WO2001034772A2 - Methodes et compositions permettant de diagnostiquer et de traiter des troubles lies au chromosome 18q - Google Patents

Methodes et compositions permettant de diagnostiquer et de traiter des troubles lies au chromosome 18q Download PDF

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WO2001034772A2
WO2001034772A2 PCT/US2000/030819 US0030819W WO0134772A2 WO 2001034772 A2 WO2001034772 A2 WO 2001034772A2 US 0030819 W US0030819 W US 0030819W WO 0134772 A2 WO0134772 A2 WO 0134772A2
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fsh23
ofthe
gene
disorder
expression
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PCT/US2000/030819
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WO2001034772A9 (fr
WO2001034772A3 (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 AU14797/01A priority Critical patent/AU1479701A/en
Publication of WO2001034772A2 publication Critical patent/WO2001034772A2/fr
Publication of WO2001034772A3 publication Critical patent/WO2001034772A3/fr
Publication of WO2001034772A9 publication Critical patent/WO2001034772A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates first, to polynucleotides of a region of human chromosome 18q which is associated with neuropsychiatric disorders.
  • the invention relates to a novel gene referred to herein as fsh23, which maps to the relevant portion of human chromosome 18q.
  • the sequences ofthe present invention can be used for diagnostic evaluation, genetic testing and/or prognosis of a s ⁇ 23-related disorder, e.g., a neuropsychiatric disorder, and/or can be used to map human chromosome 18q.
  • the invention encompasses s ⁇ 23 nucleic acids, recombinant DNA molecules, cloned genes and degenerate variants thereof, vectors containing such fsh23 nucleic acids, and hosts that have been genetically engineered to express and/or contain such molecules.
  • the invention further relates to fsh2 '3 gene products and antibodies directed against such gene products.
  • the invention further relates to methods for the identification of compounds that modulate the expression, synthesis and activity of such fsh23 genes. Still further, the invention relates to methods of using compounds, such as those identified herein, as therapeutic agents for modulation of s/j23-mediated process or in the treatment of a symptom of s/?23-related 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.
  • the invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of a sA23-related 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.
  • 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 ofthe 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 ofthe 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-H).
  • 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, first, the nucleotide sequence of a 116 kilobase interval of human chromosome 18q (i.e., nucleotides 28441-144419 of FIG. IB), and its flanking regions (nucleotides 1-28440 and 144420-160271 of FIG. lB),associated with neuropsychiatric disorders in humans, e.g., schizophrenia, attention deficit disorders, schizoaffective disorders, bipolar affective disorders, and/or unipolar affective disorders.
  • the present invention relates to a gene located within this interval, referred to herein as fsh23, which is involved in such disorders.
  • fsh23 nucleic acids recombinant DNA molecules, cloned genes or degenerate variants thereof are provided herein.
  • the invention also provides vectors, including expression vectors, containing ?/ * ⁇ .? nucleic acid molecules, and hosts that have been genetically engineered to express and/or contain such fsh23 gene products.
  • the invention further relates to novel fsh23 gene products and to antibodies directed against such gene products, or variants or fragments thereof.
  • the invention further relates to methods for modulation of s&23-mediated processes and for the treatment of/sA2.?-related disorders, such as neuropsychiatric disorders, including the amelioration or prevention of at least one symptom ofthe disorders, wherein such methods comprise administering a compound which modulates the expression of a fsh23 gene and/or the synthesis or activity of afsh23 gene product.
  • the invention relates to methods for the use of a novel fsh23 gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against afsh23 gene product, to treat or ameliorate a symptom of such disorders.
  • Such neuropsychiatric disorders include disorders relating to the central nervous system (CNS) and/or peripheral nervous system (PNS) including, but not limited to, 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-M), 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
  • ' ⁇ * !23-related disorder refers to a disorder involving afsh23 gene or gene product, or involving an aberrant level o ⁇ fsh23 gene expression, gene product synthesis and/or gene product activity relative to levels found in normal, unaffected, unimpaired individuals, levels found in clinically normal individuals, and/or levels found in a population whose levels represent a baseline, average fsh23 levels.
  • ' ⁇ s ⁇ 23-mediated processes include processes dependent and/or responsive, either directly or indirectly, to the level of expression, gene product synthesis and/or gene product activity of afsh23 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.
  • such methods can comprise modulating the level of expression or the activity of afsh23 gene or gene product in a cell such that the fsh23 -mediated process or the disorder is treated, e.g., a symptom is ameliorated.
  • such methods can comprise supplying a nucleic acid molecule encoding afsh23 gene product to increase the level, expression or activity of the fsh23 gene product within the cell such that the s&25-mediated process or the disorder is treated, e.g., a symptom is ameliorated.
  • the nucleic acid molecule encoding the fsh23 gene product can encode a normal or mutant fsh23 gene product, e.g., one with increased activity or expression levels.
  • nucleic acids and polypeptides ofthe 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 ofthe 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 detection of nucleic acids encoding a polypeptide ofthe invention.
  • 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, 6, 7, 9, 10, 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, the nucleotide sequence ofthe cDNA insert of a clone deposited with ATCC ® as Accession Number PTA-451, PTA-452 (the "cDNA of ATCC ® PTA-451, PTA-452”) 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, 6, 7, 9, 10, 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, the nucleotide sequence ofthe cDNA insert of a clone deposited with ATCC ® as Accession Number PTA-451, PTA-452 (the "cDNA of ATCC ® PTA-451, PTA-452”), 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 ofthe 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 of SEQ ID Nos. 4, 7, 10, the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, PTA-452, 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%, 75%, 85%, 95%, 98, or 99%) identical to the amino acid sequence of SEQ ID Nos. 4, 7, 10, the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, PTA-452, 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 ofthe 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, PTA-452 wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting ofthe nucleotide sequence ofthe cDNA of ATCC ® PTA- 451, PTA-452, 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, PTA-452, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting ofthe nucleotide sequence ofthe cDNA of ATCC ® PTA- 451, PTA-452, 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 ofthe invention.
  • the isolated nucleic acid molecules encode an extracellular, transmembrane, or cytoplasmic domain of a FSH23 polypeptide ofthe invention.
  • the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid ofthe invention.
  • Another aspect ofthe invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule ofthe invention.
  • the invention provides host cells containing such a vector or a nucleic acid molecule ofthe invention.
  • the invention also provides methods for producing a polypeptide ofthe invention by culturing, in a suitable medium, a host cell ofthe invention containing a recombinant expression vector such that a polypeptide is produced.
  • Another aspect of this invention features isolated or recombinant proteins and polypeptides ofthe 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 ofthe 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 ofthe protein with a second protein.
  • polypeptides ofthe 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 ofthe invention such as monoclonal or polyclonal antibodies.
  • the polypeptides ofthe invention or biologically active portions thereof, or antibodies ofthe invention can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides methods for detecting the presence ofthe activity or expression of a polypeptide ofthe 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 ofthe invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide ofthe invention such that activity or expression in the cell is modulated.
  • the agent is an antibody that specifically binds to a polypeptide ofthe invention.
  • the agent modulates expression of a polypeptide ofthe invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide ofthe 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 ofthe invention.
  • the present invention also provides methods to treat a subject having a disorder characterized by aberrant activity of a polypeptide ofthe invention or aberrant expression of a nucleic acid ofthe invention by administering an agent which is a modulator ofthe activity of a polypeptide ofthe invention or a modulator ofthe expression of a nucleic acid ofthe invention to the subject.
  • the modulator is a protein ofthe invention.
  • the modulator is a nucleic acid ofthe 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 ofthe invention, (ii) mis- regulation of a gene encoding a polypeptide ofthe invention, and (iii) aberrant post- translational modification ofthe invention wherein a wild- type form ofthe gene encodes a protein having the activity ofthe polypeptide ofthe invention.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide ofthe invention.
  • such methods entail measuring a biological activity ofthe polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity ofthe polypeptide.
  • the invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid ofthe invention by measuring the expression ofthe polypeptide or nucleic acid in the presence and absence ofthe compound.
  • the invention still further relates to methods for modulation of s ⁇ 23-mediated processes or the treatment of s ⁇ 23-related disorders, such as neuropsychiatric disorders, including but not limited to disorders resulting from fsh23 gene mutations, and/or an abnormal level of fsh23 expression or activity and disorders involving afsh23 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 fsh23 gene product such that the unimpaired/s/z23 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 fsh23 gene product such that the cell expresses the unimpaired fsh23 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 a fsh23 gene or gene product.
  • 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 a fsh23 gene or gene product.
  • the present invention is directed to methods that utilize s/j23 gene sequences and/or fsh23 gene product sequences for the diagnostic evaluation, genetic testing and/or prognosis of a sA2 -related disorder, such as a neuropsychiatric disorder.
  • a sA2 -related disorder such as a neuropsychiatric disorder.
  • the invention relates to methods for diagnosing s ⁇ 23-related disorders, e.g., neuropsychiatric disorders, wherein such methods can comprise gene expression in a patient sample, or detecting afsh23 mutation that correlates with the presence or development of such a disorder, in the genome of a mammal suspected of exhibiting such a disorder.
  • fsh23 gene sequences and/or fsh23 gene products can also be utilized as markers for mapping ofthe region ofthe long arm of human chromosome 18 spanned by chromosomal markers BAD18ct22 and BAD18cagl. These markers are identified below, and described in Section 6.2.
  • the invention still further relates to methods for identifying compounds capable of modulating the expression of a fsh2 '3 gene and/or the synthesis or activity of a fsh23 gene product, wherein such methods comprise contacting a compound with a cell that expresses afsh23 gene, measuring the levels of fsh23 gene expression, gene product expression or gene product activity, and comparing such levels to the levels o ⁇ fsh23 gene expression, gene product, or gene product activity produced by the cell in the absence ofthe compound, such that if the level obtained in the presence ofthe compound differs from that obtained in its absence, a compound capable of modulating the expression of the fsh23 gene and/or the synthesis or activity of the fsh23 gene product has been identified.
  • bipolar affective disorder BP bipolar mood disorder
  • BP- ⁇ bipolar affective (mood) disorder with hypomania and major depression bp
  • base pair(s) dbEST expressed sequence tag data base
  • allele which is used interchangeably herein with “allelic variant” refers to alternative forms of a 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.
  • allelic variant of a polymorphic region of an fsh23 gene refers to a region of an fsh23 gene having one of several nucleotide sequences found in that region ofthe gene in other individuals, as well as to polypeptides encoded by nucleic acid molecules comprising said sequences.
  • 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 ofthe invention, e.g., an epitope of a polypeptide ofthe invention.
  • a molecule which specifically binds to a given polypeptide ofthe 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 ofthe 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 ofthe protein also exist. Such soluble isoforms can arise through variable splicing of the fsh23 gene or alternatively as a result of proteo 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 fsh23 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 ofthe encoded polypeptide yet still encode a protein with the same biological activity.
  • the term "gene” or “recombinant gene”, as applied to fsh23, refers to a polynucleotide or nucleic acid molecule comprising an open reading frame encoding one ofthe fsh23 polypeptides ofthe 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. In one embodiment, the term 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 ofthe 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 ofthe number of identical amino acids at positions shared by the amino acid sequences.
  • a degree of identity of nucleic acid sequences is a function ofthe 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 ofthe number of conserved amino acids at positions shared by the amino acid sequences.
  • a sequence which is "unrelated" or “non-homologous" with one ofthe human fsh23 sequences ofthe present invention typically is a sequence which shares less than 40 % identity, though preferably less than 25 % identity with one ofthe human fsh23 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.
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • Another preferred, non-limiting example of 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 ofthe GCG sequence alignment software package.
  • ALIGN program version 2.0
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • 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.
  • an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID Nos. 1, 2, 3, 4, 6, 7, 9, 10, 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or complement thereof, corresponds to a naturally-occurring nucleic acid molecule.
  • FIG. 1 A-B depicts the gene structure and the nucleotide sequence ofthe 116 kb interval of the long arm of human chromosome 18 spanned by BAD 18ct22 and BAD l ⁇ cagl.
  • A A diagrammatic representation ofthe 18q interval, showing known genetic and physical markers in this interval. In particular, the location and orientation o ⁇ fsh23 within this interval is shown.
  • B The nucleotide sequence of 160 kb of chromosome 18q, which includes thel8q interval associated with neuropsychiatric disorders, located from position 28441-144419.
  • BAD18ct22 and BAD 18cagl markers are shown in boxes between primers.
  • the fsh23 gene corresponds to nucleotides 51458 to 63491 (bounded by " ⁇ >").
  • FIG. 2A-E fsh23 genomic nucleotide sequence. Exons are included within “[]”, “ ⁇ , "()”, or "o”.
