WO2004040016A2

WO2004040016A2 - Genetic markers on chromosome 12 associated to bipolar or unipolar disorders

Info

Publication number: WO2004040016A2
Application number: PCT/GB2003/004684
Authority: WO
Inventors: Gursharan Kalsi; Andrew Mcquillin; Hugh Malcolm Douglas Gurling; Birte Degn; Ole Mors; Torben Kruse; Mikkel Dybro Lundorf; Henrik Ewald
Original assignee: Ucl Biomedica Plc; EWALD, Marianne, Varnich
Priority date: 2002-10-31
Filing date: 2003-10-31
Publication date: 2004-05-13
Also published as: AU2003276427A1; AU2003276427A8; GB0225360D0; WO2004040016A3

Abstract

The present invention is concerned with the genetic causes for bipolar and genetically related unipolar affective disorders, providing markers and methods of treating and diagnosing bipolar and genetically related unipolar affective disorders and related neuropsychiatric disorders, and providing methods for identifying markers and compounds for use as part of therapeutic and/or diagnostic methods. The present inventors have identified and fine mapped a locus for bipolar disorder and related unipolar affective disorders on chromosome 12 for the first time and have been able to pin point several genes that are carrying mutations increasing susceptibility to bipolar disorder. The region identified is approximately two million base pairs of DNA in the chromosomal region 12q24.3 on the short arm of chromosome 12 between markers D12S2705 and D12S340.

Description

GENETIC MARKERS

TECHNICAL FIELD''

The present invention is directed to methods of detecting the presence of bipolar and genetically related unipolar affective disorder susceptibility mutations in an affected or unaffected individual .

BACKGROUND ART

Bipolar Affective disorder is a common psychiatric condition with an age corrected lifetime risk of between 0.3-1.5 %. The disorder is characterised by episodes of mania and depression.

The vast majority of psychiatric disorders are believed to involve subtle and/or undetectable changes, at the cellular and/or molecular levels, in nervous system structure and function. This lack of detectable neurological defects distinguishes "neuropsychiatric" disorders, such as bipolar and genetically related unipolar affective disorders, attention deficit disorders, schizoaffective disorder, bipolar affective disorders, or unipolar affective disorder, from neurological disorders, in which anatomical or biochemical pathologies are manifest. Hence, identification of the causative defects and the molecular pathologies of neuropsychiatric disorders are needed in order to enable clinicians to evaluate and prescribe appropriate courses of treatment to cure or ameliorate the symptoms of these disorders .

Currently, individuals are typically evaluated for bipolar and genetically related unipolar affective disorders using the criteria set forth in the most current version of the International Classification of diseases or the 7Λmerican Psychiatric Association's Diagnostic and Statistical Manual of

Mental Disorders (DSM) . While many drugs have been used to treat individuals diagnosed with bipolar and genetically related unipolar affective disorders many drugs are inadequate. For example, many drug treatments are effective in only approximately a third to a half of individuals diagnosed with bipolar and genetically related unipolar affective disorders, a further group respond partially and a third respond poorly. Previously it was impossible to predict which drug treatments will be effective in, particular bipolar and unipolar affective disorder patients. Commonly, after 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 a chronic depressed state and the presence of possible dangerous suicidal or homicidal behaviour.

Family, twin, and adoption studies have demonstrated that genetic factors contribute strongly to the aetiology of bipolar (BPAD) and genetically related .unipolar affective disorders (Rifkin and Gurling 1991) . However, to date no specific gene or DNA sequence variation has been identified. Linkage studies have identified a number of candidate regions including those at 4pl6 (Blackwood et al. 1996), 18ρ (Berrettini et al. 1994), 18q (Stine et al. 1995; Freimer et al. 1996), 21q (Straub et al. 1994) and Xq26-28 (Pekkarinen et al . 1995). Some these loci have subsequently been replicated in second independent studies.

The observation that bipolar disorder cosegregated with Darriers disease suggested that some bipolar families could be linked to a chromosome 12 locus, and this has been the subject of various studies (Dawson et al. 1995; Ewald et al . 1998; Barden and Morissette 1999; Detera-Wadleigh 1999; Detera-Wadleigh et al. 1999a; Detera-Wadleigh et al. 1999b; Curtis et al 2002; Degn et al. 2001) . Some of these studies support the possibility of a bipolar affective disorder locus at 12q23-12q24.

However fine mapping genes for common diseases such as bipolar and genetically related unipolar affective disorders is complicated by the variable definition of affective disorder phenotypes, by aetiologic heterogeneity, and by uncertainty about the mode of genetic transmission of the disease trait. With neuropsychiatric disorders there is ambiguity in distinguishing individuals who likely carry an affected genotype from those who are genetically unaffected.

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 and genetically related unipolar affective disorders and related disorders is of great importance for genetic counselling, diagnosis and treatment of individuals in affected families .

DISCLOSURE OF the INVENTION

The present invention is concerned with the identification of one or more of the genetic causes for bipolar and genetically related unipolar affective disorders, providing methods of treating and diagnosing bipolar and genetically related unipolar affective disorders and related neuropsychiatric disorders, and providing methods for identifying compounds for use as part of therapeutic and/or diagnostic methods.

The present inventors have identified and fine mapped a locus for bipolar disorder and related unipolar affective disorders on chromosome 12 for the first time and have been able to pin point seven putative genes that are carrying mutations increasing susceptibility to bipolar disorder. These results enable bipolar disorder to be subdivided into genetic subtypes which can be used to improve diagnosis, prognosis and understanding treatment responsiveness. Genotyping with the markers described herein as well as additional markers permits inter alia confirmation of the phenotypic classification of bipolar and genetically related unipolar affective disorders and related diagnoses and can assist with ambiguous clinical phenotypes which make it difficult to distinguish between bipolar and genetically related unipolar affective disorders and other possible psychiatric illnesses. The results enable the identification of a protein and its variants that cause affective disorders through the cloning and sequencing of its gene. The protein encoded by this gene may be targeted to develop new drug treatment for bipolar and unipolar affective disorders. The research will also enable the identification of which brain system as a whole is involved in bipolar disorder and thus can lead on to the identification of yet further protein targets involved upstream and downstream in the brain pathways involved in affective disorders.

Briefly, the present inventors genotyped 21 newly described and previously published microsatellite markers in a sample of 381 Danish and British bipolar patients and compared the frequency of marker alleles to a carefully selected matched control group of 477 Danish and British normal controls. Differences in allele frequencies, which were highly statistically significant, after correction for the use of markers with multiple alleles, were found for ten of these markers. The pattern of allelic association between bipolar disorders and specific markers overlapped in the Danish and UK samples when analysed separately and remained highly significant when pooled together.

C12 candida te genes region

As used herein the term "the C12 candidate genes region" describes the approximately two million base pairs of DNA in the chromosomal region 12q24.3 on the long arm of chromosome 12q24 between D12S2705, and D12S340 (see Table 3) . This region includes the 5' locus control regions, exons and introns of several chromosome 12 candidate genes (see Table 5) .

More preferably, in any of the aspects of the invention, the region analysed will be within about, or exactly, 1 megabase either side of D12S307 at the cytogenetic location 12q24.3. More preferably it is the 2 megabase region on the long arm of chromosome 12 from base number 125,850,000 to base number 127,850,000 (numbering starts from the telomere of the short arm of chromosome 12 towards the telomere of the long arm)

More preferably, the region analysed will include the markers 1634TET, D12S307, 307CA2 to D12SDK1 and a region of approximately 500 Kb flanking this.

More preferably, the region analysed is defined as that from 1634TET to D12SDK1 (which is around 278 kB) .

Most preferably the region analysed is no more than 500kb of any of those defined above, and includes at least one C12 candidate genes region marker (see below).

C12 candida te genes region markers

A "C12 candidate genes region marker" is a marker present in the C12 candidate gene region, which may be used in the present invention as described below.

Preferred markers are shown in Tables 3, 4 or 6.

Preferred markers are any one or more of:

D12S1634 307CA2

1634TET

307GT4

D12S307

D12SDK1 1634GT2

D12S340

SNP GIN (see Table 4) SNP G2M (see Table 4) SNP G3 exon M insertion T (insT) (see Table 4)

However other markers disclosed herein, or in linkage disequilibrium with those discussed above, may also be used in the present invention. These may include in particular mutations in the five prime and three prime locus control regions, the exons and introns of the C12 Candidate genes (see below) .

C12 candida te genes

As used herein the term "C12 candidate genes" refers to the genes defined in Table 5.

Preferred C12 candidate genes are any of those termed Gl, G2, G3, or G4 and the Calsequestrin homologue.

In various embodiments, the invention embraces the nucleic acid sequences of the C12 candidate genes which have never before been associated with any neuropsychiatric disorders. For example embodiments relate to the methods and materials of the invention of any of:

(a) a nucleic acid molecule containing a DNA sequence of a gene shown in Table 5. These genes have not been well characterised before. Preferred sequences are labeled Gl G2 G4 and G4 and a sequence likely to have function related to calcium and is called the "Calsequestrin" sequence in table 5.

(b) any DNA sequence that encodes a polypeptide transcribed from the DNA sequences referred to either singly or in combination as shown in table 5.

(c) any DNA sequence that hybridizes to the complement of the DNA sequences described in table 5, under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M and washing in 0.2 x SSC/0.1% SDS at 42° C. (Ausubel et al . , 1989), and encodes a gene product functionally equivalent to a C12 candidate genes product. The term "functionally equivalent to a C12 candidate gene product, " as used herein, refers to a gene product that exhibits at least one of the biological activities of an endogenous, unimpaired C12 candidate genes gene. In one embodiment, a functionally equivalent C12 candidate gene product is one that, when present in an appropriate cell type, is capable of ameliorating, preventing or delaying the onset of one or more symptoms of a C12 candidate genes bipolar, unipolar or related neuropsychiatric disorder.

