WO2001034841A1 - Procedes et compositions servant a diagnostiquer et a traiter des troubles lies au chromosome 18q - Google Patents

Procedes et compositions servant a diagnostiquer et a traiter des troubles lies au chromosome 18q Download PDF

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Publication number
WO2001034841A1
WO2001034841A1 PCT/US2000/030824 US0030824W WO0134841A1 WO 2001034841 A1 WO2001034841 A1 WO 2001034841A1 US 0030824 W US0030824 W US 0030824W WO 0134841 A1 WO0134841 A1 WO 0134841A1
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fsh26
gene
disorder
expression
nucleic acid
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PCT/US2000/030824
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English (en)
Inventor
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 AU13643/01A priority Critical patent/AU1364301A/en
Publication of WO2001034841A1 publication Critical patent/WO2001034841A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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 fsh26, which maps to the relevant portion of human chromosome 18q.
  • the sequences of the present invention can be used for diagnostic evaluation, genetic testing and/or prognosis of a fsh26- related disorder, e.g., a neuropsychiatric disorder, and/or can be used to map human chromosome 18q.
  • the invention encompasses fsh26 nucleic acids, recombinant DNA molecules, cloned genes and degenerate variants thereof, vectors containing such fsh26 nucleic acids, and hosts that have been genetically engineered to express and/or contain such molecules.
  • the invention further relates to fsh26 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 fsh26 genes.
  • the invention relates to methods of using compounds such as those identified herein as therapeutic agents for modulation of s/22r5-mediated process or in the treatment of a symptom of s/ 2(5-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.
  • a symptom of s/ 2(5-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/s/z2(5-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.
  • a/s/z2(5-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 bipolar mood disorder
  • BP-I r . 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 etio logically 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).
  • 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.
  • 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 f) transmission of the disease trait. With neuropsychiatric disorders there is even greater ambiguity in distinguishing individuals who likely carry an affected genotype from those who are genetically unaffected. For example, one can define an affected phenotype for BAD by including one or more of the broad grouping of diagnostic classifications that constitute the mood disorders: BP-I, SAD-M, MDD, and bipolar affective (mood) disorder j , with hypomania and major depression (BP-II).
  • ⁇ - 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
  • the present invention relates to a novel gene located within this interval, referred to
  • fsh26 15 herein as fsh26, which is involved in such disorders.
  • fsh26 nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof are provided herein.
  • the invention also provides vectors, including expression vectors, containing7sA2 ⁇ 5 nucleic acid molecules, and hosts that have been genetically engineered to express and/or contain such fsh26 gene products. 0
  • the invention further relates to novel fsh26 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/*2 ⁇ 5-mediated processes and for the treatment of s/j2 ⁇ 5-related disorders, such as neuropsychiatric disorders, including the amelioration or prevention of at least one symptom of the disorders, 5 wherein such methods comprise administering a compound which modulates the expression of afsh26 gene and or the synthesis or activity of afsh26 gene product, h one embodiment, the invention relates to methods for the use of a novel fsh26 gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against a fsh26 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, Huntingdon'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 5 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-II); or a unipolar affective
  • CNS-related and/or PNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is inco ⁇ orated herein by reference in its entirety.
  • DSM Diagnostic and Statistical manual of Mental Disorders
  • y_s , /z2 ⁇ 5-related disorder refers to a disorder
  • Such 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 afsh26 gene or gene product in a cell such that the s/z26-mediated process or the disorder is treated, e.g., a symptom is ameliorated.
  • such methods can comprise supplying a nucleic acid molecule encoding afsh26 gene product to increase the level, expression or activity of the fsh26 gene jc product within the cell such that the/s/z2(5-mediated process or the disorder is treated, e.g., a symptom is ameliorated.
  • the nucleic acid molecule encoding the fsh26 gene product can encode a normal or mutant fsh26 gene product, e.g., one with increased activity or expression levels.
  • nucleic acids and polypeptides of the present invention are useful as r . modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention.
  • nucleic acid molecules which are at least 45% (or
  • the invention features nucleic acid molecules which are at least 45% (or
  • nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
  • the invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45 % (or 55%, 65%o, 75%, 85%, 95%, 98%, or 99%) identical to the amino acid sequence
  • nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%o, 75%, 85%, 95%, 98, or 99%) identical to the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, PTA-452, or a complement thereof, wherein the protein
  • nucleotide sequence also exhibits at least one structural and/or functional feature of a polypeptide of the invention.
  • the invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA of ATCC ® PTA-451, PTA-452 wherein the nucleic acid molecule hybridizes to a
  • ⁇ r nucleic acid molecule consisting of the nucleotide sequence of the 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
  • nucleic acid molecule consisting of the nucleotide sequence of the 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 of the invention.
  • the isolated nucleic acid molecules encode an
  • the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.
  • vectors e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention.
  • the invention provides host cells containing such a vector or a nucleic acid
  • the invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide is produced.
  • Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention.
  • Preferred proteins and polypeptides possess at least one
  • an activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo, or in vitro, according to standard techniques.
  • activities can be a direct activity, such as an association with or an
  • ⁇ enzymatic activity on a second protein or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein.
  • polypeptides of the present invention can be operably linked to a heterologous amino acid sequence to form fusion proteins.
  • the invention further features antibodies that specifically bind a polypeptide of f) the invention such as monoclonal or polyclonal antibodies.
  • the polypeptides of the invention or biologically active portions thereof, or antibodies of the invention can be inco ⁇ orated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides methods for detecting the
  • ⁇ ⁇ - presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample.
  • the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates
  • ⁇ (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated.
  • the agent is an antibody that specifically binds to a polypeptide of the invention.
  • the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a r polypeptide of the invention.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA encoding a polypeptide of the invention.
  • the modulator is a protein of the invention.
  • the modulator is a nucleic acid of the invention.
  • the modulator is a peptide, peptidomimetic, or other small organic molecule.
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis-regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post- translational modification of the invention wherein a wild-type form of the gene encodes a
  • ⁇ protein having the activity of the polypeptide of the invention.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention.
  • such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the yr, activity of the polypeptide.
  • the invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound.
  • the invention still further relates to methods for modulation of fsh26- ⁇ - mediated processes or the treatment of s/?2 ⁇ 5-related disorders, such as neuropsychiatric disorders, including but not limited to disorders resulting from fsh26 gene mutations, and/or an abnormal level of fsh26 expression or activity and disorders involving afsh26 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
  • such methods can comprise supplying a mammal in need of treatment with a cell comprising a nucleic acid molecule that encodes an unimpaired j /_s ⁇ 2 ⁇ 5 gene product such that the cell expresses the
  • 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 fsh26 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 fsh26 gene or gene product.
  • the present invention is directed to methods that utilize _s7z2 ⁇ ' gene sequences and or fsh26 gene product sequences for the diagnostic evaluation, genetic
  • the invention relates to methods for diagnosing s/z26-related disorders, e.g., neuropsychiatric disorders, wherein such methods can comprise measu ⁇ ng fsh26 gene expression in a patient sample, or detecting afsh26 mutation that correlates with the presence or development of such a disorder, in the genome of a mammal suspected of
  • fsh26 gene sequences and/or fsh26 gene products can also be utilized as markers for mapping of the region of the long arm of human chromosome 18 spanned by chromosomal markers BAD18ct22 and BADl ⁇ cagl. 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 fsh26 gene and/or the synthesis or activity of a fsh26 gene product, wherein such methods comprise contacting a compound with a cell that expresses afsh26 gene, measuring the levels of fsh26 gene expression, gene product expression or gene product activity, and comparing such levels to the levels of fsh26 gene
  • _-, ⁇ expression, gene product, or gene product activity produced by the cell in the absence of the compound such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the fsh26 gene and or the synthesis or activity of the fsh26 gene product has been identified.
  • bipolar affective disorder BP bipolar mood disorder
  • BP-II bipolar affective (mood) disorder with hypomania and major depression bp
  • base pair(s) dbEST base pair(s) dbEST
  • expressed sequence tag data base _ (National Center for Biotechnology Information)
  • allele which is used interchangeably herein with “allelic variant” refers to alternative forms of a 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
  • the subject is said to be homozygous for the gene or allele.
  • 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 polymo ⁇ hic region of an fsh26 gene refers to a region of an fsh26 gene having one of several nucleotide sequences found in that region of the gene in other individuals, as well as to polypeptides encoded by nucleic acid molecules comprising said sequences.
  • antibody refers to immunoglobulin molecules
  • immunoglobulin molecules i. e. , molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention.
  • a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g. , a biological sample, which
  • ⁇ n naturally contains the polypeptide.
  • immuno logically 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
  • Cells 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
  • 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
  • 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).
  • sterols e.g., cholesterol
  • lipids e.g., a cationic lipid, virosome or liposome
  • viruses e.g., adenovirus, adeno-associated virus, and retrovirus
  • 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
  • the complex is cleavable under appropriate conditions within the cell so that the gene, protein, polypeptide or peptide is released in a functional form. It is also possible that soluble forms of the protein also exist. Such soluble isoforms can arise through variable splicing of the fsh26 gene or alternatively as a result of proteolysis 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 fsh26 polypeptide" may thus refer to one or more genes
  • allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
  • ⁇ refers to a polynucleotide or nucleic acid molecule comprising an open reading frame encoding one of the fsh26 polypeptides of the present invention. In one embodiment, these terms relate to a cDNA sequence including, but not limited to, a polynucleotide or nucleic acid sequence obtained via reverse transcription of an mRNA molecule. In one embodiment, the term nucleic acid or polynucleotide is a nucleic acid molecule which is not
  • 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 or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined
  • a degree of homology or similarity or identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of
  • ⁇ identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences.
