CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority of U.S. provisional patent application No. 61/088,180 filed August 12, 2008, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with U.S. government support under grant number POl NS026630-19 awarded by the National Institutes of Health. The U.S. government may have certain rights in the invention.

FIELD OF THE INVENTION

[0003] Embodiments of the invention relate to risk assessment for diseases and disorders associated with obsessive compulsive disorder. In particular, identification of variants in SAPAP3 provide for risk assessment, new targets for drug therapies and identification of new drugs for therapies.

BACKGROUND

[0004] Obsessive-compulsive disorder (OCD) and the spectrum of associated conditions, such as trichotillomania (TTM), Tourette syndrome and body dysmorphic disorder, affect about 2-4% of the world population (Karno M, et al. Arch Gen Psychiatry 1988; 45: 1094- 1099; Torres AR, et al. Am J Psychiatry 2006; 163: 1978-1985). Clinically OCD spectrum disorders are characterized by persistent intrusive thoughts (obsessions), repetitive actions (compulsions) and excessive anxiety. Although heritability studies in OCD have shown a 3- 12 times increased risk for first degree relatives and twin studies revealed higher concordance amongst monozygotic twins (80-90%) compared to dizygotic twins (47-50%), the identification of the underlying risk-conferring genetic variation by means of classic genetic association studies has proven to be difficult (Hemmings SM, Stein DJ. Psychiatr Clin North Am 2006; 29: 411-444). SUMMARY

[0005] Novel nonsynonymous heterozygous variants were detected. Heterozygous SAPAP3 variants were present in 4.2% of diagnosed TTM/OCD patients, but only in 1.1% of controls. The majority of changes presented missense mutations; one variant was an in-frame insertion of five amino acids, A148insGPAGA. In silico analysis of the missense variants applying PMut and PolyPhen predicted two, or three, respectively, variants as of functional relevance. The remaining polymorphisms were considered benign, including the two changes detected in controls. The combined analyses of 2766 alleles showed that all changes were rare, with minor allele frequencies between 0.00036 (T523K, K910R) and 0.002 (Al 89V).

[0006] Available pedigrees from TTM/OCD mutation carriers were enriched for a diverse set of psychiatric conditions, including panic disorder, ADHD, depression, bipolar disorder and substance abuse as well as OCD spectrum disorders.

[0007] Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figures IA- IB show the identified rare nonsynonymous polymorphisms in synapse-associated protein 90/postsynaptic density-95 -associated protein 3 {SAP AP 3). Figure IA is a schematic SAPAP3 of SAPAP 3, showing the 10 coding exons (blue boxes). Seven rare changes were identified in trichotillomania and obsessive-compulsive disorder (OCD) patients (upper part), but only two in controls (lower part). Most mutations fell into exon 1; however, three changes affected the conserved GKAP domain. Figure IB shows the sequences of SAPAP '3 which is highly conserved between species (97% identical amino acids between human and mouse). Accordingly the identified rare changes affected conserved residues. Red boxes — OCD cases, green boxes — controls.

[0009] Figure 2 is a schematic representation showing pedigrees and clinical details of the identified index patients with rare SAPAP3 variants. Segregation of the variants is variable in different pedigrees and often hampered by not available genotypic data. The majority of families show a increased prevalence of a OCD and related psychiatric conditions. All index patients were Caucasian. Filled symbols indicate OCD and trichotillomania patients. Numbers in symbols indicate the current age of the individuals. Arrows depict the index patients. Boxes indicate obtained genotypes for the identified rare variants. Table 2 summarizes available data across families. GAD- generalized anxiety disorder; PTSD - posttraumatic stress syndrome; BPD - bipolar disorder; MDD - major depressive disorder; ADHD - attention deficit hyperactivity disorder.

DETAILED DESCRIPTION

[0010] Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

[0011] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes or nucleic acid sequences are human.

Definitions

[0012] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0013] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0014] As used herein "SAPAP3" is inclusive of all family members, isoforms, precursors, mutants, alleles, fragments, species, variants, orthologs, sense and antisense polynucleotide strands, etc.

