WO2001033965A1 - METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS - Google Patents

Abstract

The present invention relates to the mammalian fsh25 gene, a gene associated with bipolar affective disorder (BAD) in humans. The invention encompasses fsh25 nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof, fsh25 gene products and antibodies directed against such gene products, cloning vectors containing mammalian fsh25 gene molecules, and hosts that have been genetically engineered to express such molecules. The invention further relates to methods for the identification of compounds that modulate the expression of fsh25 genes and gene products and to using such compounds as therapeutic agents in the treatment of fsh25-related disorders, e.g., neuropsychiatric disorders. The invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of fsh25-related disorders, e.g., neuropsychiatric disorders, including schizophrenia, attention deficit disorder, a schizoaffective disorder, a bipolar affective disorder or a unipolar affective disorder, and to methods and compositions for the treatment of these disorders.

Description

METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING CHROMOSOME 18q RELATED DISORDERS

Cross Reference to Related Applications This is a continuation-in-part application which claims the benefit of priority under 35 U.S.C. § 119(e) of co-pending United States Patent provisional Application No. 60/164,038, filed November 8, 1999, the entire contents of which are incoφorated herein by reference in its entirety.

Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development

This invention was made with government support under grant numbers R01MH49499 and K02MH01375 from the National Institutes of Health. The government has certain rights in the invention.

1. INTRODUCTION

The present invention relates, first, to polynucleotides of a region of human chromosome 18q which is associated with neuropsychiatric disorders. Second, the invention relates to a gene refened to herein as fsh25, 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 fsh25- related disorder, e.g., a neuropsychiatric disorder, and/or can be used to map human chromosome 18q. The invention encompasses fsh25 nucleic acids, recombinant DNA molecules, cloned genes and degenerate variants thereof, vectors containing such fsh25 nucleic acids, and hosts that have been genetically engineered to express and/or contain such molecules. The invention further relates to fsh25 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 fsh25 genes. Still further, the invention relates to methods of using compounds such as those identified herein as therapeutic agents for modulation of s"/z25-mediated process or in the treatment of a symptom of s/z25-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 _? .25-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.

2. BACKGROUND OF THE INVENTION

One of the most prevalent and potentially devastating of neuropsychiatric

_<- disorders is bipolar affective disorder (BAD), 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). The most severe and clinically distinctive forms of BAD are BP-I (severe bipolar affective (mood) disorder), which affects 2-3 million people in the United States, and SAD-

_{1 r}. M (schizoaffective disorder manic type). They are characterized by at least one full episode of mania, with or without episodes of major depression (defined by lowered mood, or depression, with associated disturbances in rhythmic behaviors such as sleeping, eating, and sexual activity). BP-I often co-segregates in families with more etiologically heterogeneous syndromes, such as with a unipolar affective disorder such as unipolar major depressive

- _<- disorder (MDD), which is a more broadly defined phenotype (Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease, Rosenberg, et al, eds., Butterworths, New York, pp. 951-965; Mclnnes and Freimer, 1995, Cun. 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 _jr. courses, and segregate together in family studies (Rosenthal, et al, 1980, Arch. General Psychiat. 37, 804-810; Levinson and Levitt, 1987, Am. J. Psychiat. 144, 415-426; Goodwin, et al, 1990, Manic Depressive Illness, Oxford University Press, New York). Hence, methods for distinguishing neuropsychiatric disorders such as these are needed in order to effectively diagnose and treat afflicted individuals. _<- While many drugs have been used to treat individuals diagnosed with BAD, including lithium salts, carbamazepine and valproic acid, none of the cunently available drugs are adequate. For example, drug treatments are effective in only approximately 60- 70%) of individuals diagnosed with BP-I. Moreover, it is cunently impossible to predict which drug treatments will be effective in, for example, particular BP-I affected individuals.

~_r. Commonly, upon diagnosis, affected individuals are prescribed one drug after another until one is found to be effective. Early prescription of an effective drug treatment, therefore, is critical for several reasons, including the avoidance of extremely dangerous manic episodes, substantial side effects of cunent treatment, and the risk of progressive deterioration if effective treatments are not found.

- ,- The existence of a genetic component for BAD is strongly supported by segregation analyses and twin studies (Bertelson, et al, 1977, Br. J. Psychiat. 130, 330-351; Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease,

Rosenberg, et al, eds., Butterworths, New York, pp. 951-965; Pauls, et al, 1992, Arch. Gen. Psychiat. 49, 703-708). Efforts to identify the chromosomal location of genes that might be involved in BP-I, however, have yielded disappointing results in that reports of linkage between BP-I and markers on chromosomes X and 11 could not be independently

,- replicated nor confirmed in the re-analyses of the original pedigrees, indicating that with BAD linkage studies, even extremely high lod scores at a single locus, can be false positives (Baron, et al, 1987, Nature 326, 289-292; Egeland, et al, 1987, Nature 325, 783-787; Kelsoe, et al, 1989, Nature 342, 238-243; Baron, et al, 1993, Nature Genet. 3, 49-55).

Recent investigations have suggested possible localization of BAD genes on

₁₀ chromosomes 18p and 21q (Benettini, et al, 1994, Proc. Natl Acad. Sci. USA 91, 5918- 5921; Munay, et al, 1994, Science 265, 2049-2054; Pauls, et al, 1995, Am. J. Hum. Genet. 57, 636-643; Maier, et al, 1995, Psych. Res. 59, 7-15; Straub, et al, 1994, Nature Genet. 8, 291-296). With respect to 21q, the proposed candidate region is not well defined and no unequivocal support exists for the presence of a BAD gene in this location.

. _. Mapping genes for common diseases believed to be caused by multiple genes, such as BAD, may be complicated by the typically imprecise definition of phenotypes, by etiologic heterogeneity, and by uncertainty about the mode of genetic transmission of the disease trait. With neuropsychiatric disorders there is even greater ambiguity in distinguishing individuals who likely carry an affected genotype from those

-_π who are genetically unaffected. For example, one can define an affected phenotype for BAD by including one or more of the broad grouping of diagnostic classifications that constitute the mood disorders: BP-I, SAD-M, MDD, and bipolar affective (mood) disorder with hypomania and major depression (BP-II).

Thus, one of the greatest difficulties facing psychiatric geneticists is

-_<- uncertainty regarding the validity of phenotype designations, since clinical diagnoses are based solely on clinical observation and subjective reports. Cunently, individuals are typically evaluated for BAD using the criteria set forth in the most cunent version of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM). Also, with complex traits such as neuropsychiatric disorders, it is difficult to

^ genetically map the trait-causing genes because: (1) neuropsychiatric disorder phenotypes do not exhibit classic Mendelian recessive or dominant inheritance patterns attributable to a single genetic locus, (2) there may be incomplete penetrance, i.e., individuals who inherit a predisposing allele may not manifest disease; (3) a phenocopy phenomenon may occur, i.e., individuals who do not inherit a predisposing allele may nevertheless develop disease due to

, environmental or random causes; (4) genetic heterogeneity may exist, in which case mutations in any one of several genes may result in identical phenotypes. Despite these difficulties, however, identification of the chromosomal location, sequence and function of genes and gene products responsible for causing neuropsychiatric disorders such as bipolar affective disorders is of great importance for genetic counseling, diagnosis and treatment of individuals in affected families.

3. SUMMARY OF THE INVENTION

The present invention encompasses, first, the nucleotide sequence of a 116 kilobase interval of human chromosome 18q (i.e., nucleotides 28441-144419 of FIG. IB), and its flanking regions (nucleotides 1-28440 and 144420-10271 of FIG. IB), associated with neuropsychiatric disorders in humans, e.g., schizophrenia, attention deficit disorders, schizoaffective disorders, bipolar affective disorders, and/or unipolar affective disorders. Second, the present invention relates to a gene located within this interval, refened to herein as fsh25, which is involved in such disorders. In addition, fsh25 nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof are provided herein. The invention also provides vectors, including expression vectors, containing fsh25 nucleic acid molecules, and hosts that have been genetically engineered to express and/or contain such fsh25 gene products.

The invention further relates to novel fsh25 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/z25-mediated processes and for the treatment of s 225-related disorders, such as neuropsychiatric disorders, including the amelioration or prevention of at least one symptom of the disorders, wherein such methods comprise administering a compound which modulates the expression of a fsh25 gene and/or the synthesis or activity of a fsh25 gene product. In one embodiment, the invention relates to methods for the use of a novel fsh25 gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against a fsh25 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 sleep disorders, and neuropsychiatric disorders that include, but are not limited to schizophrenia, schizoaffective disorder, such as schizoaffective disorder manic type (S AD- 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 disorder e.g., unipolar major depressive disorder (MDD). Further 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 cunent version of which is incoφorated herein by reference in its entirety.

The term

disorder" as used herein refers to a disorder involving afsh25 gene or gene product, or involving an abenant level of fsh25 gene expression, gene product synthesis and/or gene product activity relative to levels found in

, _r. normal, unaffected, unimpaired individuals, levels found in clinically normal individuals, and/or levels found in a population whose levels represent a baseline, average sΛ25 levels.

The term 'yϊ/?25-mediated processes" as used herein include processes dependent and/or responsive, either directly or indirectly, to the level of expression, gene product synthesis and/or gene product activity of a fsh25 gene or gene product. 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.

In another embodiment, such methods can comprise modulating the level of expression or the activity of afsh25 gene or gene product in a cell such that the

-^ _/?25-mediated process or the disorder is treated, e.g., a symptom is ameliorated. In another embodiment, such methods can comprise supplying a nucleic acid molecule encoding afsh25 gene product to increase the level, expression or activity of the fsh25 gene product within the cell such that the s/?25-mediated process or the disorder is treated, e.g., a symptom is ameliorated. The nucleic acid molecule encoding the fsh25 gene product can _jc encode a normal or mutant fsh25 gene product, e.g., one with increased activity or expression levels.

The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the

~r. 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.

The invention features nucleic acid molecules which are at least 45% (or 55%), 65%,, 75%,, 85%), 95%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID

₃₅ Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC^® as Accession Number PTA- 451 (the "cDNA of ATCC^® PTA-451") or a complement thereof.

The invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

₅ 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC^® as Accession Number PTA- 451 (the "cDNA of ATCC^® PTA-451"), or a complement thereof, wherem such nucleic acid molecules encode polypeptides or proteins that exhibit at least one stmctural and/or functional feature of a polypeptide of the invention.

, r. The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45%) (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to the amino acid sequence encoded by the cDNA of ATCC^® PTA-451, or a complement thereof.

The invention also features nucleic acid molecules which include a

₁ ^ nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%), 75%>, 85%, 95%, 98, or 99%) identical to the amino acid sequence encoded by the cDNA of ATCC^® PTA-451, or a complement thereof, wherein the protein encoded by the nucleotide sequence also exhibits at least one stmctural and/or functional feature of a polypeptide of the invention.

-_π The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA of ATCC^® PTA-451, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of the cDNA of ATCC^® PTA-451, or a complement thereof under stringent conditions. - The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA of ATCC^® PTA-451, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of the cDNA of ATCC^® PTA-451, or a complement thereof under stringent conditions, wherein such nucleic acid molecules encode _n polypeptides or proteins that exhibit at least one stmctural and/or functional feature of a polypeptide of the invention.

In other embodiments, the isolated nucleic acid molecules encode an extracellular, transmembrane, or cytoplasmic domain of a FSH25 polypeptide of the invention.

- _ In another embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention. Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In another embodiment, the invention provides host cells containing such a vector or a nucleic acid molecule of the invention. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention _<- containing a recombinant expression vector such that a polypeptide is produced.

Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Prefened proteins and polypeptides possess at least one biological activity possessed by the conesponding naturally-occuning human polypeptide. An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of

- _π 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. Such 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.

. - The polypeptides of the present invention, or biologically active portions thereof, can be operably linked to a heterologous amino acid sequence to form fusion proteins. The invention further features antibodies that specifically bind a polypeptide of the invention such as monoclonal or polyclonal antibodies. In addition, the polypeptides of the invention or biologically active portions thereof, or antibodies of the invention, can be

-_π incoφorated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

In another aspect, 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 _j , of activity such that the presence of activity is detected in the biological sample.

In another aspect, 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. In one embodiment, the agent is an antibody

-_p. that specifically binds to a polypeptide of the invention.

In another embodiment, the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention, hi yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA

- _- encoding a polypeptide of the invention. The present invention also provides methods to treat a subject having a disorder characterized by abenant activity of a polypeptide of the invention or abenant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject. In one embodiment, the modulator is a protein of the

_.- invention. In another embodiment, the modulator is a nucleic acid of the invention. In other embodiments, 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)

, _π abenant 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) abenant 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.

In another aspect, the invention provides a method for identifying a

, ^ compound that binds to or modulates the activity of a polypeptide of the invention. In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.

The invention also features methods for identifying a compound which _jr. 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 fsh25- mediated processes or the treatment of s/z25-related disorders, such as neuropsychiatric disorders, including but not limited to disorders resulting from fsh25 gene mutations, and/or _jr an abnormal level of fsh25 expression or activity and disorders involving afsh25 gene and/or gene product, wherein treatment includes the amelioration or prevention of at least one symptom of such disorders. In one embodiment, such methods can comprise supplying a mammal in need of treatment with a nucleic acid molecule encoding an unimpaired fsh25 gene product such that the unimpaired s"/?25 gene product is expressed and the disorder is

-.„ treated, e.g., a symptom is ameliorated. In another embodiment, such methods can comprise supplying a mammal in need of treatment with a cell comprising a nucleic acid molecule that encodes an unimpaired fsh25 gene product such that the cell expresses the unimpaired fsh25 gene product and the disorder is treated, e.g. , a symptom is ameliorated. In yet another embodiment, 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 fsh25 gene or gene product.

In addition, the present invention is directed to methods that utilize sA25 gene sequences and/or fsh25 gene product sequences for the diagnostic evaluation, genetic testing and/or prognosis of

disorder, such as a neuropsychiatric disorder. For example, the invention relates to methods for diagnosing sA2J-related disorders, e.g., neuropsychiatric disorders, wherein such methods can comprise

gene expression in a patient sample, or detecting afsh25 mutation that conelates with the presence or development of such a disorder, in the genome of a mammal suspected of exhibiting such a disorder.

, _rs fsh25 gene sequences and/or fsh25 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 BAD18cagl . These markers are identified below, and described in Section 6.2.

The invention still further relates to methods for identifying compounds

. , capable of modulating the expression of afsh25 gene and/or the synthesis or activity of a fsh25 gene product, wherein such methods comprise contacting a compound with a cell that expresses afsh25 gene, measuring the levels of fsh25 gene expression, gene product expression or gene product activity, and comparing such levels to the levels of fsh25 gene expression, gene product, or gene product activity produced by the cell in the absence of the _rr 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 fsh25 gene and/or the synthesis or activity of the fsh25 gene product has been identified.