  • a degree of identity of nucleic acid sequences is a function of the number of identical nucleic acids at positions shared by the nucleic acid sequences.
  • a degree of homology or similarity of amino acid sequences is a
  • a sequence which is "unrelated" or “non-homologous" with one of the human fsh26 sequences of the present invention typically is a sequence which shares less than 40 % identity, though preferably less than 25 % identity with one of the human fsh26 sequences of the present invention.
  • the sequences are aligned for optimal comparison pu ⁇ oses (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 r compared.
  • 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
  • Gapped BLAST can be utilized as described in Altschul et al. (1997)
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. Id.
  • BLAST Altschul et al.
  • Gapped BLAST Altschul et al.
  • PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • 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 f) 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
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or complement thereof,
  • ⁇ corresponds to a naturally-occurring nucleic acid molecule.
  • FIG. 1 A-B depicts the gene structure and the nucleotide sequence of the 116 kb interval of ⁇ .c the long arm of human chromosome 18 spanned by BAD18ct22 and BAD18cagl.
  • A The gene structure of the 116 kb interval. In particular, the location of the fsh26 gene within this interval is shown, the orientation of which is indicated by the direction of the arrow. Also shown are other known genetic and physical markers in this interval.
  • B The nucleotide sequence of 160 kb of chromosome 18q, which includes thel ⁇ q interval associated with neuropsychiatric disorders, located from position 28441-144419.
  • BAD18ct22 and BADl ⁇ cagl Primers used for mapping the 18q interval, and which can be used to amplify sequences comprising the BAD18ct22 and BADl ⁇ cagl are underlined: the BADct22 primer set, at positions 28547-28572 and 28384-28405, and the BAD18cagl primer set, at positions 144302-144325 and 144477- 144501.
  • BAD18ct22 and BADl ⁇ cagl markers are shown in boxes between primers.
  • the fsh26 gene corresponds to the reverse complement of nucleotides 60473 to 61150 (bounded by " ⁇ >").
  • FIG. 2 depicts afsh26 cDNA sequence.
  • 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 fsh26, which is involved in such disorders.
  • Sections 5.1, 5.2, and 5.3 describe the 18q 116 kb region, includmg/5 ⁇ 2(5 nucleic acid molecules, as well as vectors comprising these molecules, host cells engineered to contain and/or express such molecules, fsh26 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 , /*2 ⁇ ' -related processes and for treatment of s/z2 ⁇ 5-related disorders, such as neuropsychiatric disorders, are described.
  • Section 5.7 describes screening assays for compounds that interact with a fsh26 gene or gene product, or modulate fsh26 gene or gene product activity.
  • Methods of treatment of a s/22d-related disorders using the compositions of the invention and compositions such as those identified by the methods of the invention are described in Section 5.8. 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. 1A 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
  • the nucleic acid molecules of the invention further include:
  • nucleic acid molecule containing the DNA sequence of thel ⁇ q interval (nucleotides 28441-144419 of FIG. IB), or its flanking DNA (nucleotides 1-28440 and
  • nucleic acid molecule comprising afsh26 nucleic acid sequence (e.g., the nucleic acid sequences depicted in FIG. 2 or a fragment thereof);
  • nucleic acid molecule comprising afsh26 sequence that encodes a mutant of afsh26 gene product in which all or a part of a domain is deleted or altered, as well as fragments thereof; j r, nucleic acid molecules that encode fusion proteins comprising afsh26 gene product or a fragment thereof, fused to a heterologous polypeptide;
  • telomere sequences flanking the fsh26 gene i.e., the 18q genomic interval described in (a)
  • afsh26-related disorder such as a neuropsychiatric disorder, e.g., BAD, or can be used for mapping the human chromosome 18q region flanking markers
  • afsh26- ⁇ eXate ⁇ disorder such as a neuropsychiatric disorder, e.g., BAD.
  • the nucleotide sequences of the 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 of the invention also include nucleotide sequences greater than 20, 30, 40, 50, 60, 70, 80, 90,100, or more base pairs long that have at least 80%, 85%, 90%>, 95%, 98%, or more nucleotide sequence identity to the nucleotide sequences of (a)-(h) above.
  • nucleic acid molecules of the invention do not include nucleic acid molecules that consist solely of the nucleotide sequence of dbEST sequence accession nos. AA813620, AA995246, AI017933, AA995246, AA813620, AA972889, AI028677, AA416989, AA626032, AI218484, or
  • the nucleic acid molecules of the 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%, or higher amino acid sequence identity to the polypeptides encoded by the fsh26 nucleotide sequences of (a)-(h) above.
  • sequences are aligned for optimal comparison pu ⁇ oses (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
  • % identity # of identical overlapping positions/total # of overlapping positions x 100%).
  • the two sequences are the same length.
  • 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
  • BLAST protein searches can be performed with
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Re_s.2J:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997,
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps.
  • the nucleic acid molecules of the invention further include: (a) any nucleotide sequence that hybridizes to afsh26 nucleic acid molecule of the 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
  • 1 c 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. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., j rs and John Wiley & sons, Inc., New York, at pp. 6.3.1-6.3.6 and 2.10.3).
  • the fsh26 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 afsh26 gene product.
  • nucleic acid molecules that hybridize under conditions (a) and (b), above encode gene products, e.g., gene products functionally equivalent to a j c fsh26 gene product.
  • the nucleic acids of the invention are human.
  • functionally equivalent fsh26 gene products include naturally occurring/s/z2 ⁇ 5 gene products present in the same or different species.
  • fsh26 gene sequences in non- human species map to chromosome regions syntenic to the human 18q chromosome r s location within which the particular human ⁇ ?26 ' lies.
  • Functionally equivalent fsh26 gene products also include gene products that retain at least one of the biological activities of a fsh26 gene product, and/or which are recognized by and bind to antibodies (polyclonal or monoclonal) directed against afsh26 gene product.
  • nucleic acid molecules of the invention are deoxyoligonucleotides
  • 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).
  • the nucleic acid molecules of the invention further comprise the complements of the nucleic acids described above.
  • Such molecules can, for example, act as antisense molecules, useful, for example, in fsh26 gene regulation, and/or as antisense primers in amplification reactions of fsh26 gene nucleic acid sequences.
  • Nucleic acid sequences of the invention encoding afsh26 gene product or
  • ribozyme 1 may be used as part of ribozyme and/or triple helix sequences, also useful for fsh26 gene regulation.
  • nucleic acid molecules of the invention may be used as components of diagnostic methods whereby, for example, the presence of a particular fsh26 allele involved in afsh26-related disorder, e.g., a neuropsychiatric disorder, such as BAD, ⁇ may be detected, or whereby the methods involve mapping the human 18q chromosomal region spanned by chromosomal markers BAD18ct22 and BAD18cagl.
  • diagnostic methods whereby, for example, the presence of a particular fsh26 allele involved in afsh26-related disorder, e.g., a neuropsychiatric disorder, such as BAD, ⁇ may be detected, or whereby the methods involve mapping the human 18q chromosomal region spanned by chromosomal markers BAD18ct22 and BAD18cagl.
  • Fragments of the fsh26 nucleic acid molecules refer to fsh26 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,
  • the fragments can comprise sequences that encode at least 10, 20, 30, 40, 50, 60, 70, 80 or more contiguous amino acid residues of the fsh26 gene products.
  • the fsh26 nucleic acid molecule encodes a gene product exhibiting at least one biological activity of a corresponding7s/22 ⁇ 5 gene product, e.g., afsh26 gene product.
  • Fragments of the fsh26 nucleic acid molecules can also refer to fsh26 exons or introns, and, further, can refer to portions of fsh26 coding regions that encode domains of, or mature fsh26 gene products.
  • a nucleic acid molecule of the invention preferably comprises at least one of the following nucleotide sequences of the 18q interval: 28441-29265 (SEQ ID No. 4),
  • nucleic acid molecules of the invention comprise at least 10, 12, 15, 20,
  • fsh26 nucleotide sequences and nucleotide sequences of the 116 kb interval of human chromosome 18q can be readily obtained, for example, by utilizing standard sequencing and bacterial artificial chromosome (BAC) technologies.
  • BAC bacterial artificial chromosome
  • DNA sequence polymo ⁇ hisms of a fsh26 gene will exist within a population of individual organisms (e.g., within a human population). Such polymo ⁇ hisms may exist, for example, among . c 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. Such natural allelic variations can result in 1-5%), 5-20%, or 20-50% variance in the nucleotide sequence of a given gene.
  • An allele is
  • mutant gene refers to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention.
  • the term can further include nucleic acid molecules comprising upstream and/or exon intron sequences and structure.
  • any and all nucleotide variations and resulting amino acid polymo ⁇ hisms or variations that are the result of natural allelic variation of the fsh26 gene are intended to be within the scope of the
  • Allelic variants or polymo ⁇ hism include, but are not limited to, ones that do not alter the functional activity of the fsh26 gene product.
  • the isolated fsh26 gene sequences disclosed herein may be labeled and used to screen a cDNA library constructed from mRNA
  • the hybridization conditions used should generally be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived, and can routinely be determined based on, e.g. , relative relatedness of the target and reference organisms.