[0015] As used herein, "SAPAP3 molecules" is inclusive of oligonucleotides, polynucleotides, peptides, polypeptides, RNA, DNA, family members, isoforms, precursors, mutants, alleles, fragments, species, variants, orthologs, sense and antisense polynucleotide strands, etc.

[0016] As used herein, "obsessive-compulsive disorder (OCD) and the spectrum of associated conditions," comprise trichotillomania (TTM), Tourette syndrome, body dysmorphic disorder etc.

[0017] By "antisense oligonucleotides" or "antisense compound" is meant an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et al., 1991 Ann. Rev. Biochem. 60, 631- 652). An antisense oligonucleotide can upregulate or downregulate expression and/or function of a particular polynucleotide. The definition is meant to include any foreign RNA or DNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0018] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term "oligonucleotide", also includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), ed nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, Hoόgsteen or reverse Hoόgsteen types of base pairing, or the like.

[0019] As used herein, two nucleic acid sequences (or a region of the sequences) are substantially homologous when the amino acid sequences are at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, homologous. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid "identity" is equivalent to nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0020] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970) J. MoI. Biol. 48:444-453 algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available atwww.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0021] The percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (1989) CABIOS 4:11-17, which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0022] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention are variants of wild type gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0023] The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.

[0024] Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl, or amino group. Derivatives, e.g., derivative oligonucleotides, may comprise non-naturally-occurring portions, such as altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art. Derivative nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0025] A "derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reduction/alkylation, acylation, chemical coupling, or mild formalin treatment. A derivative may also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.

[0026] As used herein, the term "animal" or "patient" is meant to include, for example, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.

[0027] "Mammal" covers warm blooded mammals that are typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.

[0028] "Treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, e.g., arresting it development; and/or (c) relieving the disease- state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc.). SAPAP3 and Variants Thereof

[0029] Many diseases, besides OCD, have been found to be hereditary. The ability to screen populations for risks in developing such diseases would be important in identifying and promoting preventative healthcare in such populations, both in terms of economics and the health of the individuals. Thus, there exists a need in the art to identify risk-conferring genetic variations in various diseases which may have a basis in genetic mechanisms.

[0030] In brief, the data shown here novel non-synonymous heterozygous variants were identified. The majority of changes presented missense mutations; one variant was an in- frame insertion of 5 amino acids, A148insGPAGA. It may be that specific variants were not by themselves disease-causing abnormalities, but still leaves open the possibility that an aggregate of susceptibility variants may prove contributory to disease, as indicated for some other disorders including autism as well as OCD. The combined analyses of 2766 alleles showed that all changes were very rare, with minor allele frequencies between 0.00036 (T523K, K910R) and 0.002 (Al 89V). In summary, on the background of an intriguing SAPAP3-OCD mouse model the present data supported a role for SAPAP3 in trichotillomania and OCD.

[0031] In a preferred embodiment, a method of providing an assessment in individuals at risk for developing OCD and/or an OCD spectrum associated disease comprises obtaining a sample from an individual; identifying variants of SAPAP3 polynucleotides and/or polypeptides as compared to controls.

[0032] In a preferred embodiment, variants are combined as a mutation load and compared to case controls and analyzed by prediction algorithms. Preferably, an increase in the mutation load or an increase in the types of variants is a prediction as to an increased risk of developing OCD and/or an OCD spectrum associated disease.

[0033] In another preferred embodiment, a method of diagnosing an obsessive compulsive disorder (OCD) and/or an OCD spectrum associated disease comprises obtaining a sample from an individual; identifying variants of SAPAP3 polynucleotides and/or polypeptides as compared to controls. Preferably, diagnosis of obsessive compulsive disorder (OCD) and/or an OCD spectrum associated disease comprises identification of at least one SAPAP3 variant in a patient sample and the identified variants are combined as a mutation load and compared to case controls. [0034] In another preferred embodiment, an increase in the mutation load and/or types of variants comprising the mutation load is diagnostic of OCD and/or an OCD spectrum associated disease.