3.1. DEFINITIONS

As used herein, the following terms shall have the abbreviations indicated.

25

BAC, bacterial artificial chromosome

BAD, bipolar affective disorder BP, bipolar mood disorder

BP-I, severe bipolar affective (mood) disorder - „ BP-II, bipolar affective (mood) disorder with hypomania and major depression bp, base pair(s) dbEST, expressed sequence tag data base

(National Center for Biotechnology Information) - , EST, expressed sequence tag lod, logarithm of odds MDD, unipolar major depressive disorder RT-PCR, reverse transcriptase PCR SSCP, single-stranded conformational polymoφhism SAD-M, schizoaffective disorder manic type SNP, single nucleotide polymoφhism _<- STS, sequence tag site

YAC, yeast artificial chromosome

For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.

. _π The term "allele", which is used interchangeably herein with "allelic variant" refers to alternative forms of a gene, nucleic acid or portions thereof, as well as to a polypeptide encoded by said gene, nucleic acid, or portion thereof. Nucleic acid alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When

, _c a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.

The term "allelic variant of a polymoφhic region of an fsh25 gene" refers to ς. a region of an fsh2 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.

The term "antibody" as used herein refers to immunoglobulin molecules and immuno logically active portions of immunoglobulin molecules, i.e., molecules that contain

- _<- an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of

-^ immunoglobulin molecules include F(ab) and F(ab')₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.

^c "Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

"Complementary" sequences as used herein refer to sequences which have

_- sufficient complementarity to be able to hybridize, forming a stable duplex.

A "delivery complex" shall mean a targeting means (e.g., a molecule that results in higher affinity binding of a gene, protein, polypeptide or peptide to a target cell surface and/or increased cellular uptake by a target cell). Examples of targeting means include: sterols (e.g., cholesterol), lipids (e.g., a cationic lipid, virosome or liposome),

. _π viruses (e.g., adenovims, adeno-associated vims, and retrovirus) or target cell specific binding agents (e.g., ligands recognized by target cell specific receptors). Prefened complexes are sufficiently stable in vivo to prevent significant uncoupling prior to intemalization by the target cell. However, 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 fsh25 gene or alternatively as a result of proteo lysis of a membranous isoform.

As is well known, genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical r. 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 fsh25 polypeptide" may thus refer to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or _jc may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.

As used herein, the term "gene" or "recombinant gene", as applied to fsh25, refers to a polynucleotide or nucleic acid molecule comprising an open reading frame encoding one of the fsh25 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 genomic but is a cDNA derived from a contiguous coding region which includes, but is not limited to, reverse transcribed cDNA. In another embodiment, the term nucleic acid or

-.j- polynucleotide refers to a nucleic acid molecule which comprises contiguous nucleotide codons. In yet another embodiment, the term 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 by comparing a position in each sequence which may be aligned for puφoses of _c comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of homology or similarity or identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of identity of amino acid sequences is a function of the number of identical amino acids at

, ^ positions shared by the amino acid sequences. Likewise, 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.

Furthermore, a degree of homology or similarity of amino acid sequences is a function of the number of conserved amino acids at positions shared by the amino acid

. , sequences. A sequence which is "unrelated" or "non-homologous" with one of the human fsh25 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 fsh25 sequences of the present invention.

To determine the percent identity of two amino acid sequences or of two _j^ nucleic acids, the sequences are aligned for optimal comparison puφoses (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 conesponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid _<- residue or nucleotide as the conesponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100).

Preferably, the determination of percent identity between two sequences is _{o 0} accomplished using a mathematical algorithm. A prefened, 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 incoφorated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol

₃₅ Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison puφoses, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an

_<- iterated search which detects distant relationships between molecules. Id. When utilizing BLAST, Gapped BLAST, and 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. Another prefened, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17.

, _r. Such an algorithm is incoφorated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using

, _r techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%) (65%>, 70%, preferably 75%) or more) identical to each other typically remain

-_Λ hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NN. (1989), 6.3.1-6.3.6. A prefened, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 2.0 X SSC at 50° C. (low stringency) or 0.2 X SSC, 0.1% SDS at

-_<- 50-65°C (high stringency). In one embodiment, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID Νos. 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, conesponds to a naturally-occu ing nucleic acid molecule.

30

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A-B depicts the gene stmcture and the nucleotide sequence of the 116 kb interval of the long arm of human chromosome 18 spanned by BAD18ct22 and BAD18cagl. A. The ■jr gene structure of the 116 kb interval In particular, the location of the fsh25 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 thelδq interval associated with neuropsychiatric disorders, located from position 28441-144419. Primers used for mapping the 18q interval, and which can be used to amplify sequences comprising the BAD18ct22 and BADlδcagl 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 BAD18cagl markers are shown in boxes between primers. The fsh25 gene conesponds to reverse complement nucleotides 116254 to 116523 (bounded by "< >"). Designation of ambiguity codons is as follows: R= A or G; Y=C or T; K= G or T; M=A or C; S= C or G; W = A or T; B=not A; D=Not C; H=Not G; V=not T; and N= any base.

FIG. 2 depicts afsh25 cDNA sequence.

5. DETAILED DESCRIPTION OF THE INVENTION 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 gene has been identified within this region, refened to herein as fsh25, which is involved in such disorders. In particular, Sections 5.1, 5.2, and 5.3 describe the 18q 116 kb region, including s *25 nucleic acid molecules, as well as vectors comprising these molecules, host cells engineered to contain and/or express such molecules,^s .25 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/*25-related processes and for treatment of .Λ25-related disorders, such as neuropsychiatric disorders, are described. Section 5.7 describes screening assays for compounds that interact with a fsh25 gene or gene product, or modulate fsh25 gene or gene product activity. Methods of

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. Finally, in Section 5.11, pharmaceutical compositions for the use with the invention are described. 5.1. NUCLEIC ACID MOLECULES OF THE INVENTION

The genomic organization of the region of human chromosome 18q between the markers BAD 18ct22 and BAD18cagl has been determined (shown schematically in

FIG. 1A), the nucleotide sequence of which, and its flanking DNA, is shown in FIG. IB.

As used herein, the term "18q interval" refers to the region of chromosome 18q between the

₅ markers BAD18ct22 and BAD18cagl, i.e., nucleotides 28441-144419 of FIG.IB. A novel gene,fsh23, has been mapped to this region (see FIG. 1 A).

The nucleic acid molecules of the invention further include:

(a) a nucleic acid molecule containing the DNA sequence of thelδq interval (nucleotides 28441-144419 of FIG. IB), or its flanking DNA (nucleotides 1-28440 and

₁₀ 144420-160271 of FIG. IB), and fragments thereof;

(b) a nucleic acid molecule comprising afsh25 nucleic acid sequence (e.g., the nucleic acid sequences depicted in FIG. 2 or a fragment thereof);

(c) a nucleic acid molecule that encodes afsh25 gene product;

(d) a nucleic acid molecule that comprises s/j2.5 gene sequences of upstream

, r untranslated regions, intronic regions, and/or downstream untranslated regions, or fragments thereof, of the fsh25 nucleotide sequences in (b) above;

(e) a nucleic acid molecule comprising afsh25 sequence that encodes a mutant of a fsh25 gene product in which all or a part of a domain is deleted or altered, as well as fragments thereof;

~_π (f) nucleic acid molecules that encode fusion proteins comprising afsh25 gene product or a fragment thereof, fused to a heterologous polypeptide;

(g) nucleic acid molecules within afsh25 sequence described in (b), above

(e.g., primers), or within chromosomal nucleotide sequences flanking the fsh2 gene, i.e., the 18q genomic interval described in (a), and which can be utilized as part of the methods ~_c of the invention for identifying and diagnosing individuals at risk for or exhibiting afsh25- related disorder, such as a neuropsychiatric disorder, e.g., BAD, or can be used for mapping the human chromosome 18q region flanking markers; and

(h) nucleic acid molecules within afsh25 sequence described in (b), above, or within chromosomal nucleotide sequences flanking the fsh2 gene, i.e., the 18q genomic ~_r. interval described in (a), which conelate or cosegregate with a sΑ25-related disorder, such as a neuropsychiatric disorder, e.g., BAD.

The nucleotide sequences of the invention further include nucleotide sequences conesponding 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. However, it is understood that the 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

₅ AI018359.

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 fsh25 nucleotide sequences of (a)-(h) above.

. „ To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison puφoses (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 conesponding amino acid positions or nucleotide positions are then

, ,- compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the conesponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical overlapping positions/total # of overlapping positions x 100%). In one

-_π embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A prefened, 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

₂₅ and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incoφorated into the NBLAST and XBLAST programs of Altschul, et al, 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with

, the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison puφoses, Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res.2J:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al, 1997,

- _c supra). When utilizing BLAST, Gapped BLAST, and 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).

Another prefened, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17. Such an algorithm is incoφorated into the ALIGN program (version 2.0) which is _c part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating

, percent identity, typically only exact matches are counted.

The nucleic acid molecules of the invention further include: (a) any nucleotide sequence that hybridizes to afsh25 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

₁₅ 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 0.1 x 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, Cunent Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc.,

-_n and John Wiley & sons, Inc., New York, at pp. 6.3.1-6.3.6 and 2.10.3). Preferably the fsh25 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 afsh25 gene product. In a prefened embodiment, nucleic acid molecules that hybridize under conditions (a) and (b), above, encode gene products, e.g., gene products functionally equivalent to a _j,. fsh25 gene product.

Preferably, the nucleic acids of the invention are human. However, functionally equivalent fsh25 gene products include naturally

gene products present in the same or different species. In one embodiment, fsh25 gene sequences in non- human species map to chromosome regions syntenic to the human 18q chromosome

-_π location within which the particular human fsh25 lies. Functionally equivalent fsh25 gene products also include gene products that retain at least one of the biological activities of a fsh25 gene product, and/or which are recognized by and bind to antibodies (polyclonal or monoclonal) directed against afsh25 gene product.

Among the nucleic acid molecules of the invention are deoxyoligonucleotides

,-,_<- ("oligos") which hybridize under highly stringent or stringent conditions to the fsh25 nucleic acid molecules described above. In general, for probes between 14 and 70 nucleotides in length the melting temperature (Tm) is calculated using the formula: Tm(°C) = 81.5 + 16.6 (log [monovalent cations (molar)]) + 0.41 (% G+Q -(500/N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature is calculated using the equation Tm(°C) = 81.5 + 16.6 (log[monovalent cations (molar)]) + 0.41(% G+C) -(0.61% formamide) -(500/N) where N is the length of the

_<- probe. In general, 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).

. n. 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 fsh25 gene regulation, and/or as antisense primers in amplification reactions of fsh25 gene nucleic acid sequences.

Nucleic acid sequences of the invention encoding afsh25 gene product or

, , complements thereof, may be used as part of ribozyme and/or triple helix sequences, also useful for fsh25 gene regulation.

Still further, the nucleic acid molecules of the invention may be used as components of diagnostic methods whereby, for example, the presence of a particular J/. 5 allele involved in a fsh25 -related disorder, e.g. , a neuropsychiatric disorder, such as BAD, may be detected, or whereby the methods involve mapping the humanl 8q chromosomal region spanned by chromosomal markers BAD18ct22 and BADlδcagl.

Fragments of the fsh25 nucleic acid molecules refer to fsh2 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,

₂₅ 3500, 3750, 4000, 4250, 4500, 4750, 5000, or more contiguous nucleotides in length.

Alternatively, 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 fsh25 gene products. In one embodiment, the fsh25 nucleic acid molecule encodes a gene product exhibiting at least one biological activity of a conesponding s/z2J gene product, e.g., afsh25 gene product.

^ Fragments of the fsh25 nucleic acid molecules can also refer to fsh25 exons or introns, and, further, can refer to portions of fsh25 coding regions that encode domains of, or mature fsh25 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 ED No. 4),

₃₅ 29683-39587 (SEQ ID No. 5), 40284-43253 (SEQ ID No. 6), 43518-46075 (SEQ ID No. 7), 47264-52284 (SEQ ID No. 8), 52672-56935 (SEQ ID No. 9), 57032-57726 (SEQ ID No. 10), 58065-59057 (SEQ ID No. 11), 59815-60471 (SEQ ID No. 12), 60870-62451 (SEQ ID No. 13), 62543-63268 (SEQ ID No. 14), 63494-66959 (SEQ ED No. 15), 67964-69670 (SEQ ID No. 16), 70643-70749 (SEQ ID No. 17), 71051-72295 (SEQ ID No. 18), 72858- 76408 (SEQ ED No. 19), 76797-77123 (SEQ ID No. 20), 77663-78170 (SEQ ID No. 21), 78463-80173 (SEQ ID No. 22), 80466-81519 (SEQ ID No. 23), 81888-85946 (SEQ ID No.

₅ 24), 86346-87569 (SEQ ID No. 25), 88674-89188 (SEQ ID No. 26), 89459-89745 (SEQ ED No. 27), 90436-92299 (SEQ ED No. 28), 92406-94789 (SEQ ED No. 29), 95556-100121 (SEQ ED No. 30), 100530-101382 (SEQ ED No. 31), 101798-103865 (SEQ ED No. 32), 104486-109841 (SEQ ED No. 33), 109953-110561 (SEQ ED No. 34), 111000-113482 (SEQ ED No. 35), 113774-116253 (SEQ ED No. 36), 116846-117907 (SEQ ED No. 37), 117999-

₁₀ 118623 (SEQ ED No. 38), 118865-122881 (SEQ ED No. 39), 122978-186088 (SEQ ED No. 40), 129508-130413 (SEQ ED No. 41), 131138-134228 (SEQ ED No. 42), 134517-135473 (SEQ ED No. 43), 135815-139983 (SEQ ED No. 44), and 140683-144419 (SEQ ED No. 45) of the nucleotide sequence of FIG. IB. In an alternative prefened embodiment, the nucleic acid molecules of the invention comprise at least 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90,

₁₅ 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, or more contiguous nucleotides of at least one of the above nucleotide sequences.

With respect to identification and isolation of fsh25 nucleotide sequences and nucleotide sequences of the 116 kb interval of human chromosome 18q, such sequences can _jr. be readily obtained, for example, by utilizing standard sequencing and bacterial artificial chromosome (BAC) technologies.

As will be appreciated by those skilled in the art, DNA sequence polymoφhisms of a fsh25 gene will exist within a population of individual organisms (e.g., within a human population). Such polymoφhisms may exist, for example, among _{j t}- individuals within a population due to natural allelic variation. Such polymoφhisms include ones that lead to changes in amino acid sequence. As used herein, the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a gene product encoded by that nucleotide sequence. Such natural allelic variations can result in 1-5%), 5-20%), or 20-50% variance in the nucleotide sequence of a given gene. An allele is

-^ one of a group of genes which occur alternatively at a given genetic locus, in this case between the markers BAD18ct22 and BADlδcagl on human chromosome 18q. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. As used herein, the terms "gene" and

- _<- "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. The term can further include nucleic acid molecules comprising upstream and/or exon intron sequences and stmcture.