  • FIG. 3 A-C fsh23 cDNA nucleotide sequences and amino acid sequences for alternatively spliced mRNA transcripts: A.fsh23-Vl (altl); B.fsh31-V2 (alt2); and C.fsh31-V3 (alt3).
  • FIG. 4 The fsh23 gene and a diagram o ⁇ fsh23 RNA transcripts showing alternative splice forms.
  • FIG. 5 A-C fsh23 intronic structure.
  • FIG. 6A-C Physical-structural analysis o ⁇ fsh23 amino acid sequences. Shown at the left are plots predicting secondary structure, hydropathy, amphipathy, flexibility, antigenicity and surface probabilities (according to the rule or algorithm indicated at right) of Fsh23 amino acid sequence corresponding to the coordinates shown at top left.
  • FIG. 7 The nucleotide sequence of fsh23 comprising Exon 1.
  • FIG. 8 The nucleotide sequence of fsh23 Exon 2.
  • FIG. 9 The nucleotide sequence of fsh23 Exon B.
  • FIG. 10 The nucleotide sequence of fsh23 Exon C.
  • FIG. 11 The nucleotide sequence of fsh23 Exon 3.
  • FIG. 12 The nucleotide sequence of fsh23 Exon 3' UTR
  • compositions and methods relating to nucleic acid sequences associated with an approximately 116 kb interval on the long arm of human chromosome 18q, a region associated with neuropsychiatric disorders, are described herein.
  • a novel gene has been identified within this region, referred to herein as fsh23, which is involved in such disorders.
  • Sections 5.1, 5.2, and 5.3 describe the 18q 116 kb region, including s/z23 nucleic acid molecules, as well as vectors comprising these molecules, host cells engineered to contain and/or express such molecules, fsh23 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. For example, methods for the use of particular molecules for modulation of s&23-related processes and for treatment of ⁇ 23-related disorders, such as neuropsychiatric disorders, are described.
  • Section 5.7 describes screening assays for compounds that interact with afsh23 gene or gene product, or modulate fsh23 gene or gene product activity.
  • Methods of treatment of s/z23-related disorders using the compositions ofthe invention and compositions such as those identified by the methods ofthe invention are described in Section 5.8. Sections 5.9 and 5.10, respectively, describe diagnostic methods and kits.
  • Section 5.11 pharmaceutical compositions for the use with the invention are described.
  • FIG. 1 A The genomic organization ofthe region of human chromosome 18q between the markers BAD18ct22 and BADl ⁇ cagl has been determined (shown schematically in FIG. 1 A), the nucleotide sequence of which, and its flanking DNA, is 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.
  • a novel gene, fsh23 has been mapped to this region (see FIG. 1 A).
  • fsh23 has been mapped to this region, and three fsh23 nucleotide sequences derived from mRNA molecules of three novel fsh23 cDNA splice variants have been identified, including/s/z23-Vl (altl)(FIG. 3A), fsh231-V2 (alt2)(FIG. 3B), andfsh23-V3 (alt3)(FIG. 3C).
  • fsh23 has a complex splicing pattern; the 18q nucleotide sequence comprising the fsh23 genomic sequence is shown in FIG. 2.
  • FIG. 2 also indicates exon and intron boundaries of the fsh23 gene.
  • Nucleic acid molecules comprising s ⁇ 2J exon and intron sequences are encompassed by the present invention.
  • fsh23 exon 1 comprises the 84 base pair sequence spanning nucleotides 1 to 84 ofthe sequence shown in FIG. 2, bounded by "[]”;
  • exon 2 encompasses the 169 base pair sequence spanning nucleotides 898-1,066 ofthe sequence shown in FIG. 2, bounded by " ⁇ ”;
  • exon B encompasses the 59 base pair sequence spanning nucleotides 3,065 to 3,123 ofthe sequence shown in FIG. 2;
  • exon C encompasses the 94 base pair sequence spanning nucleotides 5,483 to 5,576 ofthe sequence shown in FIG.
  • exon 3 encompasses the 86 base pair sequence spanning nucleotides 11,001 to 11,086 ofthe sequence shown in FIG. 2, bounded by " ⁇ ”; and exon 3' UTR encompasses the 225 base pair sequence spanning nucleotides 11,814 to 12,038 ofthe sequence shown in FIG. 2, bounded by " ⁇ ”.
  • fsh23 intron 1 encompasses the 813 base pair sequence spanning nucleotides 85 to 897 ofthe sequence shown in FIG. 2; intron 2 encompasses the 1998 base pair sequence spanning nucleotides 1,067 to 3,064 ofthe sequence shown in FIG.
  • FIGS. 3A-C shows three fsh23 cDNA nucleotide sequences, each comprising various alternative exons.
  • the genomic organization ofthe human fsh23 gene is depicted in FIG. 4. Three alternative splice forms produced by the various combinations ofthe six alternative exons are shown in the remaining lower panels.
  • the fsh23 exons are indicated by boxes, with the fsh23 coding regions indicated by light shading within exons 1, 2, B, C, and 3, and the 3'- untranslated regions indicated in dark shading.
  • nucleic acid molecules of the invention are nucleic acid molecules comprising, in a 5' to 3' direction: exon 1 (SEQ ID NO:13), exon 2 (SEQ ID NO:14), exon 3 (SEQ ID NO:17), and exon 3' UTR (SEQ ID NO: 60); exon 1 (SEQ ID NO:13), exon 2 (SEQ ID NO:14), exon B (SEQ ID NO:15), exon C (SEQ ID NO: 16), exon 3 (SEQ ID NO: 17), and exon 3' UTR (SEQ ID NO: 60); and exon 1 (SEQ ID NO:13), exon 2 (SEQ ID NO:14), exon C (SEQ ID NO:16), exon 3 (SEQ ID NO: 17) and exon 3'UTR (SEQ ID NO:60).
  • These splice variants encode various fragments ofthe fsh23 polypeptide, including a polypeptide comprising the 85 amino acid sequence of SEQ ID NO: 5 shown in FIG. 3 A, a polypeptide comprising the 96 amino acid sequence of SEQ ID NO:8 shown in FIG. 3B; and a polypeptide comprising the 100 amino acid sequence of SEQ ID NO:l 1 shown in FIG. 3C, respectively.
  • an alternatively spliced human fsh23 cDNA sequence referred to herein as fsh23-V I (altl)(SEQ ID NO:4) is shown in FIG. 3 A along with the amino acid sequence (SEQ ID NO: 5) ofthe human fsh23 variant gene product (i.e., thefsh23-Vl gene product) it encodes.
  • This splice variant ofthe human fsh23 gene encodes a polypeptide comprising the 85 amino acid sequence shown in FIG. 3A and in SEQ ID NO:5.
  • This alternatively spliced human fsh23 cDNA sequence is missing exons B and C (SEQ ID Nos:15 and 16, respectively).
  • the nucleotide sequence ofthe portion of the fsh23-Vl cDNA corresponding to the open reading frame encoding the fsh23-Vl gene product is depicted in SEQ ID NO:6.
  • FIG. 3B Another alternatively spliced human fsh23 cDNA sequence, referred to herein as fsh23-V2 (alt2)(SEQ ID NO:7) is shown in FIG. 3B, along with the amino acid sequence (SEQ ID NO:8) ofthe human fsh23 variant gene product (i.e., the fsh23-V2 gene product) it encodes.
  • This splice variant ofthe human fsh23 gene encodes a polypeptide comprising the 96 amino acid sequence shown in FIG. 3B and in SEQ ID NO: 8.
  • This alternatively spliced humanfsh23 cDNA sequence contains all six exons (SEQ ID Nos: 13, 14, 15, 16, 17, and 60, respectively).
  • the nucleotide sequence ofthe portion of the fsh23 -V2 cDNA corresponding to the open reading frame encoding thefsh23-V2 gene product is depicted in SEQ ID NO:9.
  • FIG. 3C Another alternatively spliced_ s z23 cDNA sequence, referred to herein as fsh23-Y3 (alt3)(SEQ ID NO: 10) is shown in FIG. 3C along with the amino acid sequence (SEQ ID NO:l 1) ofthe humanfsh23 variant gene product (i.e., the fsh23-V3 gene product) it encodes.
  • This splice variant ofthe human fsh23 gene encodes a polypeptide comprising the 100 amino acid sequence shown in FIG. 3C and in SEQ ID NO: 11.
  • This alternatively spliced human fsh23 cDNA sequence is missing exon B (SEQ ID NO: 15).
  • SEQ ID NO: 12 The nucleotide sequence ofthe portion of the fsh23-V3 cDNA corresponding to the open reading frame encoding the fsh23-V3 gene product is depicted in SEQ ID NO: 12.
  • the nucleic acid molecules ofthe invention further include:
  • nucleic acid molecule containing the DNA sequence ofthe 18q interval (nucleotides 28441-144419 of FIG. IB), or its flanking DNA (nucleotides 1-28440 and 144420-160271 of FIG. IB), and fragments thereof;
  • nucleic acid molecule comprising afsh23 nucleic acid sequence (e.g., the nucleic acid sequences depicted in FIG. 2 or FIGS. 3A-C, or a fragment thereof);
  • nucleic acid molecule that encodes afsh23 gene product such as a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence shown in FIG. 3A-C;
  • nucleic acid molecule that comprises at least one exon of afsh23 gene (i.e., nucleotides 1-84, 898- 1,066, 3,065-3,123, 5,483-5,576, 11,001- 11,086 and 1,814- 12,038 of FIG. 2); (e) a nucleic acid molecule that comprises s/z23 gene sequences of upstream untranslated regions, intronic regions, and/or downstream untranslated regions, or fragments thereof, of the fsh23 nucleotide sequences in (b) above (e.g., nucleotides 85-897, 1,067-3,064, 3,124-5,482, and 5,577-11,000 ofthe sequence shown in FIG.2);
  • nucleic acid molecule comprising afsh23 sequence that encodes a mutant of a fsh23 gene product in which all or a part of a domain is deleted or altered, as well as fragments thereof;
  • nucleic acid molecules that encode fusion proteins comprising afsh23 gene product e.g., amino acid sequences shown in FIGS. 3A-C), or a fragment thereof, fused to a heterologous polypeptide
  • a/s/z23-related disorder such as a neuropsychiatric disorder, e.g., BAD.
  • nucleotide sequences ofthe invention further include nucleotide sequences corresponding to the nucleotide sequences of (a)-(h) above wherein one or more of the exons, or fragments thereof, have been deleted.
  • the nucleic acid molecules ofthe 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 80%, 85%, 90%, 95%, 98%, or more nucleotide sequence identity to the nucleotide sequences of (a)-(h) above. However, it is understood that the nucleic acid molecules ofthe invention do not include nucleic acid molecules that consist solely ofthe nucleotide sequence of dbEST sequence accession nos.
  • nucleic acid molecules ofthe invention further include nucleotide sequences that encode polypeptides having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity to the polypeptides encoded by the nucleotide sequences of (a)-(h) 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 molecules are identical at that position.
  • the determination of percent identity between two sequences can also be 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.2J:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997, supra).
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used (see http://www.ncbi.nlm.nih.gov).
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package.
  • ALIGN program version 2.0
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • the nucleic acid molecules ofthe invention further include: (a) any nucleotide sequence that hybridizes to afsh23 nucleic acid molecule ofthe invention described in (a)-(h) above, under stringent conditions, e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45 °C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65 °C, or (b) under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6xSSC at about 45 °C followed by one or more washes in O.lx SSC/0.2% SDS at about 68 °C, or under other hybridization conditions which are apparent to those of skill in the art (see, for example, Ausubel F.M.
  • nucleic acid molecule that hybridizes under conditions described under (a) and (b), above is one that comprises the complement of a nucleic acid molecule that encodes afsh23 gene product.
  • nucleic acid molecules that hybridize under conditions (a) and (b), above encode gene products, e.g., gene products functionally equivalent to an fsh23 gene product.
  • the nucleic acids ofthe invention are human.
  • functionally equivalent fsh23 gene products include naturally occurring sA23 gene products present in the same or different species.
  • fsh23 gene sequences in non- human species map to chromosome regions syntenic to the human 18q chromosome location within which the particular human fsh23 lies.
  • Functionally equivalent fsh23 gene products also include gene products that retain at least one ofthe biological activities of a fsh23 gene product, and/or which are recognized by and bind to antibodies (polyclonal or monoclonal) directed against afsh23 gene product.
  • nucleic acid molecules ofthe invention are deoxyoligonucleotides ("oligos") which hybridize under highly stringent or stringent conditions to the fsh23 nucleic acid molecules described above.