In another embodiment, a functionally equivalent C12 candidate gene product is one that, when present in an appropriate cell type, is capable of ameliorating, preventing or delaying the onset of one or more symptoms of a neuropsychiatric disorder. In yet another embodiment, a functionally equivalent C12 candidate genes product is one that, when present in an appropriate cell type, is capable of ameliorating, preventing or delaying the onset of one or more symptoms of bipolar and genetically related unipolar affective disorders and related disorders.

C12 candidate genes sequences can include, for example either genomic DNA (gDNA) or cDNA sequences. When referring to a nucleic acid which encodes a given amino acid sequence, therefore, it is to be understood that the nucleic acid need not only be a cDNA molecule, but can also, for example, refer to a gDNA sequence from which an mRNA species is transcribed that is processed to encode the given amino acid sequence.

As used herein, C12 candidate genes may also refer to degenerate variants of DNA sequences of the genes specified in table 5. The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the DNA sequences listed in table 5. Such hybridization conditions may be highly stringent or less highly stringent, as described above. In instances wherein the nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly stringent conditions may refer, e.g., to washing in 6 times SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligos) , 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos) . These nucleic acid molecules may encode or act as C12 candidate genes antisense molecules, useful, for example, in C12 candidate genes regulation (for and/or as antisense primers in amplification reactions of C12 candidate genes nucleic acid sequences) . Parts of these nucleic acid molecules may be used as short interfering RNA' s (siRNAs, for changing C12 candidate genes regulation. With respect to C12 candidate genes regulation, such techniques can be used to regulate, for example, a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for C12 candidate gene regulation. Still further, such molecules may be used as components of diagnostic methods whereby, for example, the presence of a particular C12 candidate genes allele responsible for causing a C12 candidate genes disorder or neuropsychiatric disorder such as bipolar and genetically related unipolar affective disorders or schizoaffective disorder, may be detected.

Neuropsychia tric disorders

The neuropsychiatric disorders referred to herein can include, but are not limited to, C12 candidate genes disorders (bipolar and genetically related unipolar affective disorders) and neuropsychiatric disorders including unipolar affective disorders, schizophrenia, delusional disorders, paraphrenia, paranoid psychosis, schizotypal disorder, schizoaffective disorder, schizoaffective depressed disorder, schizoaffective manic disorder, bipolar disorder, manic disorder, periodic bipolar disorder, rapid cycling bipolar disorder, hypomanic disorder, mixed affective disorder, and related unipolar affective disorders such as depressive psychosis, depressive stupor, depressive neurosis, neurotic depression, intermittent depressive disorder, recurrent major depressive disorder, single episode major depressive disorder, primary and secondary affective disorders, pure depressive disorder, depressive spectrum disorders and other types of unipolar affective disorders .

The term "C12 candidate genes related neuropsychiatric disorder" as used herein refers to a disorder involving an aberrant level of C12 candidate gene expression, gene product synthesis and/or gene product activity relative to levels found in normal, unaffected, unimpaired individuals, levels found in clinically normal individuals, and/or levels found in a population whose level represents a baseline, average C12 candidate genes level.

As used herein, the following terms shall have the abbreviations indicated: BAC, bacterial artificial chromosomes; BP/UP bipolar and genetically related unipolar affective disorders disorder (s); bp, base pair(s); EST, expressed sequence tag; lod, logarithm of odds; MDD, unipolar major depressive disorder; RT-PCR, reverse transcriptase PCR; SSCP, single-stranded conformational polymorphism; SAM/SAD, schizoaffective disorder manic or depressed type; STS, sequence tagged site.

General discussion of the invention

In preferred embodiments the invention relates to the use of nucleic acid or genetic analysis in the C12 candidate genes region to genetically diagnose (genotype) bipolar and genetically related unipolar affective disorders in individuals to confirm phenotypic diagnoses of bipolar and genetically related unipolar affective disorders and other related disorders such as schizoaffective disorder and affective disorders in general and to determine appropriate treatments and prevention strategies for susceptibility gene carriers or patients with particular genotypic (or haplotypic -see Table 7) subtypes. Isolated polynucleotides useful for genetic diagnosis, mutation detection and for linkage and allelic association analysis of bipolar and genetically related unipolar affective disorders and methods for obtaining such isolated polynucleotides are described. The invention encompasses use of C12 candidate genes nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof, C12 candidate gene products and antibodies directed against such gene products, cloning vectors containing mammalian C12 candidate 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 C12 candidate genes and to using such compounds as therapeutic agents in the treatment of C12 candidate gene disorders and neuropsychiatric disorders. The invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of C12 candidate gene disorders and neuropsychiatric disorders.

Some aspects of the invention will now be discussed in more detail :

Genetic ma terials of the invention

The invention encompasses uses of the following:

(a) DNA vectors that contain any of the foregoing C12 candidate genes coding sequences and/or their complements (i.e., antisense) ;

(b) DNA expression vectors that contain any of the foregoing C12 candidate genes coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and

(c) genetically engineered host cells that contain any of the foregoing C12 candidate genes coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell.

As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the CRE/LOX system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid iphosphatase, and the promoters of the yeast α-mating factors.

Fragments and homologues

The invention further includes uses of fragments of any of the DNA sequences disclosed herein.

In one embodiment, the C12 candidate gene sequences of the invention are mammalian gene sequences, with human sequences being preferred.

In another embodiment, the C12 candidate gene sequences of the invention are gene sequences encoding C12 candidate gene products containing polypeptide portions corresponding to (that is, polypeptide portions exhibiting amino acid sequence similarity to) the amino acid sequences predicted from the sequences listed in table 5, wherein the corresponding portion exhibits greater than about 50% amino acid identity with the table 5 listed sequence. In yet another embodiment, the C12 candidate gene sequences of the invention are gene sequences encoding C12 candidate gene products containing polypeptide portions corresponding to (that is, polypeptide portions exhibiting amino acid sequence similiarity to) the amino acid sequence derived from the DNA sequences listed in table 5, wherein the corresponding portion exhibits greater than about 50% amino acid identity with a table 5 DNA sequence derived protein sequence.

In addition to the human C12 candidate gene sequences disclosed in table 5, additional C12 candidate gene sequences can be identified and readily isolated, without undue experimentation, by molecular biological techniques well known in the art, used in conjunction with the C12 candidate genes sequences disclosed herein. For example, additional human C12 candidate gene sequences at the same or at different genetic loci as those disclosed in table 5 can be isolated readily. There can exist, for example, genes at other genetic or physical loci within the human genome that encode proteins that have extensive homology to one or more domains of the C12 candidate gene products and that encode gene products functionally equivalent to a C12 candidate gene product. Further, homologous C12 candidate gene sequences present in other species can be identified and isolated readily.

With respect to identification and isolation of C12 candidate gene sequences present at the same genetic or physical locus as those sequences disclosed in table 5, such sequences can, for example, be obtained readily by utilizing standard sequencing and bacterial artificial chromosome (BAC) technologies.

For example, sheared libraries can be made from BAC clones'. Fragments of a convenient size, e.g., in the size range of approximately 1 kb, are cloned into a standard plasmid, and sequenced. Further C12 candidate genes sequences can then readily be identified by alignment of the BAC sequences with the C12 candidate genes sequences depicted in table 5. Alternatively, BAC subclones containing additional C12 candidate genes sequences can be identified by identifying those subclones which hybridize to probes derived from the C12 candidate genes sequences depicted in table 5.

With respect to the cloning of a C12 candidate gene homologue in human or other species (e.g., mouse), the isolated C12 candidate gene sequences disclosed herein may be labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or tissues (e.g., brain tissues) derived from the organism (e.g., mouse) of interest. The hybridization conditions used should 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.

Alternatively, the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions. Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook, et al . , 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y.; and Ausubel, et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

Further, a C12 candidate gene homologue may be isolated from, for example, human nucleic acid, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the C12 candidate genes 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 C12 candidate gene allele (such as human brain cell lines e.g., ATCC CRL-7605, ATCC CRL-7948, ATCC CRL-2060 PFSK-1, ATCC CRL-2176 SW 598, American Type Culture Collection, Rockville, Md.; cortical neuronal cell lines, e.g., Ronnett, et al.,1990, Science 248, 603-605; Ronnett, et al . , 1994, Neuroscience 63, 1081-1099; and Dunn, et al., 1996, Int. J. Dev. Neurosci. 14, 61-68; neuronal line HCN-1A, Westlund et al . , 1992, Int. J. Dev. Neurosci. 10, 361-373).

The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a C12 candidate 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 labelled 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. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express the C12 candidate genes, such as, for example, blood samples or 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 standard terminal transferase reaction, the hybrid may be digested with RNAase 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 al.,1989.

C12 candidate gene sequences may additionally be used to isolate further mutant C12 candidate gene alleles. Such mutant alleles may be isolated from individuals either known or proposed to have a genotype that contributes to the symptoms of a C12 candidate genes disorder, or a neuropsychiatric disorder such as bipolar and genetically related unipolar affective disorders or schizoaffective disorder. Mutant alleles and mutant allele products may then be utilized in the therapeutic and diagnostic systems described below.

Additionally, such C12 candidate gene sequences can be used to detect C12 candidate gene regulatory (e.g., promoter) defects which can be associated with a C12 candidate genes disorder, or a neuropsychiatric disorder such as bipolar and genetically related unipolar affective disorders.