With respect to fsh2 allelic variants or polymoφhisms, any and all nucleotide variations and resulting amino acid polymoφhisms or variations that are the result of natural allelic variation of the fsh25 gene are intended to be within the scope of the present invention. Allelic variants or polymoφhism include, but are not limited to, ones that do not alter the functional activity of the fsh25 gene product.

With respect to the cloning of additional allelic variants of the human fsh25 gene and homologous and orthologs from other species, the isolated fsh25 gene sequences disclosed herein may be labeled and used to screen a cDNA library constmcted from mRNA

, _rr obtained from appropriate cells or tissues derived from the organism of interest. The hybridization conditions used should generally be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived, and can routinely be determined based on, e.g. , relative relatedness of the target and reference organisms.

. _<- Alternatively, 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

~₀ example, Sambrook, et al, 1989, Molecular Cloning, A Laboratory Manual, Second

Edition, Cold Spring Harbor Press, N.Y.; and Ausubel, et al, 1989-1999, Cunent Protocols in Molecular Biology, Green Publishing Associates and Wiley Enterscience, N.Y., both of which are incoφorated herein by reference in their entirety.

Further, afsh25 gene allelic variant may be isolated from, for example,

~r human nucleic acid, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the fsh25 gene product disclosed herein. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue known or suspected to express a wild type or mutantfsh25 gene allele (such as, for example, brain

., cells, including brain cells from individuals having BAD). In one embodiment, the allelic variant is isolated from an individual who has a fsh25 -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 afsh25 gene nucleic acid sequence. The

-_.- PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment may be labeled and used to screen a bacteriophage cDNA library. Alternatively, 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 conesponding to alternatively spliced mRNAs of a fsh25 gene. For example, RNA may be isolated, following standard procedures, from an appropriate _t- cellular or tissue source (i.e., one known, or suspected, to express the fsh25 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

. _π standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of cloning strategies that may be used, see e.g., Sambrook et αl, 1989, supra, or Ausubel et al, supra. A cDNA of an allelic, e.g., mutant, variant of the fsh25 gene may be isolated,

, _<- for example, by using PCR, a technique that is well known to those of skill in the art. In this case, 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 fsh25 allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an

_7f) oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant^Λ25 allele to that of the normal fsh25 allele, the mutation(s) responsible for the loss or alteration of function of the mutant fsh25

-_<- gene product can be ascertained.

Alternatively, a genomic library can be constmcted using DNA obtained from an individual suspected of or known to carry a mutant fsh25 allele, or a cDNA library can be constmcted using RNA from a tissue known, or suspected, to express a mutant fsh25 allele. An unimpaired fsh25 gene or any suitable fragment thereof may then be labeled and used as

- - a probe to identify the conesponding mutant fsh25 allele in such libraries. Clones containing the mutant fsh25 gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

Additionally, an expression library can be constmcted utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express

- _- a mutant fsh25 allele in an individual suspected of or known to carry such a mutant allele.

In this manner, 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 fsh25 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.)

In cases where afsh25 mutation results in an expressed gene product with

_<- altered function (e.g., as a result of a missense or a frameshift mutation), a polyclonal set of an -fsh25 gene product antibodies are likely to cross-react with the mutant fsh25 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.

. _f. fsh25 mutations or polymoφhisms can further be detected using PCR amplification techniques. Primers can routinely be designed to amplify overlapping regions of the whole fsh2 sequence including the promoter regulating region. In one embodiment, primers are designed to cover the exon-intron boundaries such that, coding regions can be scanned for mutations.

- ,- The invention also includes nucleic acid molecules, preferably DNA molecules, that are the complements of the nucleotide sequences of the preceding paragraphs.

In certain embodiments, the nucleic acid molecules of the invention are present as part of nucleic acid molecules comprising nucleic acid sequences that contain or _j . encode heterologous (e.g., vector, expression vector, or fusion protein) sequences.

5.2. fsh25 GENE PRODUCTS

The fsh25 gene products of the invention include polypeptides, and fragments ye thereof, encoded by afsh25 nucleic acid sequence. fsh25 gene products, or peptide fragments thereof, can be prepared for a variety of uses. For example, 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/j25-related -^ disorder, such as a neuropsychiatric disorder, e.g., BAD. fsh25 gene products can also be used as components of fusion proteins to impart a Fsh25 protein characteristic to another protein of interest. For example, afsh25 gene product, or fragment thereof, could be used to facilitate the purification, localization, or recovery of the protein of interest, by providing an antigenic tag to the fusion protein. In -r another embodiment, fsh25 gene products have uses as amino acid and protein additives to foods, soaps, shampoos, cosmetics, and the like. fsh25 gene products, sometimes refened to herein as a "Fsh25 protein", includes those gene products encoded by the fsh25 gene sequences described in Section 5.1, above. In addition, fsh25 gene products may include proteins that represent functionally equivalent (see Section 5.1 for a definition) gene products. Such an equivalent fsh25 gene products may contain deletions, including internal deletions, additions, including additions

_<- yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the amino acid sequence encoded by the fsh25 gene sequences described, above, in Section 5.1, but that result in a "silent" change, in that the change produces a functionally equivalent fsh25 gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of

, ^ the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

, ^ Alternatively, where alteration of function is desired, deletion or non- conservative alterations can be engineered to produce altered fsh25 gene products. Such alterations can, for example, alter one or more of the biological functions of the fsh25 gene product. Further, such alterations can be selected so as to generate s/.25 gene products that are better suited for expression, scale up, etc. in the host cells chosen. For example, _π cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.

Peptides and/or proteins conesponding to one or more domains of a Fsh25 protein as well as fusion proteins in which a Fsh25 protein or a portion of a fsh25 protein such as a truncated Fsh25 protein or peptide or afsh25 protein domain, is fused to an _jr unrelated protein are also within the scope of this invention. Such proteins and peptides can be designed on the basis of the fsh25 nucleotide sequence disclosed in Section 5.1, above, and/or on the basis of the fsh25 amino acid sequence disclosed herein. Fusion proteins include, but are not limited to, IgFc fusions which stabilize the Fsh25 protein or peptide and prolong half life in vivo; or fusions to any amino acid sequence that allows the fusion

-,_π protein to be anchored to the cell membrane; or fusions of fsh25 protein domains to an enzyme, fluorescent protein, luminescent protein, or a flag epitope protein or peptide which provides a marker function.

Fsh25 proteins of the invention also include Fsh25 protein sequences wherein domains encoded by at least one ex on of the cDNA sequence, or fragments thereof, have _o , been deleted. The fsh25 polypeptides of the invention can further comprise posttranslational modifications, including, but not limited to glycosylations, acetylations, myristylations, and phosphorylations. If the native Fsh25 protein does not have recognition motifs that allow such modifications, it would be routine for one skilled in the art to introduce into afsh25 gene nucleotide sequences that encode motifs such as enzyme

^ recognition signals so as to produce a modified fsh25 gene product.

The fsh25 gene products, peptide fragments thereof and fusion proteins thereof, may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the fsh25 gene polypeptides, peptides, fusion peptide and fusion polypeptides of the invention by expressing nucleic acid containing /sλ 5 gene

. _π sequences are described herein. Methods that are well known to those skilled in the art can be used to constmct expression vectors containing fsh25 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 αl, 1989,

, _.- supra, and Ausubel, et al, 1989, supra. Alternatively, RNA capable of encoding s 225 gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., ERL Press, Oxford.

A variety of host-expression vector systems maybe utilized to express the r, fsh25 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 fsh25 gene product of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. .,- subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing fsh25 gene product coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the fsh25 gene product coding sequences; insect cell systems infected with recombinant vims expression vectors (e.g., baculovims) containing the fsh25 gene product coding

~_rκ sequences; plant cell systems infected with recombinant vims expression vectors (e.g., cauliflower mosaic vims, CaMV; tobacco mosaic vims, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing^/z25 gene product coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constmcts containing promoters derived from the genome of

~r mammalian cells (e.g., metallothionein promoter) or from mammalian vimses (e.g., the adenovirus late promoter; the vaccinia vims 7.5K promoter). In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the fsh25 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 fsh25 protein or for raising antibodies to fsh25 protein, for example, vectors that direct the expression of high levels of fusion protein products that are

, readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, ΕMBO J. 2, 1791), in which the 5/2 5 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; pΕN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13, 3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 264,

, _r. 5503-5509); and the like. pGΕX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsoφtion to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGΕX 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.

In an insect system, Autographa cahfornica, nuclear polyhedrosis vims (AcNPV) is used as a vector to express foreign genes. The vims grows in Spodoptera frugiperda cells. fsh25 gene coding sequences may be cloned individually into non- essential regions (for example the polyhedrin gene) of the vims and placed under control of _π an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of fsh25 gene coding sequences will result in inactivation of the polyhedrin gene and production of non-occluded recombinant vims (i.e., vims lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant vimses 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;

₂₅ Smith, U.S. Patent No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, afsh25 gene coding sequence of interest may be ligated to an adenovims transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may

~_r> then be inserted in the adenovims genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region Εl or Ε3) will result in a recombinant vims that is viable and capable of expressing s 225 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 s/225 ._c gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire fsh25 gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the fsh25 gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation r of the entire insert. These 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).

In addition, a host cell strain may be chosen that modulates the expression of

, _n the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure

, _.- the conect modification and processing of the foreign protein expressed. To this end, 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. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38. _j^ For long-term, high-yield production of recombinant proteins, stable expression is prefened. For example, cell lines that stably express the fsh25 gene product may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators,

«_<- polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells maybe allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into

- - cell lines. This method may advantageously be used to engineer cell lines that express the fsh25 gene product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the fsh25 gene product.

A number of selection systems may be used, including but not limited to the heφes simplex vims thymidine kinase (Wigler, et al, 1977, Cell 11: 223), hypoxanthine-

.,_<- guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy, et al, 1980, Cell 22: 817) genes can be employed in tk^", hgprt^" or aprt^" cells, respectively. Also, 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. USA 78: 2072); neo, which confers resistance to the aminoglycoside G-418 (Colbene-Garapin, et al, 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance to hygromycin (Santene, et

₅ al, 1984, Gene 30: 147).

Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, 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,:

_{1 π} 8972-8976). In this system, 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 vims are loaded onto Ni²⁺-nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

. _. Alternatively, the expression characteristics of an endogenous .J225 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 fsh25 gene. For example, an endogenous fsh25 gene which is normally "transcriptionally silent", i.e., afsh25 _r. 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. Alternatively, a transcriptionally silent, endogenous fsh25 gene may be activated by insertion of a promiscuous regulatory element that works across cell types. _jc A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous fsh25 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.

, fsh25 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 maybe used to generate fsh25 transgenic animals. The term "transgenic," as used herein, refers to animals

gene sequences from a different species (e.g., mice expressing

~r human fsh25 sequences), as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) fsh25 sequences or animals that have been genetically engineered to no longer express endogenous fsh25 gene sequences (i.e., "knockout" animals), and their progeny.

Any technique known in the art may be used to introduce an fsh25 gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S.

, Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (van der Putten, et al, 1985, Proc. Natl Acad. Sci., USA 82: 6148-6152); gene targeting in embryonic stem cells (Thompson, et al, 1989, Cell 56: 313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol 3: 1803-1814); and sperm-mediated gene transfer (Lavitrano et al, 1989, Cell 57: 717-723). For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl.

₁₀ Rev. Cytol. 115: 171-229).

Any technique known in the art may be used to produce transgenic animal clones containing an fsh25 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 afsh25 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 maybe 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

2_Q example, the teaching of Lasko et αl. (Lasko, et α , 1992, Proc. Natl Acad. Sci. USA 89, 6232-6236). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the fsh25 gene transgene be integrated into the chromosomal site of the endogenous fsh25 gene, gene targeting is prefened. Briefly, when such a technique is _jr to be utilized, vectors containing some nucleotide sequences homologous to the endogenous fsh25 gene are designed for the puφose of integrating, via homologous recombination with chromosomal sequences, into and dismpting the function of the nucleotide sequence of the endogenous/s^,/225 gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous fsh25 gene in only that cell type, by following, for example, the teaching of Gu, et αl. (Gu, et αl, 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.

Once transgenic animals have been generated, the phenotypic expression of the recombinant s/2 5 gene may be assayed utilizing standard techniques. Initial screening

- _<- may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques that include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR (reverse transcriptase PCR). Samples of fsh25 gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the fsh25 transgene product.

_- fsh25 gene products, or peptide fragments thereof, can be prepared for a variety of uses. For example, 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/225-related disorder, such as a neuropsychiatric disorder, e.g., BAD. Such fsh25 gene products include

, „ but are not limited to soluble derivatives such as peptides or polypeptides conesponding to one or more domains of the fsh25 gene product, particularly s/2 5 gene products, that are modified such that they are deleted for one or more hydrophobic domains. Alternatively, antibodies to the fsh25 protein or anti-idiotypic antibodies that mimic the fsh25 gene product (including Fab fragments), antagonists or agonists can be used to treat s ϊ25-related

. _c disorders, such as neuropsychiatric disorders. In yet another approach, fsh25 gene products can be directly administered to a subject to treat a s/225-related disorder, such as neuropsychiatric disorders, or a disorder of a s/225-mediated process. In another embodiment, nucleotide constructs encoding such fsh25 gene products can be used to genetically engineer host cells to express such fsh25 gene products in vivo; these genetically _jr, engineered cells can function as "bioreactors" in the body delivering a continuous supply of fsh25 gene product, fsh25 peptides, or soluble s/225 polypeptides.

5.3. ANTIBODIES TO fsh25 GENE PRODUCTS

Described herein are methods for the production of antibodies capable of - ,- specifically recognizing one or more fshl '5 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- -_r. idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of afsh25 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 fsh25 gene products, and/or for the presence of abnormal forms of such gene products. Such antibodies may also _{τ <}- 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 fsh2 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 ./225-expressing cells prior to their introduction into the patient.

Anti-fsh25 gene product antibodies may additionally be used as a method for

_<- the inhibition of abnormal s/2 5 gene product activity. Thus, such antibodies may, be utilized as part of treatment methods for a s/225-related disorder, e.g., a neuropsychiatric disorder, such as BAD.

For the production of antibodies against afsh25 gene product, various host animals may be immunized by injection with afsh25 gene product, or a portion thereof.

, _r. 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

, r potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebαcterium pαrvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as afsh25 gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, _jr, host animals such as those described above, may be immunized by injection with fsh2 gene product supplemented with adjuvants as also described above.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. Prefened polyclonal antibody compositions are ones that have been selected for antibodies directed against a

- , polypeptide or polypeptides of the invention. Particularly prefened polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or polypeptides of the invention. Particularly prefened 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. ~_r> In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides of the invention.