  • Tm melting temperature
  • 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, e.g., to 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 ofthe invention further comprise the complements ofthe nucleic acids described above.
  • Such molecules can, for example, act as antisense molecules, useful, for example, in fsh23 gene regulation, and/or as antisense primers in amplification reactions offsh23 gene nucleic acid sequences.
  • Nucleic acid sequences ofthe invention encoding afsh23 gene product or complements thereof, may be used as part of ribozyme and/or triple helix sequences, also useful for fsh23 gene regulation.
  • nucleic acid molecules ofthe invention may be used as components of diagnostic methods whereby, for example, the presence of a particular fsh23 allele involved in a fsh23 -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.
  • diagnostic methods whereby, for example, the presence of a particular fsh23 allele involved in a fsh23 -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.
  • Fragments of the fsh23 nucleic acid molecules refer to fsh23 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, 50, 60, 70, 80, 85, 90, 95, 100 or more contiguous amino acid residues of the fsh23 gene products.
  • thefsh23 nucleic acid molecule encodes a gene product exhibiting at least one biological activity of a corresponding fsh23 gene product, e.g., afsh23 gene product.
  • Fragments of the fsh23 nucleic acid molecules can also refer to fsh23 exons or introns, and, further, can refer to portions offsh.23 coding regions that encode domains of, or mature, fsh23 gene products.
  • a nucleic acid molecule ofthe invention preferably comprises at least one of the following nucleotide sequences ofthe 18q interval: 28441-29265 (SEQ ID No. 18), 29683-39587 (SEQ ID No. 19), 40284-43253 (SEQ ID No. 20), 43518-46075 (SEQ ID No. 21), 47264-52284 (SEQ ID No. 22), 52672-56935 (SEQ ID No. 23), 57032-57726 (SEQ ID No. 24), 58065-59057 (SEQ ID No. 25), 59815-60471 (SEQ ID No. 26), 60870-62451 (SEQ ID No. 27), 62543-63268 (SEQ ID No.
  • the nucleic acid molecules ofthe 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, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, or more contiguous nucleotides of at least one ofthe above nucleotide sequences.
  • nucleotide sequences and nucleotide sequences ofthe 116 kb interval of human chromosome 18q can be readily obtained, for example, by utilizing standard sequencing and bacterial artificial chromosome (BAC) technologies and BACs described herein.
  • BAC bacterial artificial chromosome
  • DNA sequence polymorphisms of fsh23 nucleic acids or genomic sequences surrounding afsh23 nucleic acid will exist within a population of individual organisms (e.g., within a human population). Such polymo ⁇ hisms may exist, for example, among individuals within a population due to natural allelic variation. Such polymo ⁇ hisms 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.
  • 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.
  • Alternative alleles 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.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide ofthe invention. The term can further include nucleic acid molecules comprising upstream and/or exon intron sequences and structure.
  • allelic variants or polymo ⁇ hisms any and all nucleotide variations and resulting amino acid polymo ⁇ hisms or variations that are the result of natural allelic variation of thefsh23 gene are intended to be within the scope ofthe present invention.
  • Allelic variants or polymo ⁇ hisms include, but are not limited to, ones that do not alter the functional activity of the fsh23 gene product.
  • fsh23 polymo ⁇ hic sites include, but are not limited to: (a) an G/A polymo ⁇ hism present in the intron of exon B at nucleotide position 54 bp 3' of exon B (This corresponds to nucleotide position 3,177 of Figure 2 and nucleotide position 54,632 of Figure IB); (b) a G/A polymo ⁇ hism present in the intron of exon B at nucleotide position 522 bp 3' of exon B (This corresponds to nucleotide position 3,645 of Figure 2 and nucleotide position 55,100 of Figure IB); (c) a seven (7) bp deletion in the intron of exon C at nucleotide position 1,210 bp 5' of exon 3 (This corresponds to nucleotide positions 9,785-9,791 of Figure 2 and nucleotide positions 61,240-61,246 of Figure IB); and (d) a th
  • variants include, but are not limited to, fsh23 variants, e.g., allelic variants, comprising the following nucleotides: (a) a "G" at position 3,177 ofthe nucleotide sequence of FIG. 2, a “G” at position 54,632 ofthe nucleotide sequence of FIG. IB, a "A” at position 3,177 ofthe nucleotide sequence shown in FIG. 2, and a "A” at position 54,632 of the nucleotide sequence shown in FIG. IB; (b) an "G” at position 3,645 ofthe nucleotide sequence shown in FIG. 2, a "G” at position 55,100 ofthe nucleotide sequence of FIG.
  • allelic variants comprising the following nucleotides: (a) a "G” at position 3,177 ofthe nucleotide sequence of FIG. 2, a “G” at position 54,632 ofthe nucleotide sequence of FIG. IB, a "A" at
  • the isolated/s/z23 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 ofthe 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.
  • Appropriate stringency conditions are well known to those of skill in the art as discussed above, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook, et al, 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y.; and Ausubel, et al, 1989-1999, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., both of which are inco ⁇ orated herein by reference in their entirety.
  • afsh23 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 fsh23 gene product disclosed herein.
  • 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 s&2.3 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 afsh.23 -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 fsh23 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 fsh23 gene, such as, for example, brain tissue samples obtained through biopsy or post-mortem).
  • a reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end ofthe 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.
  • RNase H RNase H
  • second strand synthesis may then be primed with a poly-C primer.
  • cDNA sequences upstream ofthe amplified fragment may easily be isolated.
  • a cDNA of an allelic, e.g., mutant, variant of thefsh.23 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 individual putatively carrying a mutant fsh23 allele, and by extending the new strand with reverse transcriptase.
  • the second strand ofthe cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end ofthe normal gene.
  • 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.
  • DNA sequence analysis By comparing the DNA sequence of the mutant fsh23 allele to that ofthe normal fsh23 allele, the mutation(s) responsible for the loss or alteration of function ofthe mutant fsh23 gene product can be ascertained.
  • a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant fsh23 allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant fsh23 allele.
  • An unimpaired fsh23 gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant fsh23 allele in such libraries.
  • Clones containing the mutant fsh23 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 fsh23 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 the normal fsh23 gene product, as described, below, in Section 5.3.
  • For screening techniques see, for example, Harlow and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.)
  • a polyclonal set of anti-fsh23 gene product antibodies are likely to cross-react with the mutant fsh.23 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.
  • fsh23 mutations or polymo ⁇ hisms can further be detected using PCR amplification techniques.
  • Primers can routinely be designed to amplify overlapping regions ofthe whole fsh23 sequence including the promoter regulating region. In one embodiment, 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 ofthe nucleotide sequences ofthe preceding paragraphs.
  • nucleic acid molecules ofthe 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 fsh23 gene products ofthe invention include polypeptides, and fragments thereof, encoded by afsh23 nucleic acid sequence.
  • the fsh23 gene products ofthe invention are polypeptides comprising the amino acid sequence of FIGS. 3A-3C.
  • fsh23 gene products, or peptide fragments thereof can be prepared for a variety of uses.
  • such 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 s ⁇ 23-related disorder, such as a neuropsychiatric disorder, e.g., BAD.
  • fsh23 gene products can also be used as components of fusion proteins to impart a Fsh23 protein characteristic to another protein of interest.
  • afsh23 gene product, or fragment thereof could be used to facilitate the purification, localization, or recovery of the protein of interest, by providing an anti genie tag to the fusion protein.
  • s ⁇ 25 gene products have uses as amino acid and protein additives to foods, soaps, shampoos, cosmetics, and the like.
  • fsh23 gene products sometimes referred to herein as a "Fsh23 protein" includes those gene products encoded by the fsh23 gene sequences described in Section 5.1, above.
  • fsh23 gene products may include proteins that represent functionally equivalent (see Section 5.1 for a definition) gene products.
  • Such an equivalent fsh23 gene products 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 thefsh.23 gene sequences described, above, in Section 5.1, but that result in a "silent" change, in that the change produces a functionally equivalent fsh23 gene product.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature ofthe 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 sA25 gene products.
  • Such alterations can, for example, alter one or more ofthe biological functions of the fsh23 gene product. Further, such alterations can be selected so as to generate y-? ⁇ 23 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 a fsh23 protein as well as fusion proteins in which a Fsh23 protein or a portion of afsh23 protein such as a truncated Fsh23 protein or peptide or a Fsh23 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 thefsh23 nucleotide sequence disclosed in Section 5.1, above, and/or on the basis of the fsh23 amino acid sequence disclosed herein.
  • Fusion proteins include, but are not limited to, IgFc fusions which stabilize the Fsh23 protein or peptide 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 Fsh23 protein domains to an enzyme, fluorescent protein, luminescent protein, or a flag epitope protein or peptide which provides a marker function.
  • Fsh23 proteins ofthe invention also include Fsh23 protein sequences wherein domains encoded by at least one exon ofthe cDNA sequence, or fragments thereof, have been deleted.
  • the fsh23 polypeptides ofthe invention can further comprise posttranslational modifications, including, but not limited to glycosylations, acetylations, myristylations, and phosphorylations. If the native Fsh23 protein does not have recognition motifs that allow such modifications, it would be routine for one skilled in the art to introduce into afsh23 gene nucleotide sequences that encode motifs such as enzyme recognition signals so as to produce a modif ⁇ ed sA23 gene product.
  • the fsh23 gene products, peptide fragments thereof and fusion proteins thereof may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing the fsh23 gene polypeptides, peptides, fusion peptide and fusion polypeptides ofthe invention by expressing nucleic acid containing s/225 gene sequences are described herein.
  • Methods that are well known to those skilled in the art can be used to construct expression vectors containing s ⁇ 23 gene product coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • RNA capable of encoding fsh23 gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford.
  • host-expression vector systems may be utilized to express the fsh23 gene coding sequences ofthe invention.
  • 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 fsh23 gene product ofthe invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing s ⁇ 23 gene product coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the fsh23 gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculo virus) containing the fsh23 gene product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g.
  • plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the fsh23 gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of fsh23 protein or for raising antibodies to fsh23 protein, for example, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX 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.
  • fsh23 gene coding sequences may be cloned individually into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of fsh23 gene coding sequences will result in inactivation ofthe polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the protemaceous coat coded for by the polyhedrin gene).
  • afsh23 gene coding sequence of interest may be ligated to an adenovirus transcription translation control complex, e.g. , the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region ofthe viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing s/ ⁇ J gene product in infected hosts (e.g., see Logan and Shenk, 1984, Proc.
  • Specific initiation signals may also be required for efficient translation of inserted ⁇ 2J gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire fsh23 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 portion of the fsh23 gene coding sequence is inserted, 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 ofthe desired coding sequence to ensure translation ofthe 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 ⁇ l, 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 ofthe 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 ofthe foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe 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 fsh23 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 maybe 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 thefsh.23 gene product.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the fsh23 gene product.
  • a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler, et al, 1977, Cell 11 : 223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy, et al, 1980, 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 s ⁇ 23 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 fsh23 gene.
  • a heterologous DNA regulatory element for example, an endogenous fsh23 gene which is normally "transcriptionally silent", i.e., afsh23 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 fsh23 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 fsh23 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.
  • fsh23 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 maybe used to generate fsh23 transgenic animals.
  • transgenic refers to animals expressing s ⁇ i gene sequences from a different species (e.g., mice expressing human-fsh23 sequences), as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) fsh23 sequences or animals that have been genetically engineered to no longer express endogenous/s/z23 gene sequences (i.e., "knockout” animals), and their progeny. Any technique known in the art may be used to introduce an fsh23 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. Pat. No.
  • transgenic animal clones containing an fsh23 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 380: 64-66; Wilmut, et al, Nature 385: 810-813).
  • the present invention provides for transgenic animals that carry afsh23 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 ⁇ l, 1992, Proc. Natl. Acad. Sci. USA 89, 6232-6236).
  • fsh23 gene transgene be integrated into the chromosomal site ofthe endogenous fsh23 gene
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous fsh23 gene are designed for the pu ⁇ ose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function ofthe nucleotide sequence ofthe endogenous s ⁇ 25 gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous fsh23 gene in only that cell type, by following, for example, the teaching of Gu, et ⁇ l. (Gu, et ⁇ l., 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 ofthe recombinant fsh23 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 ofthe transgene has taken place.
  • the level of mRNA expression of the transgene in the tissues ofthe 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 of fsh.23 gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the fsh23 transgene product. fsh23 gene products, or peptide fragments thereof, can be prepared for a variety of uses.
  • such 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 s ⁇ 23-related disorder, such as a neuropsychiatric disorder, e.g., BAD.
  • a s ⁇ 23-related disorder such as a neuropsychiatric disorder, e.g., BAD.
