution single molecule dilution
  • allele-specific long-range PCR Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996.
  • the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, 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.
  • the haplotype pair in an individual may be predicted from the individual's genotype for that gene using reported methods (e.g., Clark et al. 1990 Mol Bio Evol 7: 111-22; copending PCT/USOl/12831 filed April 18, 2001 ) or through a commercial haplotyping service such as offered by Genaissance Pharmaceuticals, Inc. (New Haven, CT).
  • a commercial haplotyping service such as offered by Genaissance Pharmaceuticals, Inc. (New Haven, CT).
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No.
  • the invention also provides a method for determining the frequency of an IFNG genotype, haplotype, or haplotype pair in a population.
  • the method comprises, for each member of the population, determining the genotype or the haplotype pair for the novel IFNG polymorphic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population.
  • the population may be e.g., a reference population, a family population, a same gender population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).
  • a trait population e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment.
  • frequency data for BFNG genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and an IFNG genotype, haplotype, or haplotype pair.
  • 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), haplotype(s), or haplotype pair(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 or more of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as 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), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared.
  • the frequencies of all genotypes, haplotypes, and or haplotype pairs observed in the populations are compared. If a particular IFNG genotype, haplotype, or haplotype pair is more frequent in the trait population than in the reference population at a statistically significant amount, then the trait is predicted to be associated with that IFNG genotype, haplotype or haplotype pair.
  • the IFNG genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the full- genotypes and full-haplotypes shown in Tables 3 and 4, 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 IFNG 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/or adverse response (i.e., side effects).
  • clinical population In order to deduce a correlation between clinical response to a treatment and an IFNG genotype, haplotype, or haplotype pair, 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.
  • the individuals included in the clinical population have been graded for the existence of the medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome.
  • This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.
  • 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 IFNG 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 IFNG genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their IFNG genotype or haplotype (or haplotype pair) (also referred to as a polymorphism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymorphism group are calculated.
  • a second method for finding correlations between IFNG 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 Then- 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.
  • 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 polymorphic sites in the IFNG gene.
  • ANOVA analysis of variation
  • 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).
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of IFNG 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 IFNG 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 polymorphic sites in the IFNG gene), a serological test, or a physical exam measurement.
  • a direct DNA test i.e., genotyping or haplotyping one or more of the polymorphic sites in the IFNG gene
  • serological test i.e., genotyping or haplotyping one or more of the polymorphic sites in the IFNG gene
  • a physical exam measurement i.e., a serological test, or a physical exam measurement.
  • this diagnostic method uses the predictive haplotyping method described above.
  • the invention provides an isolated polynucleotide comprising a polymorphic variant of the IFNG gene or a fragment of the gene which contains at least one of the novel polymorphic sites described herein.
  • the nucleotide sequence of a variant IFNG 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 polymorphic sites PS 1, PS2 and PS3.
  • the nucleotide sequence of a variant fragment of the IFNG gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymorphic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the IFNG gene, which is defined by haplotype 2, (or other reported IFNG sequences) or to portions of the reference sequence (or other reported IFNG sequences), except for the haplotyping and genotyping oligonucleotides described above.
  • the location of a polymorphism in a variant IFNG gene or fragment is preferably identified by aligning its sequence against SEQ ID NO:l.
  • the polymorphism is selected from the group consisting of cytosine at PSl, thymine at PS2 and guanine at PS3.
  • the polymorphic variant comprises a naturally-occurring isogene of the IFNG gene which is defined by any one of haplotypes 1 and 3-4 shown in Table 4 below.
  • Polymorphic variants of the invention may be prepared by isolating a clone containing the IFNG gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel polymorphic sites described herein.
  • Any particular variant or fragment thereof, that is claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
  • Any particular IFNG variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.
  • IFNG isogenes, or fragments thereof may be isolated using any method that allows separation of the two "copies" of the IFNG 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 U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • TIVC targeted in vivo cloning
  • Another method which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • the invention also provides IFNG genome anthologies, which are collections of at least two IFNG 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 gender population.
  • An IFNG genome anthology may comprise individual IFNG isogenes stored in separate containers such as microtest tabes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the IFNG isogenes in the anthology may be stored in separate containers.
  • a preferred IFNG genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 4 below.
  • An IFNG genome anthology is useful in providing control nucleic acids for kits of the invention.
  • An isolated polynucleotide containing a polymorphic 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 IFNG 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
  • Host cells which may be used to express the variant IFNG 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, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE- dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning.
  • 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, herpes 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: 1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • a polymorphic variant of an IFNG gene, mRNA or cDNA fragment comprises at least one novel polymorphism identified herein and has a length of at least 10 nucleotides and may range up to the full length of the gene.
  • such fragments are 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 IFNG gene or cDNA 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 polymorphic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymorphic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the IFNG genomic, mRNA and cDNA variants described herein.
  • Polynucleotides comprising a polymorphic gene variant or fragment of the invention may useful for therapeutic purposes.
  • an expression vector encoding the isoform may be administered to the patient.
  • the patient may be one who lacks the IFNG isogene encoding that isoform or may. already have at least one copy of that isogene.
  • IFNG IFNG isogene
  • Expression of an IFNG 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 or antisense RNA for the isogene or fragment thereof.
  • 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 IFNG mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of IFNG mRNA transcribed from a particular isogene.
  • the untranslated mRNA, antisense RNA or antisense 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. Alternatively, such molecules 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.
  • Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
  • Effect(s) of the polymorphisms identified herein on expression of IFNG may.be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymorphic variant of the IFNG 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 IFNG 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 IFNG 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.
  • 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.
  • IFNG IFNG co-precipitation
  • 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 IFNG 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 IFNG isogene.
  • Such recombinant cells can be used to compare the biological activities of the different protein variants.
  • Recombinant nonh ⁇ man organisms i.e., transgenic animals, expressing a variant IFNG 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 IFNG 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 IFNG isogene and producing the encoded human IFNG protein can be used as biological models for studying diseases related to abnormal IFNG 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.
  • An additional embodiment of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel IFNG isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel IFNG isogenes; an antisense oligonucleotide directed against one of the novel IFNG isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel IFNG isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • therapeutically effective amount is meant that one or more of the symptoms relating to disorders affected by expression or function of a novel IFNG isogene is reduced and/or eliminated.
  • the 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.
  • 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 IFNG gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymorphism 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 IFNG polymorphism 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).
  • polymorphism 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.
  • EXAMPLE 1 This example illustrates examination of various regions of the IFNG gene for polymorphic sites.
  • the following target regions of the IFNG gene were amplified using PCR primer pairs.
  • the primers used for each region are represented below by providing the nucleotide positions of their initial and final nucleotides, which correspond to positions in SEQ ID NO:l ( Figure 1).
  • the PCR products were purified using a WhatmanPolyfiltronics 100 microliter 384 well unifilter plate essentially according to the manufacturers protocol.
  • the purified DNA was eluted in 50 microliter of distilled water.
  • Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol.
  • the purified PCR products were sequenced in both directions using the primer sets described previously or those represented below by the nucleotide positions of their initial and final nucleotides, which correspond to positions in SEQ ID NO: 1 ( Figure 1). Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
  • Fragment 1 49-68 complement of 536-516 Fragment 2 146-165 . complement of 689-668
  • Fragment 5 4400-4421 complement of 4968-4946
  • This example illustrates analysis of the IFNG polymorphisms identified in the Index Repository for human genotypes and haplotypes.
  • the different genotypes containing these polymorphisms that were observed in unrelated members of the reference population are shown in Table 3 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol . described below.
  • Table 3 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 3 were inferred based on linkage disequilibrium and/or Mendelian inheritance.
  • haplotype pairs shown in Table 3 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in
  • 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. In the present analysis, the list of haplotypes was augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African-American family).
  • An IFNG isogene defined by a full-haplotype shown in Table 4 below comprises the regions of the SEQ ID NOS indicated in Table 4, with their corresponding set of polymorphic locations and identities, which are also set forth in Table 4.
  • d l st SEQ ID NO refers to Figure 1, with the two alternative allelic variants of each polymorphic site indicated by the appropriate nucleotide symbol; 2 nd SEQ ID NO is a modified version of the 1 st SEQ ID NO that comprises the context sequence of each polymorphic site, PS1-PS3, to facilitate electronic searching of the haplotypes; e Region examined represents the nucleotide positions defining the start and stop positions within the 1 st SEQ ID NO of the sequenced region.
  • SEQ ID NO: 1 refers to Figure 1, with the two alternative allelic variants of each polymorphic site indicated by the appropriate nucleotide symbol.
  • SEQ ID NO: 19 is a modified version of SEQ ID NO:l that shows the context sequence of each of PS1-PS3 in a uniform format to facilitate electronic searching of the IFNG haplotypes.
  • SEQ ID NO: 19 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymorphic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each polymorphic site is separated by genomic sequence whose composition is defined elsewhere herein.
  • the size and composition of the Index Repository were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population.
  • this repository contains approximately equal sample sizes of African-descent, Asian-American, European-American, and Hispanic-Latino population groups. Almost all individuals representing each group had all four grandparents with the same ethnogeographic background.
  • the number of unrelated individuals in the Index Repository provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population.
  • a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group.
  • the size and composition of the Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the IFNG gene are likely to be similar to the relative frequencies of these IFNG haplotypes and haplotype pairs in the general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity observed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.