The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using

- ^ immobilized polypeptide. If desired, 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. Alternatively, 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. For example, 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 _c 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. By a substantially purified antibody composition is meant, in this context, that the antibody

, sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired protein or polypeptide of the invention, and preferably at most 20%), yet more preferably at most 10%, and most preferably at most 5%> (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99%> of the antibodies in the composition are directed against the desired

_{1 e}- protein or polypeptide of the invention.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256, 495-497; and U.S.

2_Q 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 _jr producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently prefened method of production.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al, 1984, Proc. Natl Acad. Sci., 81, 6851-6855; Neuberger, et al, 1984, Nature 312, 604-608; Takeda, et al, 1985, Nature, 314, 452-454) by splicing the genes from

- ^ a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al, U.S. Patent No. 4,816,567; and

~_e- Boss et al, U.S. Patent No. 4,816397, which are incoφorated herein by reference in their entirety.) In addition, techniques have been developed for the production of humanized antibodies. (See, e.g., Queen, U.S. Patent No. 5,585,089, which is incoφorated herein by reference in its entirety.) An immunoglobuin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, refened 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. et al, U.S.Department of Health and Human Services (1983). Briefly, 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. Alternatively, techniques described for the production of single chain

_{1 Q} antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242, 423-426; Huston, et al, 1988, Proc. Natl Acad. Sci. USA 85, 5879-5883; and Ward, et al, 1989, Nature 334, 544-546) can be adapted to produce single chain antibodies against fsh25 gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

. , Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constmcted (Huse, et al, 1989, _π Science, 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

5.4. USES OF NUCLEIC ACIDS OF THE INVENTION, fsh25 GENE PRODUCTS. AND ANTIBODIES

25

Described herein are various applications of nucleic acids of the invention, including fsh25 nucleic acids, fsh25 nucleic acids, fsh25 gene products, including peptide fragments and fusion proteins thereof, and of antibodies directed against fsh25 gene products and peptide fragments thereof. Such applications include, for example, prognostic

~_r, and diagnostic evaluation of a/s/225-related disorder, such as a neuropsychiatric disorder, e.g., BAD, and the identification of subjects with a predisposition to such disorders, as described, below, in Section 5.5 and 5.8. Additionally, such applications include methods for the identification of compounds that modulate the expression of a fsh25 gene and/or the synthesis or activity of a fsh25 gene product, as described below, in Section 5.7, and for the

~r treatment of a s *25-related disorder, e.g. a neuropsychiatric disorder, such as BAD, as described, below, in Section 5.10. In addition, the nucleic acid sequences of the invention, including fsh25 nucleic acid sequences and gene products, including peptide fragments and fusion proteins thereof, and antibodies directed against fsh25 gene products and peptide fragments thereof, have applications for puφoses independent of the role fsh25 may have in neuropsychiatric disorders and processes. For example,/_A25 gene products, including peptide fragments, as

, well as s 225-specific antibodies, can be used for constmction of fusion proteins to facilitate recovery, detection, or localization of another protein of interest. In addition, nucleic acid molecules of the invention, including fsh25 nucleic acid sequences, and fsh25 gene products can be used for genetic mapping, i.e., refining the genetic map of chromosome 18q. For example, antibodies specific to Fsh25 can be used as probes to detect expression of human

_{1 rx} Fsh25 in somatic cell hybrids containing human chromosomes, or portions thereof.

Nucleic acids of the invention, including . Λ25 nucleic acids, and fsh25 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

. _c- with neuropsychiatric disorders. As will be apparent to the skilled artisan, nucleic acid sequences within a genetic interval such as an interval associated with a disease can be scanned for new markers, such as microsatellites. Microsatellites, also known as simple- sequence repeats (SSRs), are hypervariable tandem-sequence repeats consisting of di-, tri-, or tetranucleotide repeats of 1-5 nucleotides. Such microsatellites make excellent genetic _jr, markers for linkage studies since they are distributed ubiquitously throughout the human genome, are highly variable in repeat length, and tend to be highly polymoφhic. Relatively common microsatellites (e.g., (CA)_n dinucleotide repeats) occur approximately every 300- 500 kb. In addition to microsatellite repeats, the region can be scanned for other types of polymoφhic sites useful for fine mapping, such as minisatellites (9-64 nucleotide repeats), _j? restriction fragment length polymoφhisms (RFLPs), and single nucleotide polymoφhisms, which occur much less frequently. Once a polymoφhic site is identified in a new sequence, PCR primers that flank the polymoφhic site can be synthesized and used to amplify the microsatellite or other polymoφhic site. The length of the repeat can then determined by resolving the PCR product on a polyacrylamide sequencing gel. Genomic DNA from

~r. human populations can then be analyzed for the such simple-sequence length polymoφhisms (SSLPs) to determine the frequency and variability of the repeat. Once a high quality SSLP is found, 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

~ r hybridization and ligase-chain reaction (LCR), can be used in addition to, or in conjunction with, PCR-based methods to analyze polymoφhisms 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).

In another embodiment, afsh25 gene, protein or a fragment or domain thereof, can be used for constmction of fusion proteins. Finally, fsh25 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.

5.5. DETECTION OF NUCLEIC ACID MOLECULES OF THE INVENTION

Portions or fragments of the nucleic acid sequences identified herein can be , _r. used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) screen for fsh25 gene-specific mutations or polymoφhisms, (ii) locate and/or further nanow chromosomal regions associated with a neuropsychiatric disorder; (iii) identify an individual from a minute biological sample (tissue typing); and (iv) aid in forensic identification of a biological sample. These applications are described in the , c subsections below.

5.5.1. DETECTION OF MUTATIONS OR POLYMORPHISMS

A variety of methods can be employed to screen for the presence of fsh25 gene-specific mutations or polymoφhisms (including polymoφhisms flanking afsh25 gene, 2 e.g., ones that cosegregate with a particular fsh25 allele) and to detect and/or assay levels of fsh25 nucleic acid sequences.

Mutations or polymoφhisms within or flanking the fsh25 gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell, or any cell that expresses the fs h25 gene of interest, can be used as the starting point for such assay 2_<r techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to those of skill in the art. fsh25 nucleic acid sequences may be used in hybridization or amplification assays of biological samples to detect abnormalities involving fsh25 gene stmcture, including point mutations, insertions, deletions, inversions, translocations and chromosomal ™ reanangements. Such assays may include, but are not limited to, Southern analyses, single-stranded conformational polymoφhism analyses (SSCP), and PCR analyses.

Diagnostic methods for the detection of fsh25 gene-specific mutations or polymoφhisms can involve for example, contacting and incubating nucleic acids obtained from a sample, e.g., derived from a patient sample or other appropriate cellular source with -, j- 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 fsh25 gene. As used herein, 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 fsh25 polynucleotide or polypeptide from an individual including, but not limited to, e.g., blood, plasma, semm, ascites, pleural _. effusion, thoracentesis, spinal fluid, lymph fluid, bone manow, 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. In the analysis of whether an fsh25 gene contains a mutation, most simply, blood can be drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be

- _r, accomplished by testing fetal cells, placental cells or amniotic cells for mutations of the fsh25 gene. Alteration of a wild-type fsh25 allele, whether, for example, by point mutation or deletion, can be detected by any of the means discussed herein. When the probes are used to detect the presence of the target sequences (for example, in screening for susceptibility to 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, semm, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, bone manow, 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 _jrv constituents, may be treated, if desired, to extract the nucleic acids. The sample nucleic acid may then be prepared in various ways to facilitate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting. The targeted region of the fsh25 nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation _jr will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art.

The diagnostic methods of the present invention further encompass contacting and incubating nucleic acids for the detection of single nucleotide mutations or

_™ polymoφhisms of the fsh25 gene. Preferably, these nucleic acid reagent sequences within the fsh25 gene, or chromosome 18q nucleotide sequences flanking the fsh25 gene are 15 to 30 nucleotides in length.

After incubation, all non-annealed nucleic acids are removed. The presence of nucleic acids that have hybridized, if any such molecules exist, is then detected. Using

- , such a detection scheme, the 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. In this case, after incubation, non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled fsh25 nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The sequences, e.g.,fsh25 gene sequences, to which the nucleic acid reagents have annealed can be

_<- compared to the annealing pattern expected from a conesponding normal sequence, e.g., normal fsh25 gene sequence, in order to determine whether afsh25 gene mutation or a cosegrating polymoφhism of interest is present.

In a prefened embodiment, fsh25 mutations or polymoφhisms can be detected by using a microassay of fsh25 nucleic acid sequences immobilized to a substrate

, _r, or "gene chip" (see, e.g. Cronin, et al, 1996, Human Mutation 7:244-255).

Alternative diagnostic methods for the detection of fsh25 gene-specific nucleic acid molecules (fsh25 flanking sequences e.g., sequences present in the nucleotide sequence shown in FIG. IB), in patient samples or other appropriate cell sources, may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis,

. ^ 1987, U.S. Patent No. 4,683,202), followed by the analysis of the amplified molecules using techniques well known to those of skill in the art, such as, for example, those listed above. The resulting amplified sequences can be compared to those that would be expected if the nucleic acid being amplified contained only normal copies of the fsh25 gene in order to determine whether afsh25 gene mutation or polymoφhism in linkage disequilibrium with a _jr, disease-causing s 225 allele exists.

Additionally, well-known genotyping techniques can be performed to identify individuals carrying s'/225 gene mutations. Such techniques include, for example, the use of restriction fragment length polymoφhisms (RFLPs), which involve sequence variations in one of the recognition sites for the specific restriction enzyme used.

_2<- Further, improved methods for analyzing DNA polymoφhisms, which can be utilized for the identification of fsh25 gene-specific mutations, have been described that capitalize on the presence of variable numbers of short, tandemly repeated DNA sequences between the restriction enzyme sites. For example, Weber (U.S. Pat. No. 5,075,217) describes a DNA marker based on length polymoφhisms 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 fsh25 gene, and the diagnosis of diseases and disorders related to fsh25 mutations.

- , Also, Caskey et αl. (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 fsh25 gene, amplifying the extracted DNA, and labeling the repeat sequences to form a genotypic map of the individual's DNA.

Other methods well known in the art may be used to identify single nucleotide polymoφhisms (SNPs), including biallelic SNPs or biallelic markers which have two alleles, both of which are present at a fairly high frequency in a population. _c Conventional techniques for detecting SNPs include, e.g. , conventional dot blot analysis, single stranded conformational polymoφhism (SSCP) analysis (see, e.g., Orita et al, 1989, Proc. Natl Acad. Sci. USA 86:2166-2770), 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. 5(5:5855-5892;

, ₀ Grompe, 1993, Nature Genetics 5: 111-117). Alternative, prefened methods of detecting and mapping SNPs involve microsequencing techniques wherein an SNP site in a target DNA is detecting by a single nucleotide primer extension reaction (see, e.g., Goelet et al, PCT Publication No. WO92/15712; Mundy, U.S. Patent No. 4,656,127; Vary and Diamond, U.S. Patent No. 4,851,331; Cohen et al, PCT Publication No. WO91/02087; Chee et al,

_{] 5} PCT Publication No. WO95/11995; Landegren et al, 1988, Science 241:1077-1080; Nicerson et al, 1990, Proc. Natl Acad. Sci. U.S.A. 87:8923-8927; Pastinen et al., 997, Genome Res. 7:606-614; Pastinen et al, 1996, Clin. Chem. 42:1391-1397; Jalanko et a , 1992, Clin. Chem. 38:39-43; Shumaker et al, 1996, Hum. Mutation 7:346-354; Caskey et al, PCT Publication No. WO 95/00669). _jr. The level of fsh2 gene expression can also be assayed. For example, RNA from a cell type or tissue known, or suspected, to express the fsh25 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

2_- of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the fsh25 gene. Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the fsh25 gene, including activation or inactivation of fsh25 gene expression.

In one embodiment of such a detection scheme, a cDNA molecule is

~r, synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA). A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the

^_c fsh25 gene nucleic acid reagents described in Section 5.1 that contain afsh25 gene or nucleic acid sequence. The prefened lengths of such nucleic acid reagents are at least 9-30 nucleotides. For detection of the amplified product, 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 ethidium bromide staining or by utilizing any other suitable nucleic acid staining method. Additionally, it is possible to perform such fsh25 gene expression assays "in

, situ", i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents described in Section 5.1 that contain afsh25 gene or nucleic acid sequence may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY).

₁ . Alternatively, if a sufficient quantity of the appropriate cells can be obtained, standard Northern analysis can be performed to determine the level of mRNA expression of the fsh25 gene.

5.5.2. CHROMOSOME MAPPING

_{1 <}- Once the 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 location of the conesponding genes on a chromosome. For example, the nucleic acid molecules described herein can be used to map the chromosomal location of fsh25 _jr* homologues in various species. Such mapping information can be used, for example, for analysis of the activity of fsh2 transgenes in mice. The nucleic acid molecules can further be used to map the location of copies of fsh25 genes in the human chromosome, such as those caused by genetic abnormalties, e.g., translocations.

Briefly, genes can be mapped to chromosomes by preparing PCR primers _<- (preferably 15-25 bp in length) from the sequence of a gene of the invention. Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene

-_n conesponding to the gene sequences will yield an amplified fragment. For a review of this technique, see D'Eustachio et al. (1983, Science 220:919-924).

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to

- , design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al, 1990, Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes (CITE), and pre-selection by hybridization to chromosome specific cDNA libraries. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. (For a _t- review of this technique, see Verma et al, Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988.)

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 conesponding to

, , noncoding regions of the genes actually are prefened for mapping puφoses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

In another embodiment, afsh25 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. _jr. For examples of this technique, see Pajunen et αl. (1988) Cytogenet. Cell Genet. 47:37-41 and Van Keuren et αl. (1986) Hum. Genet. 74:34-40. Alternatively, the presence of the polypeptide in the somatic cell hybrids can be determined by assaying an activity or property of the polypeptide, for example, enzymatic activity, as described in Bordelon-Riser et αl. (1979 Somatic Cell Genetics 5:597-613 and Owerbach et al (1978,) Proc. Natl Acad.

₂₅ Sci. USA 75:5640-5644.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be conelated with genetic map 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.

Moreover, 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 stmctural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymoφhisms.

5

5.5.3. TISSUE TYPING

The nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymoφhism (RFLP) for

, _r, identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the cunent limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP

₁₅ (described in U.S. Patent 5,272,057).

Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These _jr. primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of conesponding 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 variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard

-^ against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of FIGS. IB 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. If a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

5.5.4. USE OF GENE SEQUENCES IN FORENSIC BIOLOGY

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 peφetrator of a crime. To make such an identification, 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.

The 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). As mentioned above, 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 polymoφhisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.