[0035] In another preferred embodiment, a method of differentiating between an obsessive compulsive disorder (OCD) and an OCD spectrum associated disease comprises obtaining a sample form a patient; identifying one or more SAPAP3 variant polynucleotides and/or polypeptides as compared to controls. Preferably, the type of SAPAP3 variants detected, the mutation load, etc, is diagnostic of obsessive compulsive disorder (OCD) versus an OCD spectrum associated disease.

[0036] In another preferred embodiment, identification of the type of SAPAP3 variants detected and which SAPAP3 variants comprise a mutation load differentiates between an obsessive compulsive disorder (OCD) versus an OCD spectrum associated disease.

[0037] In another preferred embodiment, a biomarker for diagnosing patients at risk of developing an obsessive compulsive disorder or obsessive compulsive spectrum associated disease, or diagnosing an obsessive compulsive disorder or obsessive compulsive spectrum associated disease comprising: SAPAP3 variants.

[0038] In another preferred embodiment, an SAPAP3 variant comprises at least one of: R13C (c.38OT); (A148insGPAGA, c.441_442insGGGCCAGC AGGGGCA); T156M (C.467OT); Al 89V (c.566OT); T523K (C.1569-70COAA); P606T (c.1816OA); or K910R (c.2728A>G).

[0039] In another preferred embodiment, a biomarker for identifying patients at risk of developing an obsessive compulsive disorder or obsessive compulsive spectrum associated disease, or diagnosing an obsessive compulsive disorder or obsessive compulsive spectrum associated disease, comprise detecting at least one biomarker comprising: R13C (c.38C>T); (A148insGPAGA, c.441_442insGGGCCAGC AGGGGCA); T156M (c.467OT); A189V (C.566OT); T523K (c.l569-70COAA); P606T (c.1816OA); or K910R (c.2728A>G).

[0040] In another preferred embodiment, a biomarker for diagnosing an obsessive compulsive disorder or obsessive compulsive spectrum associated disease, or diagnosing an obsessive compulsive disorder or obsessive compulsive spectrum associated disease, comprise detecting at least one biomarker comprising: R13C (c.38OT); (A148insGPAGA, c.441_442insGGGCCAGC AGGGGC A); T156M (c.467OT); Al 89V (c.566OT); T523K (C.1569-70COAA); P606T (c.1816OA); or K910R (c.2728A>G). [0041] In another preferred embodiment, any of the SAPAP 3 variant comprise changes in at least one residue of a SAPAP3 polynucleotide. Preferably the residue is a conserved residue which is conserved amongst various species.

[0042] In another preferred embodiment, the SAPAP3 variants comprise at least one nucleobase change in exon 1 (see, for example, Figure 1).

[0043] The present invention also includes variants of the SAPAP3 nucleic acid and amino acid sequences. Examples of such variants are shown in the Tables. Accordingly, the variants can contain nucleotide substitutions, deletions, inversions, and insertions.

[0044] In another preferred embodiment, an isolated oligonucleotide comprises a sequence which is complementary to at least five consecutive nucleobases of SAPAP3 polynucleotides and variants thereof.

[0045] In a preferred embodiment, contacting a cell in vitro or in vivo with one or more oligonucleotides which target one or more SAPAP3 variants modulates the expression and/or function of such variants. For example, if a variant is deemed to be pathological (e.g. Rl 3C, T523K), administration of such oligonucleotides to a patient in need thereof, decreases the expression and/or function of such variants. In another preferred embodiment, it may be desirable to up-regulate expression and/or function of SAPAP3 and/or a variant thereof. Administration of one or more of such oligonucleotides to a patient in need thereof, modulate expression and/or function of SAPAP3 and/or variants thereof as a means to treat diseases or disorders associated with the SAPAP3 and/or variants thereof.

