WO2001079240A2 - Haplotypes du gene rangap1 - Google Patents

Haplotypes du gene rangap1 Download PDF

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Publication number
WO2001079240A2
WO2001079240A2 PCT/US2001/012455 US0112455W WO0179240A2 WO 2001079240 A2 WO2001079240 A2 WO 2001079240A2 US 0112455 W US0112455 W US 0112455W WO 0179240 A2 WO0179240 A2 WO 0179240A2
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Prior art keywords
rangapl
gene
seq
haplotype
individual
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PCT/US2001/012455
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English (en)
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WO2001079240A3 (fr
Inventor
Anne Chew
Julie Y. Choi
Beena Koshy
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2001259082A priority Critical patent/AU2001259082A1/en
Publication of WO2001079240A2 publication Critical patent/WO2001079240A2/fr
Publication of WO2001079240A3 publication Critical patent/WO2001079240A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • 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)
    • 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
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • This invention relates to variation in genes that encode pharmaceutically-important proteins.
  • this invention provides genetic variants of the human Ran GTPase activating protein 1 (RANGAPl) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • RMGAPl Ran GTPase activating protein 1
  • 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 polymo ⁇ hisms. 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 J Respir Crit Care Med 161: 469-74) and drug response (Wolfe CR et al.
  • RANGAPl Ran GTPase activating protein 1
  • Ran is the key regulator of the GTP/GDP cycle of Ran, which is a nuclear Ras-like GTPase required for the bidirectional transport of proteins and ribonucleoproteins across the nuclear pore complex (NPC) (Matunis et al., J. Cell Biol. 1996; 135:1457-1470).
  • NPC nuclear pore complex
  • Ran is believed to exist in its active state as Ran-GTP during the initiation of S-phase which is activated by the nucleotide exchange factor, RCC1.
  • RANGAPl acts as an antagonist for RCC1 during mitosis by inducing the GTPase activity of Ran (Bischoff et al., Proc. Natl. Acad. Sci. U. S. A 1994; 91:2587-2591). Therefore, defects in the RANGAPl gene are likely to cause cancer and other disorders associated with irregular cell cycle events.
  • the Ran GTPase activating protein 1 gene is located on chromosome 22ql3.2-ql3.31 and contains 16 exons that encode a 587 amino acid protein.
  • a reference sequence for the RANGAPl gene is shown in Figure 1 (reverse complement of all or a portion of GenBank Accession No. AL035681.13; SEQ ID NO: 1).
  • Reference sequences for the coding sequence (GenBankAccession No. NM 002883.1) and protein are shown in Figures 2 (SEQ ID NO:2) and 3 (SEQ ID NO:3), respectively.
  • polymo ⁇ hic sites correspond to the following nucleotide positions in the reverse complement of the indicated GenBank Accession Number: 10157 (PSl), 10185 (PS2), 10224 (PS3), 10233 (PS4), 10235 (PS5), 10513 (PS6), 15313 (PS7), 21610 (PS8), 21644 (PS9), 21847 (PS10), 31483 (PSl l), 31563 (PS12), 31648 (PS13), 31776 (PS14), 31882 (PS15), 34822 (PS16), 34889 (PS17), 34901 (PS18), 38326 (PS19), 38497 (PS20), 38625 (PS21), 38655 (PS22), 38666 (PS23), 39681 (PS24), 39794 (PS25), 39908 (PS26), 40098 (PS27), 40182 (PS28), 40195 (
  • the polymo ⁇ hisms at these sites are cytosine or thymine at PSl, cytosine or thymine at PS2, adenine or thymine at PS3, guanine or adenine at PS4, cytosine or thymine at PS5, cytosine or adenine at PS6, adenine or guanine at PS7, cytosine or thymine at PS8, cytosine or thymine at PS9, guanine or adenine at PS 10, cytosine or adenine at PSl 1, thymine or cytosine at PS12, adenine or guanine at PS13, guanine or cytosine at PS14, cytosine or thymine at PS15, cytosine or thymine at PS16, cytosine or thymine at PS17, guanine or adenine at PS 18, thymine or cytosine at PS 19, cytosine
  • 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. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS1-PS39 in the RANGAPl gene, which are shown below in Tables 5 and 4, respectively. Each of these RANGAPl haplotypes defines a naturally-occurring isoform (also referred to herein as an "isogene") of the RANGAPl gene that exists in the human population.
