WO2001034771A2 - METHODES ET COMPOSITIONS PERMETTANT DE DIAGNOSTIQUER ET DE TRAITER LES TROUBLES LIES AU CHROMOSOME 18q - Google Patents

METHODES ET COMPOSITIONS PERMETTANT DE DIAGNOSTIQUER ET DE TRAITER LES TROUBLES LIES AU CHROMOSOME 18q Download PDF

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WO2001034771A2
WO2001034771A2 PCT/US2000/030637 US0030637W WO0134771A2 WO 2001034771 A2 WO2001034771 A2 WO 2001034771A2 US 0030637 W US0030637 W US 0030637W WO 0134771 A2 WO0134771 A2 WO 0134771A2
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interval
gene
disorder
expression
nucleic acid
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PCT/US2000/030637
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English (en)
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WO2001034771A3 (fr
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Hong Chen
Nelson B. Freimer
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Millennium Pharmaceuticals, Inc.
The Regents Of The University Of California
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Priority to AU15875/01A priority Critical patent/AU1587501A/en
Publication of WO2001034771A2 publication Critical patent/WO2001034771A2/fr
Publication of WO2001034771A3 publication Critical patent/WO2001034771A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to nucleic acid sequences of a region of human chromosome 18q associated with neuropsychiatric disorders.
  • the sequences of the present invention can be used for diagnostic evaluation, genetic testing and/or prognosis of a neuropsychiatric disorder, and/or can be used to map human chromosome 18q.
  • the invention encompasses nucleic acids, recombinant DNA molecules, cloned genes and degenerate variants thereof, vectors containing such nucleic acids, and hosts that have been genetically engineered to express and/or contain such molecules.
  • the invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of disorders, e.g. , neuropsychiatric disorders, including, but not limited to, schizophrenia, attention deficit disorder, a schizoaffective disorder, a bipolar affective disorder or a unipolar affective disorder, related to expression of such nucleotide sequences.
  • bipolar affective disorder also known as bipolar mood disorder (BP) or manic-depressive illness, which is characterized by episodes of elevated mood (mania) and depression (Goodwin, et al, 1990, Manic Depressive Illness, Oxford University Press, New York).
  • BP-I severe bipolar affective (mood) disorder
  • SAD- M schizoaffective disorder manic type. They are characterized by at least one full episode of mania, with or without episodes of major depression (defined by lowered mood, or depression, with associated disturbances in rhythmic behaviors such as sleeping, eating, and sexual activity).
  • BP-I often co-segregates in families with more etiologically heterogeneous syndromes, such as with a unipolar affective disorder such as unipolar major depressive disorder (MDD), which is a more broadly defined phenotype (Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease, Rosenberg, et al., eds., Butterworths, New York, pp. 951-965; Mclnnes and Freimer, 1995, Curr. Opin. Genet. Develop., 5, 376-381).
  • MDD unipolar major depressive disorder
  • BP-I and SAD-M are severe mood disorders that are frequently difficult to distinguish from one another on a cross-sectional basis, follow similar clinical courses, and segregate together in family studies (Rosenthal, et al, 1980, Arch. General Psychiat. 37, 804-810; Levinson and Levitt, 1987, Am. J. Psychiat. 144, 415-426; Goodwin, et al, 1990, Manic Depressive Illness, Oxford University Press, New York).
  • methods for distinguishing neuropsychiatric disorders such as these are needed in order to effectively diagnose and treat afflicted individuals.
  • Mapping genes for common diseases believed to be caused by multiple genes, such as BAD may be complicated by the typically imprecise definition of phenotypes, by etiologic heterogeneity, and by uncertainty about the mode of genetic transmission of the disease trait. With neuropsychiatric disorders there is even greater ambiguity in distinguishing individuals who likely carry an affected genotype from those who are genetically unaffected. For example, one can define an affected phenotype for BAD by including one or more of the broad grouping of diagnostic classifications that constitute the mood disorders: BP-I, SAD-M, MDD, and bipolar affective (mood) disorder with hypomania and major depression (BP-II).
  • neuropsychiatric disorder phenotypes do not exhibit classic Mendelian recessive or dominant inheritance patterns attributable to a single genetic locus, (2) there may be incomplete penetrance, i.e., individuals who inherit a predisposing allele may not manifest disease; (3) a phenocopy phenomenon may occur, i.e., individuals who do not inherit a predisposing allele may nevertheless develop disease due to environmental or random causes; (4) genetic heterogeneity may exist, in which case mutations in any one of several genes may result in identical phenotypes.
  • the present invention encompasses the nucleotide sequence of a 116 kilobase interval of human chromosome 18q, herein termed the "18q interval", which is associated disorders, e.g., neuropsychiatric disorders, such as BAD.
  • the invention is based, in part,
  • bipolar affective disorder (BAD) in humans.
  • the methods and compositions of the invention described herein can be used to diagnose, prognosticate, and treat neuropsychiatric disorders in humans, e.g., schizophrenia, attention deficit disorders, schizoaffective disorders, bipolar ⁇ ⁇ affective disorders, and/or unipolar affective disorders.
  • the invention encompasses nucleic acids comprising the sequences of the 18q interval (i.e., nucleotides 28441-144419 of FIG. IB), its flanking regions (nucleotides 1-28440 and 144420-160271 of FIG. IB), and fragments thereof.
  • the invention also encompasses nucleic acid molecules comprising novel nucleotide sequences of the 18q
  • interval and/or its flanking regions e.g., recombinant DNA molecules, cloned genes and degenerate variants thereof, vectors containing such nucleic acids, and hosts that have been genetically engineered to express and/or contain such molecules.
  • the invention further relates to novel 18q interval gene products and to antibodies directed against such gene products, or variants or fragments thereof.
  • the invention further relates to methods for modulation of 18q interval- mediated processes and for the treatment of an 18q interval-related disorder, such as a neuropsychiatric disorder.
  • 18q interval-mediated process include processes dependent and/or responsive, either directly or indirectly, to the level of expression, gene product synthesis and/or gene product activity of a 18q interval gene or -* gene product.
  • processes can include, but are not limited to, developmental, cognitive and autonomic neural and neurological processes, such as, for example, pain, appetite, long term memory and short term memory.
  • 18q interval-related disorder refers to a disorder involving an 18q interval gene or gene product, or involving an aberrant level of 18q
  • Such neuropsychiatric disorders include disorders relating to the central nervous system (CNS) and/or peripheral nervous system (PNS) including, but not limited
  • cognitive and neurodegenerative disorders such as Alzheimer's disease, senile dementia, Huntington's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as Gilles de la Tourette's syndrome, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders that include, but are not limited to schizophrenia, schizoaffective disorder, such as schizoaffective disorder manic type (SAD-).
  • schizoaffective disorder such as schizoaffective disorder manic type
  • attention deficit disorder e.g., attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-I), bipolar affective (mood) disorder with hypomania and major depression (BP-H); or a unipolar affective disorder e.g., unipolar major depressive disorder (MDD).
  • bipolar affective disorder e.g., severe bipolar affective (mood) disorder (BP-I), bipolar affective (mood) disorder with hypomania and major depression (BP-H); or a unipolar affective disorder e.g., unipolar major depressive disorder (MDD).
  • BP-I severe bipolar affective
  • BP-H bipolar affective disorder with hypomania and major depression
  • MDD unipolar major depressive disorder
  • CNS-related and/or PNS-related disorders include, for example, those listed in the
  • DSM Mental Disorders
  • the invention provides methods for modulation of 18q interval-mediated processes and for the treatment of an 18q interval-related disorder, including the
  • such methods comprise administering a compound which modulates the expression of an 18q interval gene and/or the synthesis or activity of a 18q interval gene product.
  • the invention relates to methods for the use of an 18q interval gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against a
  • such methods can comprise modulating the level of expression or the activity of an 18q interval gene or gene product in a cell such that the 18q interval-mediated process or the disorder is treated, e.g., a symptom is ameliorated.
  • such methods can comprise supplying a nucleic acid molecule encoding an 18q interval
  • the nucleic acid molecule encoding the 18q interval gene product can encode a normal or mutant 18q interval gene product, e.g., one with increased activity or expression levels.
  • nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the
  • nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to the nucleotide sequence of SEQ ED Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC ® as Accession Number PTA- 451 (the "cDNA of ATCC ® PTA-451”) or a complement thereof.
  • the invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC ® as Accession Number PTA- 451 (the "cDNA of ATCC ® PTA-451”), or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
  • the invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, or a complement thereof.
  • the invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%o, 75%, 85%, 95%, 98, or 99%) identical to the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, or a complement thereof, wherein the protein encoded by the nucleotide sequence also exhibits at least one structural and/or functional feature of a polypeptide of the invention.
  • the invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of the cDNA of ATCC ® PTA-451, or a complement thereof under stringent conditions.
  • the invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of the cDNA of ATCC ® PTA-451, or a complement thereof under stringent conditions, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
  • the isolated nucleic acid molecules encode an extracellular, transmembrane, or cytoplasmic domain of a 18q interval polypeptide of the invention.
  • the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.
  • vectors e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention.
  • the invention provides host cells containing such a vector or a nucleic acid molecule of the invention. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide is produced.
  • Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention.
  • Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide.
  • An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo, or in vitro, according to standard techniques.
  • activities can be a direct activity, such as an association with or an enzymatic activity on a second protein or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein.
  • polypeptides of the present invention can be operably linked to a heterologous amino acid sequence to form fusion proteins.
  • the invention further features antibodies that specifically bind a polypeptide of the invention such as monoclonal or polyclonal antibodies.
  • the polypeptides of the invention or biologically active portions thereof, or antibodies of the invention can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample.
  • the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated.
  • the agent is an antibody that specifically binds to a polypeptide of the invention.
  • the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA encoding a polypeptide of the invention.
  • the present invention also provides methods to treat a subject having a disorder characterized by aberrant activity of a polypeptide of the invention or aberrant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject.
  • the modulator is a protein of the invention.
  • the modulator is a nucleic acid of the invention.
  • the modulator is a peptide, peptidomimetic, or other small organic molecule.
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis-regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post- translational modification of the invention wherein a wild-type form of the gene encodes a protein having the activity of the polypeptide of the invention.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention.
  • such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.
  • the invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound.
  • methods are provided for modulation of 18q interval-mediated processes or the treatment of 18q interval-related disorders resulting from gene mutations, and/or an abnormal level of expression or activity and disorders involving an 18q interval gene and/or gene product, wherein treatment includes the amelioration or prevention of at least one symptom of such disorders.
  • such methods can comprise supplying a mammal in need of treatment with a nucleic acid molecule encoding an unimpaired 18q gene product such that the unimpaired 18q gene product is expressed and the disorder is treated, e.g., a symptom is ameliorated.
  • such methods can comprise supplying a mammal in need of treatment with a cell comprising a nucleic acid molecule that encodes an unimpaired 18q gene product such that the cell expresses the unimpaired gene product and the disorder is treated, e.g., a symptom is ameliorated.
  • such methods comprise supplying a mammal in need of treatment with a modulatory compound, such as, for example, a small molecule, peptide, antisense nucleic acid molecule, or antibody that is capable of modulating the activity of an 18q gene or gene product.
  • the present invention is directed to methods that utilize 18q interval gene sequences, their flanking sequences, and/or 18q interval gene product sequences for the diagnostic evaluation, genetic testing and/or prognosis of an 18q interval- related disorder, such as a neuropsychiatric disorder.
  • the invention relates to methods for diagnosing 18q interval-related disorders, e.g., neuropsychiatric disorders, wherein such methods can comprise measuring 18q interval gene expression in a patient sample, or detecting an 18q interval or flanking region polymorphism or mutation that correlates with the presence or development of such a disorder, in the genome of a mammal suspected of exhibiting such a disorder.