[0046] Details of the materials and methods have been provided in the examples section which follows. Mutation analysis, including determination of mutation load, advantageously is determined by amplification and analysis of DNA from a single cell. The DNA is amplified by any procedure that efficiently reproduces DNA from the low template concentrations obtained from a single cell. Polynucleotides of the present invention may also be obtained using standard cloning and screening techniques from a cDNA library derived from mPvNA in cells of human fetal brain and spinal cord, (see for instance, Sambrook, et at., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques. [0047] When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

[0048] Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SAPAP 3 or variants thereof, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR). Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similarity to SAPAP3 or variants thereof, typically at least 95% identity. Preferred probes and primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides.

[0049] A polynucleotide encoding a polypeptide of the present invention, including homologs from species other than human, may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having the sequence of SAPAP '3 or variants thereof, or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 42°C. in a solution comprising: 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5X Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. IXSSC at about 65°C. Thus the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SAPAP3 or variants thereof , or a fragment thereof, preferably of at least 15 nucleotides.

[0050] The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus. This is a consequence of reverse transcriptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), failing to complete a DNA copy of the mRNA template during first strand cDNA synthesis.

[0051] There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al., Proc Nat Acad Sci XJS A 85, 8998-9002, 1988).

[0052] Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.

[0053] For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Polynucleotides may be introduced into host cells by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al. Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

[0054] Representative examples of appropriate hosts include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

[0055] A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al. Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

[0056] If a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

[0057] Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification. [0058] Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations and mutation loads in the associated gene (see, for example, Table 1). Detection of a mutated form of the gene characterized by the polynucleotide of SAPAP3 or variants thereof in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques well known in the art.

[0059] Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled SAPAP 3 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers, et al., Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton, et al., Proc Natl Acad Sd USA (1985) 85: 4397-4401).

[0060] An array of oligonucleotides probes comprising SAPAP3 polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Such arrays are preferably high density arrays or grids. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, Chee, et al., Science, 21 A, 610-613 (1996) and other references cited therein.

[0061] Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

[0001] Microarrays: An assay using an immobilized array of nucleic acid sequences may be used for determining the sequence of an unknown nucleic acid; single nucleotide polymorphism (SNP) analysis; analysis of gene expression patterns from a particular species, tissue, cell type, etc.; gene identification; etc.

[0002] Additional diagnostic uses for oligonucleotides designed from the sequences encoding a desired gene expression product may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding the expression products, or a fragment of a polynucleotide complementary to the polynucleotides, and will be employed under optimized conditions for identification of a specific gene. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely-related DNA or RNA sequences.

[0003] In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences, may be used as targets in a microarray. The microarray can be used to monitor the identity and/or expression level of large numbers of genes and gene transcripts simultaneously to identify genes with which target genes or its product interacts and/or to assess the efficacy of candidate therapeutic agents in regulating expression products of genes that mediate, for example, neurological disorders. This information may be used to determine gene function, and to develop and monitor the activities of therapeutic agents. [0004] Microarrays may be prepared, used, and analyzed using methods known in the art (see, e.g., Brennan et al, 1995, U.S. Pat. No. 5,474,796; Schena et al, 1996, Proc. Natl. Acad. Sci. U.S.A. 93: 10614-10619; Baldeschweiler et al, 1995, PCT application WO95/251116; Shalon, et al., 1995, PCT application WO95/35505; Heller et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 2150-2155; and Heller et al., 1997, U.S. Pat. No. 5,605,662). In other embodiments, a microarray comprises peptides, or other desired molecules which can be assayed to identify a candidate agent. [0005] Preferably, identified genes, variants, fragments, or oligopeptides thereof are used for identifying agents of therapeutic interest, e.g. by screening libraries of compounds or otherwise identifying compounds of interest by any of a variety of drug screening or analysis techniques. The gene, allele, fragment, or oligopeptide thereof employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.

[0062] Nucleic acid hybridization and wash conditions are chosen such that the sample DNA specifically binds or specifically hybridizes to its complementary DNA of the array, preferably to a specific array site, wherein its complementary DNA is located, i.e., the sample DNA hybridizes, duplexes or binds to a sequence array site with a complementary DNA probe sequence but does not substantially hybridize to a site with a non-complementary DNA sequence. As used herein, one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch. Preferably, the polynucleotides are perfectly complementary (no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see, e.g., Chee et al., 1996, Science 274:610-614).