  • the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • 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
  • afsh26 gene allelic variant may be isolated from, for example,
  • 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 fsh26 gene allele (such as, for example, brain
  • allelic variant is isolated from an individual who has a s ⁇ 2 ⁇ 5-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 afsh26 gene nucleic acid sequence.
  • 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.
  • PCR technology may also be utilized to isolate full length cDNA sequences, as well as cDNA sequences corresponding to alternatively spliced mRNAs of afsh26 gene.
  • RNA may be isolated, following standard procedures, from an appropriate
  • cellular or tissue source i.e., one known, or suspected, to express the fsh26 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 of the amplified fragment for the priming of first strand synthesis.
  • the resulting RNA/DNA hybrid may then be "tailed" with guanines using a
  • the hybrid may be digested with RNase H, and second strand synthesis may then be primed with a poly-C primer.
  • cDNA sequences upstream of the amplified fragment may easily be isolated.
  • a cDNA of an allelic, e.g., mutant, variant of the fsh26 gene maybe isolated,
  • 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 fsh26 allele, and by extending the new strand with reverse transcriptase.
  • the second strand of the cDNA is then synthesized using an
  • a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant fsh26 allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant fsh26 allele. An unimpaired fsh26 gene or any suitable fragment thereof may then be labeled and used as
  • Clones containing the mutant fsh26 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
  • 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 fsh26 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 gene product antibodies are likely to cross-react with the mutant fsh26 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.
  • ⁇ fsh26 mutations or polymo ⁇ hisms can further be detected using PCR amplification techniques.
  • Primers can routinely be designed to amplify overlapping regions of the whole sA2 ⁇ 5 sequence including the promoter regulating region.
  • primers are designed to cover the exon-intron boundaries such that, coding regions can be scanned for mutations.
  • the invention also includes nucleic acid molecules, preferably DNA molecules, that are the complements of the nucleotide sequences of the preceding paragraphs.
  • nucleic acid molecules of the invention are present as part of nucleic acid molecules comprising nucleic acid sequences that contain or
  • heterologous sequences e.g., vector, expression vector, or fusion protein
  • the fsh26 gene products of the invention include polypeptides, and fragments j e - thereof, encoded by afsh26 nucleic acid sequence.
  • fsh26 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/?2 ⁇ 5-related ⁇ disorder, such as a neuropsychiatric disorder, e.g., BAD.
  • fsh26 gene products can also be used as components of fusion proteins to impart a Fsh26 protein characteristic to another protein of interest.
  • afsh26 gene product could be used to facilitate the purification, localization, or recovery of the protein of interest, by providing an antigenic tag to the fusion protein.
  • fsh26 gene products have uses as amino acid and protein additives to foods, soaps, shampoos, cosmetics, and the like.
  • fsh26 gene products sometimes referred to herein as a "Fsh26 protein" includes those gene products encoded by the fsh26 gene sequences described in Section 5.1, above.
  • fsh26 gene products may include proteins that represent functionally equivalent (see Section 5.1 for a definition) gene products.
  • Such an equivalent fsh26 gene products may contain deletions, including internal deletions, additions, including additions s yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the amino acid sequence encoded by the fsh26 gene sequences described, above, in Section 5.1, but that result in a "silent" change, in that the change produces a functionally equivalent fsh26 gene product.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of
  • 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
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • deletion or non- conservative alterations can be engineered to produce altered fsh26 gene products.
  • Such alterations can, for example, alter one or more of the biological functions of the fsh26 gene product. Further, such alterations can be selected so as to generate fsh26 gene products that are better suited for expression, scale up, etc. in the host cells chosen.
  • r. 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 Fsh26 protein as well as fusion proteins in which a Fsh26 protein or a portion of a fsh26 protein such as a truncated Fsh26 protein or peptide or afsh26 protein domain, is fused to an
  • ⁇ unrelated protein are also within the scope of this invention.
  • Such proteins and peptides can be designed on the basis of the fsh26 nucleotide sequence disclosed in Section 5.1, above, and/or on the basis of the fsh26 amino acid sequence disclosed herein.
  • Fusion proteins include, but are not limited to, IgFc fusions which stabilize the Fsh26 protein or peptide and prolong half life in vivo; or fusions to any amino acid sequence that allows the fusion
  • 3f protein to be anchored to the cell membrane; or fusions of fsh26 protein domains to an enzyme, fluorescent protein, luminescent protein, or a flag epitope protein or peptide which provides a marker function.
  • Fsh26 proteins of the invention also include Fsh26 protein sequences wherein domains encoded by at least one exon of the cDNA sequence, or fragments thereof, have
  • the fsh26 polypeptides of the invention can further comprise posttranslational modifications, including, but not limited to glycosylations, acetylations, myristylations, and phosphorylations. If the native Fsh26 protein does not have recognition motifs that allow such modifications, it would be routine for one skilled in the art to introduce into afsh26 gene nucleotide sequences that encode motifs such as enzyme
  • the fsh26 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 fsh26 gene polypeptides, peptides, fusion peptide and fusion polypeptides of the invention by expressing nucleic acid containing fsh26 gene may be produced by recombinant DNA technology using techniques well known in the art.
  • n sequences are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing s ⁇ 2 ⁇ 5 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. See, for example, the techniques described in Sambrook, et ⁇ l, 1989,
  • RNA capable of encoding fsh26 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 jr . fsh26 gene coding sequences of the 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 fsh26 gene product of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • yeast transformed with recombinant yeast expression vectors containing the fsh26 gene product coding sequences
  • insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the fsh26 gene product coding
  • telomere sequences e.g., telomere sequences
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant 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
  • 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 fsh26 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 fsh26 protein or for raising antibodies to fsh26 protein, for example, vectors that direct the expression of high levels of fusion protein products that are
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, ⁇ MBO J. 2, 1791), in which the fsh26 gene product coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13, 3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 264,
  • pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target
  • 1 ⁇ 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.
  • fsh26 gene coding sequences may be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under control of 0 an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of fsh26 gene coding sequences will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., see Smith, et al, 1983, J. Virol. 46, 584;
  • afsh26 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 of the viral genome (e.g., region ⁇ l or ⁇ 3) will result in a recombinant virus that is viable and capable of expressing fsh26 gene product in infected hosts (e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81, 3655-3659). Specific initiation signals may also be required for efficient translation of inserted fsh26
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner, et al, 1987, Methods in Enzymol. 153, 516-544).
  • a host cell strain may be chosen that modulates the expression of
  • ⁇ the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • modifications e.g., glycosylation
  • processing e.g., cleavage
  • protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
  • r j ⁇ x For long-term, high-yield production of recombinant proteins, stable expression is preferred.
  • cell lines that stably express the fsh26 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, j r polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, j r polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into
  • This method may advantageously be used to engineer cell lines that express the fsh26 gene product.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the fsh26 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-
  • 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 mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci.
  • 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
  • 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,:
  • 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.
  • the expression characteristics of an endogenous fsh26 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 fsh26 gene.
  • an endogenous fsh26 gene which is normally "transcriptionally silent", i.e., afsh26 .rs 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 _s/z2(5 gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous _>/?26 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.
  • ⁇ ⁇ fsh26 gene products can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate fsh26 transgenic animals.
  • transgenic refers to animals expressing/s z26 gene sequences from a different species (e.g. , mice expressing
  • humanj5/j2 ⁇ 5 sequences as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) fsh26 sequences or animals that have been genetically engineered to no longer express endogenous fsh26 gene sequences (i.e., "knockout” animals), and their progeny.
  • Any technique known in the art may be used to introduce an fsh26 gene transgene into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S.
  • transgenic animal clones containing an fsh26 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 afsh26 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
  • vectors containing some nucleotide sequences homologous to the endogenous fsh26 gene are designed for the pu ⁇ ose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous fsh26 gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous/s/z2 ⁇ 5 gene in only that cell type, by following, .rs 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 of the recombinant fsh26 gene may be assayed utilizing standard techniques.
  • Initial screening .r may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place.
  • the level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques that include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR (reverse transcriptase PCR). Samples of fsh26 gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the fsh26 transgene product.
  • ⁇ . fsh26 gene products, or peptide fragments thereof can be prepared for a variety of uses.
  • gene products, or peptide fragments thereof can be used for the generation of antibodies, in diagnostic assays, or for mapping and the identification of other cellular or extracellular gene products involved in the regulation of afsh26-reXate ⁇ disorder, such as a neuropsychiatric disorder, e.g., BAD.
  • fsh26 gene products include
  • soluble derivatives such as peptides or polypeptides corresponding to one or more domains of the fsh26 gene product, particularly fsh26 gene products, that are modified such that they are deleted for one or more hydrophobic domains.
  • antibodies to the fsh26 protein or anti-idiotypic antibodies that mimic the fsh26 gene product (including Fab fragments), antagonists or agonists can be used to treat s/z2 ⁇ 5-related
  • ⁇ - disorders such as neuropsychiatric disorders.
  • fsh26 gene products can be directly administered to a subject to treat a/s/z2 ⁇ 5-related disorder, such as neuropsychiatric disorders, or a disorder of a/s/z2(5-mediated process.