  • the invention further provides methods for the use of 18q interval gene sequences and/or gene products as markers for mapping of the region of the long arm of human chromosome 18 spanned by chromosomal markers BAD18ct22 and BADl ⁇ cagl.
  • the invention still further relates to methods for identifying compounds capable of modulating the expression of an 18q gene and/or the synthesis or activity of an 18q interval gene product.
  • methods comprising contacting a compound with a cell that expresses a gene of the 18q interval, measuring the level of 18q interval gene expression, gene product expression or gene product activity, and comparing such level to the level of 18q interval gene expression, gene product, or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the 18q interval gene and/or the synthesis or activity of the 18q interval gene product has been identified.
  • bipolar affective disorder BP bipolar mood disorder BP-I
  • severe bipolar affective (mood) disorder BP-II bipolar affective (mood) disorder with hypomania and major depression bp
  • base pair(s) dbEST expressed sequence tag data base (National Center for Biotechnology Information)
  • allele which is used interchangeably herein with “allelic variant” refers to alternative forms of a 18q interval gene, nucleic acid or portions thereof, as well as to a polypeptide encoded by said gene, nucleic acid, or portion thereof. Nucleic acid alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene.
  • Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides.
  • An allele of a gene can also be a form of a gene containing a mutation.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention.
  • a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies.
  • the term "monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
  • Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • Complementary sequences as used herein refer to sequences which have sufficient complementarity to be able to hybridize, forming a stable duplex.
  • a “delivery complex” shall mean a targeting means (e.g., a molecule that results in higher affinity binding of a gene, protein, polypeptide or peptide to a target cell surface and/or increased cellular uptake by a target cell).
  • targeting means include: sterols (e.g., cholesterol), lipids (e.g., a cationic lipid, virosome or liposome), viruses (e.g. , adenovirus, adeno-associated virus, and retrovirus) or target cell specific binding agents (e.g., ligands recognized by target cell specific receptors).
  • Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell.
  • the complex is cleavable under appropriate conditions within the cell so that the gene, protein, polypeptide or peptide is released in a functional form. It is also possible that soluble forms of the protein also exist. Such soluble isoforms can arise through variable splicing or alternatively as a result of pro teo lysis of a membranous isoform.
  • genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity.
  • the term "DNA sequence encoding an 18q interval polypeptide" may thus refer to one or more genes within a particular individual.
  • certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
  • the term “gene” or “recombinant gene” refers to a polynucleotide or nucleic acid molecule comprising an open reading frame encoding one of the polypeptides of the present invention. In one embodiment, these terms relate to a cDNA sequence including, but not limited to, a polynucleotide or nucleic acid sequence obtained via reverse transcription of an mRNA molecule.
  • nucleic acid or polynucleotide is a nucleic acid molecule which is not genomic but is a cDNA derived from a contiguous coding region which includes, but is not limited to, reverse transcribed cDNA.
  • nucleic acid or polynucleotide refers to a nucleic acid molecule which comprises contiguous nucleotide codons.
  • nucleic acid or polynucleotide is a nucleic acid molecule which is genomic but which excludes intronic sequences.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position.
  • a degree of homology or similarity or identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences.
  • a degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences.
  • a degree of identity of nucleic acid sequences is a function of the number of identical nucleic acids at positions shared by the nucleic acid sequences.
  • a degree of homology or similarity of amino acid sequences is a function of the number of conserved amino acids at positions shared by the amino acid sequences.
  • a sequence which is "unrelated" or “non-homologous" with one of the human 18q interval sequences of the present invention typically is a sequence which shares less than 40 % identity, though preferably less than 25 % identity with one of the human 18q interval sequences of the present invention.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences is accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol 215:403-410.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. Id.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75% or more) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 2.0 X SSC at 50° C. (low stringency) or 0.2 X SSC, 0.1% SDS at 50-65°C (high stringency).
  • FIG. 1 A-B depicts the gene structure and the nucleotide sequence of the 116 kb interval of the long arm of human chromosome 18 spanned by BAD18ct22 and BADl ⁇ cagl.
  • A A diagrammatic representation of the 18q interval, showing genetic and physical markers in this interval.
  • B The 160 kB nucleotide sequence of chromosome 18q, which includes thel ⁇ q interval associated with neuropsychiatric disorders, located from position 28441- 144419.
  • BAD18ct22 and BAD18cagl markers are shown in boxes between the primers.
  • compositions and methods relating to nucleic acid sequences associated with an approximately 116 kb interval and its flanking region on the long arm of human chromosome 18q, a region associated with neuropsychiatric disorders are described herein.
  • Sections 5.1, 5.2, and 5.3 describe the 18q 116 kb region, herein called the "18q interval", including nucleic acid molecules, as well as vectors comprising these molecules, host cells engineered to contain and/or express such molecules, gene products, and antibodies that specifically recognize such gene products.
  • Sections 5.4, 5.5, and 5.6 describe various uses of these nucleic acids, polypeptides, and antibodies, as well as methods for their detection.
  • Section 5.7 describes screening assays useful for identification of compounds that modulate 18q interval gene expression or activity.
  • Sections 5.8 and 5.9 describe methods for diagnosis and prognostication of 18q interval related disorders and kits that can be used for such diagnostic methods.
  • Methods of treatment of 18q interval-related disorders using the compositions of the invention and compositions such as those identified by the methods of the invention are described in Section 5.10.
  • Section 5.11 pharmaceutical compositions for the use with the invention are described.
  • FIG. 1 A The genomic organization of the region of human chromosome 18q between the markers BAD18ct22 and BADl ⁇ cagl associated with neuropsychiatric disorders has been determined (shown schematically in FIG. 1 A), the nucleotide sequence of which, and its flanking DNA, are shown in FIG. IB.
  • 18q interval refers to the region of chromosome 18q between the markers BAD18ct22 and BAD18cagl, i.e., nucleotides 28441-144419 of FIG.1B.
  • the nucleic acid molecules of the invention include:
  • nucleic acid molecules comprising the DNA sequence of thel ⁇ q interval (nucleotides 28441-144419 of FIG. IB) and fragments thereof; and (b) a nucleic acid molecule comprising the DNA sequence of 18q interval flanking DNA (nucleotides 1-28440 and 144420-160271 of FIG. IB), and fragments thereof.
  • These nucleic acid molecules can be utilized, e.g., as part of the methods of the invention for identifying and diagnosing individuals at risk for or exhibiting an 18q-interval disorder such as a neuropsychiatric disorder, e.g., BAD, or can be used for mapping the human chromosome 18q region.
  • the nucleic acid molecules of the invention further include polynucleotides comprising the nucleotide sequences of (a)-(b) which encode a polypeptide involved in an 18q associated disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a polypeptide involved in an 18q associated disorder e.g., a neuropsychiatric disorder, such as BAD.
  • Protein-coding regions, such as an open reading frame encoding such a polypeptide can be readily determined by one of skill in the art. For example, using a mathematical algorithm, sequences (a)-(b) above can be compared to sequences present in public and private databases of expressed sequences, such as cDNAs and expressed sequence tag (ESTs), and thereby identify expressed sequences within the 18q interval.
  • expressed sequences such as cDNAs and expressed sequence tag (ESTs)
  • Such public and private databases include, but are not limited to: dbest [GenBank database of ESTs]; htgs [GenBank databases HTGS (high-throughput genomic sequences) and GSS (Genome Survey Sequences)]; patn (database for non-redundant geneseq and "patent preview”); and nuc (all GenBank nucleotide sequences except EST, HTGS, and GSS divisions; see Benson et al, 1999, Nucleic Acids Res., 1999, 1:12-17).
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al., 1990, J. Mol. Biol. 215:403-410.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res .25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997, supra).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used (see http://www.ncbi.nlm.nih.gov).
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the expressed sequences within the 18q interval can be analyzed for the presence of an open reading frame (ORF).
  • ORF open reading frame
  • Algorithms for identifying and analyzing open reading frames are well knownin the art. For example, PROTEAN ORF analyzer (DNASTAR, Inc. http://www.dnastar.com) can be used.
  • PROTEAN ORF analyzer DNASTAR, Inc. http://www.dnastar.com
  • the nucleic acid molecules of the invention also include nucleotide sequences greater than 20, 30, 40, 50, 60, 70, 80, 90,100, or more base pairs long that have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more nucleotide sequence identity to the nucleotide sequences of (a)-(b) above.
  • the nucleic acid molecules of the invention do not include nucleic acid molecules that consist solely of the nucleotide sequence dbEST sequence accession nos. AA813620, AA995246, AI017933, AA995246, AA813620, AA972889, AI028677, AA416989, AA626032, AI218484, or AI018359.
  • nucleic acid molecules of the invention further include nucleotide sequences that encode polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or higher amino acid sequence identity to the polypeptides encoded by the nucleotide sequences of (a)-(b) above.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the o molecules are identical at that position.
  • the percent identity between two sequences can be determined using 5 techniques similar to those described above, with or without allowing gaps, such as BLAST, NBLAST, PSI-BLAST, Gapped BLAST, or ALIGN as discussed above (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877; Altschul et al, 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res .25:3389-3402; Myers and Miller, 1988, CABIOS 0 4:11-17).
  • the nucleic acid molecules of the invention further include:
  • nucleic acid molecule of the invention described in (a) - (b) above, under stringent conditions, e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45 °C followed by 0 one or more washes in 0.2xSSC/0.1% SDS at about 50-65 °C, or
  • stringent conditions e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45 °C followed by 0 one or more washes in 0.2xSSC/0.1% SDS at about 50-65 °C, or
  • nucleic acid molecule that hybridizes under conditions described under (c) and (d), above is one that comprises the complement of a nucleic acid molecule that encodes a gene product of the 18q-interval.
  • nucleic acid molecules that hybridize under conditions (c) and (d), above encode gene products, e.g. , gene products functionally equivalent to a gene product encoded by nucleotide sequences of the 18q interval.
  • the nucleic acids of the invention are human.
  • functionally equivalent gene products include naturally occurring gene products present in the same or different species.
  • gene sequences in non-human species rnap to chromosome regions syntenic to the human 18q chromosome location.
  • Functionally equivalent gene products also include gene products that retain at least one of the biological activities of an 18q interval gene product, and/or which are recognized by and bind to antibodies (polyclonal or monoclonal) directed against an 18q interval gene product.
  • oligos deoxyoligonucleotides
  • Tm(°C) 81.5 + 16.6 (log [monovalent cations (molar)]) + 0.41 (% G+C) -(500/N) where N is the length of the probe.
  • hybridization is carried out at about 20-25 degrees below Tm (for DNA- DNA hybrids) or 10-15 degrees below Tm (for RNA-DNA hybrids).
  • Exemplary highly stringent conditions may refer to, e.g., washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for about 14-base oligos), 48°C (for about 17-base oligos), 55 °C (for about 20-base oligos), and 60°C (for about 23-base oligos).
  • nucleic acid molecules of the invention further comprise the complements of the nucleic acids described above.
  • Such molecules can, for example, act as antisense molecules, useful, for example, in regulation of expression of genes in the 18q interval, and/or as antisense primers in amplification reactions of nucleic acid sequences of the 18q interval.
  • Nucleic acid sequences of the invention encoding a gene product, or complements thereof, maybe used as part of ribozyme and/or triple helix sequences, also useful for gene regulation. Still further, the nucleic acid molecules of the invention may be used as components of diagnostic methods whereby, for example, the presence of a particular allele involved in an 18q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD, may be detected, or whereby the methods involve mapping the humanl ⁇ q chromosomal region spanned by chromosomal markers BAD18ct22 and BAD18cagl.
  • an 18q interval-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • Fragments of the 18q-interval nucleic acid molecules refer to nucleic acid
  • sequences that can be at least 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, or more contiguous nucleotides in length.