[0063] Arrays containing double-stranded probe DNA situated thereon are preferably subjected to denaturing conditions to render the DNA single-stranded prior to contacting with the sample DNA. Arrays containing single-stranded probe DNA (e.g., synthetic oligodeoxyribonucleic acids) need not be denatured prior to contacting with the sample DNA.

[0064] Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, DNA) of probe and sample nucleic acids. General parameters for specific (i.e., stringent) hybridization conditions for nucleic acids are described in Sambrook et al., supra, and in Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York.

[0065] Detection of Hybridization: Hybridization to the array may be detected by any method known to those of skill in the art. In a particular embodiment, the hybridization of fluorescently labeled sample nucleotides is detected by laser scanner. When two different fluorescent labels are used, the scanner is preferably one that is able to detect fluorescence of more than one wavelength, the wavelengths corresponding to that of each fluorescent label, preferably simultaneously or nearly simultaneously. Antibodies

[0066] In another preferred embodiment, an antibody is specific for SAPAP3 or variants thereof. The antibodies can be used in a variety of assays for diagnostic purposes, purification of molecules, identifying possible molecular associations, inhibitors, and the like.

[0067] In another preferred embodiment, aptamers are generated against SAPAP3 or variants thereof.

[0068] Antibodies of the invention may bind to SAPAP3 or variants thereof or SAPAPS interacting proteins provided by the invention to prevent normal interactions of the SAPAP3 encoded proteins and SAPAP 3 interacting proteins. Binding of antibodies to SAPAP 3 encoded proteins can interfere or enhance biological activities associated with SAPAP3 or variants thereof.

[0069] The antibodies of the invention can be used in any subject in which it is desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy. The antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and noncompetitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

[0070] The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding to an epitopic determinant present in an invention polypeptide. Such antibody fragments retain some ability to selectively bind with its antigen or receptor.

[0071] Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference). Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al, Nature, 256:495, 1975).

[0072] Antibodies which bind to an invention polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. For example, it may be desirable to produce antibodies that specifically bind to the N- or C-terminal domains of an invention polypeptide. The polypeptide or peptide used to immunize an animal is derived from translated cDNA or chemically synthesized and can be conjugated to a carrier protein, if desired. Commonly used carrier proteins which may be chemically coupled to the immunizing peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), tetanus toxoid, and the like.

[0073] Invention polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See, for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated by reference). The antibodies of the invention can be bound to many different carriers and used to detect the presence of an antigen comprising the polypeptides of the invention. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. T he nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.

[0074] There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation.

[0075] Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, puridoxal, and fluorescein, which can react with specific anti-hapten antibodies.

[0076] In using the monoclonal and polyclonal antibodies of the invention for the in vivo detection of antigen, e.g., SAPAP 3 encoded proteins, the detectably labeled antibody is given a dose which is diagnostically effective. The term "diagnostically effective" means that the amount of detectably labeled antibody is administered in sufficient quantity to enable detection of the site having the antigen comprising a polypeptide of the invention for which the antibodies are specific.

[0077] The concentration of detectably labeled antibody which is administered should be sufficient such that the binding to those cells having the polypeptide is detectable compared to the background. Further, it is desirable that the detectably labeled antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.

[0078] As a rule, the dosage of detectably labeled antibody for in vivo treatment or diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. Such dosages may vary, for example, depending on whether multiple injections are given, antigenic burden, and other factors known to those of skill in the art.

SAPAP3 Encoded Proteins, Peptides, or Variants thereof

[0079] In another preferred embodiment, a peptide comprises at least five amino acid residues corresponding to a segment of SAPAP3 or variants thereof. Preferably, the peptide comprises at least one functional activity of SAPAP '3 or variants thereof.

[0080] In another preferred embodiment, the SAPAP3 encoded proteins or variants thereof are used as antigens for the preparation of antibodies.