A cDNA of a mutant C12 candidate 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 the mutant C12 candidate genes allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an 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 C12 candidate genes allele to that of the normal C12 candidate genes allele, the mutation (s) responsible for the loss or alteration of function of the mutant C12 candidate gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant C12 candidate genes allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant C12 candidate genes allele. An unimpaired C12 candidate gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant C12 candidate genes allele in such libraries. Clones containing the mutant C12 candidate 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 constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant C12 candidate genes 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 C12 candidate genes product, as described below (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 a C12 candidate genes 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 anti-C12 candidate gene product antibodies are likely to cross- react with the mutant C12 candidate 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. C12 candidate genes mutations can further be detected using PCR amplification techniques. Primers can routinely be designed to amplify overlapping regions of the whole C12 candidate genes sequence including the promoter region. In one embodiment, primers are designed to cover the exon-intron boundaries such that, first, coding regions can be scanned for mutations. Genomic DNA isolated from lymphocytes of normal and affected individuals is used as PCR template. PCR products from normal and affected individuals are compared, either by single strand conformational polymorphism (SSCP) mutation detection techniques and/or by sequencing. The mutations responsible for the loss or alteration of function of the mutant C12 candidate genes product can then be ascertained.

Protein products of genes & host cells

C12 candidate 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 the identification of other cellular or extracellular gene products involved in the regulation of a C12 candidate genes disorder, or a neuropsychiatric disorder such as bipolar and genetically related unipolar affective disorders.

Thus the invention further relates to novel human C12 candidate gene region products and to antibodies directed against such human C12 candidate gene region products, or conserved variants or fragments thereof. C12 candidate genes nucleic acid and amino acid sequences are provided herein. The invention also relates to vectors, including expression vectors, containing mammalian C12 candidate gene molecules, and hosts that have been genetically engineered to express such C12 candidate gene region products.

The amino acid sequence or sequences that can be derived from sequences Gl G2 G3 G4 and Calsequestrin (table 5) represent preferred example C12 candidate gene products. The C12 candidate gene product, sometimes referred to herein as a "C12 candidate genes protein", includes those gene products encoded by the C12 candidate gene sequences described above in table 5.

In addition, C12 candidate gene products may include proteins that represent functionally equivalent gene products. Such an equivalent C12 candidate gene product may contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the the amino acid sequence encoded by the C12 candidate gene sequences described, above, but that result in a "silent" change, in that the change produces a functionally equivalent C12 candidate gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the a phipathic 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 C12 candidate gene products. Such alterations can, for example, alter one or more of the biological functions of the C12 candidate gene product. Further, such alterations can be selected so as to generate C12 candidate gene products that are better suited for expression, scale up, etc. in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges .

The C12 candidate 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 C12 candidate gene polypeptides, peptides, fusion peptide and fusion polypeptides of the invention by expressing nucleic acid containing C12 candidate gene sequences are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing C12 candidate 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 al . , 1989, supra, and Ausubel, et al., 1989, supra. Alternatively, RNA capable of encoding C12 candidate gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in

"Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford.

A variety of host-expression vector systems may be utilized to express the C12 candidate 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 C12 candidate 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 C12 candidate gene product coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the C12 candidate gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the C12 candidate gene product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing C12 candidate gene product coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harbouring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the C12 candidate 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 C12 candidate genes protein or for raising antibodies to C12 candidate genes 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, EMBO J. 2, 1791) , in which the C12 candidate gene product coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13, 3101-3109; Van

Heeke and Schuster, 1989, J. Biol. Chem. 264, 5503-5509); and the like. PGEX 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 adsorption to glutathione- agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica, nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The C12 candidate genes coding sequence may be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under control of an ACNPV promoter (for example the polyhedrin promoter) . Successful insertion of C12 candidate gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) . These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g., see Smith, et al., 1983, J. Virol. 46, 584; Smith, U.S. Pat. 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, the C12 candidate gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing C12 candidate 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 C12 candidate gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire C12 candidate 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 C12 candidate 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 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 the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. 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.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the C12 candidate 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 may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the C12 candidate gene product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the C12 candidate gene product.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 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 methotrexate (Wigler, et al . , 1980, Natl. Acad. Sci. USA 77, 3567; O'Hare, et al . , 1981, Proc. Natl. Acad. Sci. USA 78, 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78, 2072); neo, which confers resistance to the aminoglycoside G-418

(Colberre-Garapin, et al., 1981, J.Mol. Biol. 150, 1); and hygro, which confers resistance to hygromycin (Santerre, et al.,1984, Gene 30, 147) .

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, 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 virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

Transgenic animals

The C12 candidate gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate C12 candidate genes transgenic animals. The term "transgenic," as used herein, refers to animals expressing C12 candidate gene sequences from a different species (e.g., mice expressing human C12 candidate gene sequences), as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) C12 candidate gene sequences or animals that have been genetically engineered to no longer express endogenous C12 candidate gene sequences (i.e., "knock-out" animals), and their progeny.

Any technique known in the art may be used to introduce a C12 candidate 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. Rev. Cytol . 115; 171-229).

Any technique known in the art may be used to produce transgenic animal clones containing a C12 candidate gene 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 a C12 candidate gene transgene in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, et al., 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 C12 candidate gene transgene be integrated into the chromosomal site of the endogenous C12 candidate gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous C12 candidate gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous C12 candidate gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous C12 candidate gene in only that cell type, by following, for example, the teaching of Gu, et al. (Gu, et al . , 1994, Science 265, 103-106). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant C12 candidate 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 C12 candidate gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the C12 candidate gene transgene product.

Antibodies and uses

Described herein are methods for the production of antibodies capable of specifically recognizing one or more C12 candidate gene product epitopes or epitopes of conserved variants or peptide fragments of the C12 candidate gene products.

Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a C12 candidate gene product in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal levels of C12 candidate gene products, and/or for the presence of abnormal forms of such gene products. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, below for the evaluation of the effect of test compounds on C12 candidate gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described, below for example, evaluate the normal and/or engineered C12 candidate gene-expressing cells prior to their introduction into the patient. Anti-Cl2 candidate gene product antibodies may additionally be used as a method for the inhibition of abnormal C12 candidate gene product activity. Thus, such antibodies may, therefore, be utilized as part of treatment methods for a C12 candidate gene disorder or a neuropsychiatric disorder.

For the production of antibodies against a C12 candidate gene product, various host animals may be immunized by injection with a C12 candidate gene product, or a portion thereof. Such host animals may include, but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and otentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a C12 candidate gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with C12 candidate gene product supplemented with adjuvants as also described above.

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. Pat. 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 producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of "chimeric antibodies" (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 chimaeric 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. Pat. No. 4,816,567; and Boss et al.,U.S. Pat. No. 4,816397).

In addition, techniques have been developed for the production of humanized antibodies. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) An immunoglobuin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs) . 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 antibodies (U.S. Pat. No. 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 C12 candidate 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 constructed (Huse, et al . , 1989, Science, 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Described herein are various applications of C12 candidate gene sequences, C12 candidate gene products, including peptide fragments and fusion proteins thereof, and of antibodies directed against C12 candidate gene products and peptide fragments thereof. Such applications include, for example, prognostic and diagnostic evaluation of a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, and the identification of subjects with a predisposition to such disorders, as described below. Additionally, such applications include methods for the treatment of a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, as describe below and for the identification of compounds that modulate the expression of the C12 candidate gene and/or the synthesis or activity of the C12 candidate gene product, as described below. Such compounds can include, for example, other cellular products that are involved in mood and cognitive function regulation and in C12 candidate genes disorders and neuropsychiatric disorders, such as bipolar and genetically related unipolar affective disorders. These compounds can be used, for example, in the amelioration of C12 candidate gene disorders and neuropsychiatric disorders, such as bipolar and genetically related unipolar affective disorders.

Diagnosis & susceptibility

In addition, the present invention is directed to a method of diagnosing and detecting the presence of bipolar and genetically related unipolar affective disorders and related disorders in an individual comprising analyzing a sample of DNA from said individual with the PCR primers defined from sequences defined in the attached tables for the presence of mutations, aetiological variants or quantitative trait nucleotides in the C12 candidate genes region.

In addition, the present invention is directed to methods that utilize the C12 candidate gene and/or gene product sequences for the diagnostic evaluation, genetic testing and prognosis of a C12 candidate genes related neuropsychiatric disorder. For example, the invention relates to methods for diagnosing C12 candidate genes related neuropsychiatric disorders, wherein such methods comprise measuring C12 candidate genes expression in a patient sample, or detecting a C12 candidate genes mutation in the genome of the human individual or mammal suspected of exhibiting such a disorder.

Additionally, the invention provides a method of predicting a patient's likelihood of developing bipolar or genetically related unipolar affective disorders by determining a patient's genotype in the entire C12 candidate genes region by determining the presence or absence of allelic variants and mutations in a DNA sample derived from a patient.

Additionally, the invention provides a method of predicting the susceptibility to bipolar and genetically related unipolar affective disorders in unaffected individuals and relatives of affected individuals by correlating the presence or absence of the mutations, DNA polymorphisms and other gene variants within the C12 candidate genes region with a phenotypic diagnosis of bipolar and genetically related unipolar affective disorders and related disorders for any given individual and for family members wherein the correlation is indicative of a bipolar and genetically related unipolar affective disorder or other related disorder susceptibility gene locus.

In preferred embodiments this is carried out by a) typing blood relatives of said individual for mutations and DNA polymorphisms within the C12 candidate genes region on chromosome 12, and b) analyzing a DNA sample from said individual for the presence of said mutations or DNA polymorphisms, wherein the presence of said DNA polymorphisms in said region in an individual is indicative of an increased likelihood that the individual will develop a bipolar or unipolar affective disorder.