  • fsh23 gene products include but are not limited to soluble derivatives such as peptides or polypeptides corresponding to one or more domains of the fsh23 gene product, particularly fsh23 gene products, that are modified such that they are deleted for one or more hydrophobic domains.
  • fsh23 gene products can be directly administered to a subject to treat a fsh23-related disorder, such as neuropsychiatric disorders, or a disorder of a fsh23-mediated process.
  • nucleotide constructs encoding such fsh23 gene products can be used to genetically engineer host cells to express such fsh23 gene products in vivo; these genetically engineered cells can function as "bioreactors" in the body delivering a continuous supply of fsh23 gene product, fsh23 peptides, or soluble- s z23 polypeptides.
  • antibodies capable of specifically recognizing one or more fsh23 gene product epitopes or epitopes of conserved variants or peptide fragments ofthe 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 ofthe above.
  • Such antibodies may be used, for example, in the detection of a fsh23 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 fsh23 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 ofthe effect of test compounds on fsh2 '3 gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described below, in Section 5.10.2, to, for example, evaluate the normal and/or engineered s ⁇ 2J-expressing cells prior to their introduction into the patient.
  • Anti-fsh23 gene product antibodies may additionally be used as a method for the inhibition of abnormal fsh23 gene product activity.
  • a ⁇ A23-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • Fsh23 For the production of antibodies against afsh23 gene product, various host animals may be immunized by injection with afsh23 gene product, or a portion thereof.
  • An antigenic portion of Fsh23 can be readily predicted by algorithms known in the art (e.g., see FIGS. 3A-3C).
  • polypeptides comprising amino acids 1 to 10, 30 to 45, or 78 to 84, or fragments thereof, ofthe Fsh23 amino acid sequence shown in FIGS. 3A-3C can be used as an antigen for production of such antibodies.
  • Such host animals may include, but are not limited to rabbits, mice, and rats, to name but a few.
  • Various 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 Coryneb ⁇ cterium p ⁇ rvum.
  • BCG Bacille Calmette-Guerin
  • Coryneb ⁇ cterium p ⁇ rvum bacille Calmette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as afsh23 gene product, or an antigenic functional derivative thereof.
  • an antigen such as afsh23 gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, may be immunized by injection -with fsh23 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe invention, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) ofthe sample is contaminating antibodies.
  • a purified antibody composition means that at least 99% ofthe antibodies in the composition are directed against the desired protein or polypeptide ofthe 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.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al, 1984, Proc. Natl. Acad. Sci., 81 :, 6851-6855; Neuberger, et al, 1984, Nature 312:, 604-608; Takeda, et al, 1985, Nature, 314: 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • 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.
  • 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 ofthe 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 ofthe 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 ofthe antibody molecule and the Fab fragments, which can be generated by reducing the disulfide bridges ofthe F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse, et al, 1989, Science, 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • nucleic acids ofthe invention including fsh23 nucleic acids, fsh23 gene products, including peptide fragments and fusion proteins thereof, and of antibodies directed against fsh23 gene products and peptide fragments thereof.
  • Such applications include, for example, prognostic and diagnostic evaluation of a fsh23-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 Section 5.5 and 5.8.
  • such applications include methods for the identification of compounds that modulate the expression of afsh23 gene and/or the synthesis or activity of afsh23 gene product, as described below, in Section 5.7, and for the treatment of a s * /z23-related disorder, e.g. a neuropsychiatric disorder, such as BAD, as described, below, in Section 5.10.
  • a s * /z23-related disorder e.g. a neuropsychiatric disorder, such as BAD, as described, below, in Section 5.10.
  • nucleic acid sequences ofthe invention including s/?2.? nucleic acid sequences and gene products, including peptide fragments and fusion proteins thereof, and antibodies directed against fsh23 gene products and peptide fragments thereof, have applications for pu ⁇ oses independent ofthe role fsh23 may have in neuropsychiatric disorders and processes.
  • s ⁇ 3 gene products including peptide fragments, as well as/s * -23-specific antibodies, can be used for construction of fusion proteins to facilitate recovery, detection, or localization of another protein of interest.
  • nucleic acid molecules ofthe invention including fsh23 nucleic acid sequences, and fsh23 gene products can be used for genetic mapping, i.e, refining the genetic map of chromosome 18q.
  • nucleic acid molecules ofthe invention including fsh23 nucleic acid sequences, and fsh23 gene products can be used for genetic mapping, i.e, refining the genetic map of chromosome 18q.
  • antibodies specific to Fsh23 can be used as probes to detect expression of human Fsh23 in somatic cell hybrids containing human chromosomes, or portions thereof.
  • Nucleic acids ofthe invention including fsh23 nucleic acids, and fsh23 gene sequences and gene products can be used for mapping and refining the map of chromosome 18.
  • the sequence of the 116 kb region of the human chromosome 18q can used to develop new genetic markers that can be used to further refine 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 microsatelhtes.
  • Microsatelhtes also known as simple- sequence repeats (SSRs), are hypervariable tandem-sequence repeats consisting of di-, tri-, or tetranucleotide repeats of 1-5 nucleotides. Such microsatelhtes 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 polymo ⁇ hic. Relatively common microsatelhtes (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 polymo ⁇ hic sites useful for fine mapping, such as minisatellites (9-64 nucleotide repeats), restriction fragment length polymo ⁇ hisms (RFLPs), and single nucleotide polymo ⁇ hisms, which occur much less frequently.
  • minisatellites 9-64 nucleotide repeats
  • RFLPs restriction fragment length polymo ⁇ hisms
  • single nucleotide polymo ⁇ hisms which occur much less frequently.
  • Genomic DNA from human populations can then be analyzed for the such simple-sequence length polymo ⁇ hisms (SSLPs) to determine the frequency and variability ofthe repeat.
  • SSLP simple-sequence length polymo ⁇ hisms
  • the interval can be refined by linkage analysis on an affected population to determine whether an 18q-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 polymo ⁇ hisms 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).
  • afsh23 gene, protein or a fragment or domain thereof can be used for construction of fusion proteins.
  • fsh23 nucleic acids and gene products have generic uses, such as supplemental sources of nucleic acids, proteins and amino acids for food additives or cosmetic products.
  • fsh23 polypeptides, nucleic acids, and modulators thereof can be used to modulate the function, mo ⁇ hology, proliferation and/or differentiation of cells in the tissues in which it is expressed.
  • Such molecules can be used to treat disorders associated with abnormal or aberrant metabolism or function of cells in the tissues in which it is expressed.
  • Tissues in which fsh23 is expressed include, for example, without limitation, brain, testis, trachea, mammary gland, and uterus.
  • fsh23 polypeptides, nucleic acids, or modulators thereof can be used to treat disorders ofthe brain, such as cerebral edema, hydrocephalus, brain herniations, iatrogenic disease (due to, e.g., infection, toxins, or drugs), inflammations (e.g., bacterial and viral meningitis, encephalitis, and cerebral toxoplasmosis), cerebrovascular diseases (e.g., hypoxia, ischemia, and infarction, intracranial hemorrhage and vascular malformations, and hypertensive encephalopathy), and tumors (e.g., neuroglial tumors, neuronal tumors, tumors of pineal cells, meningeal tumors, primary and secondary lymphomas, intracranial tumors, and medulloblastoma), and to treat injury or trauma to the brain.
  • disorders ofthe brain such as cerebral edema, hydrocephalus, brain herniations, iatrogenic disease (due
  • testicular disorders such as unilateral testicular enlargment (e.g., nontuberculous, granulomatous orchitis), inflammatory diseases resulting in testicular dysfunction (e.g., gonorrhea and mumps), and tumors (e.g., germ cell tumors, interstitial cell tumors, androblastoma, testicular lymphoma and adenomatoid tumors).
  • unilateral testicular enlargment e.g., nontuberculous, granulomatous orchitis
  • inflammatory diseases resulting in testicular dysfunction e.g., gonorrhea and mumps
  • tumors e.g., germ cell tumors, interstitial cell tumors, androblastoma, testicular lymphoma and adenomatoid tumors.
  • the FSH23 polypeptides, nucleic acids and/or modulators thereof can be used to modulate the function, mo ⁇ hology, proliferation and/or differentiation of cells in the tissues in which it is expressed.
  • the FSH23 polypeptides, nucleic acids and/or modulators thereof can be used modulate the function, mo ⁇ hology, proliferation and/or differentiation ofthe ovaries.
  • such molecules can be used to treat or modulate disorders associated with the ovaries, including, without limitation, ovarian tumors, McCune- Albright syndrome (polyostotic fibrous dysplasia).
  • the FSH23 polypeptides, nucleic acids and/or modulators can be used in the treatment of infertility.
  • fsh23 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, differentiation, and/or function of bone and cartilage cells, e.g., chondrocytes and osteoblasts, and to treat bone and/or cartilage associated diseases or disorders.
  • bone and/or cartilage diseases and disorders include bone and/or cartilage injury due to for example, trauma (e.g., bone breakage, cartilage tearing), degeneration (e.g., osteoporosis), degeneration of joints, e.g., arthritis, e.g., osteoarthritis, and bone wearing.
  • fsh23 is expressed in the kidney
  • the fsh23 polypeptides, nucleic acids and/or modulators thereof can be used to modulate the function, mo ⁇ hology, proliferation and/or differentiation of cells in the tissues in which it is expressed.
  • Such molecules can also be used to treat disorders associated with abnormal or aberrant metabolism or function of cells in the tissues in which it is expressed.
  • Such molecules can be used to treat or modulate renal (kidney) disorders, such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal prohferative glomerulonephritis, glomerular lesions associated with systemic disease, such as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug and toxin induced tubulointerstitial nephritis, hypercalcemic nephropathy, and hypokale
  • fsh23 polypeptides, nucleic acids, or modulators thereof can be used to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis) hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis) cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), or malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).
  • hepatic (liver) disorders such as jaundice, hepatic failure, her
  • fsh23 nucleic acids, proteins, and modulators thereof can be used to treat cardiovascular disorders, such as ischemic heart disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, pulmonary heart disease, valvular heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., valvular and vascular obstructive lesions, atrial or ventricular septal defect, and patent ductus arteriosus), or myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy), atherosclerosis, hypertension, and angina pectoris.
  • ischemic heart disease e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease
  • 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 fsh23 gene-specific mutations or polymo ⁇ hisms, (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. 5.5.1. DETECTION OF MUTATIONS OR POLYMORPHISMS
  • a variety of methods can be employed to screen for the presence of fsh2 '3 gene-specific mutations or polymo ⁇ hisms (including polymo ⁇ hisms flanking afsh23 gene, e.g., ones that cosegregate with a particular fsh23 allele) and to detect and/or assay levels of fsh23 nucleic acid sequences.
  • Mutations or polymo ⁇ hisms within or flanking the fsh23 gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell, or any cell that expresses the fsh23 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. fsh23 nucleic acid sequences may be used in hybridization or amplification assays of biological samples to detect abnormalities involving fsh23 gene structure, including point mutations, insertions, deletions, inversions, translocations and chromosomal rearrangements. Such assays may include, but are not limited to, Southern analyses, single-stranded conformational polymo ⁇ hism analyses (SSCP), and PCR analyses.
  • SSCP single-stranded conformational polymo ⁇ hism analyses
  • Diagnostic methods for the detection of fsh23 gene-specific mutations or polymo ⁇ hisms 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 fsh23 gene.
  • patient sample biological sample or appropriate cellular source refers to a sample of tissue or fluid suspected of containing a mutated or non-mutated fsh23 polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentisis, spinal fluid, lymph fluid, bone marrow, the external sections ofthe 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 fsh23 polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentisis, spinal fluid, lymph fluid, bone marrow, the external sections ofthe skin, respiratory,
  • fsh23 gene contains a mutation
  • blood can be drawn and DNA extracted from the cells ofthe blood.
  • prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for mutations ofthe fsh23 gene.
  • Alteration of a wild-type fsh23 allele, whether, for example, by point mutation or deletion, can be detected by any ofthe means discussed herein.
  • the biological sample to be analyzed such as blood, plasma, serum, ascites, pleural effusion, thoracentisis, spinal fluid, lymph fluid, bone marrow, the external sections ofthe 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 ofthe target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting.
  • the targeted region of the fsh23 nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence ofthe 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 ofthe present invention further encompass contacting and incubating nucleic acids for the detection of single nucleotide mutations or polymo ⁇ hisms of the fsh23 gene.
  • these nucleic acid reagent sequences within the fsh23 gene, or chromosome 18q nucleotide sequences flanking the fsh23 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 ofthe 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.,fsh23 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 fsh23 gene sequence, in order to determine whether afsh23 gene mutation or a cosegrating polymo ⁇ hism of interest is present.