[0081] In another preferred embodiment, the peptides are used in the treatment of patients suffering from obsessive compulsive disorder (OCD) versus an OCD spectrum associated diseases or disorders.

[0082] In another preferred embodiment, an SAPAP3 peptide comprises at least one non- native amino acid residue or a non-amino acid molecule. A "non-native" amino acid residue comprises any change to an amino acid which is encoded by the SAPAP3 nucleic acid sequence or by the variants thereof. Thus, a non-native amino acid residue or non-amino acid molecule comprises, without limitation: a chemical equivalent, analog, synthetic molecule, derivative, variant, substitution, peptide nucleic acid, a linker molecule, inorganic molecule etc.

Antisense SAPAP3 Oligonucleotides

[0083] In another preferred embodiment, the functions and/or expression of SAPAP '3 in a cell or patient are modulated by targeting SAPAP3 molecules or variants thereof. [0084] In a preferred embodiment, an oligonucleotide comprises at least five consecutive bases complementary to nucleic acid sequences of SAPAP3 molecules or variants thereof, wherein the oligonucleotide specifically hybridizes to and modulates expression and/or function of SAPAP 3 vain vivo or in vitro. In another preferred embodiment, the oligomeric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.

[0085] In some embodiments, homology, sequence identity or complementarity, between the oligonucleotide and target is from about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

[0086] In another preferred embodiment, an oligonucleotide comprises combinations of phosphorothioate internucleotide linkages and at least one internucleotide linkage selected from the group consisting of: alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and/or combinations thereof.

[0087] In another preferred embodiment, an oligonucleotide optionally comprises at least one modified nucleobase comprising, peptide nucleic acids, locked nucleic acid (LNA) molecules, analogues, derivatives and/or combinations thereof. [0088] An oligonucleotide is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target nucleic acid sequences under conditions in which specific binding is desired. Such conditions include, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment, and conditions in which assays are performed in the case of in vitro assays.

[0089] An oligonucleotide, whether DNA, RNA, chimeric, substituted etc, is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarily to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0090] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0091] In embodiments of the present invention oligomeric oligonucleotides, particularly oligonucleotides, bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene. The functions of DNA to be interfered comprise, for example, replication and transcription. The functions of RNA to be interfered comprise all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The functions may be up-regulated or inhibited depending on the functions desired. [0092] The oligonucleotides, include, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds. [0093] In another preferred embodiment, the antisense oligonucleotides bind to coding and/or non-coding regions of a target polynucleotide and modulate the expression and/or function of the target molecule.

[0094] In another preferred embodiment, the antisense oligonucleotides bind to natural antisense polynucleotides and modulate the expression and/or function of the target molecule. [0095] In another preferred embodiment, the antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule. [0096] Alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts or "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. [0097] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0098] Variants can be produced through the use of alternative signals to start or stop transcription. Pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also embodiments of target nucleic acids.

[0099] The locations on the target nucleic acid to which the antisense compounds hybridize are defined as at least a 5-nucleobase portion of a target region to which an active antisense compound is targeted.

[00100] While the specific sequences of certain exemplary target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure. [00101] Target segments 5-100 nucleobases in length comprising a stretch of at least five (5) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[00102] Target segments can include DNA or RNA sequences that comprise at least the 5 consecutive nucleobases from the 5 '-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5 '-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 5 consecutive nucleobases from the 3 '-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3 '-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleobases). One having skill in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[00103] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. [00104] In embodiments of the invention the oligonucleotides bind to an antisense strand of a particular target. The oligonucleotides are at least 5 nucleotides in length and can be synthesized so each oligonucleotide targets overlapping sequences such that oligonucleotides are synthesized to cover the entire length of the target polynucleotide. The targets also include coding as well as non coding regions.

[00105] According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these. These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double- stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleobase positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, the gene expression or function is up regulated. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.