  • the fragments can comprise sequences that encode at least 10, 20, 30, 40, or more contiguous amino acid residues of gene products encoded by sequences of the region.
  • the 18q-interval nucleic acid molecule encodes a gene product exhibiting at least one biological activity of a corresponding 18q interval gene product, e.g., an 18q interval gene product. Fragments of the 18q nucleic acid molecules can also refer to exons or introns, and, further, can refer to portions of coding regions that encode domains of, or mature, 18q interval gene products.
  • a nucleic acid molecule of the invention preferably comprises at least one of the following nucleotide sequences of the 18q interval: 28441-29265 (SEQ ID No. 4), 29683-39587 (SEQ ID No. 5), 40284-43253 (SEQ ID No. 6), 43518-46075 (SEQ ED No. 7), 47264-52284 (SEQ ID No. 8), 52672-56935 (SEQ ED No. 9), 57032-57726 (SEQ ED No. 10), 58065-59057 (SEQ ED No. 11), 59815-60471 (SEQ ED No. 12), 60870-62451 (SEQ ED
  • nucleic acid molecules of the invention comprise at least 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500,
  • nucleotide sequences of the 116 kB interval of human chromosome 18q such sequences can be readily obtained, for example, by utilizing standard sequencing, bacterial artificial chromosome (BAC) technologies and BACs described herein.
  • BAC bacterial artificial chromosome
  • DNA sequence polymorphisms of an 18q interval nucleic acid or genomic sequences surrounding an 18q interval nucleic acid will exist within a population of individual organisms (e.g., within a human population).
  • Such polymorphisms may exist, for example, among individuals within a population due to natural allelic variation. Such polymorphisms include ones that lead to changes in amino acid sequence.
  • allelic variant refers to a nucleotide sequence which occurs at a given locus or to a gene product encoded by that nucleotide sequence. Such natural allelic variations can result in 1-5%, 5-20%, or 20-50% variance in the nucleotide sequence of a given gene.
  • An allele is one of a group of genes which occur alternatively at a given genetic locus, in this case between the markers BAD18ct22 and BADl ⁇ cagl on human chromosome 18q.
  • Genes can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals.
  • gene and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. The term can further include nucleic acid molecules comprising upstream and/or exon/intron sequences and structure.
  • allelic variants or polymorphisms any and all nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation of the gene are intended to be within the scope of the present invention.
  • Allelic variants or polymorphisms include, but are not limited to, ones that do not alter the functional activity of the gene product.
  • the isolated gene sequences disclosed herein may be labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or tissues derived from the organism of interest.
  • the hybridization conditions used should generally be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived, and can routinely be determined based on, e.g. , relative relatedness of the target and reference organisms.
  • the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • an 18q-interval gene allelic variant may be isolated from, for example, human nucleic acid, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the gene product.
  • the template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue known or suspected to express a wild type or mutant gene allele (such as, for example, brain cells, including brain cells from individuals having BAD).
  • the allelic variant is isolated from an individual who has a n 1 ⁇ q interval-mediated disorder. Such variants are described in the examples below.
  • the PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an 1 ⁇ q interval gene nucleic acid sequence.
  • the PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods.
  • the amplified fragment may be labeled and used to screen a bacteriophage cDNA library.
  • the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.
  • RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express the l ⁇ q interval gene, such as, for example, brain tissue samples obtained through biopsy or postmortem).
  • a reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed with a poly-C primer.
  • cDNA sequences upstream of the amplified fragment may easily be isolated.
  • a cDNA of an allelic, e.g., mutant, variant of the 18q interval gene may be isolated, for example, by using PCR, a technique that is well known to those of skill in the art.
  • the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an
  • the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art.
  • a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant allele, or a cDNA library can be
  • RNA from a tissue known, or suspected, to express a mutant allele constructed using RNA from a tissue known, or suspected, to express a mutant allele.
  • An unimpaired gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant allele in such libraries.
  • Clones containing the mutant gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.
  • an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant allele in an individual suspected of or known to carry such a mutant allele.
  • gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against
  • 18q interval gene product antibodies are likely to cross-react with the mutant gene product.
  • Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art. Mutations or polymorphisms within the 18q interval can further be detected using PCR amplification techniques. Primers can routinely be designed to amplify
  • primers are designed to cover the exon-intron boundaries such that, coding regions can be scanned for mutations.
  • the invention also includes nucleic acid molecules, preferably DNA molecules, that are the complements of the nucleotide sequences of the preceding paragraphs.
  • nucleic acid molecules of the invention are present as part of nucleic acid molecules comprising nucleic acid sequences that contain or encode heterologous (e.g., vector, expression vector, or fusion protein) sequences.
  • heterologous e.g., vector, expression vector, or fusion protein
  • the l ⁇ q interval gene products of the invention include polypeptides, and fragments thereof, encoded by an 18q interval nucleic acid sequence.
  • 18q interval gene products, or peptide fragments thereof can be prepared for a variety of uses.
  • gene products, or peptide fragments thereof can be used for the generation of antibodies, in diagnostic assays, or for mapping and the identification of other cellular or extracellular gene products involved in the regulation of an l ⁇ q interval-related disorder, such as a neuropsychiatric disorder, e.g., BAD.
  • l ⁇ q interval gene products can also be used as components of fusion proteins to impart an 1 ⁇ q interval protein characteristic to another protein of interest.
  • an l ⁇ q interval gene product, or fragment thereof could be used to facilitate the purification, localization, or recovery of the protein of interest, by providing an antigenic tag to the fusion protein.
  • 18q-interval gene products have uses as amino acid and protein additives to foods, soaps, shampoos, cosmetics, and the like.
  • 18q-interval gene products include those gene products encoded by the 1 ⁇ q interval gene sequences described in Section 5.1, above.
  • l q interval gene products may include proteins that represent functionally equivalent (see Section 5.1 for a definition) gene products.
  • Such an equivalent l ⁇ q-interval gene product may contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the amino acid sequence encoded by the l ⁇ q interval gene sequences described, above, in Section 5.1, but that result in a "silent" change, in that the change produces a functionally equivalent l q interval gene product.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • deletion or non- conservative alterations can be engineered to produce altered l ⁇ q interval gene products.
  • Such alterations can, for example, alter one or more of the biological functions of the l q interval gene product. Further, such alterations can be selected so as to generate 1 ⁇ q interval gene products that are better suited for expression, scale up, etc. in the host cells chosen.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
  • Peptides and/or proteins corresponding to one or more domains of an l ⁇ q interval protein as well as fusion proteins in which an 1 ⁇ q protein or a portion of an 1 ⁇ q interval protein such as a truncated l ⁇ q interval protein or peptide or a 18q interval protein domain, is fused to an unrelated protein are also within the scope of this invention.
  • Such proteins and peptides can be designed on the basis of the l ⁇ q interval nucleotide sequence disclosed in Section 5.1, above, and/or on the basis of the l ⁇ q interval amino acid sequence disclosed herein.
  • Fusion proteins include, but are not limited to, IgFc fusions which stabilize the l ⁇ q interval polypeptide and prolong half life in vivo; or fusions to any amino acid sequence that allows the fusion protein to be anchored to the cell membrane; or fusions of 18q interval protein domains to an enzyme, fluorescent protein, luminescent protein, or a flag epitope protein or peptide which provides a marker function.
  • a nucleic acid sequence encoding a signal sequence of the invention can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate.
  • the signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
  • the protein can then be readily purified from the extracellular medium by art recognized methods.
  • the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
  • the 18q interval polypeptides of the invention can further comprise posttranslational modifications, including, but not limited to glycosylations, acetylations, myristylations, and phosphorylations. If the native 18q interval protein does not have recognition motifs that allow such modifications, it would be routine for one skilled in the art to introduce into an 18q interval gene nucleotide sequences that encode motifs such as enzyme recognition signals so as to produce a modified gene product.
  • the 18q interval gene products, peptide fragments thereof and fusion proteins thereof may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the 18q interval gene polypeptides, peptides, fusion peptide and fusion polypeptides of the invention by expressing nucleic acid containing 18q
  • RNA capable of encoding 18q interval gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., -RL Press, Oxford.
  • a variety of host-expression vector systems maybe utilized to express the
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the 18q interval gene product of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria 0 (e-g- > E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing l q interval gene product coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the l ⁇ q interval gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the l ⁇ q interval gene 5 product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing l ⁇ q interval gene product coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the
  • a number of expression vectors may be advantageously selected depending upon the use intended for the 1 ⁇ q interval gene product being expressed.
  • vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, ⁇ MBO J. 2, 1791), in which the 18q intervalgene product coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced;
  • pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free
  • the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica, nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • 18q intervalgene coding sequences may be cloned individually into non- 15 essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of 1 ⁇ q intervalgene coding sequences will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • non-occluded recombinant virus i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene.
  • a number of viral -based expression systems may be utilized.
  • a 1 ⁇ q intervalgene coding sequence of interest may be ligated to an adenovirus transcription translation control
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region ⁇ l or ⁇ 3) will result in a recombinant virus that is viable and capable of expressing l q interval gene product in infected hosts, (e.g., See Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81, 3655-
  • Specific initiation signals may also be required for efficient translation of inserted 18q interval gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire l q interval gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a
  • exogenous translational control signals including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner, et al, 1987, Methods in Enzymol. 153, 516-544).
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
  • cell lines that stably express the l ⁇ q interval gene product may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines that express the 18q interval gene product.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the 18q interval gene product.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al, 1977, Cell 11, 223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 4 ⁇ : 2026), and adenine phosphoribosyltransferase (Lowy, et al, 19 ⁇ 0, Cell 22, 817) genes can be employed in tk “ , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al, 1980, Natl. Acad. Sci. USA 77, 3567; O'Hare, et al, 1981, Proc. Natl. Acad. Sci. USA 78, 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78, 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al, 1981, J. Mol. Biol. 150, 1); and hygro, which confers resistance to hygromycin (Santerre, et al, 1984, Gene 30, 147).
  • any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed.
  • a system described by Janknecht, et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al, 1991, Proc. Natl. Acad. Sci. USA 88, 8972- 8976).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino- terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+ -nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
  • an endogenous 18q interval gene within a cell, cell line, or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous 18q interval gene.
  • a heterologous DNA regulatory element for example, an endogenous l ⁇ q interval gene which is normally "transcriptionally silent", i.e., an l ⁇ q interval gene which is normally not expressed, or is expressed only at very low levels in a cell, cell line, or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell, cell line, or microorganism.
  • a transcriptionally silent, endogenous 18q interval gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous l ⁇ q interval gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described e.g., in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.
  • l ⁇ q interval gene products can also be expressed in transgenic animals.
  • mice Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate l ⁇ q interval transgenic animals.
  • transgenic refers to animals expressingl ⁇ q interval gene sequences from a different species (e.g., mice expressing human l q interval sequences), as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) l ⁇ q interval sequences or animals that have been genetically engineered to no longer express endogenous l q interval gene sequences (i.e., "knock-out” animals), and their progeny. Any technique known in the art may be used to introduce an l ⁇ q interval gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S.
  • transgenic animal clones containing an 1 ⁇ q interval transgene for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal or adult cells induced to quiescence (Campbell, et al, 1996, Nature 3 ⁇ 0, 64-66; Wilmut, et al, Nature 3 ⁇ 5, 810-813).
  • the present invention provides for transgenic animals that carry a l ⁇ q interval transgene in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., mosaic animals.
  • the transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et ⁇ l (Lasko et al, 1992, Proc. Natl. Acad. Sci. USA ⁇ 9, 6232-6236).