A variety of methods can be employed for the diagnostic and prognostic evaluation of C12 candidate gene disorders and neuropsychiatric disorders, such as Bipolar and genetically related unipolar affective disorders, and for the identification of subjects having a predisposition to such disorders.

Such methods may, for example, utilize reagents such as the C12 candidate gene nucleotide sequences described above, and antibodies directed against C12 candidate gene products, including peptide fragments thereof, as described, above. Specifically, such reagents may be used, for example, for:

(1) the detection of the presence of C12 candidate gene gene mutations, or the detection of either over- or under-expression of C12 candidate gene mRNA relative to the state of a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders;

(2) the detection of either an over- or an under-abundance of C12 candidate gene product relative to the unaffected state; and

(3) the detection of an aberrant level of C12 candidate gene product activity relative to the unaffected state.

C12 candidate gene nucleotide sequences can, for example, be used to diagnose a C12 candidate gene or neuropsychiatric disorder using, for example, the techniques for C12 candidate gene mutation detection described above.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific C12 candidate gene nucleic acid or anti-Cl2 candidate gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting abnormalities of a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders.

For the detection of C12 candidate gene mutations, any nucleated cell can be used as a starting source for genomic nucleic acid. For the detection of C12 candidate gene expression or C12 candidate gene products, any cell type or tissue in which the C12 candidate gene is expressed may be utilized.

Nucleic acid-based detection techniques and peptide detection techniques are described below.

Detection of nucleic acids

A variety of methods can be employed to screen for the presence of C12 candidate gene mutations and to detect and/or assay levels of C12 candidate gene nucleic acid sequences.

Mutations within the C12 candidate gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to those of skill in the art.

C12 candidate gene nucleic acid sequences may be used in hybridization or amplification assays of biological samples to detect abnormalities involving C12 candidate gene structure, including point mutations, insertions, deletions, inversions, translocations and chromosomal rearrangements. Such assays may include, but are not limited to, Southern analyses, single- stranded conformational polymorphism analyses (SSCP) , and PCR analyses, including but not limited to High PressureLiquid Chromatography (HPLC) methods of detecting DNA sequence mutation or DNA sequence variations.

Diagnostic methods for the detection of C12 candidate gene- specific mutations can involve for example, contacting and incubating nucleic acids including recombinant DNA molecules, cloned gene or degenerate variants thereof, obtained from a sample, e.g., derived from a patient sample or other appropriate cellular source, with one or more labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, as described below, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the C12 candidate gene. Preferably, the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed from the nucleic acid: C12 candidate gene molecule hybrid. 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 microtitre plate or polystyrene beads. In this case, after incubation, non-annealed, labeled nucleic acid reagents are easily removed. Detection of the remaining, annealed, labeled C12 candidate genes nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The C12 candidate gene sequences to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a normal C12 candidate gene sequence in order to determine whether a C12 candidate gene mutation is present.

Alternative diagnostic methods for the detection of C12 candidate gene specific nucleic acid molecules, 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. Pat. No. 4,683,202), followed by the detection of the amplified molecules using techniques well known to those of skill in the art. 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 C12 candidate gene in order to determine whether a C12 candidate gene mutation exists.

Additionally, well-known genotyping techniques can be performed to identify individuals carrying C12 candidate gene mutations. Such techniques include, for example, the use of restriction fragment length polymorphisms (RFLPs) , which involve sequence variations in one of the recognition sites for the specific restriction enzyme used.

Additionally, improved methods for analyzing DNA polymorphisms, which can be utilized for the identification of C12 candidate gene 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 polymorphisms 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 C12 candidate gene, and the diagnosis of diseases and disorders related to C12 candidate gene mutations.

Also, Caskey et al.(U.S. Pat. No. 5,364,759) describe a DNA profiling assay for detecting short tri and tetra nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the C12 candidate gene, amplifying the extracted DNA, and labelling the repeat sequences to form a genotypic map of the individual's DNA.

The level of C12 candidate gene expression can also be assayed. For example, RNA from a cell type or tissue known, or suspected, to express the C12 candidate gene, such as brain, may be isolated and tested utilizing hybridization or PCR techniques such as are described, above. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the C12 candidate gene. Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the C12 candidate gene, including activation or inactivation of C12 candidate gene expression.

In one embodiment of such a detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA) . A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the C12 candidate gene nucleic acid reagents described herein. The preferred 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 C12 candidate 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 such as those described herein 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).

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 C12 candidate gene.

Detection of gene products

Antibodies directed against unimpaired or mutant C12 candidate gene products or conserved variants or peptide fragments thereof, which are discussed, above may also be used as diagnostics and prognostics for a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, as described herein. Such methods may be used to detect abnormalities in the level of C12 candidate gene product synthesis or expression, or abnormalities in the structure, temporal expression, and/or physical location of C12 candidate gene product. The antibodies and immunoassay methods described below have, for example, important in vitro applications in assessing the efficacy of treatments for C12 candidate gene disorders or neuropsychiatric disorders, such as bipolar and genetically related unipolar affective disorders. 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 C12 candidate gene expression and C12 candidate gene peptide production. The compounds that have beneficial effects on a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, can be identified, and a therapeutically effective dose determined.

In vitro immunoassays may also be used, for example, to assess the efficacy of cell-based gene therapy for a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. Antibodies directed against C12 candidate gene peptides may be used in vitro to determine, for example, the level of C12 candidate gene expression achieved in cells genetically engineered to produce C12 candidate gene peptides. In the case of intracellular C12 candidate gene products, such an assessment is done, preferably, using cell lysates or extracts. Such analysis will allow 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 C12 candidate 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 C12 candidate gene.

Preferred diagnostic methods for the detection of C12 candidate gene products or conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the C12 candidate gene products or conserved variants or peptide fragments are detected by their interaction with an anti-C12 candidate gene product-specific antibody.

For example, antibodies, or fragments of antibodies, such as those described above, useful in the present invention, may be used to quantitatively or qualitatively detect the presence of C12 candidate gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below, this Section) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred for C12 candidate 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 C12 candidate gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labelled 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 C12 candidate 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 C12 candidate gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells, that have been incubated in cell culture, in the presence of a detectably labeled antibody capable of identifying C12 candidate 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 biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled C12 candidate 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.

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 may have virtually any possible structural 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. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation .

The binding activity of a given lot of anti-C12 candidate 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 C12 candidate gene peptide- specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme im unoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2, 1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller, A. et al., 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, Fla.,; Ishikawa, E. et al., (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 lable the antibody include, but are not limited to, malate 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 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 labelling the antibodies or antibody fragments, it is possible to detect C12 candidate 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, o- phthaldehyde 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 (DTPA) 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 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 systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase, green fluorescent protein And aequorin.

Screening for modulators

The invention further relates to methods for identifying compounds capable of modulate the expression of the mammalian C12 candidate gene and/or the synthesis or activity of the mammalian C12 candidate gene products, wherein such methods comprise contacting a compound to a cell that expresses a C12 candidate gene, measuring the level of C12 candidate gene expression, gene product expression or gene product activity, and comparing this level to the level of C12 candidate gene expression, gene product expression or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the mammalian C12 candidate gene and/or the synthesis or activity of the mammalian C12 candidate gene products has been identified.

The following assays are designed to identify compounds that bind to a C12 candidate gene product, intracellular proteins or portions of proteins that interact with a C12 candidate gene product, compounds that interfere with the interaction of a C12 candidate gene product with intracellular proteins and compounds that modulate the activity of C12 candidate gene (i.e., modulate the level of C12 candidate gene expression and/or modulate the level of C12 candidate gene product activity) . Assays may additionally be utilized that identify compounds that bind to C12 candidate gene regulatory sequences (e.g., promoter sequences; see e.g., Platt, 1994, J. Biol. Che . 269, 28558-28562), and that may modulate the level of C12 candidate 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 C12 candidate gene or some other gene involved in a C12 candidate genes regulatory pathway, or intracellular proteins .

Methods for the identification of such intracellular proteins are described below. Such intracellular proteins may be involved in the control and/or regulation of mood. Further, among these compounds are compounds that affect the level of C12 candidate gene expression and/or C12 candidate gene product activity and that can be used in the therapeutic treatment of C12 candidate genes disorders or neuropsychiatric disorders such as bipolar and genetically related unipolar affective disorders, as described below.

Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to, Ig- tailed fusion peptides, and members of random peptide libraries; (see, e.g., Lam, et al . , 1991, Nature 354, 82-84; Houghten, et al . , 1991, Nature 354, 84-86), and combinatorial chemistry- derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al., 1993, Cell 72, 767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and Fab expression library fragments, and epitope-binding fragments thereof) , and small organic or inorganic molecules.

Such compounds may further comprise compounds, in particular drugs or members of classes or families of drugs, known to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder such as bipolar and genetically related unipolar affective disorders with the use of lithium salts, atypical antipsychotics such as ziprasadone, risperidone, clozapine, quetiapine, olanzapine, butyrophenone derivatives such as haloperidol and droperidol, phenothiazaine derivatives such as chlorpromazine, prochloperazine, promazine, trifluopromazine, thioxanthine derivatives such as flupenthixol, chlorprothixene and dibenzodiazepines and antipsychotic antiepileptic drugs such as carbamazepine, and valproic acid. Antidepressant drugs such imipramine,. amitryptiline, nortryptiline, prothiaden, doxapine, other tricyclic antidepressants, tetracyclic antidepressants, serotonin reuptake inhibitor antidepressants such as fluoxetine, paroxetine, cipromil, venlafaxine, monoamine oxidase inhibitor antidepressants such as phenelzine, tranylcypromine, isocaboxazid, selegiline, and moclobamide. In addition psychotogenic drugs such as bromocriptine, apomorphine, amphetamine, methylphenidate, methylamphetaime, ketamine, may also be used to study C12 candidate gene. Many of these drugs can be or have been used in combination.

Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the C12 candidate gene product, and for ameliorating C12 candidate genes disorders or neuropsychiatric disorders, such as bipolar and genetically related unipolar affective disorders. Assays for testing the effectiveness of compounds, identified by, for example, by techniques described herein are discussed below.

In vitro assays

In vitro systems may be designed to identify compounds capable of binding the C12 candidate gene products of the invention.

Compounds identified may be useful, for example, in modulating the activity of unimpaired and/or mutant C12 candidate gene products, may be useful in elaborating the biological function of the C12 candidate gene product, may be utilized in screens for identifying compounds that disrupt normal C12 candidate gene product interactions, or may in themselves disrupt such interactions .

The principle of the assays used to identify compounds that bind to the C12 candidate gene product involves preparing a reaction mixture of the C12 candidate 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 C12 candidate gene product or the test substance onto a solid phase and detecting C12 candidate gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the C12 candidate 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 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, 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 non-immobilized component is not pre- labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labelled 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 products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for C12 candidate 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 .

Assays for intracellular binding partners

Any method suitable for detecting protein-protein interactions may be employed for identifying C12 candidate genes protein- protein interactions.

Among the traditional methods that may be employed are co- immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns. Utilizing procedures such as these allows for the identification of proteins, including intracellular proteins, that interact with C12 candidate gene products. Once isolated, such a protein can be identified and can be used in conjunction with standard techniques, to identify proteins it interacts with. For example, at least a portion of the amino acid sequence of a protein that interacts with the C12 candidate gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles," W.H. Freeman & Co., N.Y., pp.34-49) . The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such proteins. Screening made be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra, and 1990, "PCR Protocols: A Guide to Methods and Applications," Innis, et al., eds . Academic Press, Inc. , New York) . Additionally, methods may be employed that result in the simultaneous identification of genes that encode a protein which interacts with a C12 candidate genes protein. These methods include, for example, probing expression libraries with labeled C12 candidate genes protein, using C12 candidate genes protein in a manner similar to the well known technique of antibody probing of lambda-gtll and lambda-gtlO 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 system has been described (Chien, et al . , 1991, Proc. Natl. Acad. Sci. USA, 88, 9578-9582) and is commercially available from Clontech (Palo Alto, Calif. ) .

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the C12 candidate gene product and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into this plasmid as part of a cDNA library. The DNA- binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot 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.

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, C12 candidate 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 C12 candidate gene product fused to the DNA-binding domain are co-transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait C12 candidate gene sequence, such as the open reading frame of the C12 candidate 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 bait C12 candidate gene product are to be detected 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 GAL4. This library can be co-transformed along with the bait C12 candidate gene-GAL4 fusion plasmid into a yeast strain that contains a lacZ gene driven by a promoter that contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait C12 candidate 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 C12 candidate gene-interacting protein using techniques routinely practiced in the art. Assays for inhibitors of interactions

C12 candidate gene products of the invention may, in vivo, interact with one or more macromolecules, including intracellular macromolecules, such as proteins. Such macromolecules may include, but are not limited to, nucleic acid molecules and those proteins identified via methods such as those described above. For purposes of this discussion, the macromolecules are referred to herein as "binding partners". Compounds that disrupt C12 candidate gene binding in this way may be useful in regulating the activity of the C12 candidate gene product, especially mutant C12 candidate gene products. Such compounds may include, but are not limited to molecules such as peptides, and the like, as described, for example above, which would be capable of gaining access to an C12 candidate gene product.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the C12 candidate gene product and its binding partner or partners involves preparing a reaction mixture containing the C12 candidate 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 C12 candidate 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 C12 candidate 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 C12 candidate gene protein and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal C12 candidate gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant C12 candidate 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 C12 candidate gene proteins.

The assay for compounds that interfere with the interaction of the C12 candidate gene products and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the C12 candidate 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 C12 candidate gene products and the binding partners, e.g., by 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 C12 candidate gene protein and interactive intracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the C12 candidate 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, microtitre 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 C12 candidate gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. 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-labelled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) . Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

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 disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the C12 candidate gene protein and the interactive binding partner is prepared in which either the C12 candidate 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. Pat. No.4,109,496 by Rubenstein which utilizes this approach for immunoassays) . The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt C12 candidate gene protein/binding partner interaction can be identified.

In a particular embodiment, the C12 candidate gene product can be prepared for immobilization using recombinant DNA techniques described above. For example, the C12 candidate genes 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.

This antibody can be labeled with the radioactive isotope ¹²⁵I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-C12 candidate genes 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 C12 candidate 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-C12 candidate gene fusion protein and the interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the C12 candidate 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 correspond to the binding domains of the C12 candidate genes 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 methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described 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, labelled 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, a C12 candidate gene product can be anchored to a solid material as described, above, in this Section by making a GST-C12 candidate genes 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-C12 candidate genes fusion protein and allowed to bind. After washing away unbound peptides, labelled bound material, representing the binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well- known methods . Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology.

Assays for compounds tha t ameliora te disorders

Compounds, including but not limited to binding compounds identified via assay techniques such as those described above can be tested for the ability to ameliorate symptoms of a C12 candidate genes disorder or a disorder of thought and/or mood, including thought disorder and neuropsychiatric disorders.

It should be noted that the assays described herein can identify compounds that affect C12 candidate gene activity by either affecting C12 candidate gene expression or by affecting the level of C12 candidate gene product activity. For example, compounds may be identified that are involved in another step in the pathway in which the C12 candidate gene and/or C12 candidate gene product is involved and, by affecting this same pathway may modulate the effect of C12 candidate genes on the development of a neuropsychiatric disorder such as bipolar and genetically related unipolar affective disorders. Such compounds can be used as 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 a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. First, cell-based systems can be used to identify compounds that may act to ameliorate symptoms of a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. Such cell systems can include, for example, recombinant or non-recombinant cell, such as cell lines, that express the C12 candidate gene.

In utilizing such cell systems, cells that express C12 candidate genes may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, 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 C12 candidate gene, e.g., by assaying cell lysates for C12 candidate genes mRNA transcripts (e.g., by Northern analysis) or for C12 candidate gene products expressed by the cell; compounds that modulate expression of the C12 candidate genes are good candidates as therapeutics. Alternatively, the cells are examined to determine whether one or more cellular phenotypes associated with an C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, 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.

In addition, animal-based systems or models for a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, which may include, for example, C12 candidate genes mice, may be used to identify compounds capable of ameliorating symptoms of the disorder. Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions that may be effective in treating such disorders. 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 C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, in the exposed animals. The response of the animals to the exposure may be monitored by assessing the reversal of such symptoms.

With regard to intervention, any treatments that reverse any aspect of symptoms of a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, 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 below.

Compounds and methods for the treaments of disorders

Described below are methods and compositions whereby a C12 candidate gene disorder or a disorder of thought and/or mood, such as bipolar and genetically related unipolar affective disorders, may be treated.

For example, such methods can comprise administering compounds which modulate the expression of a mammalian C12 candidate gene and/or the synthesis or activity of a mammalian C12 candidate gene product so symptoms of the disorder are ameliorated. Alternatively, in those instances whereby the mammalian C12 candidate gene or neuropsychiatric disorders result from C12 candidate gene mutations, such methods can comprise supplying the mammal with a nucleic acid molecule encoding an unimpaired C12 candidate gene product such that an unimpaired C12 candidate gene product is expressed and symptoms of the disorder are ameliorated.

In another embodiment of methods for the treatment of mammalian C12 candidate genes or neuropsychiatric disorders resulting from C12 candidate gene mutations, such methods can comprise supplying the mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired C12 candidate gene product such that the cell expresses the unimpaired C12 candidate gene product and symptoms of the disorder are ameliorated.

In cases in which a loss of normal C12 candidate gene product function results in the development of a C12 candidate genes disorder or neuropsychiatric disorder phenotype, an increase in C12 candidate gene product activity would facilitate progress towards an asymptomatic state in individuals exhibiting a deficient level of C12 candidate gene expression and/or C12 candidate gene product activity. Methods for enhancing the expression or synthesis of C12 candidate genes can include, for example, methods such as those described below.

Alternatively, symptoms of C12 candidate genes disorders or neuropsychiatric disorders, such as bipolar and genetically related unipolar affective disorders, may be ameliorated by administering a compound that decreases the level of C12 candidate gene expression and/or C12 candidate gene product activity. Methods for inhibiting or reducing the level of C12 candidate gene synthesis or expression can include, for example, methods such as those described below.

In one embodiment of treatment methods, the compounds administered do not comprise compounds, in particular drugs, reported to ameliorate or exacerbate the symptoms of a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. Such compounds include antidepressants such as lithium salts, flupenthixol, risperidone, clozapine, quetiapine, olanzapine, haloperidol, droperidol, chlorpromazine, prochloperazine, phenothiazaine derivatives, promazine, trifluopromazine, butyrophenone derivatives, thioxanthine derivatives such as chlorprothixene and dibenzodiazepines and antipsychotic antiepileptic drugs such carbamazepine, and valproic acid, reserpine.

Psychotogenic drugs such as LSD, bromocriptine, apomorphine, amphetamine, methylphenidate, methylamphetaime, ketamine, Many of these drugs are used in combination.