  • nucleotide constructs encoding such ⁇ s ⁇ 2 ⁇ 5 gene products can be used to genetically engineer host cells to express such fsh26 gene products in vivo; these genetically ⁇ engineered cells can function as "bioreactors" in the body delivering a continuous supply of fsh26 gene product, fsh26 peptides, or soluble fsh26 polypeptides.
  • Described herein are methods for the production of antibodies capable of 2 , specifically recognizing one or more fsh26 gene product epitopes or epitopes of conserved variants or peptide fragments of the gene products.
  • Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti- - ⁇ idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • mAbs monoclonal antibodies
  • Such antibodies may be used, for example, in the detection of afsh26 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 fsh26 gene products, and/or for the presence of abnormal forms of such gene products.
  • Such antibodies may also - , be utilized in conjunction with, for example, compound screening schemes, as described below, in Section 5.7, for the evaluation of the effect of test compounds on fsh26 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 7s j2r5-expressing cells prior to their introduction into the patient.
  • Anti-fsh26 gene product antibodies may additionally be used as a method for
  • Such antibodies may, be utilized as part of treatment methods for a s/z26-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a s/z26-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • various host animals may be immunized by injection with afsh26 gene product, or a portion thereof.
  • Such host animals may include, but are not limited to rabbits, mice, and rats, to name but a few.
  • 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.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as afsh26 gene product, or an antigenic functional derivative thereof.
  • an antigen such as afsh26 gene product, or an antigenic functional derivative thereof.
  • ⁇ host animals such as those described above, may be immunized by injection with fsh26 gene product supplemented with adjuvants as also described above.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immuno gen.
  • Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against a
  • polypeptide or polypeptides of the invention are those that contain only antibodies directed against a polypeptide or polypeptides of the invention.
  • immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention.
  • 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
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibodies specific for a protein or polypeptide of the invention can be selected for (e.g., partially purified) or purified by, e.g., affinity chromatography.
  • a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a
  • ⁇ - chromatography column The column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies.
  • a substantially purified antibody composition is meant, in this context, that the antibody
  • n sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired protein or polypeptide of the invention, and preferably at most 20%, yet more preferably at most 10%>, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies.
  • a purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired
  • 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. r.r ⁇ 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. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma may be of any immunoglobulin class including
  • 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. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and
  • 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). The extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest", Kabat, E.
  • 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.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • fragments include but are not limited to: the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse, et al, 1989,
  • nucleic acids of the invention including fsh26 nucleic acids, fsh26 nucleic acids, fsh26 gene products, including peptide fragments and fusion proteins thereof, and of antibodies directed against fsh26 gene products and peptide fragments thereof.
  • Such applications include, for example, prognostic
  • a/s/z2 ⁇ 5-related disorder such as a neuropsychiatric disorder, e.g., BAD
  • a neuropsychiatric disorder e.g., BAD
  • such applications include methods for the identification of compounds that modulate the expression of a fsh26 gene and/or the synthesis or activity of a fsh26 gene product, as described below, in Section 5.7, and for the
  • afsh26-reXated disorder e.g. a neuropsychiatric disorder, such as BAD, as described, below, in Section 5.10.
  • afsh26-reXated disorder e.g. a neuropsychiatric disorder, such as BAD, as described, below, in Section 5.10.
  • the nucleic acid sequences of the invention including s ⁇ tf nucleic acid sequences and gene products, including peptide fragments and fusion proteins thereof, and antibodies directed against fsh26 gene products and peptide fragments thereof, have applications for pu ⁇ oses independent of the role fsh26 may have in neuropsychiatric disorders and processes.
  • fsh26 gene products, including peptide fragments as
  • nucleic acid molecules of the invention including/s/22 ⁇ 5 nucleic acid sequences, and fsh26 gene products can be used for genetic mapping, i.e., refining the genetic map of chromosome 18q.
  • antibodies specific to Fsh26 can be used as probes to detect expression of human
  • Nucleic acids of the invention including s/ ⁇ d nucleic acids, and fsh26 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
  • microsatellites also known as simple- sequence repeats (SSRs)
  • SSRs simple- sequence repeats
  • Such microsatellites make excellent genetic
  • microsatellites e.g., (CA) n dinucleotide repeats
  • 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),
  • RFLPs restriction fragment length polymo ⁇ hisms
  • single nucleotide polymo ⁇ hisms which occur much less frequently.
  • human populations can then be analyzed for the such simple-sequence length polymo ⁇ hisms (SSLPs) to determine the frequency and variability of the repeat.
  • SSLPs 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 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).
  • afsh26 gene, protein or a fragment or domain thereof can be used for construction of fusion proteins.
  • fsh26 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.
  • ft Portions or fragments of the 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 fsh26 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
  • a variety of methods can be employed to screen for the presence of fsh26 jr , gene-specific mutations or polymo ⁇ hisms (including polymo ⁇ hisms flanking afsh26 gene, e.g., ones that cosegregate with a particular fsh26 allele) and to detect and/or assay levels of fsh26 nucleic acid sequences.
  • Mutations or polymo ⁇ hisms within or flanking the fsh26 gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell, or any ⁇ cell that expresses the fsh26 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. fsh26 nucleic acid sequences may be used in hybridization or amplification assays of biological samples to detect abnormalities involving fsh26 gene structure, , 0 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 fsh26 gene-specific mutations or polymo ⁇ hisms can involve for example, contacting and incubating nucleic acids obtained .e 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 fsh26 gene.
  • the term "patient sample , biological sample or appropriate cellular source” refers to a sample of tissue or fluid suspected of containing a mutated or non-mutated fsh26 polynucleotide or polypeptide from
  • an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal, and genito-urinary tracts, stool, urine, sputum, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.
  • blood can be drawn
  • prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for mutations of the fsh26 gene. Alteration of a wild-type fsh26 allele, whether, for example, by point mutation or deletion, can be detected by any of the means discussed herein.
  • the probes are used to detect the presence of the target sequences (for example, in screening for susceptibility to
  • r a neuropsychiatric disorder, including for example, without limitation, schizophrenia, attention deficit disorder, a schizoaffective disorder, a bipolar affective disorder or a unipolar affective disorder
  • the biological sample to be analyzed such as blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal, and genito-urinary tracts, stool, urine, .rs 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 biological sample to be analyzed such as blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal, and genito-urinary tracts, stool, urine, .rs sputum
  • sample nucleic acid may then be prepared in various ways to facilitate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting.
  • the targeted region of the fsh26 nucleic acid usually must be at least partially single-stranded to form hybrids with the target sequence
  • ⁇ c targeting sequence of the probe If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art.
  • the diagnostic methods of the present invention further encompasses
  • nucleic acid reagent sequences within the fsh26 gene, or chromosome 18q nucleotide sequences flanking the fsh26 gene are 15 to 30 nucleotides in length.
  • nucleic acid from the cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • non- annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled s/226 nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The sequences,
  • fsh26 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 fsh26 gene sequence, in order to determine whether afsh26 gene mutation or a cosegrating polymo ⁇ hism of interest is present.
  • fsh26 mutations or polymo ⁇ hisms can be
  • fsh26 flanking sequences e.g., sequences present in the nucleotide sequence shown in FIG. IB
  • senors involve their amplification, e.g. , by PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), followed by the analysis of the amplified molecules using techniques well known to those of skill in the art, such as, for example, those listed above.
  • the resulting amplified sequences can be compared to those that would be expected if the nucleic acid being amplified contained only normal copies of the fsh26 gene in order to ⁇ determine whether afsh26 gene mutation or polymo ⁇ hism in linkage disequilibrium with a disease-causing s ⁇ 2(5 allele exists.
  • genotyping techniques can be performed to identify individuals carrying s/z2(5 gene mutations. Such techniques include, for example, the use of restriction fragment length polymo ⁇ hisms (RFLPs), which involve sequence variations in
  • ⁇ describes a DNA marker based on length polymo ⁇ hisms in blocks of (dC-dA)n-(dG-dT)n short tandem repeats.
  • the average separation of (dC-dA)n-(dG-dT)n blocks is estimated to be 30,000-60,000 bp. 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 fsh26 gene, and the diagnosis of diseases and disorders
  • Caskey et al. (U.S. Pat.No. 5,364,759) describe a DNA profiling assay for detecting short tri and tetra nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the fsh26 gene, amplifying the extracted DNA, and labeling the repeat sequences to form a genotypic map of the individual's DNA.
  • SNPs ⁇ - nucleotide polymo ⁇ hisms
  • Conventional techniques for detecting SNPs include, e.g. , conventional dot blot analysis, single stranded conform ational polymo ⁇ hism (SSCP) analysis (see, e.g., Orita et al, 1989, Proc. Natl. Acad. Sci. USA 86:2766-2770)), denaturing gradient gel electrophoresis (DGGE),
  • heteroduplex analysis mismatch cleavage detection, and other routine techniques well known in the art (see, e.g., Sheffield et al, 1989, Proc. Natl. Acad. Sci. 56:5855-5892; Grompe, 1993, Nature Genetics 5:111-117).
  • preferred methods of detecting and mapping SNPs involve microsequencing techniques wherein an SNP site in a target DNA is detecting by a single nucleotide primer extension reaction (see, e.g., Goelet et al,
  • RNA from a cell type or tissue known, or suspected, to express the fsh26 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 fsh26 gene.
  • analyses may reveal both quantitative and qualitative aspects of the expression pattern of the fsh26 gene, including activation or
  • a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA).
  • a sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like.