The fsh25 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 Q¥ fsh25 GENE SEQUENCES IN PREDICTIVE MEDICINE

The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) puφoses to thereby treat an individual prophylactically.

_- Accordingly, one aspect of the present invention relates to diagnostic assays for determining fsh25 protein and or nucleic acid expression as well as fsh25 activity, in the context of a biological sample (e.g., blood, semm, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with abenant or unwanted fsh25 expression or activity. The invention also

, _r. provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with an fsh25 protein, nucleic acid expression or activity. For example, mutations in an fsh25 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with an

. , fsh25 protein, nucleic acid expression or activity.

As an alternative to making determinations based on the absolute expression level of selected genes, determinations may be based on the normalized expression levels of these genes. Expression levels are normalized by conecting the absolute expression level of an fsh2 gene by comparing its expression to the expression of a gene that is not an fsh2 _jr. gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-BAD-affected normal sample, or between samples from different sources. _jr Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a gene, the level of expression of the gene is determined for 10 or more samples of different cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The cell isolates are selected depending upon the tissues in which the gene of interest is

^_n expressed. For example, for fsh25 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 fsh25-mediated disease. For example, by way of illustration only, for fsh25 family members, diseases which maybe studied include, without limitation, those associated with tissues of the brain.

Preferably, the samples used in the baseline determination will be from an fsh25-mediated diseased or from non-diseased cells of tissue. The choice of the cell source

^ is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the fsh25 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 fsh25 gene can serve as a target gene. In addition, as more data is accumulated, the mean

_{1 r}, 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 fsh25 -mediated disease state.

Another aspect of the invention pertains to monitoring the influence of agents (e.g., dmgs, compounds) on the expression or activity of fsh25 in clinical trials.

15

5.6. DETECTION OF fsh25 GENE PRODUCTS

Antibodies directed against unimpaired or mutant fsh25 gene products or conserved variants or peptide fragments thereof, which are discussed, above, in Section 5.3, may also be used as diagnostics and prognostics for a s/225-related disorder, e.g., _jr. neuropsychiatric disorder, such as BAD, as described herein. Such methods may be used to detect abnormalities in the level of fsh25 gene product synthesis or expression, or abnormalities in the stmcture, temporal expression, and/or physical location of fsh25 gene product. The antibodies and immunoassay methods described below have, for example, important in vitro applications in purifying s 225 gene products and in assessing the efficacy _jr of treatments for/s 225-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 fsh25 gene expression and fsh25 peptide production. The compounds that have beneficial effects on a s/225-related disorder, e.g., a neuropsychiatric disorder, such as BAD, can be identified, and

-_r, a therapeutically effective dose determined.

In vitro immunoassays may also be used, for example, to assess the efficacy of cell-based gene therapy for a s^,/225-related disorder, e.g., a neuropsychiatric disorder, such as BAD. Antibodies directed against fsh25 peptides may be used in vitro to determine, for example, the level of fsh25 gene expression achieved in cells genetically engineered to

^r- produce fsh25 peptides. In the case of intracellular fsh25 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 fsh25 gene. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (1988,

, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the fsh25 gene.

. „ Prefened diagnostic methods for the detection of fsh25 gene products or conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the fsh25 gene products or conserved variants or peptide fragments are detected by their interaction with an anύ-fsh25 gene product-specific antibody.

For example, 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 fsh25 gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below, this section) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are _jr. especially prefened for fsh25 gene products that are expressed on the cell surface.

The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of sh25 gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological _jc 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. Through the use of such a procedure, it is possible to determine not only the presence of the fsh25 gene product, or conserved variants or peptide fragments, but also its distribution in the examined tissue. Using the present invention,

_™ those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Immunoassays for fsh25 gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a

~r 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/225 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. As used herein, 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 fsh25 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 manow, 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.

Thus, in the assessment of whether a normal individual or a BAD-affected

, r patient is expressing the secreted fsh25 gene product, or conserved variants or peptide fragments thereof, most simply, blood can be drawn and the blood sample incubated in the presence of a detectably labeled antibody capable of identifying fsh25 gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art. The range of the fsh25 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. 225 gene products or conserved variants or peptide fragments thereof is from about 1 ng/ml to about 100 ng/ml. More prefened ranges for detection of fsh25 gene products or conserved variants or peptide fragments thereof are about 10 ng/ml to about 90 _jr. 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 prefened range for the detection of fsh25 gene products or conserved variants or peptide fragments thereof is about 35 ng/ml to about 40 ng/ml

The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable _jc- of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled fsh25 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.

^_r. By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known 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 some extent or insoluble for the purposes of the present invention. The support material

_{? r} may have virtually any possible stmctural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, 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. Alternatively, the surface may be flat such as a sheet, test strip, etc. Prefened supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. r The binding activity of a given lot of anti-fsh25 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.

One of the ways in which the fsh25 gene peptide-specific antibody can be

, r, detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Irnmunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2, 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A. et αl., 1978, J. Clin. Pathol 31, 507-520; Butler, J.E., 1981, Meth. Enzymol 73, 482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,;

, ,- Ishikawa, E. et αl., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). 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 _jr, dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric _jr 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

^_r, fragments, it is possible to detect sΛ25 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). The radioactive isotope can be detected by such means as 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. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, 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).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a

, _r. chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological

_* c 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 puφoses of labeling are luciferin, luciferase and aequorin.

Further, an antibody (or fragment thereof) can be conjugated to a therapeutic _jr. 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, doxombicin, daunombicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, _jr glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thio guanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,

-^ streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (El) (DDP) cisplatin), anthracyclines (e.g., daunombicin (formerly daunomycin) and doxombicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological

-_- response, the d g moiety is not to be constmed as limited to classical chemical therapeutic agents. For example, the dmg moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, . alpha. -interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 ("IL-1"), interleukin-2 ("EL-2"), interleukin-6 ("EL-6"), _c- granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of D gs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.

_{1 Q} 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Dmg Delivery", in

Controlled Dmg Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thoφe, "Antibody Caniers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of

. .- Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thoφe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an _jr. antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

Accordingly, in one aspect, the invention provides substantially purified antibodies or fragment thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: an amino acid sequence which is encoded _j _- by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of SEQ ED No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, an amino acid sequence encoded by the cDNA of ATCC^® PTA-451 or an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule

- - consisting of the cDNA of ATCC^® PTA-451 , or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. In various embodiments, the substantially purified antibodies of the invention, or fragments thereof, can be human, non-human, chimeric and/or humanized antibodies.

In another aspect, 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, 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, an amino acid sequence encoded by the cDNA of ATCC^® PTA-451, or an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the r cDNA of ATCC^® PTA-451 , or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. Such non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies. Alternatively, the non-human antibodies of the invention can be chimeric and/or humanized antibodies. In addition, the non-human antibodies of the invention can be polyclonal

, antibodies or monoclonal antibodies.

In still a further aspect, 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

₁₅ 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, 40, 41, 42, 43, 44, 45, or an amino acid sequence encoded by the cDNA of ATCC^® PTA- 451, or an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of the cDNA of ATCC^® PTA-451, or a

-_π complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1%o SDS at 65°C. The monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.

The substantially purified antibodies or fragments thereof specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a _jr cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention. In one embodiment, the substantially purified antibodies or fragments thereof, the human or 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, the cDNA of ATCC^® PTA 451 or an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of the cDNA of ATCC^® PTA 451, or a complement thereof, under

₃₅ conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance. Non-limiting examples of detectable substances that 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. r 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 is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. In one embodiment, 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 FSH25 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

, c encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ JD No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, an amino acid sequence encoded by the cDNA of ATCC^® PTA 451, or an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the _jr. nucleic acid molecule consisting of the cDNA of ATCC^® PTA 451, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1%) SDS at 65°C. After immunization, a sample is collected from the mammal that contains an antibody that specifically recognizes an 18q interval polypeptide, or portions thereof. Preferably, the polypeptide is recombinantly produced using a non-human host _jc cell Optionally, the antibodies can be further purified from the sample using techniques well known to those of skill in the art. The method can further comprise producing a monoclonal antibody- producing cell from the cells of the mammal. Optionally, antibodies are collected from the antibody-producing cell.

30

35 5.7. SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE fsh25

GENE OR GENE PRODUCT EXPRESSION OR ACTIVITY

The following assays are designed to identify compounds that bind to afsh25 gene product, e.g., proteins or portions of proteins that interact with afsh25 gene product, compounds that interfere with the interaction of a fsh25 gene product with other proteins c and compounds that modulate the activity of fsh25 gene (i.e., modulate the level of fsh2 gene expression and/or modulate the level of fsh25 gene product activity). Assays may additionally be utilized that identify compounds that bind to fsh25 gene regulatory sequences (e.g., promoter sequences; see e.g., Platt, 1994, J. Biol. Chem. 269: 28558- 28562) and/or trans-acting factors involved in fsh25 gene expression, and that may

_{1 r}. modulate the level of fsh25 gene expression. 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 fsh25 gene or some other gene or gene product involved in afsh25 regulatory pathway.

Methods for the identification of proteins that interact with afsh25 gene or

_{1 <}- gen product are described, below, in Section 5.7.2. Such proteins may be involved in the control and/or regulation of mood. Further, among these compounds are compounds that affect the level of fsh25 gene expression and or fsh25 gene product activity and that can be used in the therapeutic treatment of fsh25 disorders, e.g., neuropsychiatric disorders such as BAD, as described, below, in Section 5.10. _j . 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 αl, 1991, Nature 354, 82-84; Houghten, et αl., 1991, Nature 354, 84-86), and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not

JΓ limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et αl., 1993, Cell 72, 767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules. Such compounds may also comprise compounds, in particular d gs or members of classes or families of d gs, known to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder such as BAD. Such compounds include antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD), p- chlorophenylalanine, ?-propyldopacetamide dithiocarbamate derivatives e.g., FLA 63; anti-

, anxiety dmgs, 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 dmgs such as phenothiazine derivatives (e.g., chloφromazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (Haldol)), thioxanthene derivatives (e.g., chloφrothixene), and dibenzodiazepines (e.g., clozapine); benzodiazepines; dopaminergic agonists and antagonists e.g., L-DOPA, cocaine,

_<- amphetamine, α-methyl-tyrosine, reseφine, tetrabenazine, benzotropine, pargyline; noradrenergic agonists and antagonists e.g. , clonidine, phenoxybenzamine, phentolamine, tropolone. Preferably such compounds are utilized in a manner (e.g., different dosage, mode of administration, and/or co-administration with one or more additional compounds) that differs from the manner in which such compounds have been administered previously.

. _r. Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the fsh25 gene product, and for ameliorating s^,/225-related disorders, e.g., neuropsychiatric disorders such as BAD. For example, compounds identified via such techniques can provide lead compounds to be tested for an ability to modulate a s/225-mediated process and/or to ameliorate symptoms of

, c a s .25-related disorder. Assays for testing the effectiveness of compounds, identified by, for example, techniques such as those described in Sections 5.7.1 - 5.7.3, are discussed, below, in Section 5.7.5.

5.7.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO fsh25 GENE PRODUCTS

20

In vitro systems may be designed to identify compounds that bind fsh25 gene products of the invention. Compounds identified may be useful, for example, in modulating the activity of unimpaired and/or mutant fsh25 gene products, maybe useful in elucidating the biological function of the fsh25 gene product, may be utilized in screens for identifying _j, compounds that dismpt normal fsh25 gene product interactions, or may in themselves dismpt such interactions, and can provide lead compounds to be further tested for an ability to modulate a s/225-mediated process and/or to ameliorate symptoms of a s/225-related disorder.

The principle of the assays used to identify compounds that bind to fsh2 _r, gene products involves preparing a reaction mixture of the fsh25 gene product and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring s/225 gene product or the test

- , substance onto a solid phase and detecting jλ 5 gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the fsh25 gene product may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

In practice, 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 c solution of the protein and drying. Alternatively, 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.

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete,

, _r, 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 c non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g. , using a labeled antibody specific for the previously non-immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction _jr. products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for fsh25 gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

25

5.7.2. ASSAYS FOR PROTEINS THAT INTERACT WITH fsh25 GENE PRODUCTS

Any method suitable for detecting protein-protein interactions may be employed for identifying 5/?25 protein-protein interactions.

Among the traditional methods that may be employed are

30 co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns. Utilizing procedures such as these allows for the identification of proteins that interact with fsh25 gene products. Once isolated, such a protein can be identified and can be used in conjunction with standard techniques, to identify proteins with which it interacts. For example, at least a portion of the amino acid sequence of a protein

35 that interacts with the fsh25 gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, "Proteins: Stmctures and Molecular Principles," W.H. Freeman & Co., NN., pp.34- 49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such proteins. Screening made be accomplished, for example, by standard hybridization or PCR

^ techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra, and 1990, "PCR Protocols: A Guide to Methods and Applications," Innis, et al, eds. Academic Press, Inc., New York).

Additionally, methods may be employed that result in the simultaneous identification of genes that encode the a protein which interacts with afsh25 protein. These

..-, methods include, for example, probing expression libraries with labeled s/225 protein, using fsh25 protein in a manner similar to the well known technique of antibody probing of λgtl 1 libraries.

One method that detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this

₁₅ system has been described (Chien, et αl, 1991, Proc. Natl Acad. Sci. USA, 88:9578-9582) and is commercially available from Clontech (Palo Alto, CA).

Briefly, utilizing such a system, plasmids are constmcted that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the fsh25 gene product and the other consists of the transcription activator protein's _jr. 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 Sαcchαromyces cerevisiαe that contains a reporter gene (e.g., HBS or lαcZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot _jr activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

₃₀ The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, fsh25 gene products may be used as the bait gene product. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait fsh25 gene product fused to _{τ <}- 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. For example, and not by way of limitation, a bait fsh25 gene sequence, such as the open reading frame of the fsh25 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.

, A cDNA library of the cell line from which proteins that interact with the bait fsh25 gene product can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAEA This library can be co-transformed along with the bait fsh25 gene-GA 4 fusion

, r, plasmid into a yeast strain that contains a lacZ gene driven by a promoter that contains GALA activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait fsh25 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

, _<- lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait fsh25 gene-interacting protein using techniques routinely practiced in the art.