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous 18q interval gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous 18q interval gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous 18q interval gene in only that cell type, by following, for example, the teaching of Gu, et al. (Gu, et al, 1994, Science 265, 103-106).
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • the phenotypic expression of the recombinant 1 ⁇ q interval gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques that include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR (reverse transcriptase PCR). Samples ofl 8q interval gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for thel ⁇ q interval transgene product.
  • l ⁇ q interval gene products can be prepared for a variety of uses.
  • gene products, or peptide fragments thereof can be used for the generation of antibodies, in diagnostic assays, or for mapping and the identification of other cellular or extracellular gene products involved in the regulation of a l ⁇ q interval-related disorder, such as a neuropsychiatric disorder, e.g., BAD.
  • Suchl q interval gene products include but are not limited to soluble derivatives such as peptides or polypeptides corresponding to one or more domains of the 18q interval gene product, particularlyl 8q interval gene products, that are modified such that they are deleted for one or more hydrophobic domains.
  • antibodies to thel ⁇ q interval protein or anti- idiotypic antibodies that mimic thel ⁇ q interval gene product can be used to treat 1 ⁇ q interval-related disorders, such as neuropsychiatric disorders.
  • nucleotide constructs encoding suchl ⁇ q interval gene products can be used to genetically engineer host cells to express suchl ⁇ q interval gene products in vivo; these genetically engineered cells can function as "bioreactors" in the body delivering a continuous supply of 18q interval gene product, peptides, or soluble polypeptides.
  • ANTIBODIES TO 18q INTERVAL GENE PRODUCTS Described herein are methods for the production of antibodies capable of specifically recognizing one or more 1 ⁇ q interval gene product epitopes or epitopes of conserved variants or peptide fragments of the gene products.
  • Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • mAbs monoclonal antibodies
  • Such antibodies may be used, for example, in the detection of a l ⁇ q interval gene product in an biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal levels of l ⁇ q interval gene products, and/or for the presence of abnormal forms of such gene products.
  • Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described below, in Section 5.7, for the evaluation of the effect of test compounds onl8q interval gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described below, in Section 5.8.2, to, for example, evaluate the normal and/or engineered 18q interval-expressing cells prior to their introduction into the patient.
  • Anti-1 ⁇ q interval gene product antibodies may additionally be used as a method for the inhibition of abnormal l ⁇ q interval gene product activity.
  • a l ⁇ q interval-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • various host animals may be immunized by injection with al ⁇ q interval gene product, or a portion thereof.
  • Such host animals may include, but are not limited to rabbits, mice, and rats, to name but a few.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as al ⁇ q interval gene product, or an antigenic functional derivative thereof.
  • an antigen such as al ⁇ q interval gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, may be immunized by injection withl ⁇ q interval gene product supplemented with adjuvants as also described above.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen.
  • Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against a polypeptide or polypeptides of the invention.
  • Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or polypeptides of the invention.
  • Particularly preferred immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention. In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides of the invention.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibodies specific for a protein or polypeptide of the invention can be selected for (e.g. , partially purified) or purified by, e.g., affinity chromatography.
  • a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column.
  • the column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies.
  • a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired protein or polypeptide of the invention, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies.
  • a purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256, 495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al, 1983, Immunology Today 4, 72; Cole et al, 1983, Proc. Natl. Acad. Sci. USA 80, 2026-2030), and the EBV-hybridoma technique (Cole et al, 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. In addition, techniques developed for the production of "chimeric antibodies"
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Patent No. 4, ⁇ 16,567; and Boss et al., U.S. Patent No. 4,816397, which are incorporated herein by reference in their entirety.)
  • An immunoglobuin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest", Kabat, E. et al., U.S.Department of Health and Human Services (1983).
  • humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries maybe constructed (Huse, et al, 1989, Science, 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. 5.4. USES OF NUCLEIC ACIDS OF THE INVENTION, 18q-INTERVAL GENE PRODUCTS. AND ANTIBODIES
  • nucleic acids of the invention including 1 ⁇ q interval nucleic acids, 18q interval gene products, including peptide fragments and fusion proteins thereof, and of antibodies directed against 18q interval gene products and peptide fragments thereof.
  • Such applications include, for example, prognostic and diagnostic evaluation of a l ⁇ q interval-related disorder, such as a neuropsychiatric disorder, e.g., BAD, and the identification of subjects with a predisposition to such disorders, as described, below, in Sections 5.5 and 5. ⁇ .
  • such applications include methods for the identification of compounds that modulate the expression of al ⁇ q interval gene and/or the synthesis or activity of a 1 ⁇ q interval gene product, as described below, in Section 5.7, and for the treatment of a l ⁇ q interval-related disorder, e.g. a neuropsychiatric disorder, such as BAD, as described, below, in Section 5. ⁇ .
  • a l ⁇ q interval-related disorder e.g. a neuropsychiatric disorder, such as BAD, as described, below, in Section 5. ⁇ .
  • nucleic acid sequences of the invention including l ⁇ q 5 interval nucleic acid sequences and gene products, including peptide fragments and fusion proteins thereof, and antibodies directed against 1 ⁇ q interval gene products and peptide fragments thereof, have applications for purposes independent of the role l ⁇ q interval may have in neuropsychiatric disorders and processes.
  • 1 ⁇ q interval gene products, including peptide fragments, as well as antibodies, can be used for construction of fusion 0 proteins to facilitate recovery, detection, or localization of another protein of interest.
  • nucleic acid molecules of the invention including l ⁇ q interval nucleic acid sequences, and l ⁇ q interval gene products, can be used for genetic mapping, i.e., refining the genetic map of chromosome l ⁇ q.
  • nucleic acids of the invention including l ⁇ q interval nucleic acids, and 1 ⁇ q interval gene products have generic uses, such as 5 supplemental sources of nucleic acids, proteins and amino acids for food additives or cosmetic products.
  • Nucleic acids of the invention including l ⁇ q interval nucleic acids, and l ⁇ q interval gene products can be used for mapping and refining the map of chromosome l ⁇ .
  • the sequence of the 116 kb region of the human chromosome 18q can used to develop new 0 genetic markers that can be used to further refme the interval of 18q that is associated with neuropsychiatric disorders.
  • nucleic acid sequences within a genetic interval such as an interval associated with a disease can be scanned for new markers, such as microsatellites.
  • Microsatellites also known as simple- sequence repeats (SSRs) are hypervariable tandem-sequence repeats consisting of di-, tri-, 5 or tetranucleotide repeats of 1-5 nucleotides. Such microsatellites make excellent genetic markers for linkage studies since they are distributed ubiquitously throughout the human genome, are highly variable in repeat length, and tend to be highly polymorphic. Relatively common microsatellites (e.g., (CA) n dinucleotide repeats) occur approximately every 300- 500 kb.
  • CA n dinucleotide repeats
  • the region can be scanned for other types of polymorphic sites useful for fine mapping, such as minisatellites (9-64 nucleotide repeats), restriction fragment length polymorphisms (RFLPs), and single nucleotide polymorphisms, which occur much less frequently.
  • minisatellites 9-64 nucleotide repeats
  • RFLPs restriction fragment length polymorphisms
  • single nucleotide polymorphisms which occur much less frequently.
  • Genomic DNA from human populations can then be analyzed for the such simple-sequence length polymorphisms (SSLPs) to determine the frequency and variability of the repeat.
  • SSLP simple-sequence length polymorphisms
  • the interval can be refined by linkage analysis an affected population to determine whether an l ⁇ q-related disorder, such as a neuropsychological disorder, e.g. , BAD, is linked to the new marker.
  • Other techniques such as Southern blot hybridization and ligase-chain reaction (LCR), can be used in addition to, or in conjunction, with, PCR-based methods to analyze polymorphisms in genomic populations (Current Protocols in Human Genetics, Dracopoli et al. (eds.) John Wiley & Sons, 1998; see also Section 5.5.2 for details of methods for chromosome mapping).
  • a 1 ⁇ q interval gene, protein or a fragment or domain thereof can be used for construction of fusion proteins.
  • nucleic acid sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) screen for 18q interval gene-specific mutations or polymorphisms, (ii) locate and/or further narrow chromosomal regions associated with a neuropsychiatric disorder; (iii) identify an individual from a minute biological sample (tissue typing); and (iv) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • a variety of methods can be employed to screen for the presence of l ⁇ q interval gene-specific mutations or polymorphisms (including polymorphisms flanking a l ⁇ q interval gene, e.g., ones that cosegregate with a particular 18q interval allele) and to detect and/or assay levels of l ⁇ q interval nucleic acid sequences. Mutations or polymorphisms within or flanking the l ⁇ q interval gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell, or any cell that expresses the l ⁇ q interval gene of interest, can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to those of skill in the art.
  • 1 ⁇ q interval nucleic acid sequences may be used in hybridization or amplification assays of biological samples to detect abnormalities involving l ⁇ q interval gene structure, including point mutations, insertions, deletions, inversions, translocations and chromosomal rearrangements.
  • assays may include, but are not limited to, Southern analyses, single-stranded conformational polymorphism analyses (SSCP), and PCR analyses.
  • Diagnostic methods for the detection of l ⁇ q interval gene-specific mutations or polymorphisms can involve for example, contacting and incubating nucleic acids obtained from a sample, e.g., derived from a patient sample or other appropriate cellular source with one or more labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, such as described in Section 5.1, above, under conditions favorable for the specific annealing of these reagents to their complementary sequences within or flanking the 1 ⁇ q interval gene.
  • a sample e.g., derived from a patient sample or other appropriate cellular source
  • labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, such as described in Section 5.1, above
  • patient sample biological sample or appropriate cellular source refers to a sample of tissue or fluid suspected of containing a mutated or non-mutated 18q interval polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal, and genito-urinary tracts, stool, urine, sputum, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.
  • a sample of tissue or fluid suspected of containing a mutated or non-mutated 18q interval polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal,
  • the biological sample to be analyzed such as blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal, and genito-urinary tracts, stool, urine, sputum, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents, may be treated, if desired, to extract the nucleic acids.
  • the sample nucleic acid may then be prepared in various ways to facilitate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting.
  • the targeted region of the l ⁇ q interval nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art.
  • the diagnostic methods of the present invention further encompass contacting and incubating nucleic acids for the detection of single nucleotide mutations or polymorphisms of the l ⁇ q interval gene.
  • these nucleic acid reagent sequences within the l ⁇ q interval gene, or chromosome 18q nucleotide sequences flanking the 18q interval gene are 15 to 30 nucleotides in length.
  • nucleic acid from the cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using standard techniques well-known to those in the art.
  • sequences e.g., l ⁇ q interval gene sequences, to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a corresponding normal sequence, e.g. , normal 18q interval gene sequence, in order to determine whether a l ⁇ q interval gene mutation or a cosegregating polymorphism of interest is present.
  • a corresponding normal sequence e.g. , normal 18q interval gene sequence
  • 18q interval mutations or polymorphisms can be detected by using a microassay of l ⁇ q interval nucleic acid sequences immobilized to a substrate or "gene chip" (see, e.g. Cronin, et al., 1996, Human Mutation 7:244-255).
  • Alternative diagnostic methods for the detection of 1 ⁇ q interval gene-specific nucleic acid molecules (or l ⁇ q interval gene flanking sequences, e.g., sequences present in the nucleotide sequence shown in FIG. IB), in patient samples or other appropriate cell sources may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), followed by the analysis of the amplified molecules using techniques well known to those of skill in the art, such as, for example, those listed above.
  • the resulting amplified sequences can be compared to those that would be expected if the nucleic acid being amplified contained only normal copies of the l ⁇ q interval gene in order to determine whether a l ⁇ q interval gene mutation or polymorphism in linkage disequilibrium with a disease-causing allele exists.