  • ⁇ - 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 fsh26 gene nucleic acid reagents described in Section 5.1 that contain afsh26 gene or nucleic acid sequence.
  • the preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides.
  • the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard
  • nucleic acid staining or by utilizing any other suitable nucleic acid staining method.
  • Nucleic acid reagents described in Section 5.1 that contain afsh26 gene or nucleic acid sequence may be
  • Standard Northern analysis can be performed to determine the level of mRNA expression of the fsh26 gene.
  • sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the
  • the nucleic acid molecules described herein can be used to map the chromosomal location of fsh26 homologues in various species. Such mapping information can be used, for example, for analysis of the activity of fsh26 transgenes in mice.
  • the nucleic acid molecules can further be used to map the location of copies of fsh26 genes in the human chromosome, such as
  • ⁇ r those caused by genetic abnormalties, e.g., translocations.
  • genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the sequence of a gene of the invention.
  • Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
  • - c particular sequence to a particular chromosome Three or more sequences can be assigned per day using a single thermal cycler.
  • oligonucleotide primers Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.
  • Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al.; 1990, Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes (CITE), and pre-selection by hybridization to chromosome specific cDNA libraries.
  • FISH Fluorescence in situ hybridization
  • Reagents for chromosome mapping can be used individually to mark a single
  • chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • afsh26 polypeptide and fragments and sequences thereof and antibodies specific thereto can be used to map the location of the gene encoding the polypeptide on a chromosome.
  • This mapping can be carried out by specifically detecting the presence of the polypeptide in members of a panel of somatic cell hybrids between cells of a first species of animal from which the protein originates and cells from a ⁇ second species of animal and then determining which somatic cell hybrid(s) expresses the polypeptide and noting the chromosome(s) from the first species of animal that it contains.
  • the presence of the polypeptide in the somatic cell hybrids can be determined by assaying an activity or
  • polypeptide for example, enzymatic activity, as described in Bordelon-Riser et ⁇ l. (1979) Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978J Proc. Natl. Acad. Sci. USA 75:5640-5644.
  • _ n data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland et al.,1987, Nature 325:783-787.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. r 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.
  • nucleic acid sequences of the present invention can also be used to generate nucleic acid sequences of the present invention.
  • sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected jr , portions of an individual's genome.
  • the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique , set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the nucleic acid sequences of the invention uniquely represent portions of the 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
  • _, - and 1C 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. 2 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification of the individual, living or dead can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a 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 of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e., another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e., another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
  • fsh26 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. 5.5.5 USE OF fsh26 GENE SEQUENCES IN PREDICTIVE
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for
  • one aspect of the present invention relates to diagnostic assays for determining fsh26 protein and or nucleic acid expression as well as fsh26 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,
  • 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 fsh26 protein, nucleic acid expression or activity. For example, mutations in an fsh26 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically
  • . r treat an individual prior to the onset of a disorder characterized by or associated with an fsh26 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 f) an fsh26 gene by comparing its expression to the expression of a gene that is not an fsh26 gene, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-BAD-affected normal sample, or between samples from r different sources.
  • the expression level can be provided as a relative expression level.
  • the level of expression of the gene is determined for 10 or more samples of different cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question.
  • cell isolates are selected depending upon the tissues in which the gene of interest is expressed. For example, for fsh26 family members, expression was observed in the brain. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question. The expression level of the gene determined for the test sample (absolute level of expression) 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 fsh26-mediated disease.
  • diseases which may be studied include, without limitation, those associated with tissues of the brain.
  • the samples used in the baseline determination will be from an fsh26-mediated diseased or from non-diseased cells of tissue.
  • fsh26 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 fsh26 gene can serve as a target gene.
  • the mean expression score is particularly important in identifying whether an fsh26 gene can serve as a target gene.
  • n . 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 of the fsh26-mediated disease state.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of fsh26 in clinical trials.
  • agents e.g., drugs, compounds
  • ⁇ n may also be used as diagnostics and prognostics for a fsh26-related disorder, e.g., neuropsychiatric disorder, such as BAD, as described herein.
  • a fsh26-related disorder e.g., neuropsychiatric disorder, such as BAD
  • Such methods may be used to detect abnormalities in the level of fsh26 gene product synthesis or expression, or abnormalities in the structure, temporal expression, and or physical location of fsh26 gene product.
  • the antibodies and immunoassay methods described below have, for example, r important in vitro applications in purifying/5/z26 ' gene products and in assessing the efficacy of treatments fory_s/z2 ⁇ 5-related disorders, e.g., neuropsychiatric disorders, such as BAD.
  • Antibodies, or fragments of antibodies, such as those described below may be used to screen potentially therapeutic compounds in vitro to determine their effects on fsh26 gene expression and fsh26
  • 3 ⁇ fsh26- ⁇ eXated disorder e.g., a neuropsychiatric disorder, such as BAD, can be identified, and a therapeutically effective dose determined.
  • a neuropsychiatric disorder such as BAD
  • In vitro immunoassays may also be used, for example, to assess the efficacy of cell-based gene therapy for afsh26-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • afsh26-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • Antibodies directed against fsh26 peptides may be used in vitro to determine,
  • fsh26 gene expression achieved in cells genetically engineered to produce fsh26 peptides.
  • intracellular fsh26 gene products such an assessment is done, preferably, using cell lysates or extracts. Such analysis allows for a determination of the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.
  • the tissue or cell type to be analyzed will generally include those that are known, or suspected, to express the fsh26 gene.
  • the protein isolation methods employed c herein may, for example, be such as those described in Harlow and Lane (1988,
  • 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
  • Preferred diagnostic methods for the detection of fsh26 gene products or conserved variants or peptide fragments thereof may involve, for example, immunoassays wherein the fsh26 gene products or conserved variants or peptide fragments are detected by their interaction with an anti-fsh26 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 fsh26 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
  • 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 fsh26 gene products or conserved variants or peptide .r fragments thereof.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • ⁇ r 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 ⁇ 2 ⁇ 5 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.
  • 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 ⁇ 2 ⁇ 5 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 fsh26 c polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone marrow, the external sections of the skin, respiratory, intestinal, and genito-urinary tracts, stool, urine, sputum, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.
  • the range of the fsh26 gene products, or conserved variants or peptide fragments thereof, which may be detected in the blood or any one of the patient samples listed supra using a detectably labeled antibody capable of identifying ⁇ s/z26 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 fsh26 gene r.rs 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 fsh26 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 c 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 fsh26 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 of the carrier can be either soluble to
  • 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.
  • binding activity of a given lot of anti-fsh26 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,
  • the detection can be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the 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 For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect fsh26 gene peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986).
  • RIA radioimmunoassay
  • the use of a gamma counter or a scintillation counter or by autoradiography It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • chemiluminescent compound 1 ⁇ chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • bioluminescent compound may be used to label the antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for pu ⁇ oses of labeling are
  • 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,
  • ⁇ n alkylating agents e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and an
  • 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,
  • lymphokines 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 r 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, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • the substantially purified antibodies of the invention, or fragments thereof can be human, non-human, chimeric and/or humanized antibodies.
  • the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
  • the non-human antibodies of the invention can be chimeric and/or humanized antibodies.
  • the non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.
  • the invention provides monoclonal antibodies or
  • ⁇ - fragments thereof which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
  • the monoclonal antibodies can be human,
  • 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 of the invention.
  • n non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the 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. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
  • Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance.
  • detectable substances that
  • antibodies of the invention can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.
  • the invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention
  • ⁇ invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
  • Still another aspect of the invention is a method of making an antibody that
  • ⁇ - specifically recognizes an fsh26 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 No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • a sample is collected from the mammal that contains an antibody that specifically recognizes an 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
  • antibodies are collected from the antibody-producing cell.
  • the following assays are designed to identify compounds that bind to afsh26 gene product, e.g., proteins or portions of proteins that interact with afsh26 gene product, compounds that interfere with the interaction of afsh26 gene product with other proteins
  • fsh26 gene regulatory sequences e.g., promoter sequences; see e.g., Platt, 1994, J. Biol. Chem. 269: 28558- 28562
  • 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 fsh26 gene or some other gene or gene product involved in afsh26 regulatory pathway.
  • fl 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 ⁇ l., 1991, Nature 354, 82-84; Houghten, et ⁇ l., 1991, Nature 354, 84-86), and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopep tides (including, but not y, limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et ⁇ l., 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
  • 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, jp-propyldopacetamide dithiocarbamate derivatives e.g., FLA 63; anti-
  • ⁇ - anxiety drugs e.g., diazepam
  • monoamine oxidase (MAO) inhibitors e.g., iproniazid, clorgyline, phenelzine and isocarboxazid
  • biogenic amine uptake blockers e.g.
  • tricyclic antidepressants such as desipramine, imipramine and amitriptyline; serotonin reuptake inhibitors e.g., fluoxetine; antipsychotic drugs such as phenothiazine derivatives (e.g., chlo ⁇ romazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (Haldol)), thioxanthene derivatives (e.g., chlo ⁇ rothixene), and dibenzodiazepines (e.g., clozapine); benzodiazepines; dopaminergic agonists and antagonists e.g., L-DOPA, cocaine,
  • antipsychotic drugs such as phenothiazine derivatives (e.g., chlo ⁇ romazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (
  • noradrenergic agonists and antagonists e.g. , clonidine, phenoxybenzamine, phentolamine, tropolone.