  • isogene also referred to herein as an "isogene
  • the invention provides a method, composition and kit for genotyping the RANGAPl gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more polymo ⁇ hic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38 and PS39 in both copies of the RANGAPl 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 RANGAPl polymo ⁇ hic sites.
  • a genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel RANGAPl polymo ⁇ hic sites. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below.
  • the invention also provides a method for haplotyping the RANGAPl gene in an individual.
  • the haplotyping method comprises determining, for one copy of the RANGAPl gene, the identity of the nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38 and PS39.
  • the haplotyping method comprises determining whether one copy of the individual's RANGAPl gene is defined by one of the RANGAPl haplotypes shown in Table 5, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises determining whether both copies of the individual's RANGAPl gene are defined by one of the RANGAPl haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof.
  • the method for establishing the RANGAPl haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with RANGAPl activity, e.g., cancer and other disorders associated with an irregular cell cycle.
  • the haplotyping method can be used by the pharmaceutical research scientist to validate RANGAPl as a candidate target for treating a specific condition or disease predicted to be associated with RANGAPl activity. Determining for a particular population the frequency of one or more of the individual RANGAPl haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue RANGAPl as a target for treating the specific disease of interest. In particular, if variable RANGAPl activity is associated with the disease, then one or more RANGAPl haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
  • variable RANGAPl 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 RANGAPl haplotype or haplotype pair, apply the information derived from detecting RANGAPl haplotypes in an individual to decide whether modulating RANGAPl activity would be useful in treating the disease.
  • the claimed invention is also useful in screening for compounds targeting RANGAPl to treat a specific condition or disease predicted to be associated with RANGAPl activity. For example, detecting which of the RANGAPl 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 most frequent RANGAPl 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.
  • the method for haplotyping the RANGAPl 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 RANGAPl activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which of the RANGAPl haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute RANGAPl 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 a RANGAPl haplotype or haplotype pair that had a previously unknown association with response to the drug being studied in the trial. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any RANGAPl haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and a RANGAPl genotype, haplotype, or haplotype pair for one or more of the novel polymo ⁇ hic sites described herein.
  • the method comprises comparing the frequency of the RANGAPl genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the RANGAPl genotype, haplotype, or haplotype pair in a reference population. A higher frequency of the RANGAPl genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the RANGAPl 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 RANGAPl haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic treatments for cancer and other disorders associated with an irregular cell cycle.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymo ⁇ hic variant of a reference sequence for the RANGAP 1 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 thymine at PSl, thymine at PS2, thymine at PS3, adenine at PS4, thymine at PS5, adenine at PS6, guanine at PS7, thymine at PS8, thymine at PS9, adenine at PS10, adenine at PSl l, cytosine at PS12, guanine at PS13, cytosine at PS 14, thymine at PS 15, thymine at PS 16, thymine at PS 17, adenine at PS 18, cytosine at PS 19, thymine at PS20, thymine at PS21, adenine at
  • a particularly preferred polymo ⁇ hic variant is an isogene of the RANGAPl gene.
  • a RANGAPl isogene of the invention comprises cytosine or thymine at PSl, cytosine or thymine at PS2, adenine or thymine at PS3, guanine or adenine at PS4, cytosine or thymine at PS5, cytosine or adenine at PS6, adenine or guanine at PS7, cytosine or thymine at PS8, cytosine or thymine at PS9, guanine or adenine at PS 10, cytosine or adenine at PSl 1, thymine or cytosine at PS 12, adenine or guanine at PS13, guanine or cytosine at PS14, cytosine or thymine at PS15, cytosine or thymine at PS 16, cytosine or thymine at PS 17, guanine or
  • the invention provides a polynucleotide comprising a polymo ⁇ hic variant of a reference sequence for a RANGAPl cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO: 2 (Fig.2) and the polymo ⁇ hic cDNA comprises at least one polymo ⁇ hism selected from the group consisting of adenine at a position corresponding to nucleotide 164, cytosine at a position corresponding to nucleotide 397, thymine at a position corresponding to nucleotide 696, thymine at a position corresponding to nucleotide 870 and guanine at a position corresponding to nucleotide 926.