5.7.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH/s/t25 GENE PRODUCT INTERACTIONS

20 fsh25 gene products of the invention may, in vivo, interact with one or more macromolecules, including cellular or extracellular macromolecules, such as proteins. Such macromolecules may include, but are not limited to, other proteins, such as cellular receptors, or nucleic acid molecules and those proteins identified via methods such as those

~r described, above, in Sections 5.7.1 - 5.7.3. For purposes of this discussion, the macromolecules are refened to herein as "binding partners". Compounds that disruptfsh25 binding in this way may be useful in regulating the activity of the fsh25 gene product, especially mutant fsh25 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,

^_r, which would be capable of gaining access to afsh25 gene product.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the fsh25 gene product and its binding partner or partners involves preparing a reaction mixture containing the fsh25 gene product, and the binding partner under conditions and for a time sufficient to allow the two to interact and

- , bind, thus forming a complex. In order to test a compound for inhibitory activity, 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 fsh25 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 fsh25 gene protein and the binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction

_<- mixture containing the test compound, indicates that the compound interferes with the interaction of the fsh25 gene protein and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal fsh25 gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant fsh25 gene protein. This comparison may be

, ₍-. important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal fsh25 gene proteins.

The assay for compounds that interfere with the interaction of the fsh25 gene products and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the fsh25 gene product or the binding

, _<- partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the fsh25 gene products and the binding partners, e.g., by _jr. competition, 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 fsh25 gene protein and interactive binding partner. Alternatively, test compounds that dismpt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding _jc the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the fsh25 gene product or the interactive binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently

~ utilized. 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 fsh25 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

^r stored. In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized

_<- species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of

. _π addition of reaction components, test compounds that inhibit complex formation or that dismpt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one

, ^- of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that dismpt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be _jr. used. In this approach, a preformed complex of the fsh25 gene protein and the interactive binding partner is prepared in which either the fsh25 gene product or its binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of _jc the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that dismpt afsh25 gene protein/binding partner interaction can be identified.

In a particular embodiment, the fsh25 gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.2. above. For

-^ example, the fsh25 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. 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

- g. ¹²⁵I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-fsh25 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. At the end of the reaction period, 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 fsh25 gene protein and the interactive binding partner can be detected by measuring the

_<- amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-fsh25 gene fusion protein and the interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose

₁ ^ beads. The test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the fsh25 gene product/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

, _<- In another embodiment of the invention, these same techniques can be employed using peptide fragments that conespond to the binding domains of the fsh25 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. These _jr. methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for dismption 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 conespond to the region of the protein involved in interactive binding. _jr Alternatively, one protein can be anchored to a solid surface using methods described in this section, above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, afsh25 gene product can be anchored to a solid material as described, above, in this section by making a GST-fsh25 fusion protein and allowing it to bind to glutathione agarose beads. The interactive binding

-_- partner obtained can be labeled with a radioactive isotope, such as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-fsh25 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.

5

5.7.4. ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE ft/ι25-RELATED DISORDERS

Compounds, including but not limited to binding compounds identified via assay techniques such as those described, above, in Sections 5.7.1 - 5.7.4, can be tested for

, r, the ability to ameliorate symptoms of a s/225-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

₁ ,- affective (mood) disorder with hypomania and major depression (BP-II); unipolar affective disorders, such as unipolar major depressive disorder (MDD), dysthymic disorder; obsessive-compulsive disorders; phobias, e.g., agoraphobia; panic disorders; generalized anxiety disorders; somatization disorders and hypochondriasis; and attention deficit disorders. It should be noted that the assays described herein can identify compounds that _jr. affect fsh25 gene activity by either affecting s 225 gene expression or by affecting the level of fsh25 gene product activity. For example, compounds may be identified that are involved in another step in the pathway in which the fsh25 gene and/or fsh25 gene product is involved and, by affecting this same pathway may modulate the effect of fsh25 on the development of a neuropsychiatric disorder such as BAD. Such compounds can be used as _jc part of a therapeutic method for the treatment of the disorder.

Described below are cell-based and animal model-based assays for the identification of compounds exhibiting such an ability to ameliorate symptoms of afsh25- related disorder, e.g., a neuropsychiatric disorder, such as BAD.

First, cell-based systems can be used to identify compounds that may act to

_™ ameliorate symptoms of a s/225-related disorder, e.g., a neuropsychiatric disorder, such as BAD. Such cell systems can include, for example, recombinant or non-recombinant cell, such as cell lines, that express the fsh25 gene.

In utilizing such cell systems, cells that express fsh25 may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of a s 225-related

₃^ disorder, e.g., a neuropsychiatric disorder, such as BAD, at a sufficient concentration and for a sufficient time to elicit such an amelioration of such symptoms in the exposed cells. After exposure, the cells can be assayed to measure alterations in the expression of the fsh25 gene, e.g., by assaying cell lysates for fsh25 mRNA transcripts (e.g., by Northern analysis) or for fsh25 gene products expressed by the cell; compounds that modulate expression of the fsh25 gene are good candidates as therapeutics. Alternatively, the cells are examined to determine whether one or more cellular phenotypes associated with a s/225-related disorder, c 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.

En addition, animal-based systems or models for a s 225-related disorder, e.g. , a neuropsychiatric disorder, such as BAD, may be used to identify compounds capable of

. _r, ameliorating symptoms of the disorder. Such animal-based systems or models may include, for example, transgenic mice, e.g., mice that have been genetically engineered to express exogenous or endogenousys 225 sequences or, alternatively, to no longer express endogenous s/225 gene sequences (i.e., "knock-out" mice). Such animal models may be used as test substrates for the identification of dmgs, pharmaceuticals, therapies and

- _<- interventions that may be effective in treating such disorders. For example, 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 s 225-related disorder, e.g., a neuropsychiatric disorder, such as BAD, in the exposed animals. The response of the animals to the exposure may be monitored by _jr. assessing the reversal of such symptoms.

With regard to intervention, any treatments that reverse any aspect of symptoms of a ϋ./225-related disorder, e.g., a neuropsychiatric disorder, such as BAD, should be considered as candidates for human therapeutic intervention in such a disorder. Dosages of test agents may be determined by deriving dose-response curves, as discussed in _j r- Section 5.10.1, below.

5.8. METHODS FOR DIAGNOSIS AND PROGNOSTICATION OF fsh25- RELATED-DISORDERS

A variety of methods can be employed for the diagnostic and prognostic 30 evaluation of 3/225-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 fsh25 gene nucleotide sequences described in Sections 5.1, and antibodies directed against fsh25 gene products, including peptide fragments thereof, as described, above, in Section 5.3. 35 Specifically, such reagents may be used, for example, for: (1) the detection of the presence of fsh2 gene mutations, the detection of polymoφhisms that cosegregate with particular fsh25 gene mutations or the detection of either over- or under-expression of fsh25 gene mRNA relative to the state of a s/225-related disorder, such as a neuropsychiatric disorder, e.g., BAD; _c (2) the detection of either an over- or an under-abundance of fsh25 gene product relative to the unaffected state; and (3) the detection of an abenant level of fsh2 gene product activity relative to the unaffected state. Nucleic acid molecules of the invention can, for example, be used to diagnose an fsh25- , _r. related or neuropsychiatric disorder using, for example, the techniques for fsh25 mutation/co-segregating polymoφhism 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-fsh25 gene antibody reagent described herein, which may be conveniently , _<- used, e.g., in clinical settings, to diagnose patients exhibiting abnormalities of a fsh25- related disorder, e.g., a neuropsychiatric disorder, such as BAD.

For the detection of fsh25 mutations, any nucleated cell can be used as a starting source for genomic nucleic acid. For the detection of fsh25 gene expression or fsh25 gene products, any cell type or tissue in which the fsh25 gene is expressed may be _jr. 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 _jr associated with abenant expression or activity of a polypeptide of the invention. For example, 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 abenant expression or activity of a polypeptide of the invention. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for -r, developing such a disease or disorder. Thus, the present invention provides a method in which a test sample is obtained from a subject and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention is detected, wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abenant expression or activity of the polypeptide. As - , used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., blood, plasma, semm, ascites, pleural effusion, thoracentesis, spinal fluid, lymph fluid, urine, sputum, tears, saliva), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other dmg candidate) to

_<- treat a disease or disorder associated with abenant expression or activity of a polypeptide of the invention. For example, 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). Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder

, _r, associated with abenant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with abenant 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 abenant expression or activity of a polypeptide of the invention. In prefened embodiments, 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 _jr. least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention. For example, such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) _jr- a chromosomal reanangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an abenant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post- translational

- - modification of the protein encoded by the gene. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a gene.

In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195

- _<- and 4,683,202), 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. (1995) Nucleic Acids Res. 23:675-682). 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 _s 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

.₀ mutations described herein.

Alternative amplification methods include: self sustained sequence replication (Guatelli et al (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q- Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), or any other nucleic acid

. ^ amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a selected gene from a sample cell _jr. can be identified by alterations in restriction enzyme cleavage patterns. For example, 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. Moreover, the use of sequence specific ribozymes _jr (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.

In other embodiments, genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density anays containing hundreds or thousands of oligonucleotides probes (Cronin et al, 1996, Human Mutation

_3Q 7:244-255; Kozal et al, 1996, Nature Medicine 2:753-759). For example, genetic mutations can be identified in two-dimensional anays containing light-generated DNA probes as described in Cronin et al, supra. Briefly, a first hybridization anay 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 anays of sequential overlapping probes.

-. , This step allows the identification of point mutations. This step is followed by a second hybridization anay that allows the characterization of specific mutations by using smaller, specialized probe anays complementary to all variants or mutations detected. Each mutation anay is composed of parallel probe sets, one complementary to the wild- type gene and the other complementary to the mutant gene.

In yet another embodiment, 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 conesponding 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,

, _r. Bio/Techniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al, 1996, Adv. Chromatogr. 36:127-162; and Griffin et al, 1993, Appl Biochem. Biotechnol 38:147-159).

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

₁₅ RNA/DNA heteroduplexes (Myers et al, 1985, Science 230:1242). In general, 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 _jr. 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. In other embodiments, 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 _{j e}- regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. (See, e.g., Cotton et al, 1988, Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al, 1992, Methods Enzymol. 217:286-295.) In a prefened embodiment, the control DNA or RNA can be labeled for detection. hi still another embodiment, the mismatch cleavage reaction employs one or

^_π more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair enzymes") in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells. For example, 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). According

~_<- to an exemplary embodiment, a probe based on a selected sequence, e.g., a wild-type 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. (See, e.g., U.S. Patent No. 5,459,039.) In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in genes. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and c 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 stmcture of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the

, _π detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary stmcture is more sensitive to a change in sequence. In a prefened embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen

₁₅ et al, 1991, Trends Genet. 7:5).

In yet another embodiment, 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). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely _jr. denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC- rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner, 1987, Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, but are _j? not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al, 1986, Nature 324:163; Saiki et al, 1989, Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific

-„ oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.

~ , Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al, 1989, Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner, 1993, Tibtech 11 :238). In addition, it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al, 1992, Mol. Cell Probes 6:1). It is anticipated that in certain

_<- embodiments 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.

- _n 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. Furthermore, any cell type or tissue, preferably

1 peripheral blood leukocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.

5.9. KITS _jr. The invention further provides kits that facilitate the use /and or detection of fsh25 genes or co-segregating polymoφhisms and fsh25 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. Furthermore, any cell type or tissue in which the _jc polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.

In one embodiment, a diagnostic test kit for identifying cells or tissues which express or mis-express s 225 genes or gene products is provided. In this embodiment, a diagnostic kit is provided, with one or more containers comprising an oligonucleotide, e.g.,

_™ a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention. In another embodiment, a kit is provided, with one or more containers comprising a pair of primers useful for amplifying afsh25 nucleic acid molecule encoding afsh25 polypeptide of the invention. In various other embodiments, the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit

.,,. can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instmctions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with abenant 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.

In another embodiment, the invention provides kits for detecting the presence of a polypeptide of the invention in a biological sample (a test sample). Such kits can be

, _r. used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with abenant expression of a polypeptide of the invention as discussed, for example, in sections above relating to uses of the sequences of the invention. In this embodiment, a kit is provided, with one or more containers comprising: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and,

, r optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent. Such kits can be used to determine if a subject is suffering from or is at increased risk of a fsh-related disorder, such as a neuropsychiatric disorder, e.g., BAD.

20

5.10. COMPOSITIONS AND METHODS FOR TREATMENT OF fsh25- RELATED DISORDERS

Described herein are methods and compositions whereby a s/225-mediated process can be modulated and/or whereby a symptom of a s/225-related disorder, e.g., a _jc symptom of a neuropsychiatric disorder, such as a cognitive or mood disorder, for example, BAD, may be treated.

Such a method can comprise administering a compound which modulates the expression of afsh25 gene and/or the expression or activity of afsh25 gene product, so that the process is modulated or a symptom of the disorder is ameliorated.

-. . In some instances, a/s 225-related disorder phenotype or symptom can occur as a result of a decrease in expression or activity of a component of a Fsh25-mediated pathway, such as a Fsh25 receptor, ligand, or an upstream or downstream component of a Fsh25 signal transduction pathway. In such cases, increase in the level of fsh25 gene expression and/or fsh25 gene product expression or activity could facilitate the progress

„ towards an asymptomatic state. For example, in one embodiment, a method for treating such a s/225-related disorder phenotype can comprise administering to a subject a Fsh25 peptide or polypeptide, or fragment, analog or mimetic thereof, to ameliorate at least one symptom of a s^,/225-related disorder phenotype. En another embodiment, a method for treating such a . 225-related disorder phenotype can comprise administering to a subject a compound that modulates the activity or expression of afsh25 gene or gene product to ameliorate at least one symptom of a s/225-related disorder phenotype.

_<- Likewise, in those instances where decrease Fsh25 expression or activity leading to a sΑ25-related disorder, e.g., neuropsychiatric disorder, is the result of a fsh25 gene mutation(s), such methods can comprise administering a compound that increases the activity or expression of a fsh25 gene or gene product. In such case in which a loss of normal fsh25 gene product function results in the development of a/^, 225-related disorder

, _r, phenotype, e.g., a neuropsychiatric disorder phenotype, an increase in fsh2 gene product activity would facilitate progress towards an asymptomatic state in individuals exhibiting a deficient level of sh25 gene expression and/or fsh25 gene product activity.

In another embodiment, a method for treating such a s 225-related disorder phenotype can comprise supplying a subject with a nucleic acid molecule encoding an

- _<- unimpaired s 225 gene product, such that an unimpaired fsh25 gene product is expressed and symptoms of the disorder are ameliorated.

In another embodiment, methods for the treatment of mammalian fsh25- related disorder, e.g., a neuropsychiatric disorders, such methods can comprise supplying a mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired fsh25 _jr. gene product such that the cell expresses the unimpaired fsh25 gene product and symptoms of the disorder are ameliorated.

In another embodiment, methods for enhancing the expression or synthesis of afsh25 gene or gene product can include, for example, methods such as those described below, in Section 5.10.2. _jr Alternatively, symptoms of a s/225-related disorder phenotype, e.g., a neuropsychiatric disorder, such as BAD, may be ameliorated by administering a compound that decreases the level of fsh25 gene expression andlorfsh25 gene product activity. In one embodiment, such a method comprises administering an anti-Fsh25 antibody to a subject to ameliorate at least one symptom of s/225-related disorder phenotype. In another

~r, embodiment, 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 fsh25- related disorder phenotype.