  • 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 fsh26 gene product, and for ameliorating 5 , ?2 ⁇ 5-related disorders, e.g., neuropsychiatric disorders such as BAD.
  • compounds identified via such techniques can provide lead compounds to be tested for an ability to modulate a/s ⁇ 2 ⁇ 5-mediated process and/or to ameliorate symptoms of
  • In vitro systems may be designed to identify compounds that bind fsh26 gene products of the invention.
  • Compounds identified may be useful, for example, in modulating the activity of unimpaired and/or mutant fsh26 gene products, may be useful in elucidating the biological function of the fsh26 gene product, maybe utilized in screens for identifying j r compounds that disrupt normal fsh26 gene product interactions, or may in themselves disrupt such interactions, and can provide lead compounds to be further tested for an ability to modulate ay_s ⁇ 26-mediated process and or to ameliorate symptoms of afsh26-reXated disorder.
  • ⁇ r gene products involves preparing a reaction mixture of the fsh26 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.
  • assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring s/z2 ⁇ 5 gene product or the test
  • the fsh26 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
  • 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. Where the previously
  • 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).
  • reaction can be conducted in a liquid phase, the reaction
  • Any method suitable for detecting protein-protein interactions may be employed for identifying s/!2 ⁇ 5 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
  • methods include, for example, probing expression libraries with XaheXedfsh26 protein, using fsh26 protein in a manner similar to the well known technique of antibody probing of ⁇ gtl 1 libraries.
  • plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the fsh26 gene product and the other consists of the transcription activator protein's 0 activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into this plasmid as part of a cDNA library.
  • the DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast S ⁇ cch ⁇ romyces cerevisi ⁇ e that contains a reporter gene (e.g., HBS or l ⁇ cZ) whose regulatory region contains the transcription activator's binding site.
  • a reporter gene e.g., HBS or l ⁇ cZ
  • the two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product.
  • fsh26 gene products may be used as the bait gene product.
  • Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
  • 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 fsh26 gene sequence such as the open reading frame of the fsh26 gene, can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.
  • cDNA library of the cell line from which proteins that interact with the bait fsh26 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 GAL4.
  • This library can be co-transformed along with the bait fsh26 gene-GAL4 fusion
  • n 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 fsh26 gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies that express HIS3 can be detected by their growth on petri dishes containing semi-solid agar based media
  • the cDNA can then be purified from these strains, and used to produce and isolate the bait fsh26 gene-interacting protein using techniques routinely practiced in the art.
  • 20 fsh26 gene products of the 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
  • binding partners Compounds that disrupt _> ?2 ⁇ 5 binding in this way may be useful in regulating the activity of the fsh26 gene product, especially mutant fsh26 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,
  • the basic principle of the assay systems used to identify compounds that interfere with the interaction between the fsh26 gene product and its binding partner or partners involves preparing a reaction mixture containing the fsh26 gene product, and the binding partner under conditions and for a time sufficient to allow the two to interact and
  • the reaction mixture is prepared in the presence and absence of the 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 fsh26 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 fsh26 gene protein and the binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction
  • reaction mixtures containing the test compound and normal fsh26 gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant fsh26 gene protein. This comparison may be
  • the assay for compounds that interfere with the interaction of the fsh26 gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the fsh26 gene product or the binding
  • test compounds that interfere with the interaction between the fsh26 gene products and the binding partners can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the fsh26 gene protein and interactive binding partner.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding
  • test compound to the reaction mixture after complexes have been formed.
  • the various formats are described briefly below.
  • either the fsh26 gene product or the interactive binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
  • microtiter plates are conveniently
  • 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 fsh26 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
  • the partner of the 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.
  • 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).
  • test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.
  • reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one
  • 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
  • the fsh26 gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.2. above.
  • the fsh26 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
  • the GST-fsh26 fusion protein can be anchored to glutathione-agarose beads.
  • the interactive binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
  • unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
  • the interaction between the fsh26 gene protein and the interactive binding partner can be detected by measuring the
  • the GST-fsh26 gene fusion protein and the interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose
  • 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 fsh26 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 fsh26 protein and/or the interactive or binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites.
  • ⁇ methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding.
  • one protein can be anchored to a solid surface using methods described in this section, above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding
  • segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.
  • afsh26 gene product can be anchored to a solid material as described, above, in this section by making a GST-fsh26 fusion protein and allowing it to bind to glutathione agarose beads.
  • 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-fsh26 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 z2(5-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
  • MDD unipolar major depressive disorder
  • phobias e.g., agoraphobia
  • panic disorders generalized anxiety disorders
  • somatization disorders and hypochondriasis and attention deficit disorders.
  • J ⁇ affect fsh26 gene activity by either affecting s j2 ⁇ 5 gene expression or by affecting the level of fsh26 gene product activity.
  • compounds may be identified that are involved in another step in the pathway in which the fsh26 gene and/or fsh26 gene product is involved and, by affecting this same pathway may modulate the effect of sh26 on the development of a neuropsychiatric disorder such as BAD.
  • Such compounds can be used as c part of a therapeutic method for the treatment of the disorder.
  • afsh26-related disorder e.g., a neuropsychiatric disorder, such as BAD.
  • cell-based systems can be used to identify compounds that may act to
  • Such cell systems can include, for example, recombinant or non-recombinant cell, such as cell lines, that express the fsh26 gene.
  • cells that express ⁇ s/j26 may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of afsh26-related
  • disorder e.g., a neuropsychiatric disorder, such as BAD
  • BAD neuropsychiatric disorder
  • the cells can be assayed to measure alterations in the expression of the fsh26 gene, e.g., by assaying cell lysates for fsh26 mRNA transcripts (e.g., by Northern analysis) or for fsh26 gene products expressed by the cell; compounds that modulate expression of the fsh26 gene are good candidates as therapeutics.
  • the cells are examined to determine whether one or more cellular phenotypes associated with ay_s/z2 ⁇ 5-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.
  • ay_s/z2 ⁇ 5-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • animal-based systems or models for a s i2(5-related disorder e.g. , a neuropsychiatric disorder, such as BAD, may be used to identify compounds capable of
  • 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 fsh26 sequences or, alternatively, to no longer express endogenous fsh26 gene sequences (i.e., "knock-out" mice).
  • transgenic mice e.g., mice that have been genetically engineered to express exogenous or endogenous fsh26 sequences or, alternatively, to no longer express endogenous fsh26 gene sequences (i.e., "knock-out" mice).
  • Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and
  • animal models may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms, at a sufficient concentration and for a sufficient time to elicit such an amelioration of symptoms of a fsh26-r elated disorder, e.g., a neuropsychiatric disorder, such as BAD, in, the exposed animals.
  • a fsh26-r elated disorder e.g., a neuropsychiatric disorder, such as BAD
  • the response of the animals to the exposure may be monitored by j rs assessing the reversal of such symptoms.
  • any treatments that reverse any aspect of symptoms of a s ⁇ d-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.
  • a variety of methods can be employed for the diagnostic and prognostic 0 evaluation of/_s/?2(5-related disorders, such as neuropsychiatric disorders, e.g., BAD, and for the identification of subjects having a predisposition to such disorders.
  • Such methods may, for example, utilize reagents such as the fsh26 gene nucleotide sequences described in Sections 5.1, and antibodies directed against s ⁇ 2 ⁇ 5 gene products, including peptide fragments thereof, as described, above, in Section 5.3.
  • such reagents may be used, for example, for: (1) the detection of the presence of fsh26 gene mutations, the detection of polymo ⁇ hisms that cosegregate with particular fsh26 gene mutations or the detection of either over- or under-expression of fsh26 gene mRNA relative to the state of a/ ?26 ' -related disorder, such as a neuropsychiatric disorder, e.g., BAD; - (2) the detection of either an over- or an under-abundance of fsh26 gene product relative to the unaffected state; and (3) the detection of an aberrant level of fsh26 gene product activity relative to the unaffected state.
  • a neuropsychiatric disorder e.g., BAD
  • Nucleic acid molecules of the invention can, for example, be used to diagnose an fsh26- .
  • ⁇ related or neuropsychiatric disorder using, for example, the techniques for fsh26 mutation/co-segregating polymo ⁇ hism detection described above.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific gene nucleic acid of the invention or anti-fsh26 gene antibody reagent described herein, which may be conveniently 1 r used, e.g., in clinical settings, to diagnose patients exhibiting abnormalities of a fsh26- related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a fsh26- 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 fsh26 gene is expressed may be 7f) 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 j e associated with aberrant expression or activity of a polypeptide of the invention.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • the prognostic assays can be utilized to identify a subject having or at risk for - ⁇ developing such a disease or disorder.
  • test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g. , blood, plasma, serum, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, urine, sputum, tears, saliva), cell sample, or tissue.
  • 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 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 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 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 determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,
  • ⁇ - treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the polypeptide).
  • agents of a type which decrease activity of the polypeptide e.g., agents of a type which decrease activity of the polypeptide.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder
  • polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide).
  • the methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at
  • a chromosomal rearrangement of the gene 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 o r 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 of the sample, contacting the nucleic acid sample with one or more primers which specifically r hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting
  • 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
  • mutations in a selected gene from a sample cell r. 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 are used to identify mutations in the sample DNA.