  • fsh23 mutations or polymo ⁇ hisms can be detected by using a microassay of sh23 nucleic acid sequences immobilized to a substrate or "gene chip" (see, e.g. Cronin, et al., 1996, Human Mutation 7:244-255).
  • fsh23 flanking sequences e.g., sequences present in the nucleotide sequence shown in FIG. IB
  • PCR the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,2 ' 02
  • analysis ofthe 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 fsh23 gene in order to determine whether afsh23 gene mutation or polymo ⁇ hism in linkage disequilibrium with a disease-causing/ ⁇ :? allele exists.
  • genotyping techniques can be performed to identify individuals carrying s ⁇ 2J gene mutations. Such techniques include, for example, the use of restriction fragment length polymo ⁇ hisms (RFLPs), which involve sequence variations in one ofthe recognition sites for the specific restriction enzyme used.
  • RFLPs restriction fragment length polymo ⁇ hisms
  • Markers that are so closely spaced exhibit a high frequency co-inheritance, and are extremely useful in the identification of genetic mutations, such as, for example, mutations within the fsh23 gene, and the diagnosis of diseases and disorders related to fsh23 mutations.
  • Caskey et ⁇ l. (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 fsh23 gene, amplifying the extracted DNA, and labeling the repeat sequences to form a genotypic map ofthe individual's DNA.
  • SNPs single nucleotide polymo ⁇ hisms
  • 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 polymo ⁇ hism (SSCP) analysis (see, e.g., Orita et al, 1989, Proc. Natl. Acad. Sci.
  • 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. 56:5855-5892; Grompe, 1993, N ⁇ twre Genetics 5: 11-117).
  • preferred methods of detecting and mapping S ⁇ Ps involve microsequencing techniques wherein an S ⁇ P site in a target D ⁇ A 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 fsh23 gene expression can also be assayed.
  • RNA from a cell type or tissue known, or suspected, to express the fsh23 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 fsh23 gene.
  • Such analyses may reveal both quantitative and qualitative aspects ofthe expression pattern of the fsh23 gene, including activation or inactivation of fsh23 gene expression.
  • a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription ofthe 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 fsh23 gene nucleic acid reagents described in Section 5.1 that contain afsh23 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.
  • fsh23 gene expression assays "in situ", i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
  • Nucleic acid reagents described in Section 5.1 that contain afsh23 gene or nucleic acid sequence maybe used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, “PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY).
  • Standard Northern analysis can be performed to determine the level of mRNA expression of the fsh23 gene.
  • nucleic acid molecules described herein or fragments thereof can be used to map the location ofthe corresponding genes on a chromosome.
  • nucleic acid molecules described herein can be used to map the chromosomal location of fsh23 homologues in various species. Such mapping information can be used, for example, for analysis ofthe activity of fsh23 transgenes in mice.
  • the nucleic acid molecules can further be used to map the location of copies of fsh23 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 ofthe invention.
  • Computer analysis ofthe sequence of a gene ofthe 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 ofthe 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 ofthe genes actually are preferred for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • afsh23 polypeptide and fragments and sequences thereof and antibodies specific thereto can be used to map the location ofthe gene encoding the polypeptide on a chromosome.
  • This mapping can be carried out by specifically detecting the presence ofthe 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 ofthe polypeptide in the somatic cell hybrids can be determined by assaying an activity or property ofthe polypeptide, for example, enzymatic activity, as described in Bordelon-Riser et al. (1919) 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 ofthe invention can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe 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 polymo ⁇ hisms.
  • the nucleic acid sequences ofthe 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 polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • 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 ofthe present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences ofthe 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 ofthe 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 ofthe present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the nucleic acid sequences ofthe invention uniquely represent portions ofthe 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 ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses.
  • the noncoding sequences of FIG. IB can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those shown in FIG. 3 A, 3B, and 3C are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • 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 pe ⁇ etrator 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 compared to a standard, thereby allowing identification ofthe origin ofthe biological sample.
  • sequences ofthe 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 polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the nucleic acid sequences ofthe invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
  • the fsh23 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) pu ⁇ oses to thereby treat an individual prophylactically. Accordingly, one aspect ofthe present invention relates to diagnostic assays for determining fsh23 protein and/or nucleic acid expression as well as fsh23 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 fsh23 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 fsh23 protein, nucleic acid expression or activity. For example, mutations in an fsh23 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with an fsh23 protein, nucleic acid expression or activity.
  • determinations may be based on the normalized expression levels of these genes.
  • Expression levels are normalized by correcting the absolute expression level of an fsh23 gene by comparing its expression to the expression of a gene that is not an fsh23 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 ofthe 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.
  • the expression level can be provided as a relative expression level.
  • the level of expression ofthe gene is determined for 10 or more samples of different cell isolates, preferably 50 or more samples, prior to the determination ofthe 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 fsh23 family members, expression was observed in the brain.
  • the mean expression level of each ofthe 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 ofthe gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of a fsh23-mediated disease.
  • diseases which may be studied include, without limitation, those associated with tissues ofthe brain.
  • the samples used in the baseline determination will be from an fsh23-mediated diseased or from non-diseased cells of tissue.
  • the choice ofthe cell source is dependent on the use ofthe relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the fsh23 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 fsh23 gene can serve as a target gene.
  • the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from brain cells provides a means for grading the severity ofthe fsh23 -mediated disease state.
  • Another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of fsh23 in clinical trials.
  • agents e.g., drugs, compounds
  • Antibodies directed against unimpaired or mutant fsh23 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 /s/z23-related disorder, e.g., neuropsychiatric disorder, such as BAD, as described herein.
  • a /s/z23-related disorder e.g., neuropsychiatric disorder, such as BAD, as described herein.
  • Such methods may be used to detect abnormalities in the level of ' fsh23 gene product synthesis or expression, or abnormalities in the structure, temporal expression, and/or physical location of fsh23 gene product.
  • the antibodies and immunoassay methods described below have, for example, important in vitro applications in purifying s/z25 gene products and in assessing the efficacy of treatments for s ⁇ 23-related disorders, e.g., neuropsychiatric disorders, such as BAD.
  • Antibodies, or fragments of antibodies, such as those described below maybe used to screen potentially therapeutic compounds in vitro to determine their effects on fsh23 gene expression and fsh23 peptide production.
  • the compounds that have beneficial effects on a s ⁇ 23-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 s ⁇ 23-related disorder, e.g. , a neuropsychiatric disorder, such as BAD.
  • Antibodies directed against fsh23 peptides may be used in vitro to determine, for example, the level of fsh23 gene expression achieved in cells genetically engineered to produce sA2.? peptides.
  • intracellular fsh23 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 ofthe gene replacement protocol.
  • the tissue or cell type to be analyzed will generally include those that are known, or suspected, to express the fsh23 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 fsh23 gene.
  • Preferred diagnostic methods for the detection of fsh23 gene products or conserved variants or peptide fragments thereof may involve, for example, immunoassays wherein the fsh23 gene products or conserved variants or peptide fragments are detected by their interaction with an anti-fsh23 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 fsh23 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.
  • fluorescently labeled antibody see below, this section
  • fluorimetric detection are especially preferred for fsh23 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 fsh23 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 ofthe present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays for fsh23 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 s/z2.? 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 fsh23 polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentisis, spinal fluid, lymph fluid, bone marrow, the external sections ofthe 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 s , ⁇ 23 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 the fsh23 gene products, or conserved variants or peptide fragments thereof, which may be detected in the blood or any one ofthe patient samples listed supra using a detectably labeled antibody capable of identifying s/?25 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 fsh23 gene products or conserved variants or peptide fragments thereof are about 10 ng/ml to about 90 ng/ml, about 20 ng/ml to about 80 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 fsh23 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 a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins.
  • a 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 fsh23 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 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 ofthe carrier can be either soluble to some extent or insoluble for the pu ⁇ oses ofthe 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 anti-fsh23 gene product antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • 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, 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 be accomplished by visual comparison ofthe extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • 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 ofthe 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 chemilummescent compound. The presence ofthe chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemilummescent 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 ofthe chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for pu ⁇ oses 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 (IJ) (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 conjugates ofthe invention can be used for modifying a given biological response, 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 ("IL-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.
  • IL-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 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 SEQ ID No. 1, 2, 3, 4, 6, 7, 9, 10, 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, the amino acid sequence of SEQ ID Nos.
  • the substantially purified antibodies ofthe 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, 6, 7, 9, 10, 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, the amino acid sequence of SEQ ID Nos.
  • non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
  • the non-human antibodies ofthe invention can be chimeric and/or humanized antibodies.
  • the non-human antibodies ofthe 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 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, 6, 7, 9, 10, 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, the amino acid sequence of SEQ ID Nos.
  • the monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.
  • the 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 ofthe invention.
  • the substantially purified antibodies or fragments thereof, the human or non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, ofthe invention specifically bind to a secreted sequence or an extracellular domain of an amino acid sequence encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID No.
  • any ofthe antibodies ofthe invention can be conjugated to a therapeutic moiety or to a detectable substance.
  • detectable substances that can be conjugated to the antibodies ofthe 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 ofthe invention conjugated to a detectable substance, and instructions for use.
  • Still another aspect ofthe invention is a pharmaceutical composition comprising an antibody ofthe invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains an antibody ofthe invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
  • Still another aspect ofthe invention is a method of making an antibody that specifically recognizes an fsh23 gene product, 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 ID Nos.
  • a sample is collected from the mammal that contains an antibody that specifically recognizes an FSH26 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 monoclonal antibody- producing cell from the cells ofthe mammal.
  • antibodies are collected from the antibody-producing cell.
  • the following assays are designed to identify compounds that bind to afsh23 gene product, e.g., proteins or portions of proteins that interact with afsh23 gene product, compounds that interfere with the interaction of afsh23 gene product with other proteins and compounds that modulate the activity of fsh2 '3 gene (i.e., modulate the level of sh23 gene expression and or modulate the level of fsh23 gene product activity).
  • Assays may additionally be utilized that identify compounds that bind to fsh23 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 fsh23 gene or some other gene or gene product involved in afsh23 regulatory pathway.
  • proteins that interact with afsh23 gene or gene product are described, below, in Section 5.7.2. Such proteins may be involved in the control and/or regulation of mood. Further, among these compounds are compounds that affect the level of fsh23 gene expression and/or fsh23 gene product activity and that can be used in the therapeutic treatment of sh23 disorders, e.g., neuropsychiatric disorders such as BAD, as described, below, in Section 5.10.
  • sh23 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 fsh23 gene product, and for ameliorating s ⁇ 23-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 afsh2 J-mediated process and/or to ameliorate symptoms of a JA23-related disorder.
  • Assays for testing the effectiveness of compounds identified by, for example, techniques such as those described in Sections 5.7.1 - 5.7.3, are discussed, below, in Section 5.7.5.
  • In vitro systems may be designed to identify compounds that bind fsh23 gene products ofthe invention.
  • Compounds identified may be useful, for example, in modulating the activity of unimpaired and/or mutant fsh23 gene products, may be useful in elucidating the biological function of the fsh23 gene product, may be utilized in screens for identifying compounds that disrupt normal fsh23 gene product interactions, or may in themselves disrupt such interactions, and can provide lead compounds to be further tested for an ability to modulate a fsh23 -mediated process and/or to ameliorate symptoms of a s/*25-related disorder.
  • the principle ofthe assays used to identify compounds that bind to fsh2 '3 gene products involves preparing a reaction mixture of the fsh23 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 s/z2 ? gene product or the test substance onto a solid phase and detecting s/j2.5 gene product/test compound complexes anchored on the solid phase at the end ofthe reaction.
  • the fsh23 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 ofthe 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 fsh23 gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component ofthe possible complex to detect anchored complexes.
  • Any method suitable for detecting protein-protein interactions may be employed for identifying ⁇ 2J protein-protein interactions.
  • 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 afsh23 protein. These methods include, for example, probing expression libraries with labeled s/*23 protein, using fsh23 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 ofthe DNA-binding domain of a transcription activator protein fused to the fsh23 gene product and the other consists ofthe 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 ofthe yeast S ⁇ cch ⁇ romyces 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.
  • fsh23 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 fsh23 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 fsh23 gene sequence such as the open reading frame of the fsh23 gene, can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain ofthe 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 plasmids.
  • a cDNA library ofthe cell line from which proteins that interact with the bait fsh23 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 activation domain of GALA
  • This library can be co-transformed along with the bait fsh23 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 fsh23 gene product will reconstitute an active GAL4 protein and thereby drive expression ofthe HIS3 gene.