  • a particularly preferred polymo ⁇ hic cDNA variant comprises the coding sequence of a RANGAPl isogene defined by haplotypes 2-28.
  • Polynucleotides complementary to these RANGAPl genomic and cDNA variants are also provided by the invention. It is believed that polymo ⁇ hic variants of the RANGAPl -gene will be useful in studying the expression and function of RANGAPl, and in expressing RANGAPl protein for use in screening for candidate drugs to treat diseases related to RANGAPl activity.
  • 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 RANGAPl 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 RANGAPl protein.
  • the reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymo ⁇ hic variant comprises at least one variant amino acid selected from the group consisting of histidine at a position corresponding to amino acid position 55, glutamine at a position corresponding to amino acid position 133 and glycine at a position corresponding to amino acid position 309.
  • a polymo ⁇ hic variant of RANGAPl is useful in studying the effect of the variation on the biological activity of RANGAPl as well as on the binding affinity of candidate drugs targeting RANGAPl for the treatment of cancer and other disorders associated with an irregular cell cycle.
  • the present invention also provides antibodies that recognize and bind to the above polymo ⁇ hic RANGAPl protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.
  • the present invention also provides nonhuman transgenic animals comprising one of the RANGAPl polymo ⁇ hic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the RANGAPl isogenes in vivo, for in vivo screening and testing of drugs targeted against RANGAPl protein, and for testing the efficacy of therapeutic agents and compounds for cancer and other disorders associated with an irregular cell cycle in a biological system.
  • the present invention also provides a computer system for storing and displaying polymo ⁇ hism data determined for the RANGAPl 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 RANGAPl gene in a reference population.
  • the computer system is capable of producing a display showing RANGAPl haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the RANGAPl gene that is the reverse complement of all or a portion of Genbank Accession Number AL035681.13 (contiguous lines; SEQ ID NO:l), 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 polymo ⁇ hic site(s) and polymo ⁇ hism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
  • Figure 2 illustrates a reference sequence for the RANGAPl coding sequence (contiguous lines; SEQ ID NO:2) with the polymo ⁇ hic site(s) and polymo ⁇ hism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
  • Figure 3 illustrates a reference sequence for the RANGAPl protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymo ⁇ hism(s) of Figure 2 positioned below the polymo ⁇ hic site in the sequence.
  • the present invention is based on the discovery of novel variants of the RANGAPl gene.
  • the inventors herein discovered 28 isogenes of the RANGAPl gene by characterizing the RANGAPl gene found in genomic DNAs isolated from an Index Repository that contains immortalized cell lines from one chimpanzee and 93 human individuals.
  • the human v 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),
  • 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 CEPH Utah cohort) and one two-generation African- American family.
  • the RANGAPl isogenes present in the human reference population are defined by haplotypes for 39 polymo ⁇ hic sites in the RANGAPl gene, all of which are believed to be novel.
  • the novel RANGAPl polymo ⁇ hic sites identified by the inventors are referred to as PS1-PS39 to designate the order in which they are located in the gene (see Table 3 below).
  • PS1-PS39 to designate the order in which they are located in the gene (see Table 3 below).
  • the inventors herein also determined the pair of haplotypes for the RANGAPl gene present in individual human members of this repository.
  • the human genotypes and haplotypes found in the repository for the RANGAPl gene include those shown in Tables 4 and 5, respectively.
  • the polymo ⁇ hism and haplotype data disclosed herein are useful for validating whether RANGAPl is a suitable target for drugs to treat cancer and other disorders associated with an irregular cell cycle, 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 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.
  • Full-genotype The unphased 5' to 3' sequence of nucleotide pairs found at all known polymo ⁇ hic sites 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 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.