  • 2 ⁇ - (see, e.g., U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al, 1996, Human Mutation
  • 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 anays 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 c the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977, Proc. Natl. Acad. Sci. USA 74:560 or Sanger, 1977, Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (1995,
  • RNA/RNA RNA/RNA
  • Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or
  • RNA/DNA heteroduplexes 15 RNA/DNA heteroduplexes (Myers et al., 1985, Science 230:1242).
  • the technique of mismatch cleavage entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to n 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 of the mismatched
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or
  • DNA mismatch repair enzymes proteins that recognize mismatched base pairs in double-stranded DNA
  • 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 r wild type nucleic acids (Orita et al., 1989, Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton, 1993, Mutat. Res. 285:125-144; Hayashi, 1992, Genet. Anal. Tech. Appl. 9:73-79). 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
  • RNA rather than DNA
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen
  • 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
  • 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).
  • 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).
  • oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • r Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al., 1989, Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner, 1993, Tibtech 11:238).
  • it maybe desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al., 1992, Mol.
  • amplification may also be performed using Taq ligase for amplification (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g. , in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.
  • any cell type or tissue preferably any cell type or tissue, preferably
  • peripheral blood leukocytes in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
  • kits that facilitate the use /and or detection of fsh26 genes or co-segregating polymo ⁇ hisms and fsh26 gene products described herein.
  • the kits described herein may be conveniently used, e.g. , in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.
  • any cell type or tissue in which the jc 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/s/z26 ' genes or gene products.
  • a diagnostic kit is provided, with one or more containers comprising an oligonucleotide, e.g. ,
  • kits comprising a pair of primers useful for amplifying afsh26 nucleic acid molecule encoding afsh26 polypeptide of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • kits can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained.
  • Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.
  • 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 of the invention in a biological sample can be
  • kits comprising: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and, . optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • a fsh-related disorder such as a neuropsychiatric disorder, e.g., BAD.
  • afsh26-mediated process can be modulated and or whereby a symptom of a fsh26-r eXated disorder, e.g., a
  • a neuropsychiatric disorder such as a cognitive or mood disorder, for example, BAD
  • a cognitive or mood disorder for example, BAD
  • Such a method can comprise administering a compound which modulates the expression of afsh26 gene and/or the expression or activity of afsh26 gene product, so that the process is modulated or a symptom of the disorder is ameliorated.
  • afsh26-reXated disorder phenotype or symptom can occur as a result of a decrease in expression or activity of a component of a Fsh26-mediated pathway, such as a Fsh26 receptor, ligand, or an upstream or downstream component of a Fsh26 signal transduction pathway.
  • a component of a Fsh26-mediated pathway such as a Fsh26 receptor, ligand, or an upstream or downstream component of a Fsh26 signal transduction pathway.
  • increase in the level of fsh26 gene expression and or fsh26 gene product expression or activity could facilitate the progress
  • a method for treating such a ⁇ A26 ' -related disorder phenotype can comprise administering to a subject a Fsh26 peptide or polypeptide, or fragment, analog or mimetic thereof, to ameliorate at least one symptom of afsh26-related disorder phenotype.
  • a method for treating such a/s/?2 ⁇ 5-related disorder phenotype can comprise administering to a subject a compound that modulates the activity or expression of afsh26 gene or gene product to ameliorate at least one symptom of a s/*2(5-related disorder phenotype.
  • such methods can comprise administering a compound that increases the activity or expression of afsh26 gene or gene product.
  • a loss of normal fsh26 gene product function results in the development of a s/z2 ⁇ 5-related disorder
  • ⁇ phenotype e.g., a neuropsychiatric disorder phenotype
  • an increase in fsh26 gene product activity would facilitate progress towards an asymptomatic state in individuals exhibiting a deficient level of fsh26 gene expression and/or fsh26 gene product activity.
  • a method for treating such a ⁇ s ?2(5-related disorder phenotype can comprise supplying a subject with a nucleic acid molecule encoding an
  • methods for the treatment of mammalian fsh26-related disorder e.g. , a neuropsychiatric disorders
  • methods for the treatment of mammalian fsh26-related disorder can comprise supplying a mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired fsh26
  • ⁇ 0 gene product such that the cell expresses the unimpaired ⁇ s/z2 ⁇ 5 gene product and symptoms of the disorder are ameliorated.
  • methods for enhancing the expression or synthesis of afsh26 gene or gene product can include, for example, methods such as those described below, in Section 5.10.2. . c
  • symptoms of a yA2 ⁇ 5-related disorder phenotype e.g., a neuropsychiatric disorder, such as BAD
  • a neuropsychiatric disorder such as BAD
  • such a method comprises administering an anti-Fsh26 antibody to a subject to ameliorate at least one symptom of s/z2(5-related disorder phenotype.
  • any of the compounds identified by the screening methods described in Section 5.7, above may be administered to an individual to treat a symptom of a fsh26- related disorder phenotype.
  • Methods for inhibiting or reducing the level of fsh26 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), -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
  • MAO monoamine oxidase
  • ⁇ - blockers e.g., tricyclic antidepressants such as desipramine, imipramine and amitriptyline; serotonin reuptake inhibitors e.g., fluoxetine; antipsychotic drugs such as phenothiazine derivatives (e.g., chlo ⁇ romazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (Haldol)), thioxanthene derivatives (e.g., chlo ⁇ rothixene), and dibenzodiazepines (e.g., clozapine); benzodiazepines; dopaminergic agonists and
  • antagonists e.g., L-DOPA, cocaine, amphetamine, ⁇ -methyl-tyrosine, rese ⁇ ine, tetrabenazine, benzotropine, pargyline; noradrenergic agonists and antagonists e.g. , clonidine, phenoxybenzamine, phentolamine, tropolone. If such treatment methods do comprise such compounds, preferably such compounds are utilized in a manner (e.g., different dosage, mode of administration, and/or co-administration with one or more
  • symptoms of certain fsh26-reXated disorders such as neuropsychiatric disorders, e.g., BAD
  • fsh26-reXated disorders such as neuropsychiatric disorders, e.g., BAD
  • symptoms of certain fsh26-reXated disorders maybe ameliorated by decreasing the level of fsh26 gene expression and/or fsh26 gene product activity by using fsh26 nucleic acid sequences in conjunction with well-known antisense, gene "knock-out,” ribozyme and/or triple helix methods to decrease the level of fsh26 gene expression.
  • fsh26-reXated disorders such as neuropsychiatric disorders, e.g., BAD
  • fsh26 gene 25 exhibit the ability to modulate the activity, expression or synthesis of the fsh26 gene, including the ability to ameliorate the symptoms of a/sA2 ⁇ 5-related disorder, e.g., a neuropsychiatric disorder, such as BAD, are antisense, ribozyme, and triple helix molecules.
  • a/sA2 ⁇ 5-related disorder e.g., a neuropsychiatric disorder, such as BAD
  • 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
  • Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA
  • 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 of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base 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
  • oligonucleotides complementary to coding or non- coding regions of the fsh26 gene can be used in an antisense approach to inhibit translation of endogenous fsh26 mRNA.
  • Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides 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 of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between f) antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g. , 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. 86, 6553-6556; Lemaitre, et al, 1987, Proc. Natl. Acad.
  • 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,
  • 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-carboxymethylamino
  • 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-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose,
  • 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.
  • oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier, et al, 1987, Nucl. Acids Res. 15, 6625-6641).
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue, et al, 1987, Nucl. Acids Res. 15, 6131-6148), or a
  • Oligonucleotides of the invention maybe synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl. Acids Res.
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451), etc.
  • Antisense nucleotides complementary to the target fsh26 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)
  • 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
  • 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
  • vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters
  • 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 of the metallothionein gene r x (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.
  • 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. (For a review, see Rossi, 1994, Current Biology 4, 469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme
  • 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 ribozyme is engineered so that the cleavage recognition site is
  • the target gene mRNA located near the 5' end of the target gene mRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described j r 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. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47, 207-216).
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes
  • 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 oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo.
  • modified oligonucleotides e.g., for improved stability, targeting, etc.
  • 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 of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Endogenous target gene expression can also be reduced by inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene r) (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body (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).
  • deoxyribonucleotide sequences complementary to the regulatory region of the target gene r i.e., the target gene promoter and/or enhancers
  • Nucleic acid molecules to be used in triplex helix formation for the inhibition j r of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences maybe pyrimidine-based, which will result in TAT and CGC + triplets across the three associated
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine
  • 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
  • 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,
  • c 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 of the invention may be prepared by any method known in the art for the synthesis of DNA and
  • 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 j r vectors that inco ⁇ orate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, 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 afsh26 gene product can be utilized for the treatment of afsh26-reXated disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • afsh26-reXated 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 fsh26 gene or a portion of the fsh26 gene that directs the production of a fsh26 gene product exhibiting normal fsh26 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.
  • fsh26 genes can be expressed in the brain, such gene replacement c therapy techniques should be capable delivering _> j2(5 gene sequences to these cell types within patients.
  • techniques that are well known to those of skill in the art can be used to enable j/z2t5 gene sequences to cross the blood-brain barrier readily and to deliver the sequences to cells in the brain. With respect to delivery that is capable of crossing the
  • viral vectors such as, for example, those described above, are preferable.
  • techniques for delivery involve direct administration of such fsh26 gene sequences to the site of the cells in which the fsh26 gene sequences are to be expressed.
  • Additional methods that may be utilized to increase the overall level of fsh26 gene expression and/or fsh26 gene product activity include the introduction of appropriate 7s z2(5-expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to ameliorate the symptoms of afsh26-reXated disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • afsh26-reXated disorder e.g., a neuropsychiatric disorder, such as BAD.