Methods for inhibiting or reducing the level of sh25 synthesis or expression can include, for example, methods such as those described in Section 5.10.1.

.,_<- In one embodiment of treatment methods, the compounds administered do not comprise compounds, in particular dmgs, reported to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder, such as BAD. Such compounds include antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD), ?-chlorophenylalanine, >-propyldopacetamide dithiocarbamate derivatives e.g., FLA 63; anti-anxiety dmgs, 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 dmgs such as phenothiazine derivatives (e.g., chloφromazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (Haldol)), thioxanthene derivatives (e.g., chloφrothixene), and dibenzodiazepines (e.g., clozapine); benzodiazepines; dopaminergic agonists and

. _r, antagonists e.g., L-DOPA, cocaine, amphetamine, -methyl-tyrosine, reseφine, 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

, _<- additional compounds) that differs from the manner in which such compounds have been administered previously.

5.10.1. INHIBITORY ANTISENSE, RIBOZYME, AND TRIPLE HELIX APPROACHES

20 In another embodiment, symptoms of certain s 225-related disorders, such as neuropsychiatric disorders, e.g., BAD, may be ameliorated by decreasing the level of fsh2 gene expression and/or fsh25 gene product activity by using fsh25 nucleic acid sequences in conjunction with well-known antisense, gene "knock-out," ribozyme and/or triple helix methods to decrease the level of fsh25 gene expression. Among the compounds that may

25 exhibit the ability to modulate the activity, expression or synthesis of the fsh25 gene, including the ability to ameliorate the symptoms of a sλ25-related disorder, e.g., a neuropsychiatric disorder, such as BAD, are antisense, ribozyme, and triple helix molecules. Such molecules may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are

30 well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA

35 transcripts and prevent translation. Absolute complementarity, although prefened, is not required.

A sequence "complementary" to a portion of an RNA, as refened to herein, 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

. ^ standard procedures to determine the melting point of the hybridized complex.

In one embodiment, oligonucleotides complementary to coding or non- coding regions of the fsh25 gene can be used in an antisense approach to inhibit translation of endogenous fsh25 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.

, c In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

Regardless of the choice of target sequence, it is prefened that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is prefened that these studies utilize controls that distinguish between _jr. antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also prefened 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. It is prefened that the control oligonucleotide is of approximately the same _jr length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The

_™ oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g. , 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.

₃₅ Sci. 84, 648-652; PCT Publication No. WO88/09810, published December 15, 1988) or the blood-brain banier (see, e.g., PCT Publication No. WO89/10134, published April 25, 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al, 1988, BioTechniques 6, 958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5, 539-549). To this end, 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, _{ι n} 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 '-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,

. ^ 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-

5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, _jr. xylulose, and hexose. r yet another embodiment, 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. _jt- In yet another embodiment, the antisense oligonucleotide is an -anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands mn 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

_3Q chimeric RNA-DNA analogue (Inoue, et al, 1987, FEBS Lett. 215, 327-330).

Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl. Acids Res.

~r 16, 3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al, 1988, Proc. Natl Acad. Sci. U.S.A. 85, 7448-7451), etc.

While antisense nucleotides complementary to the target fsh25 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)

. r. can be administered systemically.

However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous rnRNAs. Therefore a prefened 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 constmct

. _<- to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA. For example, 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 _jr. integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constmcted 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 _j , can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290, 304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma vims (Yamamoto, et al, 1980, Cell 22, 787-797), the heφes thymidine kinase promoter (Wagner, et al, 1981, Proc. Natl Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of the metallothionein gene

,₀ (Brinster, et al, 1982, Nature 296, 39-42), etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA constmct which can be introduced directly into the tissue site. Alternatively, viral vectors can be used that selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).

-_<- Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and, therefore, expression of target gene product. (See, e.g., PCT Enternational 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, Cunent Biology 4, 469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme r molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Patent No. 5,093,246, which is incoφorated herein by reference in its entirety.

, r. While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs, the use of hammerhead ribozymes is prefened. 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 constmction and

. _- production of hammerhead ribozymes is well known in the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially fig. 4, page 833) and in Haseloff and Gerlach, 1988, Nature, 334, 585-591, which is incoφorated herein by reference in its entirety.

Preferably the ribozyme is engineered so that the cleavage recognition site is _jr. 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 endoribonuc leases (hereinafter "Cech-type ribozymes") such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 EVS RNA) and that has been extensively described _{j t}- 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 _n place. The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in the target gene.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo. A prefened method of delivery involves using a

-_c- DNA constmct "encoding" the ribozyme under the control of a strong constitutive pol III 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

₅ (e.g., see Smithies, et al, 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al, 1989, Cell 5, 313-321; each of which is incoφorated by reference herein in its entirety). For example, a mutant, non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used,

. r, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA constmct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g. , see Thomas and

, ,- Capecchi, 1987 and Thompson, 1989, supra). However this approach can be adapted for use in humans provided the recombinant DNA constmcts are directly administered or targeted to the required site in vivo using appropriate viral vectors.

Alternatively, endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene _jr, (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 Dmg Des., 6(6), 569-584; Helene, et al, 1992, Ann. NN. Acad. Sci., 660, 27- 36; and Maher, 1992, Bioassays 14(12), 807-815).

Nucleic acid molecules to be used in triplex helix formation for the inhibition _{j c} of transcription should be single stranded and composed of deoxynucleo tides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing mles, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC⁺ triplets across the three associated

^r. strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, 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

-_c- residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex. Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

^ In instances wherein the antisense, ribozyme, and/or triple helix molecules described herein are utilized to inhibit mutant gene expression, it is possible that 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

, r. be lower than is necessary for a normal phenotype. In such cases, to ensure that substantially normal levels of target gene activity are maintained, therefore, 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,

. g. ribozyme, or triple helix treatments are being utilized. Alternatively, in instances whereby 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 _jr. RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, 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 incoφorated into a wide variety of _{j e}- vectors that incoφorate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constmcts that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

30 5.10.2. GENE REPLACEMENT THERAPY

With respect to an increase in the level of normal fsh25 gene expression and/or fsh25 gene product activity, nucleic acid sequences encoding afsh25 gene product, for example, can be utilized for the treatment of a s/225-related disorder, e.g. , a neuropsychiatric disorder, such as BAD. Such treatment can be administered, for example, ~_- in the form of gene replacement therapy. Specifically, one or more copies of a normal s/225 gene or a portion of the fsh25 gene that directs the production of a fsh25 gene product exhibiting normal fsh25 gene function, may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to adenovims, adeno-associated vims, and retrovims vectors, in addition to other particles that introduce DNA into cells, such as liposomes.

Because/s/225 genes can be expressed in the brain, such gene replacement r therapy techniques should be capable

gene sequences to these cell types within patients. Thus, in one embodiment, techniques that are well known to those of skill in the art (see, e.g., PCT Publication No. WO89/10134, published April 25, 1988) can be used to enable fsh25 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

, , blood-brain banier, viral vectors such as, for example, those described above, are preferable.

In another embodiment, techniques for delivery involve direct administration of such fsh25 gene sequences to the site of the cells in which the fsh25 gene sequences are to be expressed.

. r Additional methods that may be utilized to increase the overall level of fsh25 gene expression and/or fsh25 gene product activity include the introduction of appropriate /s/225-expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to ameliorate the symptoms of afsh25-τelated disorder, e.g., a neuropsychiatric disorder, such as BAD. Such cells may be either recombinant or non- _jr, recombinant.

Among the cells that can be administered to increase the overall level of fsh25 gene expression in a patient are normal cells, preferably brain cells, that express the fsh25 gene.

Alternatively, cells, preferably autologous cells, can be engineered to express _jc fsh25 gene sequences, and may then be introduced into a patient in positions appropriate for the amelioration of the symptoms of a s/225-related disorder, e.g., a neuropsychiatric disorder, such as BAD. Alternately, cells that express an unimpaired/s/225 gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells. The expression of the fsh25 gene sequences is controlled by the appropriate gene _r, regulatory sequences to allow such expression in the necessary cell types. Such gene regulatory sequences are well known to the skilled artisan. Such cell-based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson, U.S. Patent No. 5,399,349.

When the cells to be administered are non-autologous cells, they can be

- r administered using well known techniques that prevent a host immune response against the introduced cells from developing. For example, 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.

Additionally, compounds, such as those identified via techniques such as those described, above, in Section 5.8, that are capable of modulating fsh25 gene product _c activity can be administered using standard techniques that are well known to those of skill in the art. In instances in which the compounds to be administered are to involve an interaction with brain cells, the administration techniques should include well known ones that allow for a crossing of the blood-brain barrier.

₁₀ 5.10.3. PHARMACOGENOMICS

Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with abenant activity of the polypeptide. In

, _c conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or dmg) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active dmg. Thus, the pharmacogenomics of the _jr. individual permits the selection of effective agents (e.g., dmgs) 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. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual _jr can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variations in the response to dmgs due to altered dmg disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266. In general, two types of .r. pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way dmgs act on the body are refened to as "altered dmg action." Genetic conditions transmitted as single factors altering the way the body acts on dmgs are refened to as "altered dmg metabolism". These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. For example, glucose-6-phosphate

- _c dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant dmgs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of dmg metabolizing enzymes is a major determinant of both the intensity and duration of dmg action. The discovery of genetic polymoφhisms of dmg metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)

_- and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected dmg effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a dmg. These polymoφhisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among

. r. different populations. For example, the gene coding for CYP2D6 is highly polymoφhic 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 dmg response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as

. demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite moφhine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the activity of a polypeptide of the invention, expression of a nucleic _jr. 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. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding dmg-metabolizing enzymes to the identification of an individual's dmg responsiveness phenotype. This knowledge, when _jc applied to dosing or dmg selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.

₃₀ 5.10.4. MONITORING OF EFFECTS DURING CLINICAL TRIALS

Monitoring the influence of agents (e.g., dmgs, compounds) on the expression or activity of a polypeptide of the invention (e.g., the ability to modulate abenant cell proliferation chemotaxis, and/or differentiation) can be applied not only in basic dmg screening, but also in clinical trials. For example, 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. Alternatively, 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. In such clinical trials, expression or activity of a polypeptide of the invention and preferably, that of other polypeptide that have been implicated in for example, a cellular proliferation disorder, can be used as a marker of the immune responsiveness of a particular cell.

For example, and not by way of limitation, genes, including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, dmg or

-, , small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, 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

₁ r 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. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response _jr. state may be determined before, and at various points during, treatment of the individual with the agent.

In a prefened embodiment, 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 dmg _jc candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the ₀ post-administration samples; (v) comparing the level of the polypeptide or nucleic acid of the invention in the pre-administration sample with the level of the polypeptide or nucleic acid of the invention in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of the

₃₅ polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.

5.11. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION r fsh25 gene products or compounds that are determined to affect fsh25 gene expression or gene product activity, can be administered to a patient at therapeutically effective doses to treat or ameliorate a/sA25-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.

10

5.11.1. EFFECTIVE DOSE

As defined herein, a therapeutically effective amount (i.e., an effective dosage) of protein, polypeptide, or anti-Fsh25 antibody, 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. _jr. Moreover, 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. In a prefened example, 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. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression or

- activity. An agent may, for example, be a small molecule. For example, such 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 organometaUic 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.

It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the _t- 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

, _π about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.

, , When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, 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. In addition, it is understood that the specific dose level for any particular animal subject will _jr. 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 dmg combination, and the degree of expression or activity to be modulated.

Toxicity and therapeutic efficacy of such compounds can be determined by

2_- standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50%> of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices are prefened. While compounds that

,, _n exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies

- _<- preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, 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 IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such r 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.

5.11.2. FORMULATIONS AND USE

Pharmaceutical compositions for use in accordance with the present

, r. invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

Thus, 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.

. _<- For oral 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, polyvinylpynolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium _jr. stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. 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 _{j c}- 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,

^_r, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

~_- For administration by inhalation, 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. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the _c 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

- _π in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as

. _c cocoa butter or other glycerides.

In addition to the formulations described previously, 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. Thus, for example, the compounds may be formulated with suitable polymeric or _jr. 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.

The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or _jc dispenser device may be accompanied by instructions for administration.

6. EXAMPLE: IDENTIFICATION OF fsh25 GENE AND GENE PRODUCTS

-_π In the Example presented in this section, studies are described that, first, define an interval approximately 116 kb on the long arm of human chromosome 18 within which lies a region associated with a neuropsychiatric disorder is located and, second, identify a gene, refened to herein as fsh25, which lies within this region and which can be involved in . 225-related disorders, e.g., neuropsychiatric disorders.

35 6.1. MATERIALS AND METHODS

Linkage Disequilibrium Linkage studies were performed using DNA from samples of neuropsychiatric disorder (BP-I) patients of two large pedigrees from Costa

Rica. The initial sample and linkage analysis techniques were as described in Freimer et al,

1996, Nature Genetics 12:436-441. The present study took advantage of the additional

_<- physical markers identified via the physical mapping techniques described below.

Yeast artificial chromosome (NAC mapping For physical mapping, yeast artificial chromosomes (YACs) containing human sequences were mapped to the region being analyzed based on publicly available maps (Cohen et al, 1993, C.R. Acad. Sci. 316, 1484-1488). The YACs were then ordered and contig reconstmcted by performing standard

, r, short tag sequence (STS)-content mapping with microsatellite markers and non- polymoφhic STSs available from databases that sunound the genetically defined candidate region.

Bacterial artificial chromosome (BAC) mapping The STSs from the region were used to screen a human BAC library (Research Genetics, Huntsville, AL). The ends of

. .- the 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 DΝA 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 sublibrary _jr. production. Microsatellite sequences from the subhbraries were identified by conesponding microsatellite probes. Sequences around such repeats were obtained to enable development of PCR primers for genomic DΝA.

Radiation hybrid (RH mapping Standard RH mapping techniques were applied to a Stanford G3 RH mapping panel (Research Genetics, Huntsville, AL) to order

J_Γ all microsatellite markers and non-polymoφhic STSs in the region being analyzed.

Sample sequencing Random sheared libraries were made from all the BACs within the defined genetic region. Approximately 6,000 subclones within the approximately 116 kb region were sequenced with vector primers in order to achieve a 6-fold sequence coverage of the region. All sequences were processed through an automated sequence

-r. analysis pipeline that assessed quality, removed vector sequences and masked repetitive sequences. Sequences within the 116 kb candidate region, as defined by the disease haplotype shared by the affected individuals, were assembled into sequence clusters using the Sequencher program. Sequencing primers were designed from both ends of each cluster and used to sequence the conesponding BACs to extend the contigs. Eventually, all

- , clusters were assembled together to produce a single continuous DΝA 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 canied out as described by Rommens, et al. (1994, in Identification of Transcribed Sequences, Hochgeschwender and Gardiner, eds., Plenum Press, New York, pp. 65-79).