  • 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 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 invention also provides compositions and methods for detecting the novel RANGAPl polymo ⁇ hisms and haplotypes identified herein.
  • compositions comprise at least one RANGAPl genotyping oligonucleotide.
  • a RANGAPl 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.
  • the term "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.
  • 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.
  • 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 a RANGAPl polynucleotide, i.e., a RANGAPl 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-RANGAPl 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 “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.
  • 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 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 polymo ⁇ hic 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 RANGAPl gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • CTACTCT CCCGAGA SEQ ID NO 6
  • TCCAGTGRTTTATCT SEQ ID NO 10 and its complement
  • CTCATTAYAGAGAAA SEQ ID NO 11 and its complement
  • CTCTGGAYCATGAGC SEQ ID NO 12 and its complement
  • GGAGAAGRGGCTCCT (SEQ ID NO 21 and its complement
  • CAGCCCAYTCCTGTG (SEQ ID NO 22 and its complement
  • AGCAGGGRCTCCTCT (SEQ ID NO 26 and its complement
  • CTGGACCMTTGCTTC SEQ ID NO 27 and its complement
  • CAACCCARAGCTGTT (SEQ ID NO 40 and its complement
  • a preferred ASO primer for detecting RANGAPl gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • TCTGCGCGCGGTGYG SEQ ID NO:43
  • CCCGTCCCATGGCRC SEQ ID NO:44
  • AGTTCGCGGTGCGYG (SEQ ID NO:45); AGCGCGATGGGGCRC (SEQ ID NO:46);
  • ACCGGACTACTCTWC SEQ ID NO: 47
  • GCGGCCTCTCGGGWA SEQ ID NO: 48
  • CTCTACCCGAGAGRC SEQ ID NO: 49
  • CCCGCTCGCGCGGYC SEQ ID NO: 50
  • CTACCCGAGAGGCYG SEQ ID NO 51
  • GCCCCGCTCGCGCRG SEQ ID NO 52
  • GCTCGATTCCCCCMA SEQ ID NO 53
  • GGCGCCGCCGCGTKG SEQ ID NO 54
  • TTAACACTCATTAYA SEQ ID NO 57
  • AGATGATTTCTCTRT SEQ, ID NO 58
  • TCTGTACTCTGGAYC SEQ ID NO 59
  • AGACAGGCTCATGRT SEQ D NO 60
  • GGCGCCCGGTTCCRG SEQ ID NO- 107) GGGCTGGGCCTCCYG (SEQ ID NO 108
  • 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 RANGAPl gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • GCGCGCGGTG (SEQ ID NO: 121); GTCCCATGGC (SEQ ID NO:122) TCGCGGTGCG (SEQ ID NO: 123); GCGATGGGGC (SEQ ID NO:124) GGACTACTCT (SEQ ID NO 125) ; GCCTCTCGGG (SEQ ID NO 126)
  • CCCGAGAGGC SEQ ID NO 129
  • CCGCTCGCGC SEQ ID NO 130
  • TCCAGGCATA (SEQ ID NO 141) TGGGGGATGC (SEQ ID NO 142)
  • CTGGGTGTCT SEQ ID NO 145
  • GTCAGTTTCA SEQ ID NO- 1436
  • CTGGGAGAAG SEQ ID NO- 155
  • GGCAGGAGCC SEQ ID NO: 156
  • CTGGGGCTCC SEQ ID NO 163
  • CCCTGCTAAA SEQ ID NO 164
  • GCCCCACACC (SEQ ID NO 189)
  • CCTGTCCACA (SEQ ID NO: 190)
  • CTCGGGGGCC SEQ ID NO 197) and CCACGTACAA (SEQ ID NO 198)
  • 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.
  • RANGAPl 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 RANGAPl 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.
  • the above described oligonucleotide compositions and kits are useful in methods for genotyping and/or haplotyping the RANGAPl gene in an individual.
  • the terms "RANGAPl genotype” and “RANGAPl 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 RANGAPl 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 sample comprising the two copies of the RANGAPl gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymo ⁇ hic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38 and PS39 in the two copies to assign a RANGAPl genotype to the individual.