  • genotyping techniques can be performed to identify individuals carrying l ⁇ q interval gene mutations. Such techniques include, for example, the use of restriction fragment length polymorphisms (RFLPs), which involve sequence variations in one of the recognition sites for the specific restriction enzyme used.
  • RFLPs restriction fragment length polymorphisms
  • Caskey et al. (U.S. Pat.No. 5,364,759) describe a DNA profiling assay for detecting short tri and tetra nucleotide repeat sequences.
  • the process includes extracting the DNA of interest, such as the l ⁇ q interval gene, amplifying the extracted DNA, and labeling the repeat sequences to form a genotypic map of the individual's DNA.
  • SNPs single nucleotide polymorphisms
  • biallelic SNPs or biallelic markers which have two alleles, both of which are present at a fairly high frequency in a population.
  • Conventional techniques for detecting SNPs include, e.g., conventional dot blot analysis, single stranded conformational polymorphism (SSCP) analysis (see, e.g., Orita et al, 1989, Proc. Natl. Acad. Sci.
  • SSCP single stranded conformational polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • heteroduplex analysis mismatch cleavage detection
  • other routine techniques well known in the art (see, e.g., Sheffield et al, 1989, Proc. Natl. Acad. Sci. 86:5855-5892; Grompe, 1993, Nature Genetics 5:111-117).
  • preferred methods of detecting and mapping SNPs involve microsequencing techniques wherein an SNP site in a target DNA is detecting by a single nucleotide primer extension reaction (see, e.g., Goelet et al, PCT Publication No. WO92/15712; Mundy, U.S. Patent No.
  • the level of 18q interval gene expression can also be assayed.
  • RNA from a cell type or tissue known, or suspected, to express the 1 ⁇ q interval gene, such as brain may be isolated and tested utilizing hybridization or PCR techniques such as are described, above.
  • the isolated cells can be derived from cell culture or from a patient.
  • the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the l ⁇ q interval gene.
  • Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the l ⁇ q interval gene, including activation or inactivation of 18q interval gene expression.
  • a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA).
  • a sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like.
  • the nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the nucleic acid reagents described in Section 5.1 that contain a l ⁇ q interval gene or nucleic acid sequence.
  • nucleic acid reagents are at least 9-30 nucleotides.
  • the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides.
  • enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
  • Nucleic acid reagents described in Section 5.1 that contain a l ⁇ q interval gene or nucleic acid sequence may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, “PCR i Situ Hybridization: Protocols And Applications", Raven Press, NY).
  • nucleic acid molecules described herein or fragments thereof can be used to map the location of the corresponding genes on a chromosome.
  • nucleic acid molecules described herein can be used to map the chromosomal location of 18q interval homologues in various species. Such mapping information can be used, for example, for analysis of the activity of 18q interval transgenes in mice.
  • the nucleic acid molecules can further be used to map the location of copies of l ⁇ q interval genes in the human chromosome, such as those caused by genetic abnormalties, e.g., translocations.
  • genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the sequence of a gene of the invention.
  • Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
  • These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gene sequences will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al., 1990, Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes (CITE), and pre-selection by hybridization to chromosome specific cDNA libraries.
  • in situ hybridization described in Fan et al., 1990, Proc. Natl. Acad. Sci. USA 87:6223-27
  • CITE labeled flow-sorted chromosomes
  • Fluorescence in situ hybridization of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • FISH Fluorescence in situ hybridization
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • a 1 ⁇ q interval polypeptide and fragments and sequences thereof and antibodies specific thereto can be used to map the location of the gene encoding the polypeptide on a chromosome.
  • This mapping can be carried out by specifically detecting the presence of the polypeptide in members of a panel of somatic cell hybrids between cells of a first species of animal from which the protein originates and cells from a second species of animal and then determining which somatic cell hybrid(s) expresses the polypeptide and noting the chromosome(s) from the first species of animal that it contains.
  • somatic cell hybrid(s) expresses the polypeptide and noting the chromosome(s) from the first species of animal that it contains.
  • the presence of the polypeptide in the somatic cell hybrids can be determined by assaying an activity or property of the polypeptide, for example, enzymatic activity, as described in Bordelon-Riser et al. (1979J Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978 . ) Proc. Natl. Acad. Sci. USA 75:5640-5644.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the nucleic acid sequences of the invention uniquely represent portions of the 5 human genome.
  • allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. 0 Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification 5 database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology.
  • forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene.
  • the amplified sequence can then be 5 compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e., another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e., another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
  • the 1 ⁇ q interval nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining 18q interval-encoded protein and/or nucleic acid expression as well as 18q interval protein activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted 18q interval gene expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with an l ⁇ q interval gene protein, nucleic acid expression or activity. For example, mutations in an l ⁇ q interval gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with an 1 ⁇ q interval protein, nucleic acid expression or activity. As an alternative to making determinations based on the absolute expression level of selected genes, determinations may be based on the normalized expression levels of these genes.
  • Expression levels are normalized by correcting the absolute expression level of an 18q interval gene by comparing its expression to the expression of a gene that is not an 18q interval gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-BAD-affected normal sample, or between samples from different sources. Alternatively, the expression level can be provided as a relative expression level.
  • the level of expression of the gene is determined for 10 or more samples of different cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question.
  • the cell isolates are selected depending upon the tissues in which the gene of interest is expressed. For example, for 1 ⁇ q interval family members, expression was observed in the brain.
  • the mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question.
  • the expression level of the gene determined for the test sample absolute level of expression
  • diseases which may be studied include, without limitation, those associated with tissues of the brain.
  • the samples used in the baseline determination will be from an l ⁇ q interval-mediated diseased or from non-diseased cells of tissue.
  • the choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the 1 ⁇ q interval gene assayed is cell-type specific for the tissues in which expression is observed versus the expression found in normal cells. Such a use is particularly important in identifying whether an l ⁇ q interval gene can serve as a target gene.
  • Expression data from brain cells provides a means for grading the severity of the l q interval-mediated disease state.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of l ⁇ q interval gene products in clinical trials.
  • Antibodies directed against unimpaired or mutant 1 ⁇ q interval gene products or conserved variants or peptide fragments thereof, which are discussed, above, in Section 5.3, may also be used as diagnostics and prognostics for a l ⁇ q interval-related disorder, e.g., neuropsychiatric disorder, such as BAD, as described herein. Such methods may be used to detect abnormalities in the level of l ⁇ q interval gene product synthesis or expression, or abnormalities in the structure, temporal expression, and/or physical location of 1 ⁇ q interval gene product.
  • a l ⁇ q interval-related disorder e.g., neuropsychiatric disorder, such as BAD, as described herein.
  • Such methods may be used to detect abnormalities in the level of l ⁇ q interval gene product synthesis or expression, or abnormalities in the structure, temporal expression, and/or physical location of 1 ⁇ q interval gene product.
  • the antibodies and immunoassay methods described below have, for example, important in vitro applications in purifying 18q interval gene products and in assessing the efficacy of treatments for 18q interval-related disorders, e.g., neuropsychiatric disorders, such as BAD.
  • 18q interval-related disorders e.g., neuropsychiatric disorders, such as BAD.
  • Antibodies, or fragments of antibodies, such as those described below may be used to screen potentially therapeutic compounds in vitro to determine their effects on l ⁇ q interval gene expression and 18q interval peptide production.
  • the compounds that have beneficial effects on a l ⁇ q interval-related disorder e.g., a neuropsychiatric disorder, such as BAD, can be identified, and a therapeutically effective dose determined.
  • In vitro immunoassays may also be used, for example, to assess the efficacy of cell-based gene therapy for a l ⁇ q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • Antibodies directed against 18q interval peptides may be used in vitro to determine, for example, the level of 18q interval gene expression achieved in cells genetically engineered to produce l q interval peptides.
  • intracellular 18q interval gene products such an assessment is done, preferably, using cell lysates or extracts. Such analysis allows for a determination of the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.
  • the tissue or cell type to be analyzed will generally include those that are known, or suspected, to express the l ⁇ q interval gene.
  • the protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • the isolated cells can be derived from cell culture or from a patient.
  • the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the 18q interval gene.
  • Preferred diagnostic methods for the detection of l ⁇ q interval gene products or conserved variants or peptide fragments thereof may involve, for example, immunoassays wherein the 1 ⁇ q interval gene products or conserved variants or peptide fragments are detected by their interaction with an anti-18q interval gene product-specific antibody.
  • antibodies, or fragments of antibodies, such as those described, above, in Section 5.3, useful in the present invention may be used to quantitatively or qualitatively detect the presence of 18q interval gene products or conserved variants or peptide fragments thereof.
  • This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below, this section) coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • Such techniques are especially preferred for 1 ⁇ q interval gene products that are expressed on the cell surface.
  • the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of 18q interval gene products or conserved variants or peptide fragments thereof.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays for 1 ⁇ q interval gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells, that have been incubated in cell culture, in the presence of a detectably labeled antibody capable of identifying l ⁇ q interval gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
  • the term "patient sample”, “sample”, “biological sample” or “appropriate cellular source” refers to a sample of tissue or fluid suspected of containing a mutated or non-mutated l ⁇ q interval polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal, and genito-urinary tracts, stool, urine, sputum, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.
  • blood can be drawn and the blood sample incubated in the presence of a detectably labeled antibody capable of identifying l ⁇ q interval gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
  • the range of thel8q o interval gene products, or conserved variants or peptide fragments thereof, which may be detected in the blood or any one of the patient samples listed supra using a detectably labeled antibody capable of identifying 18q interval gene products or conserved variants or peptide fragments thereof is from about 1 ng/ml to about 100 ng/ml. More preferred ranges for detection of 1 ⁇ q interval gene products or conserved variants or peptide fragments 5 thereof are about 10 ng/ml to about 90 ng/ml, about 20 ng/ml to about ⁇ 0 ng/ml, about 25 ng/ml to about 70 ng/ml, and about 30 ng/ml to about 60 ng/ml. The most preferred range for the detection of 1 ⁇ q interval gene products or conserved variants or peptide fragments thereof is about 35 ng/ml to about 40 ng/ml.
  • the biological sample may be brought in contact with and immobilized onto 0 solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled l ⁇ q interval gene specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on solid support may then be 5 detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to 0 some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • binding activity of a given lot of l ⁇ q interval gene product antibody may be determined according to well known methods. Those skilled in the art will be able to
  • EIA enzyme immunoassay
  • enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycerophosphate, dehydrogenase, triose phosphate isomerase, 0 horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, ⁇ - galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • RLA radioimmunoassay
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • an antibody can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti -mitotic agents (e.g
  • the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, .alpha. -interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example,
  • lymphokines interleukin-1 ("EL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • EL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophase colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the invention provides substantially purified antibodies or fragment thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid
  • the substantially purified antibodies of the invention, or fragments thereof can be human, non-human, chimeric and/or humanized antibodies.
  • the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID No. 1, 2, 3, 4, 5, 6, 7, ⁇ , 9, 10, 11, 12, 13, 14, 15, 16, 17, l ⁇ , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
  • an amino acid sequence encoded by the cDNA of ATCC ® PTA-451 or an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the cDNA of ATCC ® PTA-451, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C.
  • non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
  • the non-human antibodies of the invention can be chimeric and/or humanized antibodies.
  • the non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.
  • the invention provides monoclonal antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide
  • amino acid sequence selected from the group consisting of: an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ED No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or an amino acid sequence encoded by the cDNA of ATCC ® PTA-
  • the monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.
  • substantially purified antibodies or fragments thereof specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention.
  • the substantially purified antibodies or fragments thereof, the human or non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention.
  • the substantially purified antibodies or fragments thereof, the human or non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention.
  • nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ED No. 1, 2, 3, 4, 5, 6, 7, ⁇ , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, the cDNA of ATCC ® PTA 451 or an amino acid
  • Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance.