Antisense, ribozyme and triple helix embodiments

In another embodiment, symptoms of certain C12 candidate gene disorders or neuropsychiatric disorders, such as bipolar and genetically related unipolar affective disorders may be ameliorated by decreasing the level of C12 candidate gene expression and/or C12 candidate gene product activity by using C12 candidate gene sequences in conjunction with well-known antisense, gene "knock-out," ribozyme and/or triple helix methods to decrease the level of C12 candidate gene expression. Among the compounds that may exhibit the ability to modulate the activity, expression or synthesis of the C12 candidate gene, including the ability to ameliorate the symptoms of a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders, 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 well known to those of skill in the art.

Antisense RNA, short interfering RNA (siRNAs)and DNA molecules act to directly block the translation of mRNA by hybridizing to targetted mRNA and preventing protein translation. Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.

A sequence "complementary" to a portion of an RNA, as referred 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 non-coding regions of the C12 candidate gene could be used in an antisense approach to inhibit translation of endogenous C12 candidate genes 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. 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 preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., 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. Sci. 84, 648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 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, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5 ' -methoxycarboxymethyluracil, 5- methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- oxyacetic acid (v) , wybutoxosine, pseudouracil, queosine, 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, xylulose, and hexose.

In 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.

In yet another embodiment, the antisense oligonucleotide is an α- anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual .beta . -units, the strands run parallel to each other (Gautier, et al., 1987, Nucl . Acids Res. 15, 6625- 6641). The oligonucleotide is a 2 ' -O-methylribonucleotide (Inoue, et al., 1987, Nucl. Acids Res. 15, 6131-6148), or a chimeric RNA- DNA analogue (Inoue, et al., 1987, FEBS Lett. 215, 327-330).

Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl. Acids Res. 16, 3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al . , 1988, Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451), etc.

While antisense nucleotides complementary to the target gene coding region sequence could be used, those complementary to the transcribed, untranslated region are most preferred. For example, antisense oligonucleotides having the following sequences can be utilized in accordance with the invention: Antisense molecules should be delivered to cells that express the target gene in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically .

However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation 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 integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290, 304- 310) , the promoter contained in the 31 long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22, 787-797), the herpes thymidine kinase promoter (Wagner, et al . , 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of the metallothionein gene (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 construct 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 International Publication WO90/11364, published Oct. 4, 1990; Sarver, et al., 1990, Science 247, 1222- 1225) .

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4, 469-471) . The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme 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. Pat. No. 5,093,246, which is incorporated herein by reference in its entirety.

While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially Figure. 4, page 833) and in Haseloff and Gerlach, 1988, Nature, 334, 585-591, which is incorporated herein by reference in its entirety.

Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts. For example, hammerhead ribozymes having the following sequences can be utilized. The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described 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 place.

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 preferred method of delivery involves using a DNA construct "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 (e.g., see Smithies, et al., 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al . , 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety) . 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, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. 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 constructs 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 (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells in the body. (See generally, Helene, 1991, Anticancer Drug Des . , 6(6), 569-584; Helene, et al., 1992, Ann. N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12), 807-815).

Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides . The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC⁺ triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. 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 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 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 that do not contain sequences susceptible to whatever antisense, 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 siRNAs, DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. 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 incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Gene replacement therapy

With respect to an increase in the level of normal C12 candidate gene expression and/or C12 candidate gene product activity, C12 candidate gene nucleic acid sequences described above can, for example, be utilized for the treatment of a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. Such treatment can be administered, for example, in the form of gene replacement therapy. Specifically, one or more copies of a normal C12 candidate gene or a portion of the C12 candidate gene that directs the production of a C12 candidate gene product exhibiting normal C12 candidate gene function, may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.

Because the C12 candidate gene is expressed in the brain, such gene replacement therapy techniques should be capable delivering C12 candidate 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 Apr. 25, 1988) can be used to enable C12 candidate 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 barrier, viral vectors such as, for example, those described above, are preferable. Also included are methods using liposomes either in vivo ex vivo or in vitro. Wherein C12 candidate gene sense or antisense DNA is delivered to the cytoplasm and nucleus of target cells. Liposomes can deliver C12 candidate gene sense or nonsense RNA to humans and the human brain or in mammals through intrathecal delivery either as part of a viral vector or as DN conjugated with nuclear localizing proteins or other proteins that increase take up into the cell nucleus.

In another embodiment, techniques for delivery involve direct administration of such C12 candidate gene sequences to the site of the cells in which the C12 candidate gene sequences are to be expressed. Additional methods that may be utilized to increase the overall level of C12 candidate gene expression and/or C12 candidate gene product activity include the introduction of appropriate C12 candidate gene-expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to ameliorate the symptoms of a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. Such cells may be either recombinant or non-recombinant .

Among the cells that can be administered to increase the overall level of C12 candidate gene expression in a patient are normal cells, preferably brain cells and also choroid plexus cells within the CNS which are accessible through intrathecal injections. Alternatively, cells, preferably autologous cells, can be engineered to express C12 candidate gene sequences, and may then be introduced into a patient in positions appropriate for the amelioration of the symptoms of a C12 candidate gene disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. Alternately, cells that express an unimpaired C12 candidate gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells. The expression of the C12 candidate gene sequences is controlled by the appropriate gene 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. Pat. No. 5,399,349.

When the cells to be administered are non-autologous cells, they can be administered using well known techniques that prevent a host immune response against the introduced cells from developing. 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, that are capable of modulating C12 candidate gene product 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 such as intrathecal injection and conjugation with compounds that allow transfer across the blood brain barrier.

Pharmaceuticals

The compounds that are determined to affect C12 candidate gene expression or gene product activity can be administered to a patient at therapeutically effective doses to treat or ameliorate a C12 candidate genes disorder or a neuropsychiatric disorder, such as bipolar and genetically related unipolar affective disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of such a disorder.

Effective dose

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the 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 preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects .

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

Formula tions and use Pharmaceutical compositions for use in accordance with the present 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 intrathecal, 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, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate) ; or wetting agents (e.g., sodium lauryl sulphate). 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 be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats) ; emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid) . The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

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 compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or- emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. 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 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 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 dispenser device may be accompanied by instructions for administration.

Drug trea tment

The invention also provides a method of predicting a patient's likelihood to respond to drug treatment for bipolar and genetically related unipolar affective disorders and related psychiatric disorders comprising determining a patient's genotype in the C12 candidate genes region and in flanking regions on the long arm of chromosome 12q24.3 as an indication of an increased likelihood that a drug treatment for bipolar and genetically related unipolar affective disorders will be effective.

Trea tmen t response

The invention also provides a method for predicting treatment response of bipolar and genetically related unipolar affective disorders patients using genotype data i.e. the patient's genotype in the C12 candidate genes region is determined and compared with previously determined genotypes and mutations found in other individuals previously diagnosed with bipolar and genetically related unipolar affective disorders.

Once an individual is genotyped as having a bipolar and genetically related unipolar affective disorder susceptibility gene in the 12q24.3 region, the individual is treated with any of the known methods effective in treating at least certain individuals affected with bipolar and genetically related unipolar affective disorders.

These known methods include the administration of drugs or modulators such as those described above.

Studies can be conducted correlating effective treatments with bipolar and genetically related unipolar affective disorder genotypes and mutations in the C12 candidate genes region and flanking regions on 12q24.3 to determine the most effective treatments for particular genotypes. Bipolar and genetically related unipolar affective disorder patients can then be genotyped in the C12 candidate genes region and flanking regions and the statistically most effective treatment can be determined as a first course of therapy.

Imaging

The invention also provides a method for for predicting brain imaging and other neurophysiological changes in bipolar and genetically related unipolar affective disorders and genetically related disorders, which method comprisses use of DNA markers and mutations defined in the C12 candidate genes region and flanking regions, genotyped in individuals with or without bipolar and genetically related unipolar affective disorders so that magnetic resonance imaging, functional magnetic resonance imaging, single photon emission tomography, labelled deoxyglucose or labeled oxygen positron emission tomography and evoked EEG responses such as the P300 or P50 responses can be predicted for the purposes of diagnosing, predicting or designing new therapeutic agents for the treatment of bipolar and genetically related unipolar affective disorders and other related psychiatric disorders.

The invention will now be further described with reference to the following non-limiting Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these .