[00106] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-like" (i.e., generally having one or more 2'-hydroxyl or 2'-modified sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are "DNA-like" and those which have A- form-like structure are "RNA-like." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions. [00107] In another preferred embodiment, the desired oligonucleotides or antisense compounds, comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof. [00108] dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs). It is currently not known whether RNAa is conserved in other organisms.

[00109] Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA) and microRNA (miRNA), have been found to be the trigger of an evolutionary conserved mechanism known as RNA interference (RNAi). RNAi invariably leads to gene silencing via remodeling chromatin to thereby suppress transcription, degrading complementary mRNA, or blocking protein translation. dsRNAs may also act as small activating RNAs (saRNA). Without wishing to be bound by theory, by targeting sequences in gene promoters, saRNAs would induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation (RNAa).

[00110] RNA interference (RNAi) has become a powerful tool for blocking gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA (siRNA) either as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed to siRNAs. This system enables efficient transport of the pre-siRNAs to the cytoplasm where they are active and permit the use of regulated and tissue specific promoters for gene expression. [00111] In a preferred embodiment, an oligonucleotide or antisense compound comprises an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often desired over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[00112] According to the present invention, the oligonucleotides or "antisense compounds" include antisense oligonucleotides (e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA- like, RNA- like, or mixtures thereof, or may be mimetics of one or more of these. These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double- stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleobase positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines (Hammond et al, Nat. Rev. Genet., 1991, 2, 110-119; Matzke et al, Curr. Opin. Genet. Dev., 2001, 11, 221-227; Sharp, Genes Dev., 2001, 15, 485-490). When formed from two strands, or a single strand that takes the form of a self- complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.

Identification of Novel Therapies

[00113] In another preferred embodiment, a method of identifying candidate therapeutic agents for treatment of diseases associated with variants in SAPAP3 genes comprises providing sample, such as for example, a cell comprising an SAPAP3 gene having at least one variant as compared to wild type SAPAP3; contacting the cell with a candidate therapeutic agent and, measuring the biological effects of the candidate therapeutic agents on the cell by correlating the SAPAP3 variant with an OCD and OCD spectrum associated disease and/or comparing the level or SAPAP3 biological activity in the cell after administration of the candidate therapeutic agent as compared to the level or SAPAP3 biological activity in the cell prior to administering the candidate therapeutic agent, wherein modulation of the level or SAPAP3 biological activity is indicative of a therapeutic agent for the treatment of diseases associated with variants in SAPAP 3 genes. In some embodiments, the SAPAP 3 gene comprise one or more variants as compared to wild type SAP AP 3.

[00114] In another preferred embodiment, a method of identifying candidate therapeutic agents for treatment of diseases associated with variants in SAPAP3 polynucleotides and/or peptides comprising: providing a sample comprising an SAP AP 3 polynucleotide and/or peptide having at least one variant as compared to wild type SAPAP3; administering a candidate therapeutic agent to the sample; assaying for an effect of the candidate therapeutic agent on a SAPAP3 biological activity. Preferably, the SAPAP3 gene comprise one or more variants as compared to wild type SAP AP 3.

[00115] In another preferred embodiment, a method of identifying targets of candidate therapeutic agents for treating OCD and OCD spectrum associated diseases in an SAPAP3 polynucleotide comprises obtaining a biological sample from an individual; identifying variants of SAPAP3 polynucleotides and/or polypeptides as compared to controls; contacting a sample with a candidate therapeutic agent; assaying biological effects of the candidate therapeutic agents on SAPAP3 associated biological activity in the sample after administration of the candidate therapeutic agent as controls. Preferably, the targets are variants detected in an SAPAP3 polynucleotide from an individual at risk or having OCD and OCD spectrum associated diseases as compared to wild type SAPAP3 polynucleotide.

[00116] The SAP AP 3 nucleic acid targets of the present invention and appropriate variants can be used in high-throughput screens to assay candidate compounds for the ability to modulate the translational profile of the target nucleic acid in a wild-type or SAPAP 3 variant expressing cell. Compounds can be identified that increase or decrease the translational profile. Such modulatory methods can be performed in vitro (e.g., by culturing a cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).