  • 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 fsh23 gene-interacting protein using techniques routinely practiced in the art.
  • fsh23 gene products ofthe invention may, in vivo, interact with one or more macromolecules, including cellular or extracellular macromolecules, such as proteins.
  • macromolecules may include, but are not limited to, other proteins, such as cellular receptors, or nucleic acid molecules and those proteins identified via methods such as those described, above, in Sections 5.7.1 - 5.7.3.
  • binding partners the macromolecules are referred to herein as "binding partners”.
  • Bind partners Compounds that disrupt fsh23 binding in this way may be useful in regulating the activity of the fsh23 gene product, especially mutant fsh23 gene products.
  • 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 afsh23 gene product.
  • the basic principle ofthe assay systems used to identify compounds that interfere with the interaction between the fsh23 gene product and its binding partner or partners involves preparing a reaction mixture containing the fsh23 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 ofthe test compound.
  • the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the fsh23 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 fsh23 gene protein and the binding partner is then detected.
  • complex formation within reaction mixtures containing the test compound and normal fsh23 gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant fsh23 gene protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal fsh23 gene proteins.
  • the assay for compounds that interfere with the interaction of the fsh23 gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the fsh23 gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end ofthe 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 fsh23 gene products and the binding partners can be identified by conducting the reaction in the presence ofthe test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the fsh23 gene protein and interactive binding partner.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one ofthe 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 fsh23 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 fsh23 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.
  • the partner ofthe immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and 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.
  • 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).
  • the antibody in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody.
  • 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 ofthe test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one ofthe 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 fsh23 gene protein and the interactive binding partner is prepared in which either the fsh23 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 ofthe species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt afsh23 gene protein/binding partner interaction can be identified.
  • the fsh23 gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.2. above.
  • the fsh23 coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, 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-fsh23 fusion protein can be anchored to glutathione-agarose beads.
  • the interactive binding partner can then be added in the presence or absence ofthe 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 the fsh23 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 ofthe interaction by the test compound will result in a decrease in measured radioactivity.
  • the GST-fsh23 gene fusion protein and the interactive binding partner can be mixed together in liquid in the absence ofthe 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 fsh23 gene product/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
  • these same techniques can be employed using peptide fragments that correspond to the binding domains of the fsh23 protein and/or the interactive or binding partner (in cases where the binding partner is a protein), in place of one or both ofthe full length proteins.
  • Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis ofthe gene encoding one ofthe 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 ofthe genes encoding the respective proteins will reveal the mutations that correspond to the region ofthe 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 ofthe protein, which can then be tested for binding activity and purified or synthesized.
  • a proteolytic enzyme such as trypsin
  • afsh23 gene product can be anchored to a solid material as described, above, in this section by making a GST-fsh23 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-fsh23 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.
  • a s ⁇ 23-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-LI); unipolar affective disorders, such as unipolar major depressive disorder (MDD), dysthymic disorder; obsessive-compulsive disorders; phobias, e.g., agoraphobia; panic disorders; generalized anxiety disorders; somatization disorders and hypo
  • 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
  • the assays described herein can identify compounds that affect s ⁇ 23 gene activity by either affecting s , /z23 gene expression or by affecting the level of fsh23 gene product activity.
  • compounds may be identified that are involved in another step in the pathway in which the fsh23 gene and/or fsh23 gene product is involved and, by affecting this same pathway may modulate the effect of fsh23 on the development of a neuropsychiatric disorder such as BAD.
  • Such compounds can be used as part of a therapeutic method for the treatment ofthe disorder.
  • a fsh23-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 sA23-related disorder, e.g. , a neuropsychiatric disorder, such as BAD.
  • a sA23-related disorder e.g. , 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 fsh23 gene.
  • cells that express fsh23 may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of a s/z2.?-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.
  • a neuropsychiatric disorder such as BAD
  • the cells can be assayed to measure alterations in the expression of the fsh23 gene, e.g., by assaying cell lysates for fsh23 mRNA transcripts (e.g., by Northern analysis) or for fsh23 gene products expressed by the cell; compounds that modulate expression of the fsh23 gene are good candidates as therapeutics.
  • the cells are examined to determine whether one or more cellular phenotypes associated with a s/*23-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 s/*23-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • animal-based systems or models for a_/s ⁇ 23-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • 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 fsh23 sequences or, alternatively, to no longer express endogenous fsh23 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 afsh23- related disorder, e.g., a neuropsychiatric disorder, such as BAD, in the exposed animals.
  • afsh23-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • the response ofthe animals to the exposure maybe monitored by assessing the reversal of such symptoms.
  • any treatments that reverse any aspect of symptoms of a/s/z23-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • a neuropsychiatric disorder such as BAD
  • Dosages of test agents may be determined by deriving dose-response curves, as discussed in Section 5.10.1, below. 5.8. METHODS FOR DIAGNOSIS AND PROGNOSTICATION OF fsh23 - RELATED-DISORDERS
  • a variety of methods can be employed for the diagnostic and prognostic evaluation of s ⁇ 23-related disorders, such as neuropsychiatric disorders, e.g., BAD, and for the identification of subjects having a predisposition to such disorders.
  • s ⁇ 23-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 fsh23 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 fsh23 gene products, including peptide fragments thereof, as described, above, in Section 5.3.
  • reagents may be used, for example, for:
  • fsh2 '3 gene mutations the detection of the presence of fsh2 '3 gene mutations, the detection of polymo ⁇ hisms that cosegregate with particular fsh23 gene mutations or the detection of either over- or under-expression of fsh23 gene mRNA relative to the state of a s/z23-related disorder, such as a neuropsychiatric disorder, e.g., BAD;
  • a neuropsychiatric disorder e.g., BAD
  • Nucleic acid molecules ofthe invention can, for example, be used to diagnose an fsh23- related or neuropsychiatric disorder using, for example, the techniques for fsh23 mutation/co- segregating polymo ⁇ hism detection described above.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one specific nucleic acid ofthe invention or anti- fsh23 gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting abnormalities of a s/z23-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a s/z23-related disorder e.g., 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 fsh23 gene is expressed may be utilized.
  • Nucleic acid-based detection techniques are described, above, in Section 5.5.
  • Peptide detection techniques are described, above, in Section 5.6.
  • 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 ofthe 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 ofthe 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, thoracentisis, 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 ofthe 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 ofthe
  • 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 ofthe invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence ofthe 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 ofthe polypeptide).
  • the methods ofthe invention can also be used to detect genetic lesions or mutations in a gene ofthe invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide ofthe 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 ofthe invention, or the mis-expression ofthe gene encoding the polypeptide ofthe 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 ofthe gene; 4) a chromosomal rearrangement ofthe gene; 5) an alteration in the level of a messenger RNA transcript ofthe gene; 6) an aberrant modification ofthe gene, such as ofthe methylation pattern ofthe genomic DNA; 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript ofthe gene; 8) a non- wild type level of a the protein encoded by the gene; 9) an allelic loss ofthe gene; and 10) an inappropriate post-translational modification ofthe protein encoded by the gene.
  • assay techniques known in the art which can be used for detecting lesions in a gene.
  • detection ofthe 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 4,683,202), 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 ofthe 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 ofthe gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe 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 ofthe 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 ofthe 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 ohgonucleotides 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 ohgonucleotides 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 ofthe 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. 38:147-159).
  • 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.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • 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.
  • either 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 ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. (See, e.g., Cotton et al, 1988, Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al., 1992, Methods Enzymol. 217:286-295.)
  • the 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.
  • single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al., 1989, Proc. Natl.
  • Single-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • 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).
  • allele specific ohgonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the ohgonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.
  • Ohgonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization) (Gibbs et al., 1989, Nucleic Acids Res.
  • 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 ofthe 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 prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which maybe 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 ofthe invention.
  • any cell type or tissue, preferably peripheral blood leukocytes, in which the polypeptide ofthe invention is expressed may be utilized in the prognostic assays described herein.
  • kits that facilitate the use /and or detection of fsh23 genes or co-segregating polymo ⁇ hisms and fsh23 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 ofthe 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 sA23 genes or gene products is provided.
  • 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 ofthe invention.
  • a kit is provided, with one or more containers comprising a pair of primers useful for amplifying afsh23 nucleic acid molecule encoding afsh23 polypeptide ofthe 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 ofthe kit is usually enclosed within an individual container and all ofthe 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 ofthe polypeptide.
  • Such a kit 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.
  • kits for detecting the presence of a polypeptide ofthe invention in a biological sample 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 ofthe invention as discussed, for example, in sections above relating to uses ofthe sequences ofthe invention.
  • a kit is provided, with one or more containers comprising: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide ofthe 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.
  • kits can be used to determine if a subject is suffering from or is at increased risk of a/s z23-related disorder, such as a neuropsychiatric disorder, e.g., BAD. 5.10. COMPOSITIONS AND METHODS FOR TREATMENT OF fsh23- RELATED DISORDERS
  • a s ⁇ 23-mediated process can be modulated and/or whereby a symptom of a s&23-related disorder, e.g., a symptom of a neuropsychiatric disorder, such as a cognitive or mood disorder, for example, BAD, may be treated.
  • a symptom of a s&23-related disorder e.g., a symptom of a neuropsychiatric disorder, such as a cognitive or mood disorder, for example, BAD, may be treated.
  • Such a method can comprise administering a compound which modulates the expression of afsh23 gene and/or the expression or activity of afsh23 gene product, so that the process is modulated or a symptom ofthe disorder is ameliorated.
  • a s ⁇ 23-related disorder phenotype or symptom can occur as a result of a decrease in expression or activity of a component of a Fsh23-mediated pathway, such as a Fsh23 receptor, ligand, or an upstream or downstream conponent of a Fsh23 signal transduction pathway.
  • a component of a Fsh23-mediated pathway such as a Fsh23 receptor, ligand, or an upstream or downstream conponent of a Fsh23 signal transduction pathway.
  • increasing the level of sh23 gene expression and/or fsh23 gene product expression or activity could facilitate the progress towards an asymptomatic state.
  • a method for treating such afsh23- related disorder phenotype can comprise administering to a subject a Fsh23 peptide or polypeptide, or fragment, analog or mimetic thereof, to ameliorate at least one symptom of a fsh23-related disorder phenotype.
  • a method for treating such afsh23- related disorder phenotype can comprise administering to a subject a compound that modulates the activity or expression of afsh23 gene or gene product to ameliorate at least one symptom of a s ⁇ 23-related disorder phenotype.
  • such methods can comprise administering a compound that increases the activity or expression of a fsh23 gene or gen product.
  • a loss of normal fsh23 gene product function results in the development of a/s ⁇ 23-related disorder phenotype, e.g., a neuropsychiatric disorder phenotype
  • an increase in fsh23 gene product activity would facilitate progress towards an asymptomatic state in individuals exhibiting a deficient level of fsh23 gene expression and/or fsh23 gene product activity.
  • a method for treating such a fsh23 -related disorder phenotype can comprise supplying a subject with a nucleic acid molecule encoding an unimpaired fsh23 gene product, such that an unimpaired fsh23 gene product is expressed and symptoms ofthe disorder are ameliorated.
  • methods for the treatment of mammalian fsh23 -related disorder e.g. , a neuropsychiatric disorders
  • methods for the treatment of mammalian fsh23 -related disorder can comprise supplying a mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired fsh23 gene product such that the cell expresses the unimpaired/s/z23 gene product and symptoms ofthe disorder are ameliorated.
  • methods for enhancing the expression or synthesis of a fsh23 gene or gene product can include, for example, methods such as those described below, in Section 5.10.2.
  • symptoms of a s ⁇ 23-related disorder phenotype may be ameliorated by administering a compound that decreases the level of fsh23 gene expression and/or fsh23 gene product activity.
  • a method comprises administering an anti-Fsh23 antibody to a subject to ameliorate at least one symptom of s/*23-related disorder phenootype.
  • any ofthe compounds identified by the screening methods described in Section 5.7, above may be administered to an individual to treat a symptom of a s ⁇ 23-related disorder phenotype.
  • Methods for inhibiting or reducing the level of fsh23 synthesis or expression can include, for example, methods such as those described in Section 5.10.1.
  • 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), 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
  • antipsychotic drugs such as lithium salts,
  • symptoms of certain s ⁇ 23-related disorders such as neuropsychiatric disorders, e.g. , BAD
  • symptoms of certain s ⁇ 23-related disorders may be ameliorated by decreasing the level of fsh23 gene expression and/or fsh23 gene product activity by using fsh23 nucleic acid sequences in conjunction with well-known antisense, gene "knock-out,” ribozyme and/or triple helix methods to decrease the level of ' fsh23 gene expression.