  • 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 PS1-PS39.
  • the nucleic acid sample 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 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 RANGAPl gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in introns or in 5' and 3' untranslated regions. If a RANGAPl 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 sample containing only one of the two copies of the RANGAPl 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 polymo ⁇ hic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38 and PS39 in that copy to assign a RANGAPl haplotype to the individual.
  • the nucleic acid may be isolated using any method capable of separating the two copies of the RANGAPl gene or fragment such as one of the methods described above for preparing RANGAPl isogenes, with targeted in vivo cloning being the preferred approach.
  • any individual clone will only provide haplotype information on one of the two RANGAPl gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional RANGAPl 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 RANGAPl gene in an individual.
  • the nucleotide at each of PS1-PS39 is identified.
  • the haplotyping method comprises determining whether an individual has one or more of the RANGAPl haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies of the individual's RANGAPl gene, the phased sequence of nucleotides present at each of PS1-PS39.
  • the present invention also contemplates that typically only a subset of PS1-PS39 will need to be directly examined to assign to an individual one or more of the haplotypes shown in Table 5. This is because at least one polymo ⁇ hic site in a gene is frequently in strong linkage disequilibrium with one or more other polymo ⁇ hic sites in that gene (Drysdale, CM et al.
  • a RANGAPl haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymo ⁇ hic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38 and PS39 in each copy of the RANGAPl gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS39 in each copy of the RANGAPl 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 RANGAPl 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 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 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
  • 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 RANGAPl gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene, 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 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) (WarteU 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).
  • 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 WO91/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283.
  • Extended primers containing a polymo ⁇ hism 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. 17:8392, 1989; Ruano et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995).
  • 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).
  • the identity of the allele(s) present at any of the novel polymo ⁇ hic sites described herein may be indirectly determined by genotyping another polymo ⁇ hic site that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest.
  • 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.
  • an individual's RANGAPl haplotype pair is predicted from its RANGAPl genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying a RANGAPl genotype for the individual at two or more RANGAPl polymo ⁇ hic sites described herein, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing RANGAPl haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data.
  • the reference haplotype pairs include the RANGAPl haplotype pairs shown in Table 4.
  • 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. Hartl et al., Principles of Population Genomics, Sinauer Associates (Sunderland, MA), 3 rd Ed., 1997) postulates that the frequency of finding the haplotype pair H, I H 2 is equal to
  • a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System TM 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 System TM technology U.S. Patent No. 5,866,404
  • single molecule dilution single molecule dil
  • 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) or through 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. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., supra).
  • the invention also provides a method for determining the frequency of a RANGAPl 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 RANGAPl polymo ⁇ hic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population.
  • the population may be a reference population, a family population, a same sex 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).
  • frequency data for RANGAPl 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 a RANGAPl 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 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), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared.
  • the trait is predicted to be associated with that RANGAPl genotype, haplotype, or haplotype pair.
  • the RANGAPl 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 4 and 5, 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 RANGAPl 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 a RANGAPl 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 aheady 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 RANGAPl 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 RANGAPl genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their RANGAPl 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.
  • a second method for finding correlations between RANGAPl 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.
  • 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 RANGAPl gene.
  • ANOVA analysis of variation
  • PCT/USOO/17540 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 RANGAPl 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 RANGAPl 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 RANGAPl 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 RANGAPl gene
  • a 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.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the RANGAPl 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 RANGAPl 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 PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS23, PS24, PS25, PS26, PS27, PS28, PS29, PS30, PS31, PS32, PS33, PS34, PS35, PS36, PS37, PS38 and PS39.
  • nucleotide sequence of a variant fragment of the RANGAPl 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 of the RANGAPl gene, which is defined by haplotype 1, (or other reported RANGAPl sequences) or to portions of the reference sequence (or other reported RANGAPl sequences), except for genotyping oligonucleotides as described above.