  • detectable substances that
  • antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.
  • the invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention
  • the 10 invention is a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
  • Still another aspect of the invention is a method of making an antibody that
  • the method comprising immunizing a mammal with a polypeptide.
  • the polypeptide used as an immungen comprises an amino acid sequence selected from the group consisting of: an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ED No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • a sample is collected from the mammal that contains an antibody that specifically recognizes an 18q interval polypeptide, or portions thereof.
  • the polypeptide is recombinantly produced using a non-human host cell.
  • the antibodies can be further purified from the sample using techniques well known to those of skill in the art.
  • the method can further comprise producing a
  • the following assays are designed to identify compounds that bind to an l ⁇ q interval gene product, e.g., proteins or portions of proteins that interact with an l ⁇ q interval gene product, compounds that interfere with the interaction of an l ⁇ q interval gene product with other proteins and compounds that modulate the activity of l ⁇ q interval gene (i.e., modulate the level of 1 ⁇ q interval gene expression and/or modulate the level of 1 ⁇ q interval gene product activity).
  • Assays may additionally be utilized that identify compounds that bind to l q interval gene regulatory sequences (e.g., promoter sequences; see e.g., Platt, 1994, J. Biol. Chem.
  • Compounds may include, but are not limited to, small organic molecules, such as ones that are able to cross the blood-brain barrier, gain entry into an appropriate cell and affect expression of the 18q interval gene or some other gene or gene product involved in a l ⁇ q interval regulatory pathway.
  • Such intracellular proteins may be involved in the control and/or regulation of mood.
  • compounds that affect the level of 1 ⁇ q interval gene expression and/or 1 ⁇ q interval gene product activity and that can be used in the therapeutic treatment of l ⁇ q interval disorders e.g., neuropsychiatric disorders such as BAD, as described, below, in Section 5.10.
  • Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to, Ig-tailed fusion peptides, and members of random peptide libraries; (see, e.g., Lam, et al, 1991, Nature 354, 82-84; Houghten, et al, 1991, Nature 354, 84-86), and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al, 1993, Cell 72, 767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorgan
  • Such compounds may also comprise compounds, in particular drugs or members of classes or families of drugs, known to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder such as BAD.
  • antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD), p- chlorophenylalanine, -propyldopacetamide dithiocarbamate derivatives e.g., FLA 63
  • anti- anxiety drugs e.g., diazepam
  • monoamine oxidase (MAO) inhibitors e.g., iproniazid, clorgyline, phenelzine and isocarboxazid
  • biogenic amine uptake blockers e.g., tricyclic antidepressants such as desipramine, imipramine and amitriptyline
  • serotonin reuptake inhibitors e.g., fluoxetine
  • Such compounds are utilized in a manner (e.g., different dosage, mode of administration, and/or co-administration with one or more additional compounds) that differs from the manner in which such compounds have been administered previously.
  • Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the l ⁇ q interval gene product, and for ameliorating l ⁇ q interval-related disorders, e.g., neuropsychiatric disorders such as BAD.
  • compounds identified via such techniques can provide lead compounds to be tested for an ability to modulate a 18q interval-mediated process and/or to ameliorate symptoms of a l ⁇ q interval-related disorder.
  • Assays for testing the effectiveness of compounds identified by, for example, techniques such as those described in Sections 5.7.1 - 5.7.4, are discussed, below, in Section 5.7.5.
  • In vitro systems may be designed to identify compounds that bind 1 ⁇ q interval gene products of the invention.
  • Compounds identified may be useful, for example, in modulating the activity of unimpaired and/or mutant l ⁇ q interval gene products, may be useful in elucidating the biological function of the 1 ⁇ q interval gene product, may be utilized in screens for identifying compounds that disrupt normal 1 ⁇ q interval gene product interactions, or may in themselves disrupt such interactions, and can provide lead compounds to be further tested for an ability to modulate an 1 ⁇ q interval-mediated process and/or to ameliorate symptoms of a 1 ⁇ q interval-related disorder.
  • the principle of the assays used to identify compounds that bind to l ⁇ q interval gene products involves preparing a reaction mixture of the l q interval gene product and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture.
  • These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring 1 ⁇ q interval gene product or the test substance onto a solid phase and detecting 1 ⁇ q interval gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the 1 ⁇ q interval gene product may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.
  • microtiter plates may conveniently be utilized as the solid phase.
  • the anchored component may be immobilized by non-covalent or covalent attachments.
  • Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface.
  • the surfaces may be prepared in advance and stored.
  • the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non-immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for 1 ⁇ q interval gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
  • Any method suitable for detecting protein-protein interactions may be employed for identifying 18q interval protein-protein interactions.
  • traditional methods that may be employed are co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns. Utilizing procedures such as these allows for the identification of proteins that interact with 18q interval gene products. Once isolated, such a protein can be identified and can be used in conjunction with standard techniques, to identify proteins with which it interacts.
  • amino acid sequence of a protein that interacts with the 18q interval gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles," W.H. Freeman & Co., N.Y., pp.34-49).
  • the amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such proteins. Screening made be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra, and 1990, "PCR Protocols: A Guide to Methods and Applications,” Innis, et al, eds. Academic Press, Inc., New York).
  • methods may be employed that result in the simultaneous identification of genes that encode the a protein which interacts with a l ⁇ q interval protein. These methods include, for example, probing expression libraries with labeled 18q interval protein, using 1 ⁇ q interval protein in a manner similar to the well known technique of antibody probing of ⁇ gtl 1 libraries.
  • plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the 18q interval gene product and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into this plasmid as part of a cDNA library.
  • the DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site.
  • a reporter gene e.g., HBS or lacZ
  • the two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product.
  • 18q interval gene products may be used as the bait gene product.
  • Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
  • This library and a plasmid encoding a hybrid of a bait 18q interval gene product fused to the DNA-binding domain are co-transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene.
  • a bait 18q interval gene sequence such as the open reading frame of the 18q interval gene, can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library 5 plasmids.
  • a cDNA library of the cell line from which proteins that interact with the bait 18q interval gene product can be made using methods routinely practiced in the art.
  • the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional 0 activation domain of GA-L4.
  • This library can be co-transformed along with the bait 18q interval gene-GAL4 fusion plasmid into a yeast strain that contains a lacZ gene driven by a promoter that contains GAL4 activation sequence.
  • a cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait l ⁇ q interval gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene.
  • HIS3 5 Colonies that express HIS3 can be detected by their growth on petri dishes containing semi- solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait l ⁇ q interval gene-interacting protein using techniques routinely practiced in the art.
  • 1 ⁇ q interval gene products of the invention may, in vivo, interact with one or more macromolecules, including cellular or extracellular macromolecules, such as proteins.
  • Such macromolecules may include, but are not limited to, other proteins, such as cellular 5 receptors, or nucleic acid molecules and those proteins identified via methods such as those described, above, in Sections 5.7.1 - 5.7.3.
  • the 18q interval gene product may interact with a receptor as a peptide hormone or neuropeptide.
  • the macromolecules are referred to herein as "binding partners".
  • Binding partners Compounds that disrupt 18q interval binding in this way may be useful in regulating the activity of the l ⁇ q interval gene product, especially mutant l ⁇ q interval gene products.
  • such compounds may interfere with the interaction of the l ⁇ q interval gene product, with its receptor.
  • Such compounds may include, but are not limited to molecules such as peptides, and the like, as described, for example, in Section 5.7.2 above, which would be capable of gaining access to a 18q interval gene product.
  • the basic principle of the assay systems used to identify compounds that o interfere with the interaction between the 18q interval gene product and its binding partner or partners involves preparing a reaction mixture containing the 18q interval gene product, and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex.
  • the reaction mixture is prepared in the presence and absence of the test compound. 5
  • the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the l q interval gene product and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the 18q interval gene protein and the binding partner is then detected.
  • complex formation within reaction mixtures containing the test compound and normal 18q interval gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant 18q interval gene protein. 5 This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal 18q interval gene proteins.
  • the assay for compounds that interfere with the interaction of the 1 ⁇ q interval gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the 1 ⁇ q interval gene 0 product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction.
  • homogeneous assays the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested.
  • test compounds that interfere with the interaction between the 18q interval gene products and 5 the binding partners can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the 18q interval gene protein and interactive intracellular binding partner.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
  • the various formats are described briefly below.
  • either the l ⁇ q interval gene product or the interactive binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
  • the anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the l q interval gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound.
  • any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds that inhibit complex or that disrupt preformed complexes can be identified.
  • a homogeneous assay can be used.
  • a preformed complex of the l q interval gene protein and the interactive binding partner is prepared in which either the 1 ⁇ q interval gene product or its binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays).
  • the addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt a l ⁇ q interval gene protein/binding partner interaction can be identified.
  • the 18q interval gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.2. above.
  • the 18q interval coding region can be fused to a glutathione-S-transferase (GST) o gene using a fusion vector, such as pGEX-5X- 1 , in such a manner that its binding activity is maintained in the resulting fusion protein.
  • GST glutathione-S-transferase
  • the interactive binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above, in Section 5.3. This antibody can be labeled with the radioactive isotope 125 I, for example, by methods routinely practiced in the art.
  • the GST- fusion protein in a heterogeneous assay, e.g., 5 the GST- fusion protein can be anchored to glutathione-agarose beads.
  • the interactive binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
  • unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
  • the interaction between 0 the 1 ⁇ q interval gene protein and the interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
  • the GST-fusion protein and the interactive binding partner can 5 be mixed together in liquid in the absence of the solid glutathione-agarose beads.
  • the test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the l ⁇ q interval gene product/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity 0 associated with the beads.
  • these same techniques can be employed using peptide fragments that correspond to the binding domains of the 18q interval protein and/or the interactive or binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins.
  • Any number of methods 5 routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding.
  • one protein can be anchored to a solid surface using methods described in this section, above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.
  • a proteolytic enzyme such as trypsin
  • a l ⁇ q interval gene product can be anchored to a solid material as described, above, in this section by making a GST- fusion protein and allowing it to bind to glutathione agarose beads.
  • the interactive binding partner obtained can be labeled with a radioactive isotope, such as 35 S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology.
  • ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE 18q INTERVAL-RELATED DISORDERS Compounds, including but not limited to binding compounds identified via assay techniques such as those described, above, in Sections 5.7.1 - 5.7.4, can be tested for the ability to ameliorate symptoms of a l ⁇ q interval-related disorder, e.g., a neuropsychiatric disorder, such as a disorder of thought and/or mood, including thought disorders such as schizophrenia, schizotypal personality disorder; psychosis; mood disorders, such as schizoaffective disorders (e.g., schizoaffective disorder manic type (SAD- M); bipolar affective (mood) disorders, such as severe bipolar affective (mood) disorder (BP-I), bipolar affective (mood) disorder with hypomania and major depression (BP-EI); unipolar affective disorders, such as unipolar major depressive disorder (MDD), dysthymic disorder; obse
  • the assays described herein can identify compounds that affect 18q interval gene activity by either affecting 18q interval gene expression or by affecting the level of 18q interval gene product activity.
  • compounds may be identified that are involved in another step in the pathway in which the l ⁇ q interval gene and/or l q interval gene product is involved and, by affecting this same pathway may modulate the effect of 18q interval on the development of a neuropsychiatric disorder such as BAD.
  • Such compounds can be used as part of a therapeutic method for the treatment of the disorder.
  • a l ⁇ q interval-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • cell-based systems can be used to identify compounds that may act to ameliorate symptoms of a l ⁇ q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a neuropsychiatric disorder such as BAD
  • Such cell systems can include, for example, recombinant or non-recombinant cell, such as cell lines, that express the 1 ⁇ q interval gene.