  • a s ?23-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • antisense, ribozyme, and triple helix molecules Such molecules may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
  • 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 ohgonucleotides that are complementary to a target gene mRNA. The antisense ohgonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand ofthe 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 ofthe antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point ofthe hybridized complex.
  • ohgonucleotides complementary to coding or non-coding regions of the fsh23 gene can be used in an antisense approach to inhibit translation of endogenous fsh23 mRNA.
  • Antisense nucleic acids should be at least six nucleotides in length, and are preferably ohgonucleotides ranging from 6 to about 50 nucleotides in length.
  • 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 ofthe 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 ohgonucleotides. It is also preferred that these studies compare levels ofthe 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 ofthe oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the ohgonucleotides 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 ofthe molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989, Proc. Natl. Acad. Sci. U.S.A.
  • the 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.
  • Ohgonucleotides ofthe 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.). As examples, phosphorothioate ohgonucleotides maybe synthesized by the method of Stein, et al. (1988, Nucl. Acids Res.
  • methylphosphonate ohgonucleotides 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 fsh23 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 III or pol II promoter.
  • the use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation ofthe target gene mRNA.
  • a vector can be introduced e.g. , such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression ofthe 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, 787- 797), the he ⁇ es thymidine kinase promoter (Wagner, et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster, et al, 1982, Nature 296, 39-42), etc.
  • 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 ofthe 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 inco ⁇ orated 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 inco ⁇ orated herein by reference in its entirety.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end ofthe target gene mRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non- functional mRNA transcripts.
  • the ribozymes ofthe present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one that occurs naturally in Tetrahymena thermophila (known as the TVS, or L-19 INS R ⁇ A) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al, 1984, Science, 224, 574-578; Zaug and Cech, 1986, Science, 231, 470-475; Zaug, et al, 1986, Nature, 324, 429-433; published International patent application No.
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage ofthe target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in the target gene.
  • the ribozymes can be composed of modified ohgonucleotides (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 in or pol II promoter, so that transfected cells will produce sufficient quantities ofthe 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 "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 inco ⁇ orated 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 inco ⁇ orated by reference herein in its entirety).
  • a mutant, non-functional target gene flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions ofthe 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 ofthe DNA construct, via targeted homologous recombination, results in inactivation ofthe target gene.
  • ES embryonic stem
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas and Capecchi, 1987 and Thompson, 1989, supra).
  • this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region ofthe target gene (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe target gene in target cells in the body (see generally, Helene, 1991, Anticancer Drug Des., 6(6), 569-584; Helene, et al, 1992, Ann. N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12), 807-815).
  • the target gene promoter and/or enhancers i.e., the target gene promoter and/or enhancers
  • 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 ohgonucleotides 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 ofthe resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand ofthe 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 ofthe purine residues are located on a single strand ofthe 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, it may be preferable to co-administer normal target gene protein in order to maintain the requisite level of target gene activity.
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules ofthe invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors that inco ⁇ orate 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 afsh23 gene product can be utilized for the treatment of a/s/z23-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a/s/z23-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 fsh23 gene or a portion of the fsh23 gene that directs the production of afsh23 gene product exhibiting normal fsh23 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.
  • ecausefsh23 genes can be expressed in the brain, such gene replacement therapy techniques should be capable delivering s/223 gene sequences to these cell types within patients.
  • techniques that are well known to those of skill in the art see, e.g., PCT Publication No.
  • WO89/10134 published April 25, 1988
  • viral vectors such as, for example, those described above, are preferable.
  • techniques for delivery involve direct administration of such fsh23 gene sequences to the site ofthe cells in which the fsh23 gene sequences are to be expressed.
  • Additional methods that may be utilized to increase the overall level of fsh23 gene expression and/or s/z23 gene product activity include the introduction of appropriate s/z23-expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to ameliorate the symptoms of a/s/*23-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a/s/*23-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 fsh23 gene expression in a patient are normal cells, preferably brain cells, that express the fsh23 gene.
  • cells preferably autologous cells
  • fsh23 gene sequences can be engineered to express fsh23 gene sequences, and may then be introduced into a patient in positions appropriate for the amelioration ofthe symptoms of a s/z23-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a s/z23-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • cells that express an unimpaired s ⁇ 23 gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells.
  • the expression of the fsh23 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-aut ⁇ logous 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 5.8, that are capable of modulating fsh23 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 ofthe blood-brain barrier.
  • Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide ofthe invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity ofthe polypeptide.
  • the pharmacogenomics i.e., the study ofthe relationship 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 ofthe pharmacologically active drug.
  • the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide ofthe invention, expression of a nucleic acid ofthe invention, or mutation content of a gene ofthe invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe 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., Under (1997) Clin. Chem. 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a 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 polymo ⁇ hisms.
  • 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) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymo ⁇ hic 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 mo ⁇ hine. The other extreme are 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 ofthe 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 ofthe individual.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic 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 ofthe polypeptide, such as a modulator identified by one ofthe exemplary screening assays described herein.
  • MONITORING OF EFFECTS DURING CLINICAL TRIALS Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a polypeptide ofthe invention (e.g., the ability to modulate abenant cell proliferation chemotaxis, and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, 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.
  • agents e.g., drugs, compounds
  • 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 ofthe invention and preferably, that of other polypeptide that have been implicated in a/s/?23-related disorder e.g., a neuropsychiatric disorder such as BAD, can be used as a marker ofthe immune responsiveness of a particular cell.
  • genes including those ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe methods as described herein, or by measuring the levels of activity of a gene ofthe invention or other genes.
  • the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment ofthe 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 ofthe agent; (ii) detecting the level ofthe polypeptide or nucleic acid ofthe invention in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level the ofthe polypeptide or nucleic acid ofthe invention in the post- administration samples; (v) comparing the level ofthe polypeptide or nucleic acid ofthe invention in the pre-administration sample with the level ofthe polypeptide or nucleic acid of the invention in the post-administration sample or samples; and (vi) altering the administration ofthe agent to the subject accordingly.
  • an agent e.g., an agonist, antagonist,
  • increased administration ofthe agent may be desirable to increase the expression or activity ofthe polypeptide to higher levels than detected, i.e., to increase the effectiveness ofthe agent.
  • decreased administration ofthe agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness ofthe agent.
  • fsh23 gene products such as, for example, the novel Fsh23 polypeptide disclosed herein, or compounds that are determined to affect fsh23 gene expression or gene product activity, can be administered to a patient at therapeutically effective doses to treat or ameliorate a s/ ⁇ 23-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a therapeutically effective dose refers to that amount ofthe compound sufficient to result in amelioration of symptoms of such a disorder.
  • a therapeutically effective amount i.e., an effective dosage
  • protein or polypeptide for example, Fsh23 polypeptide or anti-Fsh23 antibody
  • 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 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity ofthe disease or disorder, previous treatments, the general health and
  • 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 subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • 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,.
  • 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 mole, 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 ofthe ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) ofthe small molecule will vary, for example, depending upon the identity, size, and condition ofthe 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 ofthe invention.
  • Exemplary doses include milligram or microgram amounts ofthe small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency ofthe small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • 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 ofthe specific compound employed, the age, body weight, general health, gender, and diet ofthe 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% ofthe population) and the ED 50 (the dose therapeutically effective in 50% ofthe 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 .
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • 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 ofthe test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration ofthe 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 ofthe active compound.
  • 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.
  • 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 may be formulated containing a powder mix ofthe compound and a suitable powder base such as lactose or starch.
  • 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 For physical mapping, yeast artificial chromosomes (YACs) containing human sequences were mapped to the region being analyzed based on publicly available maps (Cohen et ⁇ l., 1993, C.R. Acad. Sci. 316, 1484-1488). The YACs were then ordered and contig reconstructed by performing standard short tag sequence (STS)-content mapping with microsatellite markers and non-polymo ⁇ hic STSs available from databases that surround the genetically defined candidate region.
  • STS standard short tag sequence
  • BAC Bacterial artificial chromosome mapping
  • the STSs from the region were used to screen a human BAC library (Research Genetics, HuntsviUe, AL).
  • the ends of the BACs were cloned or directly sequenced. The end sequences were used to amplify the next overlapping BACs.
  • additional microsatelhtes were identified. Specifically, random sheared libraries were prepared from overlapping BACs within the defined genetic interval.
  • BAC DNA was sheared with a nebulizer (CIS-US Inc., Bedford, MA). Fragments in the size range of 600 to 1,000 bp were utilized for the sub library production.
  • Microsatellite sequences from the sublibraries were identified by corresponding microsatellite probes. Sequences around such repeats were obtained to enable development of PCR primers for genomic DNA.
  • RH mapping Standard RH mapping techniques were applied to a Stanford G3 RH mapping panel (Research Genetics, HuntsviUe, AL) to order all microsatellite markers and non-polymo ⁇ hic STSs in the region being analyzed.
  • Sample sequencing Random sheared libraries were made from all the BACs within the defined genetic region. Approximately 6,000 subclones within the approximately 116 kb region were sequenced with vector primers in order to achieve a 6-fold sequence coverage ofthe region. All sequences were processed through an automated sequence analysis pipeline that assessed quality, removed vector sequences and masked repetitive sequences. Sequences within the 116 kb candidate region, as defined by the disease haplotype shared by the affected individuals, were assembled into sequence clusters using the Sequencher program. Sequencing primers were designed from both ends of each cluster and used to sequence the corresponding BACs to extend the contigs. Eventually, all clusters were assembled together to produce a single continuous DNA strand which covered the whole interval of interest as defined by the haplotype.
  • cDNA selection was used as an additional method for gene identification of transcribed sequences over large regions ofthe genome. Through a combination of characterizations including physical mapping and RNA hybridization, the selected cDNAs were arranged into transcription units. The cDNA selection technique was carried out as described by Rommens, et al. (1994, in Identification of Transcribed Sequences, Hochgeschwender and Gardiner, eds., Plenum Press, New York, pp. 65-79).
  • cDNA libraries cDNA libraries purchased from Clontech (Palo Alto, CA) and were screened according to manufacturer's recommendations.
  • Northern analysis Standard Northern analysis techniques were utilized in probing human and fetal multiple tissue Northern blots purchased from Clontech (Palo Alto, CA). Different gene probes were hybridized to the northern blots. Blots were hybridized to different gene probes, which were derived by PCR from fsh23 cDNA sequences.
  • YACs were mapped to the chromosome 18 region being analyzed.
  • the region from publicly available markers D18S1161 and D18S554 which spans most ofthe D18S469- D18S554 region described above, was also mapped and contiged with BACs.
  • Sublibraries from the contiged BACs were constructed, from which microsatellite marker sequences were identified and sequenced.
  • the radiation hybrid (RH) mapping technique was independently applied to the region being analyzed.
  • RH was used to order all microsatellite markers and non-polymo ⁇ hic STSs in the region.
  • the high resolution physical map ultimately constructed was obtained using data from RH mapping and STS-content mapping.
  • BAD18ct22 is defined by a (GA) 22 di-nucleotide repeat; the BADct22 primer set used was as follows:
  • BAD18cagl is defined by a (CAG) U tri -nucleotide repeat; the following primer set was used for amplification ofthe BADl ⁇ cagl marker:
  • BAC clones within the newly identified 116 kb neuropsychiatric disorder region were further analyzed to identify specific genes within the region.
  • 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.
  • fsh23 A novel gene, termed fsh23, was found within the 116 kb interval between BAD18ct22 and BADl ⁇ cagl on the long arm of chromosome 18. fsh23, therefore, can be involved in neuropsychiatric disorders. fsh23 is positioned within the 116 kb interval between BAD 18 ct22 and BAD 18 cagl, as shown in FIG. 1A. The arrow denotes the location and direction of transcription relative to genetic markers shown above in the diagram.
  • the nucleotide sequence depicted in FIG. 2 comprises the fsh23 genomic nucleotide sequence.
  • the total genomic region of fsh23 is estimated to comprise approximately 12 kb, and produces at least 3 distinct mRNAs by alternative splicing of mRNA transcripts.
  • the fsh23 gene organization and its transcription pattern and gene products were further characterized, and is described in detail below.
  • fsh23 cDNA clones were identified and characterized by a combination of EST database mining, cDNA library screening, and DNA sequencing. TBLAST searching the EST database (dbEST) found a sequence that contained partial homology to sequences within the 116 kb interval (Altschul et al, 1990, J. Mol. Biol.
  • FIG. 3 A Two novel cDNA clones, fsh23-N2 (alt2), and fsh23-V3 (alt3), were also independently identified using a TBLAST search ofthe EST database (dbEST accession no. AI807540).