  • 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 thymine at PSl, thymine at PS2, thymine at PS3, adenine at PS4, thymine at PS5, adenine at PS6, guanine at PS7, thymine at PS8, thymine at PS9, adenine at PS 10, adenine at PSl 1, cytosine at PS 12, guanine at PS13, cytosine at PS14, thymine at PS15, thymine at PS16, thymine at PS17, adenine at PS18, cytosine at PS 19, thymine at PS20, thymine at PS21, adenine at PS22, guanine at PS23, adenine at PS24, thymine at PS25, cytosine at PS26, cytosine at PS
  • Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the RANGAPl gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel 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.
  • RANGAPl isogenes may be isolated using any method that allows separation of the two "copies" of the RANGAPl 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 RANGAP 1 genome anthologies, which are collections of RANGAPl 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.
  • a RANGAPl genome anthology may comprise individual RANGAPl 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 RANGAPl isogenes in the anthology may be stored in separate containers.
  • a preferred RANGAPl 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 RANGAPl 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 RANGAPl 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. 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: 1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • polymo ⁇ hic variants of the RANGAPl gene will produce RANGAPl mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules.
  • These mRNAs can be used for the preparation of a RANGAPl cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the RANGAPl reference coding sequence shown in Figure 2.
  • the invention also provides RANGAPl mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO: 2 (Fig.
  • a particularly preferred polymo ⁇ hic cDNA variant comprises the coding sequence of a RANGAPl isogene defined by haplotypes 2-28.
  • 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 RANGAPl 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.
  • a polymo ⁇ hic variant of a RANGAPl gene fragment comprises at least one novel polymo ⁇ hism 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 RANGAPl 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 RANGAPl 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 RANGAPl isogene encoding that isoform or may already have at least one copy of that isogene.
  • RANGAPl isogene In other situations, it may be desirable to decrease or block expression of a particular RANGAPl isogene.
  • Expression of a RANGAPl 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 RANGAPl mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of RANGAPl 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.
  • 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.
  • the invention also provides an isolated polypeptide comprising a polymo ⁇ hic variant of the reference RANGAPl amino acid sequence shown in Figure 3.
  • the location of a variant amino acid in a RANGAPl polypeptide or fragment of the invention is identified by aligning its sequence against SEQ ID NO:3 (Fig.3).
  • a RANGAPl 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 histidine at a position corresponding to amino acid position 55, glutamine at a position corresponding to amino acid position 133 and glycine at a position corresponding to amino acid position 309.
  • RANGAPl 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.
  • a RANGAPl 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 RANGAPl peptide variants, which are any fragments of a RANGAPl protein variant that contain one or more of the amino acid variations shown in Table 2.
  • a RANGAPl 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 RANGAPl peptide variants may be useful as antigens to generate antibodies specific for one of the above RANGAPl isoforms.
  • the RANGAPl peptide variants may be useful in drug screening assays.
  • a RANGAPl variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing one of the variant RANGAPl genomic and cDNA sequences as described above.
  • the RANGAPl protein variant may be isolated from a biological sample of an individual having a RANGAPl isogene which encodes the variant protein. Where the sample contains two different RANGAPl isoforms (i.e., the individual has different RANGAPl isogenes), a particular RANGAPl isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular RANGAPl isoform but does not bind to the other RANGAPl isoform.
  • the expressed or isolated RANGAPl protein may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isoform of the RANGAPl protein as discussed further below.
  • RANGAPl 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 RANGAPl gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric RANGAPl protein.
  • the non-RANGAPl 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 RANGAPl and non- RANGAPl portions so that the RANGAPl protein may be cleaved and purified away from the non- RANGAPl portion.
  • An additional embodiment of the invention relates to using a novel RANGAPl 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 RANGAPl 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 RANGAPl protein or peptide variant may be free in solution or affixed to a solid support.
  • high throughput screening of compounds for binding to a RANGAPl 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 RANGAPl protein(s) of interest and then washed. Bound RANGAPl protein(s) are then detected using methods well-known in the art.
  • a novel RANGAPl protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the RANGAPl protein.