  • cells that express 1 ⁇ q interval may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of a 1 ⁇ q interval- related disorder, e.g., a neuropsychiatric disorder, such as BAD, at a sufficient concentration and for a sufficient time to elicit such an amelioration of such symptoms in the exposed cells.
  • the cells can be assayed to measure alterations in the expression of the 1 ⁇ q interval gene, e.g. , by assaying cell lysates for 1 ⁇ q interval mRNA transcripts (e.g. , by Northern analysis) or for 1 ⁇ q interval gene products expressed by the cell; compounds that modulate expression of the l ⁇ q interval gene are good candidates as therapeutics.
  • the cells are examined to determine whether one or more cellular phenotypes associated with a 18q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD, has been altered to resemble a more normal or unimpaired, unaffected phenotype, or a phenotype more likely to produce a lower incidence or severity of disorder symptoms.
  • a neuropsychiatric disorder such as BAD
  • animal-based systems or models for a 1 ⁇ q interval-related disorder may be used to identify compounds capable of ameliorating symptoms of the disorder.
  • a neuropsychiatric disorder such as BAD
  • Such animal-based systems or models may include, for example, transgenic mice, e.g., mice that have been genetically engineered to express exogenous or endogenous 1 ⁇ q interval sequences or, alternatively, to no longer express endogenous 18q interval gene sequences (i.e., "knock-out" mice).
  • Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions that may be effective in treating such disorders.
  • animal models may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms, at a sufficient concentration and for a sufficient time to elicit such an amelioration of symptoms of a 18q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD, in the exposed animals.
  • a neuropsychiatric disorder such as BAD
  • the response of the animals to the exposure may be monitored by assessing the reversal of such symptoms.
  • any treatments that reverse any aspect of 5 symptoms of a l ⁇ q interval-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • Dosages of test agents may be determined by deriving dose-response curves, as discussed in Section 5.11.1, below,
  • a variety of methods can be employed for the diagnostic and prognostic evaluation of l ⁇ q interval-related disorders, such as neuropsychiatric disorders, e.g., BAD, and for the identification of subjects having a predisposition to such disorders.
  • l ⁇ q interval-related disorders such as neuropsychiatric disorders, e.g., BAD
  • Such methods may, for example, utilize reagents such as the nucleotide sequences described in Sections 5.1, and antibodies directed against l ⁇ q interval gene products, including peptide fragments thereof, as described, above, in Section 5.3.
  • reagents such as the nucleotide sequences described in Sections 5.1, and antibodies directed against l ⁇ q interval gene products, including peptide fragments thereof, as described, above, in Section 5.3.
  • reagents may be used, for example, for:
  • Nucleic acid molecules of the invention can, for example, be used to diagnose an 18q interval-related or neuropsychiatric disorder using, for example, the techniques for 1 ⁇ q in
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific nucleic acid of the invention or anti-18q interval gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting abnormalities of a 18q interval-
  • JJ related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • a neuropsychiatric disorder such as BAD.
  • any nucleated cell can be used as a starting source for genomic nucleic acid.
  • any cell type or tissue in which the 18q interval gene is expressed may be utilized. Nucleic acid-based detection techniques are described, above, in Section 5.5.
  • the methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder.
  • test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, urine, sputum, tears, saliva), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide).
  • the methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention.
  • such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal rearrangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non- wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post- translational modification of the protein encoded by the gene.
  • assay techniques known in the art which can be used for detecting lesions in a gene.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,6 ⁇ 3,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 : 1077- 1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q- Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a selected gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, e.g., U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al, 1996, Human Mutation 7:244-255; Kozal et al., 1996, Nature Medicine 2:753-759).
  • a sample and control nucleic acids e.g., DNA or RNA
  • high density arrays containing hundreds or thousands of oligonucleotides probes
  • genetic mutations can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
  • This step allows the identification of point mutations.
  • This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977, Proc. Natl. Acad. Sci. USA 74:560 or Sanger, 1977, Proc. Natl. Acad. Sci. USA 74:5463).
  • any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (1995, Bio/Techniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT Publication No.
  • WO 94/16101 Cohen et al., 1996, Adv. Chromatogr. 36:127-162; and Griffin et al., 1993, Appl. Biochem. Biotechnol. 3 ⁇ :147-159).
  • Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al., 1985, Science 230:1242).
  • the technique of mismatch cleavage entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions.
  • DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair enzymes") in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes proteins that recognize mismatched base pairs in double-stranded DNA
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al., 1994, Carcinogenesis 15:1657-1662).
  • a probe based on a selected sequence is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like.
  • electrophoresis protocols See, e.g., U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in genes.
  • SSCP single strand conformation polymorphism
  • SSCP single strand conformation polymorphism
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al., 1991, Trends Genet. 7:5).
  • the movement of mutant or wild- type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al., 1985, Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC- rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner, 1987, Biophys. Chem. 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al., 1986, Nature 324:163; Saiki et al., 1989, Proc. Natl. Acad. Sci. USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • allele specific amplification technology which depends on selective PCR amplification maybe used in conjunction with the instant invention.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al., 1989, Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner, 1993, Tibtech 11:238).
  • it maybe desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al., 1992, Mol. Cell Probes 6:1).
  • amplification may also be performed using Taq ligase for amplification (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.
  • any cell type or tissue, preferably peripheral blood leukocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
  • kits that facilitate the use /and or detection of 18q interval genes or co-segregating polymorphisms and 18q interval gene products described herein.
  • the kits described herein may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.
  • any cell type or tissue in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
  • a diagnostic test kit for identifying cells or tissues which express or mis-express 18q interval genes or gene products.
  • a diagnostic kit is provided, with one or more containers comprising an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention.
  • a kit is provided, with one or more containers comprising a pair of primers useful for amplifying a l ⁇ q interval nucleic acid molecule encoding a l ⁇ q interval polypeptide of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained.
  • Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.
  • kits can be used, for example, to measure the levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
  • the invention provides kits for detecting the presence of a polypeptide of the invention in a biological sample (a test sample). Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of a polypeptide of the invention as discussed, for example, in sections above relating to uses of the sequences of the invention.
  • kits comprising: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • kits can be used to determine if a subject is suffering from or is at increased risk of an l ⁇ q interval-related disorder, such as a neuropsychiatric disorder, e.g., BAD.
  • anvl ⁇ q interval- mediated process can be modulated and/or whereby an l ⁇ q interval-related disorder, e.g., a neuropsychiatric disorder, such as a cognitive or mood disorder, for example, BAD, may be treated.
  • an l ⁇ q interval-related disorder e.g., a neuropsychiatric disorder, such as a cognitive or mood disorder, for example, BAD
  • such methods can comprise administering compounds which modulate the expression of a l ⁇ q interval gene and/or the synthesis or activity of a 18q interval gene product so that the process is modulated or a symptom of the disorder is ameliorated.
  • such methods can comprise supplying a mammal with a nucleic acid molecule encoding an unimpaired l ⁇ q interval gene product, or with an unimpaired interval -related protein or polypeptide, such that an unimpaired 1 ⁇ q interval gene product is expressed and symptoms of the disorder are ameliorated.
  • such methods can comprise supplying a mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired 1 ⁇ q interval gene product such that the cell expresses the unimpaired l ⁇ q interval gene product and symptoms of the disorder are ameliorated.
  • 18q interval gene product activity In cases in which a loss of normal 18q interval gene product function results in the development of a l ⁇ q interval-related disorder phenotype, e.g., a neuropsychiatric disorder phenotype, an increase in 18q interval gene product activity would facilitate progress towards an asymptomatic state in individuals exhibiting a deficient level of 18q interval gene expression and/or 18q interval gene product activity.
  • Methods for enhancing the expression or synthesis of 18q interval can include, for example, methods such as those described below, in Section 5.10.2.
  • symptoms of l ⁇ q interval-related disorder phenotype e.g., a neuropsychiatric disorder, such as BAD
  • symptoms of l ⁇ q interval-related disorder phenotype may be ameliorated by administering a compound that decreases the level of l ⁇ q interval gene expression and/or l ⁇ q interval gene product activity.
  • Methods for inhibiting or reducing the level of 1 ⁇ q interval synthesis or expression can include, for example, methods such as those described in Section 5.10.1.
  • an l q interval peptide or polypeptide, or fragment thereof, or a l ⁇ q interval mutant, variant, or mimetic can be administered to a subject to ameliorate the symptoms of a l ⁇ q interval-related disorder phenotype.
  • an unimpaired l ⁇ q interval protein or polypeptide can be used to agonize a l ⁇ q interval-mediated pathway.
  • a mutant polypeptide can be used to antagonize or inhibit an l q interval-mediated pathway.
  • the compounds administered do not comprise compounds, in particular drugs, reported to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder, such as BAD.
  • antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD), /?-chlorophenylalanine, -propyldopacetamide dithiocarbamate derivatives e.g., FLA 63
  • anti-anxiety drugs e.g., diazepam
  • monoamine oxidase (MAO) inhibitors e.g., iproniazid, clorgyline, phenelzine and isocarboxazid
  • biogenic amine uptake blockers e.g.
  • tricyclic antidepressants such as desipramine, imipramine and amitriptyline
  • serotonin reuptake inhibitors e.g. , fluoxetine
  • antipsychotic drugs such as phenothiazine derivatives (e.g., chlorpromazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (Haldol)), thioxanthene derivatives (e.g.
  • benzodiazepines e.g., clozapine
  • benzodiazepines dopaminergic agonists and antagonists e.g., L-DOPA, cocaine, amphetamine, ⁇ -methyl-tyrosine, reserpine, tetrabenazine, benzotropine, pargyline
  • noradrenergic agonists and antagonists e.g., clonidine, phenoxybenzamine, phentolamine, tropolone.
  • treatment methods do comprise such compounds, preferably such compounds are utilized in a manner (e.g., different dosage, mode of administration, and/or co-administration with one or more additional compounds) that differs from the manner in which such compounds have been administered previously.
  • symptoms of certain 18q interval-related disorders may be ameliorated by decreasing the level of l ⁇ q interval gene expression and/or l q interval gene product activity by using 18q interval nucleic acid sequences in conjunction with well-known antisense, gene "knock- out,” ribozyme and/or triple helix methods to decrease the level of l ⁇ q interval gene expression.
  • the compounds that may exhibit the ability to modulate the activity, expression or synthesis of the 18q interval gene including the ability to ameliorate the symptoms of a 18q interval -related disorder, e.g. , a neuropsychiatric disorder, such as BAD, are antisense, ribozyme, and triple helix molecules.
  • Such molecules may be designed to • * reduce or inhibit either unimpaired, or if appropriate, mutant target gene activity.
  • Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense ⁇ approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • ⁇ means a sequence having sufficient complementarity to be able to hybodize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base in
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • oligonucleotides complementary to coding or non- coding regions of the 18q interval gene can be used in an antisense approach to inhibit
  • Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g.
  • oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueo
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier, et al, 1987, Nucl. Acids Res. 15, 6625-6641).
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue, et al, 1987, Nucl. Acids Res. 15, 6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al, 1987, FEBS Lett. 215, 327-330).
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl. Acids Res. 16, 3209)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451), etc.
  • antisense nucleotides complementary to the target l ⁇ q interval gene coding region sequence could be used, those complementary to the transcribed, untranslated region can also be utilized.
  • Antisense molecules should be delivered to cells that express the target gene in vivo.
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol HI or pol II promoter.
  • a vector can be introduced e.g., such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290, 304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, 1980, Cell 22, 181-191), the herpes thymidine kinase promoter (Wagner, et al, 1981, Proc. Natl. Acad. Sci. U.S.A.
  • plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site.
  • viral vectors can be used that selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
  • Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and, therefore, expression of target gene product. (See, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver, et al, 1990, Science 247, 1222-1225).