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross-reference. EXAMPLES

PATIENTS AND METHODS

The UK sample consisted of 301 bipolar individuals with 315 screened normal controls. The cases and controls were all asked if both their parents and grandparents were of Irish, Welsh, Scottish or English ancestry. Those subjects with two or more grandparents who had any other ancestry were excluded but subjects with a single grandparent with a European ancestry were included. After complete description of the study to the subjects, written informed consent was obtained. All subjects were interviewed by a psychiatrist using the Lifetime Version of the Schizophrenia and Affective Disorders Schedule (SADS-L) (Spitzer et al. 1978). The cases were also rated using the 90 item OPCRIT checklist, (McGuffin et al . 1991) the family history was recorded and pedigree diagram was drawn. Diagnoses were assigned using Research Diagnostic Criteria (Spitzer and Endicott 1977) . The Danish sample consisted of 81 bipolar patients and 120 psychiatrically unscreened controls. After complete description of the study to the subjects, written informed consent was obtained. All cases met the RDC and ICD-10 diagnostic criteria for bipolar disorder (1993) . Using the 90 item OPCRIT checklist (McGuffin et al. 1991) 80 patients fulfilled RDC or DSM-III-R criteria for bipolar disorder. Only patients with an age of onset below 35 years were included. Patients were interviewed using the semi-structured interview SCAN version 2.1 (1996). Two senior psychiatrists made consensus best estimate of lifetime diagnosis . For the UK sample genomic DNA was extracted from lymphocytes by SDS lysis, proteinase K digestion and phenol/chloroform extraction. For the Danish sample genomic DNA was prepared from venous blood using standard sucrose/triton-lysis protocol with sodium chloride/isopropanol precipitation. Fifteen Marker loci were initially selected from the Marshfield centre for medical genetics database http: //research.marshfieldclinic. org/genetics/ and from the Genome database http://gdbwww.gdb.org/. In addition at the region where fine mapping was to be performed, publicly available genomic sequence from this region was examined for potential microsatellite repeat structures. These sequences were amplified in a representative number of samples and the products were investigated for potential polymorphic alleles. Seven novel microsatellites were identified using this approach. All genotyping was carried out by polymerase chain reaction (PCR) amplification of microsatellites using standard methods. In both centres amplification was performed with one of the PCR primers being directly labelled with a fluorescent probe. In Denmark the samples were then automatically loaded and electrophoresed using the POP 4 protocol for ABI Prism 310 Genetic Analyser. GeneScan and GenoTyper programs were used to perform analysis of the allele sizes and allele scoring (Applied Biosystems, Foster City, CA, USA) . In the UK the amplified samples were loaded manually and electrophoresed on a Li-Cor DNA4200 sequencer (Li-Cor, Nebraska, USA) . The GenelmaglR (Li-Cor, Nebraska, USA) program was used to perform analysis of the allele sizes and for allele scoring. In both centres two researchers scored the genotypes independently.

Mutation screening of predicted exons and their flanking intronic sequence was performed using the Li-Cor infrared fluorescent sequencing system. The method involved direct sequencing of PCR products amplified from genomic DNA. M13 tagged PCR primers that amplified the entire exon and parts of the surrounding introns were designed. Amplification products were purified with Microclean reagent according to manufacturer' s instructions (Microzone, UK) . PCR products were then sequenced simultaneously in both directions with the SequiTherm Excel II DNA sequencing kit (Epicentre, USA) using two M13 primers fluorescently labelled with IRD700 and IRD800 (MWG Biotech, DE) . Sequencing products were analysed on a Li-Cor 4200 automated sequencer (Li-Cor, US) . The samples were arranged such that the guanosine dideoxy nucleotide terminated tracks from each of the samples were loaded together followed by the adenosine, thymidine and cytidine. This facilitated visual scoring of potential mutations and allowed recognition of polymorphisms in their heterozygote state. The validity of potential mutations was assessed by comparison with the gel image produced from simultaneous sequencing reactions primed from the opposite strand. SNP typing was performed using either the Taqman assay or Pyrosequnecing according to manufacturer's instructions.

STATISTICAL ANALYSIS

Allelic association between BPAD and each marker was tested using the CLUMP program (Sham and Curtis 1995) . This performs a Monte Carlo evaluation of significance levels to produce an empirical p value for the observed chi-squared statistic, since the asymptotic p value may be inaccurate for markers having large numbers of alleles. The chi-squared statistic is calculated in the usual fashion, then random configurations of alleles are generated, constrained to produce the same marginal totals. The proportion of occasions on which the chi-squared statistic generated from these random configurations is as high as the observed value provides an empirical p value for the test. The test therefore is very conservative and means that our results are very unlikely to have occurred by chance. In order to investigate whether linkage disequilibrium was present in this region in our sample, we carried out linkage disequilibrium analyses between each pair of markers using the EH program (Xie and Ott 1993) . Data from the seven SNPs and D12s307 was analysed using multilocus haplotype analysis. Analysis was done using Genecounting (Zhao et al . , 2002) which is a program that is designed to estimate haplotypes and their frequencies from case control data where there is some missing data.

RESULTS

The following published markers were genotyped; D12S1614, D12S342, AfmB332ZCl, D12S340, D12S1639, D12S324, D12S866,

D12S386, AfmB337ZD5, D12S1634, D12S307, D12S1658, GATA41E12, D12S2075 and D12S1675. In addition we identified and genotyped the following novel microsatellite markers; 307CA2, 1634GT2, 1634TET, 307GT4, 307CA1, D12SDK1 and D12SDK2. Primer sequences that were used to amplify the novel microsatellites are shown in table 1. The number of alleles observed for each marker along with the number of individuals typed for each marker is shown in table 2. Results from Clump analysis for each of the markers is shown in table 3. Clump analysis was performed on the two populations separately and then analysis of combined Danish and UK samples was performed. When the Danish samples were investigated on their own, three microsatellite markers produced p-values that were nominally significant; these were D12S1634 (T3 p = 0.016), 307CA1 (T3 p = 0.0429), D12S2075 (T3 p = 0.024). None of these markers produced significant p-values with a standard 2 x N contingency table. When the UK sample was investigated on its own, five microsatellite markers produced p-values that achieved corrected significance. These five markers all produced significant p- values with conventional 2 x N contingency tables, these markers were; 307CA2 (TI p = 0.0015), 1634TET (Tl p = 0.002), 307GT4 (Tl p •= 0.048), D12S307 (Tl p = 0.0041) and D12SDK1 (Tl p = 0.0019). The most significant p-values obtained using Clump on the UK sample alone were; 307CA2 (T4 p = 0.0007), 1634TET (T4 p = 0.0009), 307GT4 (Tl p = 0.048), D12S307 (T4 p = 0.003) and D12SDK1 (T2 p = 0.0004). When allele counts for the Danish and UK samples were combined six markers produced p-values that achieved significance. Five of these markers produced significant p-values with conventional 2 x N contingency tables, these markers were; 1634GT2 (Tl p = 0.0102), 1634TET (Tl p = 0.0075), 307GT4 (Tl p = 0.0325), D12S307 (Tl p = 0.038) and D12SDK1 (Tl p = 0.0019). The most significant p-values obtained using Clump on the combined Danish and UK samples were; D12S340 (T3 p = 0.042), 1634GT2 (T2 p = 0.0098), 1634TET (T2 p = 0.0036), 307GT4 (T3 p = 0.0209), D12S307 (T3 p = 0.001) and D12SDK1 (T4 p = 0.003) . The number of alleles observed for each marker and the number of individuals successfully typed for each marker is shown in table 2. Data from the SNPs analysed to date are shown in table 4. Two SNPs produced nominally significant results these were GIN (p = 0.0029) and G3 exon M insertion T (insT) with significance of p== 0.0288. Eight marker multilocus haplotype analysis produced a likelihood heterogeneity ratio test (LRT) statistic of 111.819824 which equates to a p-value of between 0.000000 and 0.014031 depending on the degrees of freedom that are applied (45 or 81) . Four haplotypes whose estimated allele frequencies appear to contribute most to the LRT score are shown in table 7. Three SNP haplotype analysis with SNPs GIN, G2M and G3 exon MinsT showing most association with bipolar disorder produced an LRT score of 37.560059 (7d.f.) which equates to a significance (p) of 0.000004.

DISCUSSION

The results presented here provide further evidence for a bipolar affective disorder locus on 12q24.3, furthermore the data refines the region to an interval of approximately 1Mb. Inter marker distances are shown in table 3. The region spanned by markers showing positive association spans 278 Kb and this implicates a region of approximately 500 Kb either side. The highly significant p-values observed with markers 307CA2, 1634TET, D12S307 and D12SDK1, in the UK sample and the fact that the addition of the data from the Danish samples maintained the significant results, albeit at a lower level of significance, indicates that the locus that we have detected here is likely to be present in a number of different populations. The significant results obtained from both samples means that it is also likely that any bipolar affective disorder susceptibility mutation is also likely to be common to more than just the Danish and UK samples. Results from the SNP-microsatellite haplotype analysis give additional evidence for a susceptibility gene on 12q24.3 than the single marker allelic associations.

The region implicated in this study contains the expressed genes listed in table 5. One of these potential genes (G2) bears homology to a macaque brain expressed gene. We have investigated the G2 and PUFU expressed sequences in terms of their expression in the human brain. Both are expressed in human brain. The G2 sequence was used to create a polyclonal antibody which was then applied to human brain and labeled immunochemically. Regions in the brain where G2 was expressed include the striatum and greater amygdale regions thus providing the first evidence for functional and anatomical localisation of a candidate gene region expressed sequence being expressed in part of the human brain known to control mood and emotions. Other ESTs in the candidate genes regions may be localized to brain specific regions. It is likely that the genes in the C12 candidate genes region may show a high degree of alternative splicing as we have found for the G2 and exons of the calsequestrin homologue.

Table 1 Novel genetic markers at cl2q24.3 generated for the fine mapping of bipolar and unipolar affective disorders

Table 1 Shows the names of newly characterised polymorphic markers together with the type of repeat, the size range of the alleles (in base pairs bp) and the oligonucleotide sequences that were used to amplify each of the markers. Table 2

Genetic markers and number of individuals genotyped in order to fine map the chromosome 12q24.3 candidate genes region.

Table 3

Microsatellite evidence for fine mapping the chromosome 12 bipolar and related unipolar affective disorder susceptibility locus to genes in the 12q24.3 region.

Table 4

SNP evidence for fine mapping the chromosome 12 bipolar and related unipolar affective disorder susceptibility locus to genes in the 12q24.3 region.

Table 5

Chromosome 12 candidate genes and expressed sequences as defined m the Ensembl database at http://www.ensembl.org located in the 12q24 region defined by allelic association and linkage disequilibrium studies.