  • a particular RANGAPl haplotype or group of RANGAPl haplotypes encodes a RANGAPl protein variant with an amino acid sequence distinct from that of RANGAPl protein isoforms encoded by other RANGAPl haplotypes
  • detection of that particular RANGAPl haplotype or group of RANGAPl haplotypes may be accomplished by detecting expression of the encoded RANGAPl protein variant using any of the methods described herein or otherwise commonly known to the skilled artisan.
  • the invention provides antibodies specific for and immunoreactive with one or more of the novel RANGAPl variant proteins described herein.
  • the antibodies may be either monoclonal or polyclonal in origin.
  • the RANGAP 1 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 RANGAPl 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.g., 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).
  • albumins e.g., human, bovine, fish, ovine
  • 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 protein isoforms described herein is administered to an individual to neutralize activity of the RANGAPl 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 protein isoforms described herein may be used to immunoprecipitate the RANGAPl protein variant from solution as well as react with RANGAPl protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
  • the antibodies will detect RANGAPl 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 RANGAPl 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 RANGAPl 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., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J.
  • 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.USA 86; 10029).
  • Effect(s) of the polymo ⁇ hisms identified herein on expression of RANGAPl may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the RANGAPl 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 RANGAPl 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 RANGAPl 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 RANGAPl isogene is introduced into a cell in such a way that it recombines with the endogenous RANGAPl gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired RANGAPl 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 RANGAPl isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NTH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the RANGAPl isogene. Such recombinant cells can be used to compare the biological activities of the different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant RANGAPl 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 RANGAPl 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 RANGAPl isogene and producing human RANGAPl protein can be used as biological models for studying diseases related to abnormal RANGAPl 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 RANGAPl isogene described herein may comprise any of the following active ingredients: a polynucleotide comprising one of these novel RANGAPl isogenes; an antisense oligonucleotide directed against one of the novel RANGAPl isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel RANGAPl 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.
  • 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 RANGAPl 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 RANGAPl 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).
  • 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.
  • EXAMPLE 1 This example illustrates examination of various regions of the RANGAPl gene for polymo ⁇ hic sites. Amplification of Target Regions
  • the following target regions of the RANGAPl 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 the reverse complement of the indicated GenBank Accession Number.
  • Amplification profile 97°C - 2 min. 1 cycle
  • the PCR products were purified using a Whatman/Polyfiltronics 100 ⁇ l 384 well unifilter plate essentially according to the manufacturers protocol.
  • the purified DNA was eluted in 50 ⁇ l 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 the reverse complement of the indicated GenBank Accession Number. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
  • a a PolyId is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc.
  • EXAMPLE 2 This example illustrates analysis of the RANGAPl polymo ⁇ hisms identified in the Index Repository for human genotypes and haplotypes.
  • the different genotypes containing these polymo ⁇ hisms that were observed in the reference population 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 were inferred based on linkage disequilibrium and/or Mendelian inheritance.
  • haplotype pairs shown in Table 4 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 mdividuals in a population sample.
  • 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 augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African- American family) and then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
  • Table 6 the number of chromosomes in unrelated individuals characterized by a given haplotype is shown, arranged by ethnic background of the subjects in the Index Repository.
  • Table 7 the number of unrelated subjects characterized by a given haplotype pair is shown, again arranged by ethnic background of the subjects in the Index Repository.
  • AF African or African-American
  • AS Asian
  • CA Caucasian
  • HL Hispanic-Latino
  • AM Native Americans.

Abstract

L'invention concerne des polymorphismes mononucléotidiques présents dans le gène humain de la protéine activatrice de la Ran GTPase 1 (RANGAP1). Par ailleurs, elle se rapporte à différents génotypes, haplotypes et paires d'haplotypes pour le gène RANGAP1 qui existent dans la population. L'invention concerne également des compositions et des méthodes destinées à soumettre le gène RANGAP1 à un haplotypage et/ou un génotypage chez un individu. Elle porte enfin sur des polynucléotides contenant l'un au moins des polymorphismes RANGAP1 susmentionnés.
PCT/US2001/012455 2000-04-17 2001-04-17 Haplotypes du gene rangap1 WO2001079240A2 (fr)

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US9487823B2 (en) 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification

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