  • the nucleotide sequences depicted in FIGS. 3B-C represent these novel fsh23 cDNA nucleotide sequences.
  • the sequences of these two cDNAs, as well as the nucleotide sequences of sh23 '-VI (altl) were aligned with the fsh23 genomic sequence, and the fsh23 gene structure was thereby determined.
  • FIG. 2 An annotated s&23 genomic sequence, showing sA23 exons, introns, and splice sites is shown in FIG. 2.
  • FIG. 4 A diagrammatic representation of the fsh23 gene is also shown in FIG. 4.
  • the fsh23 gene produces 3 mRNAs comprising 6 distinct exons: exon 1, exon 2, exon B, exon C, exon 3, and exon 3' UTR.
  • Splice form 1 comprises exon 1, exon 2, exon 3, and exon 3'UTR, and is exemplified by cDNA clone fsh23- VI (altl), as shown in FIG.
  • splice form 2 comprises exon 1, exon 2, exon B, exon C, exon 3, and exon 3'UTR, and is exemplified by fsh231-V2 (alt2), as shown in FIG. 3B; and splice form 3 comprises exon 1, exon 2, exon C, exon 3, and exon 3'UTR, and is exemplified by clone fsh23 -W 3 (alt3), as shown in FIG. 3C.
  • the alternatively spliced human fsh23 cDNA sequence, fsh23- VI (altl)(SEQ ID NO:4) shown in FIG. 3 A encodes a polypeptide comprising the 85 amino acid sequence shown in SEQ LD NO:5.
  • the alternatively spliced cDNA sequence, fsh23 cDNA sequence, fsh23-V2 (alt2)(SEQ ID NO:7), shown in FIG. 3B encodes a polypeptide comprising the 96 amino acid sequence shown in SEQ ID NO: 8.
  • the alternatively spliced fsh23 cDNA sequence, fsh23-V3 (alt3)(SEQ ID NO:10) shown in FIG. 3C encodes a polypeptide comprising the 100 amino acid sequence shown in SEQ LD NO:l 1.
  • FIG. 6A-C shows the results of structural analysis using PROTEAN by DNASTAR, Inc. (http://www.dnastar.com). Fsh23 structural analysis was performed by a number of methods. Secondary structures were predicted by the methods of Chou-Fasman and Gamier et al.
  • FIGS. 6A- C which indicate the physical and structural characteristics of the fsh23- VI (altl), fsh31- 2 (alt2) and fsh31-V3 (alt3) alternative splice forms.
  • fsh23 -related disorders encompass disorders ofthe above-mentioned tissues, in which fsh2 '3 is expressed.
  • BLASTX searching (Gish et al., 1993, Nature Genet. 3:266-72; Altschul et al., 1990, J. Mol. Biol. 215: 403-410) was then done using standard parameters to predict protein sequences that might be encoded by the novel gene.
  • a BLASTX search of both strands and all 6 reading frames of the fsh23 fragment against the protein database detected no significant homologies to other known proteins.
  • Fsh23 structural analysis was performed by a number of methods. Secondary structures were predicted by the methods of Chou-Fasman and Gamier et al, and hydropathy, surface probability, and chain flexibility were analyzed by the methods of Kyte and Doolitle, Emini et al., and Ka ⁇ lus and Schulz, respectively (Yang et al, 1998, J. Protein Chem. 17:61- 71). Antigenic index and amphipathic regions were determined by the methods of Eisenberg and Jameson- Wolf, respectively (Faaberg et al., 1996, Arch. Virol. 141:1337-1348; Beattie et al., 1992, Eur. J. Biochem. 210:59-66).
  • Fsh23 The characteristics of Fsh23 are shown in Figures 6A-C. Rules used for predicting secondary structure, hydropathy, amphipathy, flexibility, antigenicity and surface probabilities are indicated at right, and plots showing results for Fsh23 amino acids are shown on the left, corresponding to the coordinates for Fsh23 amino acids given above and below the plots.
  • each polymo ⁇ hic residue in the intron is shown, and, in parentheses, in the genomic sequence shown in FIG. 2.
  • such polymo ⁇ hic sites maybe used, for a number of pu ⁇ oses, for example, in linkage disequilibrium and association studies, for correlating genetic sequences with phenotypic conditions, drug response or toxicity, or to detect, diagnose, and/or to determine a predisposition or risk of developing a/s/z23-related disorder, such as a neuropsychiatric disorder.
  • fsh23 related disorders may include, but are not limited to, chromosome 18q associated disorders, neuropsychiatric disorders or BAD.
  • methods disclosed in Section 5.5.1, above may be used to identify additional polymo ⁇ hic sites useful for these pu ⁇ oses.
  • novel sequences ofthe entire 18q interval were identified that can be used with the methods ofthe invention, were identified.
  • the 116 bp was 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 ofthe 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 ofthe 18q interval: 28441-29265 (SEQ LD No. 18), 29683-39587 (SEQ LD No. 19), 40284-43253 (SEQ LD No. 20), 43518-46075 (SEQ LD No. 21), 47264-52284 (SEQ LD No. 22), 52672-56935 (SEQ LD No. 23), 57032-57726 (SEQ LD No. 24), 58065- 59057 (SEQ LD No. 25), 59815-60471 (SEQ LD No. 26), 60870-62451 (SEQ LD No. 27), 62543-63268 (SEQ LD No.
  • This Section describes, in detail, exemplary and non-limiting methods which can be used to identify variations in fsh23 among individuals, and to determine whether such variations correlate with a bipolar affective disorder.
  • the experiments described in this Section can be used to detect variations ofthe level of ' fsh23 mRNA in cell samples from BAD-affected and control (i.e., non-BAD affected) patients.
  • the cell samples are cell lines, for example, lymphoblast cell lines, from BAD-affected and control individuals.
  • the samples may be tissue samples such as brain tissue samples, from BAD-affected and control individuals.
  • any cell, cell line or tissue sample could be used in such methods.
  • Such variations can then be used, e.g. , to diagnose BAD in individuals as well as to identify individuals predisposed to BAD, by detecting the presence or absence ofthe variation in a genetic sample obtained from an individual suspected of having or of being predisposed to a BAD condition.
  • the therapeutic methods and compositions ofthe invention can also be used to treat individuals for BAD, e.g., by reversing or neutralizing the variance in fsh23 in the individual.
  • fsh23 mRNA expression levels can be evaluated, according to the following methods, in samples, e.g. , from cell lines obtained from patients suffering from BAD.
  • lymphoblast cells or other cells known to express sA23 can be isolated from patients suffering from BAD and cultured as a cell line.
  • the fsh23 mRNA expression levels in such cells can then be compared to fsh23 mRNA expression levels in cells, preferably from the same type of cells, isolated from patients not suffering from BAD (i.e., from non-affected individuals).
  • Such "control" cell lines can be readily obtained, e.g., from the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • mRNA can be extracted from such cell lines and use, e.g., in Taqman PCR experiments, to determine the amount or level of sh23 expressed in cells, e.g., by amplifying and detecting the mRNA samples under a standard program on an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems).
  • fsh23 mRNA levels are compared to a suitable internal control, such as GAPDH (glyceraldehyde-3-phosphate dehydrogenase), whose mRNA levels are measured in the same cell lines.
  • GAPDH glycogene
  • mRNA levels measured from such an internal control can then serve to normalize the fsh23 mRNA levels measured for the different cell lines.
  • Exemplary primer sequences that can be used in the PCR amplification of both fsh23 and GAPDH are provided below in Tables 2 and 3, respectively.
  • TaqMan PCR experiments using the fsh23-specific primers listed in Table 2 revealed expression brain, mammary gland, testis, trachea, and uterus.
  • Routine techniques of statistical analysis can be readily used by those skilled in the art to determine whether variations of fsh23 mRNA levels correlate with BAD. Preferably, any correlations identified by such techniques are subsequently verified, e.g., using larger, and therefore statistically more robust, samples. Differences in fsh23 mRNA expression levels that are thus identified and confirmed to correlate with BAD can then be used in both the diagnostic and prognostic evaluation of patients who are suspected of suffering from a BAD or are suspected of being predisposed to a BAD.
  • mRNA levels of fsh23 can be measured from cell lines obtained from a patient and compared to fsh23 mRNA levels both in cell lines obtained from normal individuals not suffering from or predisposed to BAD, and in cell lines obtained from individuals who are suffering from or predisposed to BAD.
  • Variations in fsh23 expression can also be exploited in the methods ofthe invention to treat BAD by reversing and/or neutralizing the variation in a patient, e.g., using the methods described, above, in Section 5.7, e.g., to either reduce or increase levels of fsh23 mRNA expressed in a patient or in an appropriate cell population or subpopulation ofthe patient.
  • Ep69104 represents a composite deposit of a mixture of two strains, each of which contains either bacterial artificial chromosome (BAC) BAC69 or BAC104.
  • the two BACs together, contain the 160 kb region of human chromosome 18 depicted FIG. IB.
  • 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.
  • B AC69 DNA yields fragments having a total length of approximately 220kb
  • BAC 104 DNA yields fragments having a total length of approximately 80kb.
  • Ep34680 represents a composite deposit of a mixture of five strains, each of which contains either fsh23, P2, P3, P4, or P5 cDNA clones in a PT7T3-PAC vector (2.9kb).
  • a strain harboring a particular cDNA clone an aliquot of the mixture can be streaked out to single colonies on nutrient media (e.g., LB plates) supplemented with 100 ⁇ g/ml chloramphenicol, single colonies grown, and then DNA can be extracted using standard procedures.
  • a sample ofthe DNA preparation can be digested with EcoRI and Not I, and the resulting products can be separated by standard gel electrophoresis techniques.
  • Liberated inserts are ofthe following approximate sizes: fsh23: 500 bp
  • insert bands should be isolated, digested with Pstl, and the resulting products should be separated using standard gel electrophoresis. fsh23 DNA will be cut at one site, yielding approximately 100 and 400 bp bands. Strain 2 insert will not be cut, yielding a 500 bp band.

Abstract

Cette invention a trait au gène mammalien fsh23, qui est un nouveau gène associé au trouble affectif bipolaire chez l'homme. Elle porte sur des acides nucléiques de fsh23, sur des molécules d'ADN de recombinaison, sur des gènes clonés ou des variants dégénérés de ceux-ci, sur des produits géniques et sur des anticorps dirigés contre ces produits géniques, sur des vecteurs de clonage contenant des molécules du gène mammalien fsh23 ainsi que sur des hôtes produits par génie génétique afin d'exprimer ces molécules. L'invention a trait, de surcroît, à des méthodes permettant d'identifier des composés modulant l'expression des gènes fsh23 et des produits géniques ainsi qu'à des techniques d'utilisation de ces composés en tant qu'agents thérapeutiques dans le traitement de troubles liés au gène fsh23, par exemple des troubles neuropsychiatriques. Elle concerne, en outre, des méthodes d'évaluation diagnostique, d'essai génétique et de pronostic de troubles liés au gène fsh23, par exemple des troubles neuropsychiatriques, notamment la schizophrénie, l'hyperactivité avec déficit de l'attention, la schizophrénie dysthimique, les troubles affectifs, bipolaire ou unipolaire, de même qu'elle concerne des méthodes et des compositions permettant de traiter ces troubles.
PCT/US2000/030819 1999-11-08 2000-11-08 Methodes et compositions permettant de diagnostiquer et de traiter des troubles lies au chromosome 18q WO2001034772A2 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1997037043A1 (fr) * 1996-03-29 1997-10-09 The Regents Of The University Of California PROCEDES DE TRAITEMENT DES TROUBLES AFFECTIFS BIPOLAIRES ASSOCIES A DES MARQUEURS SUR LE CHROMOSOME 18q

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997037043A1 (fr) * 1996-03-29 1997-10-09 The Regents Of The University Of California PROCEDES DE TRAITEMENT DES TROUBLES AFFECTIFS BIPOLAIRES ASSOCIES A DES MARQUEURS SUR LE CHROMOSOME 18q

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Title
AHERN H.: 'Biochemical, reagent kits offer scientists good return on investment' THE SCIENTIST, [Online] vol. 9, no. 15, July 1995, page 20, XP002939912 *
DATABASE GENBANK [Online] 16 June 1999 VANDERBOSCH ET AL.: 'ESTs from uninoculated roots of Medicago truncatula', XP002939911 Database accession no. AI737517 *
FREIMER ET AL.: 'Genetic mapping using haplotype, association and linkage methods suggests a locus for severe bipolar disorder (BPI) at 18q22-123' NATURE GENETICS vol. 12, April 1996, pages 436 - 441, XP002939913 *

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