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Abstract

Cette invention concerne des variants génétiques du gène Gamma de l'Interféron (IFNG) L'invention concerne divers génotypes, haplotypes et paires d'haplotypes qui existent pour le gène IFNG dans la population générale des Etats-Unis, ainsi que des compositions et des méthodes d'haplotypage et/ou de génotypage du gène IFNG chez un individu. Sont également décrits des polynucléotides définis par les haplotypes.
PCT/US2001/026678 2000-08-25 2001-08-27 Haplotypes du gene ifng WO2002016631A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007140625A1 (fr) * 2006-06-09 2007-12-13 The University Of British Columbia Polymorphismes de l'interféron gamma utilisés comme indicateurs de résultat chez des sujets gravement malades

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5521301A (en) * 1988-12-12 1996-05-28 City Of Hope Genotyping of multiple allele systems
US5851762A (en) * 1990-07-11 1998-12-22 Gene Type Ag Genomic mapping method by direct haplotyping using intron sequence analysis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5521301A (en) * 1988-12-12 1996-05-28 City Of Hope Genotyping of multiple allele systems
US5851762A (en) * 1990-07-11 1998-12-22 Gene Type Ag Genomic mapping method by direct haplotyping using intron sequence analysis

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Title
GRAY ET AL.: "Structure of the human immune interferon gene", NATURE, vol. 298, 26 August 1982 (1982-08-26), pages 859 - 863, XP002907009 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007140625A1 (fr) * 2006-06-09 2007-12-13 The University Of British Columbia Polymorphismes de l'interféron gamma utilisés comme indicateurs de résultat chez des sujets gravement malades

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