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event.
  • the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Patent No. 5,093,246, which is incorporated herein by reference in its entirety.
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs
  • the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially fig. 4, page 833) and in Haseloff and Gerlach, 1988, Nature, 334, 585-591, which is incorporated herein by reference in its entirety.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA, i.e., to increase efficiency and minimize o the intracellular accumulation of non- functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one that occurs naturally in Tetrahymena thermophila (known as the EVS, or L-19 EVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al, 1984, Science, 224, 574-578; Zaug and 5 Cech, 1986, Science, 231, 470-475; Zaug, et al, 1986, Nature, 324, 429-433; published International patent application No. WO 8 ⁇ /04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47, 207-216).
  • Cech-type ribozymes such as the one that occurs naturally in Tetrahymena thermophila (known as the EVS, or L-19 EVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al, 1984, Science, 224, 57
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair 0 active site sequences that are present in the target gene.
  • the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol HI or 5 pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Endogenous target gene expression can also be reduced by inactivating or 0 "knocking out" the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al, 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al, 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety).
  • targeted homologous recombination e.g., see Smithies, et al, 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al, 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety).
  • a mutant, non-functional target gene flanked by DNA homologous to the endogenous 5 target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene.
  • ES embryonic stem
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene ( c, the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the target gene c, the target gene promoter and/or enhancers
  • triple helical structures that prevent transcription of the target gene in target cells in the body.
  • Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called “switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the technique may so efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles that the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype.
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity may, be introduced into cells via gene therapy methods such as those described, below, in Section 5.10.2 that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized.
  • the target gene encodes an extracellular protein
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • nucleic acid sequences encoding a l ⁇ q interval gene product for example, be utilized for the treatment of a 18q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a 18q interval-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • Such treatment can be administered, for example, in the form of gene replacement therapy.
  • one or more copies of a normal l ⁇ q interval gene or a portion of the l q interval gene that directs the production of a 18q interval gene product exhibiting normal 18q interval gene function may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
  • l ⁇ q interval genes can be expressed in the brain, such gene replacement therapy techniques should be capable delivering l ⁇ q interval gene sequences to these cell types within patients.
  • techniques that are well known to those of skill in the art can be used to enable 18q interval gene sequences to cross the blood-brain barrier readily and to deliver the sequences to cells in the brain.
  • viral vectors such as, for example, those described above, are preferable.
  • techniques for delivery involve direct administration of such 18q interval gene sequences to the site of the cells in which the 18q interval gene sequences are to be expressed.
  • Additional methods that may be utilized to increase the overall level of l ⁇ q interval gene expression and/or 1 ⁇ q interval gene product activity include the introduction of appropriate 1 ⁇ q interval-expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to ameliorate the symptoms of a l ⁇ q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a l ⁇ q interval-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • Such cells may be either recombinant or non-recombinant.
  • the cells that can be administered to increase the overall level of 1 ⁇ q interval gene expression in a patient are normal cells, preferably brain cells, that express the l ⁇ q interval gene.
  • cells preferably autologous cells
  • a 18q interval -related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • cells that express an unimpaired 18q interval gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells.
  • the expression of the 18q interval gene sequences is controlled by the appropriate gene regulatory sequences to allow such expression in the necessary cell types.
  • gene regulatory sequences are well known to the skilled artisan.
  • Such cell- based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson, U.S. Patent No. 5,399,349.
  • the cells to be administered are non-autologous cells, they can be administered using well known techniques that prevent a host immune response against the introduced cells from developing.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • compounds such as those identified via techniques such as those described, above, in Section 5.7, that are capable of modulating 18q interval gene product activity can be administered using standard techniques that are well known to those of skill in the art.
  • the administration techniques should include well known ones that allow for a crossing of the blood-brain barrier.
  • PHARMACOGENOMICS Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide.
  • the pharmacogenomics i.e., the study of the relationship o between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or 5 therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic 0 treatment of the individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a 5 single factor altering the way drugs act on the body are referred to as “altered drug action.” Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism”. These pharmacogenetic conditions can occur either as rare defects or as polymorphisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) 5 and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme -ire the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply geno typing of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.
  • Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a polypeptide of the invention can be applied not only in basic drug screening, but also in clinical trials.
  • agents e.g., drugs, compounds
  • the effectiveness of an agent, as determined by a screening assay as described herein, to increase gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting decreased gene expression, protein levels, or protein activity.
  • the effectiveness of an agent, as determined by a screening assay, to decrease gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting increased gene expression, protein levels, or protein activity.
  • expression or activity of a polypeptide of the invention and preferably, that of other polypeptide that have been implicated in a l ⁇ q interval-related disorder can be used as a marker of the immune responsiveness of a particular cell.
  • genes including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • a polypeptide of the invention e.g., as identified in a screening assay described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the post-administration samples; (v) comparing the level of the polypeptide or nucleic acid of the invention in the pre-administration sample with the level of the polypeptide or nucleic acid of the invention in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g., an agonist, antagonist,
  • increased administration of the agent may be desirable to increase the expression or activity of the polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • l ⁇ q interval gene products or compounds that are determined to affect l ⁇ q interval gene expression or gene product activity can be administered to a patient at therapeutically effective doses to treat or ameliorate a l ⁇ q interval-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of such a disorder.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to ⁇ mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to ⁇ mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a ⁇ 0 subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino ⁇ acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per 5
  • organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micro grams per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.
  • appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • an animal e.g., a human
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. , containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example as an emulsion in an acceptable oil
  • ion exchange resins for example as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • yeast artificial chromosome YAC . mapping
  • yeast artificial chromosomes YACs
  • YACs yeast artificial chromosomes containing human sequences were mapped to the region being analyzed based on publicly available maps (Cohen et al, 1993, C.R. Acad. Sci. 316,
  • the YACs were then ordered and contig reconstructed by performing standard short tag sequence (STS)-content mapping with microsatellite markers and non- polymorphic STSs available from databases that surround the genetically defined candidate region.
  • STS standard short tag sequence
  • BAC DNA was sheared with a nebulizer (CIS-US Inc., Bedford,
  • RH mapping Standard RH mapping techniques were 5 applied to a Stanford G3 RH mapping panel (Research Genetics, Huntsville, AL) to order all microsatellite markers and non-polymorphic STSs in the region being analyzed.
  • Northern analysis Standard Northern analysis techniques were utilized in probing human and fetal multiple tissue Northern blots purchased from Clontech (Palo Alto, CA). Blots were hybridized to different gene probes, which were derived by PCR from l ⁇ q interval cDNA sequences.
  • the radiation hybrid (RH) mapping technique was independently applied to the region being analyzed.
  • RH was used to order all microsatellite markers and non-polymorphic STSs in the region.
  • the high resolution physical map ultimately constructed was obtained using data from RH mapping and STS-content mapping.
  • BADl ⁇ ct22 is defined by a (GA) 22 di-nucleotide repeat; the BADct22 primer set used was as follows:
  • BAD18cagl is defined by a (CAG) n tri-nucleotide repeat; the following primer set was used for amplification of the BADl ⁇ cagl marker:
  • the BAC clones within the newly identified 116 kb neuropsychiatric disorder region were further analyzed to identify specific genes within the region. Two overlapping BACs, BAC69 and BAC104, were able to cover the whole 116 kb interval.
  • a combination of sample sequencing, cDNA selection and transcription mapping analyses were combined to arrange sequences into tentative transcription units, that is, tentatively delineating the coding sequences of genes within this genomic region of interest.
  • the nucleotide sequences depicted in FIG. IB represent 18q interval genomic nucleotide sequences. Novel sequences of thel ⁇ q interval were identified that can be used with the methods of the invention. Sequences of the 116 kb interval were used as input to the NBLAST program, and public and private databases were searched, including: dbest [GenBank database of ESTs (Expressed Sequence Tags)]; htgs [GenBank databases HTGS (high-throughput genomic sequences) and GSS (Genome Survey Sequences)]; patn (database for non-redundant geneseq and "patent preview"); and nuc (all GenBank nucleotide sequences except EST, HTGS, and GSS divisions) (see Benson et al, 1999, Nucleic Acids Res., 1999, 1:12-17).
  • Second, the resulting HSPs for each database hit were examined. All non-human hits were excluded and all possible 90 bp regions of the query within each HSP were evaluated for percent identity by determining the fraction identity, matches divided by the sum of 90 plus the subject gaps in the alignment region. Sequences were determined to be novel if the fraction identity was less than 0.95 and the query sequences were at least 100 bp.
  • This method identified the following novel nucleotide sequences of the l ⁇ q interval: 2 ⁇ 441-29265 (SEQ ED No. 4), 296 ⁇ 3-395 ⁇ 7 (SEQ ED No. 5), 40284-43253 (SEQ ID No. 6), 4351 ⁇ -46075 (SEQ ID No. 7), 47264-52284 (SEQ ID No. 8), 52672-56935 (SEQ ID No. 9), 57032-57726 (SEQ ID No. 10), 58065-59057 (SEQ ID No. 11), 59815-60471 (SEQ ED No. 12), 60870-62451 (SEQ ED No. 13), 62543-6326 ⁇ (SEQ ED No.
  • Ep69104 represents a composite deposit of a mixture of two strains, each of which contains either bacterial artificial chromosome (BAC) BAC69 or BAC 104.
  • the two BACs together contain the approximately 160 kb region of human chromosome 18 depicted in FIG. IB.
  • BAC69 or BAC 104 an aliquot of the mixture can be streaked out to single colonies on nutrient media (e.g., LB plates) supplemented with lOO ⁇ g/ml ampicillin, a single colony can be grown, and then BAC DNA can be extracted from the culture using standard procedures.
  • nutrient media e.g., LB plates
  • BAC69 DNA yields fragments having a total length of approximately 220kb; BAC 104 DNA yields fragments having a total length of approximately ⁇ Okb.

Abstract

La présente invention concerne des acides nucléiques de l'intervalle 18q, des molécules d'ADN recombinant, des gènes clonés ou leurs variantes dégénérées, des produits génétiques de l'intervalle 18q et des anticorps dirigés contre ces produits génétiques, des vecteurs de clonage contenant les molécules du gène de l'intervalle 18q et des hôtes génétiquement modifiés exprimant ces molécules. L'invention concerne également des méthodes d'identification de composés modulant l'expression des gènes de l'intervalle 18q et des produits génétiques, ainsi que l'utilisation de ces composés comme agents thérapeutiques dans le traitement de troubles liés à l'intervalle 18q, tels que les troubles neuropsychiatriques. Elle se rapporte en outre à des méthodes destinées à l'évaluation diagnostique, au test génétique et au prognostique de troubles liés à l'intervalle 18q, et notamment de troubles neuropsychiatriques tels que la schizophrénie, le trouble déficitaire de l'attention, les troubles schizo-affectifs, la maladie affective bipolaire ou la maladie affective unipolaire, ainsi qu'à des méthodes et des compositions destinées au traitement de ces troubles.
PCT/US2000/030637 1999-11-08 2000-11-07 METHODES ET COMPOSITIONS PERMETTANT DE DIAGNOSTIQUER ET DE TRAITER LES TROUBLES LIES AU CHROMOSOME 18q WO2001034771A2 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAMPSON R.M. ET AL.: 'Mapping studies on a pericentric inversion (18) (p11.31q21.1) in a family with both schizophrenia and learning disability' PSYCHIATRIC GENETICS vol. 9, no. 3, 1999, pages 161 - 163, XP002940335 *
MCMAHON F.J.: 'Lingage of bipolar affective disorder to chromosome 18 markers in a new pedigree series' AM. J. HUMAN GENETICS vol. 61, 1997, pages 1397 - 1404, XP002940334 *

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