  • Such cells may be either recombinant or non- j r, recombinant.
  • the cells that can be administered to increase the overall level of fsh26 gene expression in a patient are normal cells, preferably brain cells, that express the fsh26 gene.
  • cells preferably autologous cells, can be engineered to express
  • fsh26 gene sequences may then be introduced into a patient in positions appropriate for the amelioration of the symptoms of a fsh26-r elated disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a fsh26-r elated disorder e.g., a neuropsychiatric disorder, such as BAD.
  • cells that express an unimpaired fsh26 gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells.
  • the expression of the fsh26 gene sequences is controlled by the appropriate gene
  • the cells to be administered are non-autologous cells, they can be any non-autologous cells.
  • 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 fsh26 gene product ⁇ - activity can be administered using standard techniques that are well known to those of skill in the art.
  • the administration techniques should include well known ones that allow for a crossing of the blood-brain barrier.
  • Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide.
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • the pharmacogenomics of the ⁇ individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype.
  • Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens.
  • the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual j c can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • 3n 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. For example, glucose-6-phosphate
  • G6PD 3 ⁇ - dehydrogenase deficiency
  • 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
  • CYP2D6 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
  • the activity of a polypeptide of the invention, expression of a nucleic jrx acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply geno typing of polymo ⁇ hic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype.
  • Monitoring the influence of agents (e.g. , drugs, compounds) on the expression or activity of a polypeptide of the invention can be applied not only in basic drug screening, but also in clinical trials.
  • agents e.g. , drugs, compounds
  • the effectiveness of an agent, as 35 determined by a screening assay as described herein, to increase gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting decreased gene expression, protein levels, or protein activity.
  • the effectiveness of an agent, as determined by a screening assay, to decrease gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting increased gene expression, protein levels, or protein activity.
  • expression or activity c of a polypeptide of the invention and preferably, that of other polypeptide that have been implicated in ay_s , ⁇ 26 ' -related disorder, e.g., a neuropsychiatric disorder such as BAD, can be used as a marker of the immune responsiveness of a particular cell.
  • genes including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or
  • r small molecule which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified.
  • a polypeptide of the invention e.g., as identified in a screening assay described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated in the disorder.
  • the levels of gene expression i.e., a
  • ⁇ - gene expression pattern can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response rx state may be determined before, and at various points during, treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug
  • polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent maybe desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • ⁇ - fsh26 gene products or compounds that are determined to affect fsh26 gene expression or gene product activity, can be administered to a patient at therapeutically effective doses to treat or ameliorate a ⁇ s ⁇ 2(5-related disorder, e.g., a neuropsychiatric disorder, such as BAD.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of such a disorder.
  • a therapeutically effective amount i.e., an effective dosage
  • 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
  • c 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.
  • the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • 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 jc 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
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or
  • inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per 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.
  • ⁇ - identity, size, and condition of the subject or sample being treated further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to
  • 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.
  • 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.
  • 9 ⁇ depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • Toxicity and therapeutic efficacy of such compounds can be determined by
  • LD 50 the dose lethal to 50% of the population
  • ED 50 the dose therapeutically effective in 50%
  • 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 prefened. While compounds that exhibit large therapeutic indices are prefened. While compounds that exhibit large therapeutic indices are prefened. While compounds that exhibit large therapeutic indices are prefened. While compounds that exhibit large therapeutic indices are prefened. While compounds that exhibit large therapeutic indices are prefened. While compounds that
  • 3n 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 c preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • Such ⁇ - information can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention are provided.
  • r, 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 r.rs 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 r.rs stearate, talc or silica
  • 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 ye 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,
  • Preparations for oral administration may be suitably formulated to give controlled release of the 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 of the r 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
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as
  • 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 yr x 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.
  • 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 j c dispenser device may be accompanied by instructions for administration.
  • yeast artificial chromosomes containing human sequences were mapped to the region being analyzed based on publicly available maps (Cohen et al, 1993, CR. Acad. Sci. 316, 1484-1488). The YACs were then ordered and contig reconstructed by performing standard
  • STS short tag sequence
  • BAC Bacterial artificial chromosome mapping
  • STSs from the region were used to screen a human BAC library (Research Genetics, HuntsviUe, AL). The ends of
  • BACs were cloned or directly sequenced. The end sequences were used to amplify the next overlapping BACs. From each BAC, additional microsatellites 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 r.r production. Microsatellite sequences from the sublibraries were identified by conesponding 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
  • clusters were assembled together to produce a single continuous DNA strand which covered the whole interval of interest as defined by the haplotype.
  • the resulting sequences were then compared to public DNA and protein databases using BLAST algorithms (Altschul et al, 1990, J. Mol. Biol. 215: 403-410).
  • cDNA selection cDNA selection was used as an additional method for gene identification of transcribed sequences over large regions of the genome. Through a combination of characterizations including physical mapping and RNA hybridization, the , selected cDNAs were ananged 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).
  • Northern analysis Standard Northern analysis techniques were utilized in probing human and fetal multiple tissue Northern blots purchased from Clontech (Palo Alto, , ⁇ - CA). Blots were hybridized to different gene probes, which were derived by PCR from fsh26 cDNA sequences.
  • YACs were mapped to the chromosome 18 region being analyzed.
  • the region from publicly available markers D18S1161 and D18S554 which spans most of the D18S469- 3 ⁇ - 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 To ensure development of an accurate physical map, 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 and BAD18cagl are defined by a (GA) 22 di-nucleotide repeat; the
  • BADct22 primer set used was as follows:
  • BADl ⁇ cagl is defined by a (CAG) n tri-nucleotide repeat; the following primer set was ⁇ n used for amplification of the BAD18cagl 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.
  • fsh26 A gene, termed fsh26, was found within the 116 kb interval between BAD18ct22 and BAD18cagl on the long arm of chromosome 18. fsh26, therefore, can be involved in neuropsychiatric disorders. j. fsh26 is positioned within the 116 kb interval between BAD 18 ct22 and
  • BAD 18 cagl as shown in Figure 1A.
  • AJTOWS denote the location and direction of transcript relative to genetic markers shown above in the diagram.
  • Novel sequences of the entire 18q interval were identified that can be used with the methods of the invention, were identified.
  • the 116 bp was used as input to the n NBLAST program and public and private databases were searched, including: dbest
  • Ep69104 represents a composite deposit of a mixture of two strains, each of which contains either bacterial artificial chromosome (BAC) BAC69 or BAC 104.
  • the two BACs together, contain the approximately 160 kb region of human chromosome 18 depicted in FIG. IB.
  • BAC69 or BAC 104 an aliquot of the mixture can be streaked out to single colonies on nutrient media (e.g., LB plates) supplemented with lOO ⁇ g/ml ampicillin, a single colony can be grown, and then BAC DNA can be extracted from the culture using standard procedures.
  • nutrient media e.g., LB plates
  • BAC69 DNA yields fragments having a total length of approximately 220kb
  • BAC 104 DNA yields fragments having a total length of approximately 80kb.
  • Ep34680 represents a composite deposit of a mixture of five strains, one of which contains the fsh26 cDNA clone in a PT7T3-PAC vector (2.9kb).
  • a strain harboring the fsh26 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 of the DNA preparation can be digested with EcoRl and Not I, and the resulting products can be separated by standard gel electrophoresis techniques.
  • Liberated inserts are of the following approximate sizes: 500 bp 500 bp 550 bp 600bp fsh26: 700 bp

Abstract

L'invention concerne le gène fsh26 de mammifère, un nouveau gène associé à des troubles affectifs bipolaires chez l'être humain. L'invention comprend des acides nucléiques de fsh26, des molécules d'ADN recombinées, des gènes clonés ou des variants dégénérés de ceux-ci, des produits géniques de fsh26 et des anticorps dirigés contre ces produits géniques, des vecteurs de clonage contenant des molécules de gène fsh26 de mammifère, et des hôtes mis au point génétiquement en vue d'exprimer ces molécules. L'invention concerne en outre des procédés d'identification de composés qui modulent l'expression de gènes fsh26 et de produits géniques de fsh26, et l'utilisation de ces composés comme agents thérapeutiques dans le traitement de troubles liés à fsh26, p. ex. des troubles neuropsychiatriques. L'invention a aussi trait à des méthodes d'évaluation diagnostique, d'essai génétique et de pronostic de troubles liés à fsh26, p. ex. des troubles neuropsychiatriques comprenant la schizophrénie, le déficit de l'attention, un trouble schizo-affectif, un trouble affectif bipolaire ou un trouble affectif unipolaire, et à des procédés et des compositions utiles pour le traitement de ces troubles.
PCT/US2000/030824 1999-11-08 2000-11-08 Procedes et compositions servant a diagnostiquer et a traiter des troubles lies au chromosome 18q WO2001034841A1 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DALY ET AL.: "Do multiple data sets provide support for a bipolar illness susceptibility locus on chromosome 18?", GENETIC EPIDEMIOLOGY, vol. 14, 1997, pages 599 - 604, XP002936831 *
NOTHEN ET AL.: "Evaluation of linkage of bipolar affective disorder to chromosome 18 in a sample of 57 german families", MOLECULAR PSYCHIATRY, vol. 4, 1999, pages 76 - 84, XP002936832 *

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