Transcription mapping The combination of sample sequencing and cDNA selection were ananged into tentative transcription units which provided the framework for . r. a detailed transcription map of the genomic region of interest. cDNA libraries cDNA libraries purchased from Clontech (Palo Alto, CA) and prepared in house were screened according to manufacturer's recommendations.

Northern analysis Standard Northern analysis techniques were utilized in probing human and fetal multiple tissue Northern blots purchased from Clontech (Palo Alto, , c CA). Blots were hybridized to different gene probes, which were derived by PCR from fsh25 cDNA sequences.

6.2. RESULTS

Genetic regions involved in bipolar affective disorder (BAD) human genes _jr. had previously been reported to map to broad portions of the long (18q) and short (18p) arms of human chromosome 18, including a broad 18q genetic region of about 6-7 cM between markers D18S469 and D18S554 (Freimer, et al, 1996, Neuropsychiat. Genet. 67: 254-263; Freimer, et al, 1996, Nature Genetics 12: 436-441).

Prior to attempting to identify gene sequences, studies were performed to _jc- further nanow the neuropsychiatric disorder region. Specifically, a finer haplotype analysis was performed using the same samples from the pedigrees, which took advantage of the additional physical markers identified via the physical mapping techniques described below. In order to provide the precise order of genetic markers necessary for linkage and LD mapping, and to guide new microsatellite marker development for finer mapping, a -.r, high resolution physical map of the 18q23 candidate region was developed using YAC, BAC and RH techniques.

For such physical mapping, first, YACs were mapped to the chromosome 18 region being analyzed. Using the mapped YAC contig as a framework, the region from publicly available markers D18S1161 and D18S554, which spans most of the D18S469- -.. D18S554 region described above, was also mapped and contiged with BACs. Subhbraries from the contiged BACs were constmcted, from which microsatellite marker sequences were identified and sequenced.

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-polymoφhic STSs in the region. Thus, the _- high resolution physical map ultimately constmcted was obtained using data from RH mapping and STS-content mapping.

The new markers identified via physical mapping were typed in samples from the two large pedigrees affected with bipolar affective disorder. Results of this haplotype analysis nanowed down the chromosome 18 long arm region, within which a gene involved , r. in neuropsychiatric disorders lies, to an interval of about 116 kb between two markers,

BAD18ct22 and BAD18cagl. BAD18ct22 is defined by a (GA)₂₂ di-nucleotide repeat; the

BADct22 primer set used was as follows:

forward, 5'-GTTGAGTGATAGGTAAAGACAG-3' , and reverse, 5'- AACAACAAAAAGTGACTTAATTATCG-3'

15

located at positions 28547-28572 and 28384-28405, respectively, of the nucleotide sequence shown in FIG. IB (underscored at their respective locations in this figure). BAD18cagl is defined by a (CAG)_π tri-nucleotide repeat; the following primer set was _jr. used for amplification of the BAD18cagl marker:

forward, 5'-CATTACATCAAGGATGTAGTTACC-3' . and reverse 5'- GAACCCTGTTAAAACACATATTCTG-3'

_jc located at positions 144302-144325 and 144477-144501 respectively, of the nucleotide sequence shown in FIG. IB (underscored at their respective locations in this figure). These two markers define the 18q interval of approximately 116 kb.

6.2.1. CHARACTERIZATION OF TBEfsh25 GENES AND GENE

PRODUCTS

30

The BAC clones within the newly identified 116 kb neuropsychiatric disorder region were further analyzed to identify specific genes within the region. Two overlapping

BACs, BAC69 (ATCC Deposit No. PTA-451) and BAC104 (ATCC Deposit No. PTA-

451), cover the entire 116 kb interval A combination of sample sequencing, cDNA

~_<- selection and transcription mapping analyses were combined to anange sequences into tentative transcription units, that is, tentatively delineating the coding sequences of genes within this genomic region of interest.

The fsh2 '5 was identified within the 116 kb interval between BAD18ct22 and BADlδcagl on the long arm of chromosome 18. fsh25, therefore, can be involved in neuropsychiatric disorders.

_<■ fsh25 is positioned within the 116 kb interval between BAD 18 ct22 and

BAD 18 cagl, as shown in Figure 1A. Arrows denote the location and direction of transcript relative to genetic markers shown above in the diagram. The nucleotide sequence of fsh25 is shown in FIG. 2.

Novel sequences of the entire 18q interval were identified that can be used

- r, with the methods of the invention, were identified. The 116 bp was used as input to the NBLAST program and public and private databases were searched, including: dbest [GenBank database of ESTs (Expressed Sequence Tags)]; htgs [GenBank databases HTGS (high-throughput genomic sequences) and GSS (Genome Survey Sequences)]; patn (database for non-redundant geneseq and "patent preview"); and nuc (all GenBank

, nucleotide sequences except EST, HTGS, and GSS divisions) (see Benson et al, 1999, Nucleic Acids Res., 1999, 1:12-17). NBLAST default parameter settings were used except for S=320 S2=320 which requires minimally a -90 bp stretch with 95% identity, N=-20 which increases the mismatch score, and -spanl which simplifies the output conesponding to repeated elements (see Altschul et al, 1990, J. Mol. Biol. 215: 403-410). Second, the _jr. resulting HSPs for each database hit were examined. All non-human hits were excluded and all possible 90 bp regions of the query within each HSP were evaluated for percent identity by determining the fraction identity, matches divided by the sum of 90 plus the subject gaps in the alignment region. Sequences were determined to be novel if the fraction identity was less than 0.95 and the query sequences were at least 100 bp. This method _j identified the following novel nucleotide sequences of the 116 kb region shown in FIG.IB: 28441-29265 (SEQ JD No. 4), 29683-39587 (SEQ ED No. 5), 40284-43253 (SEQ ED No. 6), 43518-46075 (SEQ ED No. 7), 47264-52284 (SEQ ED No. 8), 52672-56935 (SEQ ED No. 9), 57032-57726 (SEQ ED No. 10), 58065-59057 (SEQ ED No. 11), 59815-60471 (SEQ LD No. 12), 60870-62451 (SEQ ID No. 13), 62543-63268 (SEQ ED No. 14), 63494-66959 (SEQ ED

₃₀ No. 15), 67964-69670 (SEQ ED No. 16), 70643-70749 (SEQ ED No. 17), 71051-72295 (SEQ ED No. 18), 72858-76408 (SEQ ED No. 19), 76797-77123 (SEQ ED No. 20), 77663- 78170 (SEQ ED No. 21), 78463-80173 (SEQ ED No. 22), 80466-81519 (SEQ ID No. 23), 81888-85946 (SEQ ID No. 24), 86346-87569 (SEQ ID No. 25), 88674-89188 (SEQ ID No. 26), 89459-89745 (SEQ ID No. 27), 90436-92299 (SEQ ID No. 28), 92406-94789 (SEQ ID

₃₅ No. 29), 95556-100121 (SEQ ID No. 30), 100530-101382 (SEQ ED No. 31), 101798- 103865 (SEQ ED No. 32), 104486-109841 (SEQ ID No. 33), 109953-110561 (SEQ ID No. 34), 111000-113482 (SEQ ED No.35), 113774-116253 (SEQ ED No.36), 116846-117907 (SEQ ED No.37), 117999-118623 (SEQ ID No.38), 118865-122881 (SEQ ID No.39), 122978-186088 (SEQ ID No.40), 129508-130413 (SEQ ID No.41), 131138-134228 (SEQ ID No.42), 134517-135473 (SEQ ED No.43), 135815-139983 (SEQ ED No.44), and 140683-144419 (SEQ ED No.45) ofthe sequence shown in FIG. IB.

7. DEPOSIT OF MICROORGANISMS

The following microorganism was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Virginia 20110, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Puφoses of Patent Procedure, and complies with the criteria set forth in 37 C.F.R. § 1.801-1.809 regarding availability and permanency of deposits. The deposit was made on the date indicated and assigned the indicated accession number:

Microorganism Deposit ATCC Deposit No. Date of Deposit Ep69104 PTA-451 August 4, 1999

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. To distinguish the strains and isolate a strain harboring either 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 lOOμg/ml ampicillin, a single colony can be grown, and then BAC DNA can be extracted from the culture using standard procedures. Next, a sample of the DNA preparation can be digested with Not I and resolved via standard pulse-field gel electrophoresis procedures. BAC69 DNA yields fragments having a total length of approximately 220kb; BAC 104 DNA yields fragments having a total length of approximately 80kb.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All publications and patent applications mentioned in this specification are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference.

International Application No: PCT/ /

Claims

WHAT IS CLAIMED IS:

An isolated polynucleotide comprising: a) the nucleotide sequence of FIG. IB; b) nucleotides 1-825 of FIG. IB; 1243-11147 of FIG. IB; 11844-14813 of FIG. IB; 15078-17635 of FIG. IB; 18824-23844 of FIG. IB; 24232-28495 of FIG. IB; 28592-29286 of FIG. IB; 29625-30617 of FIG. IB; 31375-32031 of FIG. IB; 32430-34011 of FIG. IB; 34103-34828 of FIG. IB; 35054-38519 of FIG. IB; 39524-41230 of FIG. IB; 42203-42309 of FIG. IB; 42611-43855 of FIG. IB; 44418-47968 of FIG. IB; 48357-48683 of FIG. IB; 49223-49730 of FIG. IB; 50023-51733 of FIG. IB; 52026-53079 of FIG. IB; 53448-57506 of FIG. 0 IB; 57906-59129 of FIG. IB; 60234-60748 of FIG. IB; 61019-61305 of FIG. IB; 61996-63859 of FIG. IB; 63966-66349 of FIG. IB; 67116-71681 of FIG. IB; 72090-72942 of FIG. IB; 73358-75425 of FIG. IB; 76046-81401 of FIG. IB; 81513-82121 of FIG. IB; 82560-85042 of FIG. IB; 85334-87813 of FIG. IB; 88406-89467 of FIG. IB; 89559-90183 of FIG. IB; 90425-94446 of FIG. 5 IB; 94537-100765 of FIG. IB; 101067-101972 of FIG. IB; 102697-105787 of FIG. IB; 106076-107032 of FIG. IB; 107374-111541 of FIG. IB; or 112241-115977 of FIG. IB; or c) a fragment comprising at least 10 contiguous nucleotides of the nucleotide sequences of b). 0

2. An isolated polynucleotide comprising the nucleotide sequence as shown in FIG. 2.

3. An isolated polynucleotide which hybridizes under stringent conditions to the complement of the polynucleotide of Claim 1 or 2. 5

4. A vector comprising the polynucleotide of Claim 1 or 2.

5. An expression vector comprising the polynucleotide of Claim 1 or 2 in operative association with a nucleotide regulatory sequence that controls expression of the nucleotide Q sequence in a host cell.

6. A host cell genetically engineered to contain the polynucleotide of Claim 1 or 2.

7. A genetically engineered host cell that contains the nucleotide sequence of Claim 1 or 2 in operative association with a nucleotide regulatory sequence that controls expression of the nucleotide sequence in the host cell.

8. An isolated gene product comprising an amino acid sequence encoded by a nucleic acid comprising the polynucleotide of Claim 2.

9. An antibody that immunospecifically binds the gene product of Claim 8.

c 10. A method for detecting, diagnosing or determining the risk of a/s 225-related disorder in a mammal, comprising measuring s 225 gene expression in a patient sample.

11. A method for detecting, diagnosing or determining the risk of a/s/225-related disorder in a mammal, comprising detecting a polymoφhic site in the genome of the

. r. mammal.

12. A method for detecting, diagnosing or determining the risk of a/s/225-related disorder in a mammal, comprising detecting a mutation contained in the genome of the mammal.

15

13. A method for modulating a treating a/s/225-related disorder in a mammal comprising administering to the mammal a compound to the mammal that modulates the synthesis, expression or activity of a mammalian fsh25 gene or fsh25 gene product so that at least one symptom of the disorder is ameliorated.

20

14. The method of Claim 13 wherein the/s/225-related disorder is a neuropsychiatric disorder.

15. The method of Claim 13 wherein the compound increases the synthesis, expression - .. or activity of a mammalian fsh25 gene or fsh25 gene product.

16. The method of Claim 13 wherein the compound comprises any one of the polynucleo tides of Claim 1 or 2.

-_π 17. The method of Claim 13 wherein the compound is a small organic molecule.

18. The method of Claim 13 wherein the compound alters the synthesis, expression or activity of a mammalian fsh25 gene or fsh25 gene product.

- ,

19. A method for treating a/s 225-related disorder or a disorder involving a fsh25- mediated process a in a mammal comprising administering to the mammal afsh25 gene product, or a fragment, analog, or mimetic thereof, so that at least one symptom of the disorder is ameliorated.

20. A method for treating a/ 225-related disorder or a disorder involving a fsh25- mediated process a in a mammal comprising administering to the mammal a mutant form of

5 afsh25 gene product, or a fragment, analog, or mimetic thereof, so that at least one symptom of the disorder is ameliorated.

21. A method for treating a/s 225-related disorder in a mammal comprising administering to the mammal afsh25 gene product, or fragment, analog, or mimetic thereof,

. r. so that at least one symptom of the disorder is ameliorated.

22. A method of treating a s/225-related disorder resulting from a mutation in afsh25 gene, in a mammal, comprising supplying the mammal with a nucleic acid molecule that encodes an unimpaired fsh25 gene product such that an unimpaired fsh25 gene product is

. r expressed and symptoms of the disorder are ameliorated.

23. The method of Claim 23 wherein the/^, 225-related disorder is a neuropsychiatric disorder.

_jr.

24. The method of Claim 24 wherein the neuropsychiatric disorder is schizophrenia, attention deficit disorder, a schizoaffective disorder, a bipolar affective disorder or a unipolar disorder.

25. The method of Claim 24 in which the cell is engineered ex vivo to express an _jr unimpaired fsh25 protein.

26. A method for identifying an individual having or at risk of developing afsh25- related disorder comprising the step of detecting the presence or absence of a polymoφhism that conelates with afsh25 allele associated with the disorder, wherein presence of the .r. polymoφhism indicates that the individual has or is at risk of developing the s/225-related disorder.

27. A method for identifying a compound capable of modulating fsh25 activity, comprising: . a) contacting a compound to a cell that expresses afsh25 gene; b) measuring the level of fsh25 gene expression in the cell; and c) comparing the level obtained in (b) to fsh25 gene expression level obtained in the absence of the compound; such that if the level obtained in (b) differs from that obtained in the absence of the compound, a compound capable of modulating afsh25 activity has been identified.

28. A kit comprising a compound which selectively binds to a polypeptide of Claim 8 and instmctions for use.

29. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of Claim 1 or 2 and instmctions for use.