TABLE 6

_Single Nucleotide Polymorphisms (SNPs), Mutant Base Pair Changes and Insertion/Deletions found in the exonic, intronic and locus control regions of the sequences defined in table 5.

Table 7

Predicted eight marker haplotypes derived from the analysis of data from seven SNPs (listed in the order they appear in Table 4, from top to bottom) and the microsatellite D12S307 showing marked changes in frequencies between the cases and the controls.

* Haplotypes associated with bipolar isorder increasing susceptibility (A, B, C)

# Haplotype associated with lower risk of bipolar disorder (D)

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Claims

1 A method for determining the susceptibility of an individual to a neuropsychiatric disorder, or a method of diagnosis, detection or prognosis of the neuropsychiatric disorder, the method comprising use of a C12 candidate gene region marker.

2 A method as claimed in claim 1 wherein the disorder is bipolar or a genetically related unipolar affective disorder

3 A method as claimed in claim 1 or claim 2 wherein the method comprises :

(i) obtaining a nucleic acid or protein sample from the individual; (ii) determining the structure, level of expression, and\or activity of a polypeptide encoded by a C12 candidate gene.

4 A method as claimed in claim 1 or claim 2 wherein the method comprises : (i) obtaining a sample of nucleic acid from the individual, and

(ii) determining in that sample, the presence or absence of a C12 candidate gene region marker.

5 A method as claimed in claim 4 wherein the nucleic acid is RNA, cDNA or genomic DNA.

6 A method as claimed in claim 4 or claim 5 wherein the marker is present in the region defined as around 1 megabase either side of D12S307 in Table 3.

7 A method as claimed in claim 6 wherein the marker is present in the region defined from 1634TET to D12SDK1 in Table 3.

8 A method as claimed in any one of claims 4 to 7 wherein the C12 candidate gene region marker is selected from the group consisting of any shown in Tables 3, 4 or 6, or a marker which is in linkage disequilibrium with any of these. 9 A method as claimed in claim 8 wherein the C12 candidate gene region marker is an SNP selected from the group consisting of those in Table 4 or Table 6 or a polymorphic marker which is in linkage disequilibrium with any of these.

10 A method as claimed in claim 8 wherein the C12 candidate gene region marker is selected from the group consisting of

D12S1634 (see Table 3) 307CA2 (see Table 3)

1634TET(see Table 3)

307GT4(see Table 3)

D12S307 (see Table 3)

D12SDK1 (see Table 3) 1634GT2(see Table 3)

D12S340 (see Table 3)

SNP GIN (see Table 4)

SNP G2M (see Table 4)

SNP G3 exon insertion T (insT) (see Table 4), or a marker which is in linkage disequilibrium with any of these.

11 A method as claimed in any one of claims 4 to 10 wherein the C12 candidate gene region marker is within a C12 candidate gene.

12 A method as claimed in claim 11 wherein the C12 candidate gene is selected from the genes termed Gl, G2, G3, G4, or Calsequestrin homologue of Table 5.

13 A method as claimed in any one of claims 4 to 12 wherein two or more of said C12 candidate gene region markers are assessed.

14 A method as claimed in any one of claims 4 to 13 wherein a haplotype is defined.

15 A method as claimed in any of claims 4 to 14 wherein the C12 candidate gene region in assessed by determining the binding of an oligonucleotide probe to the nucleic acid sample under conditions favourable for the specific annealing of these reagents to their complementary sequences .

16 A method as claimed in claim 15 wherein the probe comprises all or part of (i) a C12 candidate gene sequence, or (ii) a polymorphic form of said C12 candidate gene, or (iii) the complement of either.

17 A method as claimed in claim 15 or claim 16 wherein the probe comprises a nucleic acid sequence which binds under stringent conditions specifically to one particular allele of a C12 candidate gene region marker and does not bind specifically to other alleles of the C12 candidate gene region marker.

18 A method as claimed in any one of claims 15 to 17 wherein the probe is labelled and binding of the probe is determined by presence of the label.

19 A method as claimed in any of claims 4 to 14 wherein the method comprises amplifying a region of the C12 candidate gene region comprising at least one C12 candidate gene region marker, optionally using a primer shown in Table 1.

20 A method as claimed in claim 19 wherein all or part of a C12 candidate gene is amplified.

21 A method as claimed in claim 20 wherein the region of the C12 candidate gene which is amplified is all or part of an exon of Gl, G2 or G3.

22 A method as claimed in any one of claims 4 to 14 wherein the C12 candidate gene region marker is assessed by a method selected from the group consisting of: strand conformation polymorphic marker analysis; heteroduplex analysis; RFLP analysis; the detection of either over- or under-expression of C12 candidate gene mRNA relative to the unaffected state; the detection of either an over- or an under-abundance of C12 candidate gene product relative to the unaffected state; the detection of an aberrant level of C12 candidate gene product activity relative to the unaffected state.

23 A method as claimed in claim 3 wherein the level of mRNA expression of the 012 candidate gene is determined by Northern analysis or reverse transcriptase PCR.

24 A method of determining the presence or absence in a test sample of a 012 candidate gene region marker of any one of claims 6 to 12 which method comprises determining the binding of an oligonucleotide probe to the nucleic acid sample, wherein the probe comprises all or part of (i) a 012 candidate gene sequence, or (ii) a polymorphic form of said 012 candidate gene, or (iii) the complement of either.

25 A method of determining the presence or absence in a test sample of a 012 candidate gene region marker of any one of claims 6 to 12 which method comprises use of two oligonucleotide primers capable of amplifying a portion of the 012 candidate gene region sequence which portion comprises at least one of said marker.

26 A method as claimed in any one of claims 4 to 24 wherein the 012 candidate gene region marker is assessed or confirmed by nucleotide sequencing.

27 An oligonucleotide probe for use in a method of any one of claims 15 to 18 or claim 24, which is specific for a 012 candidate gene region marker which is a microsatellite repeat of Table 2, or for an SNP selected from Table 6.

28 A PCR primer pair for use in a method of claim 19 or claim

25 which primer pair comprises first and second primers which hybridise to DNA in regions including or flanking the 012 candidate gene region marker.

29 A kit for determining the susceptibility of an individual to bipolar or a genetically related unipolar affective disorder, or a method of diagnosis or prognosis of bipolar or a genetically related unipolar affective disorder, the kit comprising a probe and\or primer of claim 27 or claim 28.

30 A method of bipolar or a genetically related unipolar affective disorder therapy, which method including the step of screening an individual for bipolar or a genetically related unipolar affective disorder in accordance with the method of any one of claims 1 to 23, whereby the predisposition is correlated with a 012 candidate gene region marker, and if a predisposition is identified, providing therapeutic treatment for the individual .

31 A method of predicting the susceptibility to bipolar or a genetically related unipolar affective disorder in an unaffected individual and relatives of affected individuals by (i) performing a method as claimed in any one of claims 1 to 23 on said individual and relatives,

(ii) correlating the results with a phenotypic diagnosis of bipolar and genetically related unipolar affective disorders for any given individual and for family members

32 A method for identifying or isolating genetic loci associated with susceptibility to bipolar or a genetically related unipolar affective disorder comprising screening genomic libraries with a 012 candidate gene region markers and identifying open reading frames in regions adjacent to said markers .

33 A method for mapping polymorphic markers which are associated with susceptibility to bipolar or a genetically related unipolar affective disorder, the method comprising identifying polymorphic markers which are in linkage disequilibrium with a 012 candidate gene region marker shown in Tables 3, 4 or 6.

34 A method of identifying a molecule for use in the diagnosis, prognosis or treatment of bipolar or a genetically related unipolar affective disorder, which method comprises contacting a test substance with a cell that expresses a 012 candidate gene, measuring the level of 012 candidate gene expression, gene product expression or gene product activity, and comparing this level to the level of 012 candidate gene expression, gene product expression or gene product activity produced by the cell in the absence of the test substance.

35 A method of identifying a molecule for use in the diagnosis, prognosis or treatment of bipolar or a genetically related unipolar affective disorder, which method comprises: admixing a test substance with a polypeptide encoded by a nucleic acid molecule comprising a 012 candidate gene and measuring the level of activity of the polypeptide.

36 A method of identifying a molecule for use in the diagnosis, prognosis or treatment of bipolar or a genetically related unipolar affective disorder, which method comprises: admixing a test substance with a polypeptide encoded by a nucleic acid molecule comprising a 012 candidate gene and determining the binding of the test substance to the polypeptide.

37 An antibody specific for a polypeptide encoded by a 012 candidate gene for use as a diagnostic and prognostic for bipolar or a genetically related unipolar affective disorder.

38 A transgenic animal in which a heterologous 012 candidate gene is expressed, for use in the identification of a molecule for use in the diagnosis, prognosis or treatment of bipolar or a genetically related unipolar affective disorder.

39 A nucleic acid molecule comprising a C12 candidate gene, or a DNA sequence that hybridizes to the complement of the DNA sequence of a 012 candidate gene, under highly stringent conditions for use in the diagnosis, prognosis or treatment of bipolar or a genetically related unipolar affective disorder.

40 A polypeptide which is a 012 candidate gene product, or a polypeptide which is functionally equivalent to a 012 candidate gene product, for use in the treatment of bipolar or a genetically related unipolar affective disorder.

41 A method of treatment of bipolar or a genetically related unipolar affective disorder comprising administering to a patient a substance which modulates expression from a C12 candidate gene, or administering a compound which modulates the level of activity of a 012 candidate gene product.

42 A method as claimed in claim 41 wherein the compound is a nucleic acid molecule comprising a 012 candidate gene, or a DNA sequence that hybridizes to the complement of the DNA sequence of a 012 candidate gene.