[00117] For instance, a reporter construct containing an SAPAP3 nucleic acid target can be transfected into a cell. The assay includes the steps of combining the cell with a candidate compound under conditions that allow the SAPAP3 nucleic acid target to interact with the compound, and to detect the effect of said contact upon the translation profile of the target. The effect, if any, can be determined by assays, such as for example, blotting, or simply by assaying for the reporter gene and comparing its translation to that of a cell that has not been contacted by said compound.

[00118] Test compounds can be obtained using approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one- bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des . 12:145).

[00119] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 :1303; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compounds maybe presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 97:6378-6382; Felici (1991) J. MoI. Biol. 222:301-310; Ladner supra.

[00120] Candidate compounds include, for example, 1) peptides such as soluble peptides, including 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 libraries made of D- and/or L-confϊguration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single-chain antibodies as well as Fab, F(ab') ₂ , Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries). [00121] In accordance with the methods of the present invention, at least one candidate compound as defined elsewhere herein is used to promote a positive response with respect to a cell comprising an SAPAP3 mutation load. By "positive therapeutic response" is intended an improvement in the disorder, syndrome, symptoms, or translational profile associated with the disorder.

[00122] Treating a patient comprises administration of one or more of the identified agents. In another preferred embodiment, the agents can be administered as part of a treatment regimen with one or more other therapies and medicaments.

[00123] The compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.

[00124] An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit a cysteine protease. The compounds may be administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect. Prodrugs of compounds of the present invention may be prepared by any suitable method. For those compounds in which the prodrug moiety is a ketone functionality, specifically ketals and/or hemiacetals, the conversion may be effected in accordance with conventional methods. [00125] No unacceptable toxicological effects are expected when compounds, derivatives, salts, compositions etc, of the present invention are administered in accordance with the present invention. The compounds of this invention, which may have good bioavailability, may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect. [00126] In another preferred embodiment, there is provided a pharmaceutical or veterinary composition comprising one or more SAPAP3 compounds (e.g. antisense, compounds identified through an assay described above, etc) and a pharmaceutically or veterinarily acceptable carrier. Other active materials may also be present, as may be considered appropriate or advisable for the disease or condition being treated or prevented. [00127] The carrier, or, if more than one be present, each of the carriers, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

[00128] The compounds described herein are suitable for use in a variety of drug delivery systems described above. Additionally, in order to enhance the in vivo serum half-life of the administered compound, the compounds may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended serum half-life of the compounds. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each of which is incorporated herein by reference. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.

[00129] The formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.

[00130] Such methods include the step of bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.

Kits [00131] In another preferred embodiment, a kit is provided comprising any one or more of the biomarkers comprising R13C (c.38C>T); (A148insGPAGA, c.441_442insGGGCCAGC AGGGGC A); T156M (c.467OT); Al 89V (c.566OT); T523K (C.1569-70COAA); P606T (c.1816OA); or K910R (c.2728A>G) and/or antibodies, probes etc.

[00132] For use in the applications described or suggested above, kits or articles of manufacture are also provided by the invention. Such kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a probe that is or can be detectably labeled. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter- means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.

[00133] The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above.

[00134] The kits of the invention have a number of embodiments. A typical embodiment is a kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a primary antibody that binds to the biomarkers of each molecular signature and instructions for using the antibody for evaluating the presence of biomarkers in at least one type of mammalian cell. The kit can further comprise a set of instructions and materials for preparing a tissue sample and applying antibody and probe to the same section of a tissue sample. The kit may include both a primary and secondary antibody, wherein the secondary antibody is conjugated to a label, e.g., an enzymatic label.

[00135] Another embodiment is a kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a polynucleotide that hybridizes to a complement of the polynucleotides under stringent conditions, the label on said container indicates that the composition can be used to evaluate the presence of a molecular signature in at least one type of mammalian cell, and instructions for using the polynucleotide for evaluating the presence of biomarker RNA or DNA in at least one type of mammalian cell.

[00136] Other optional components in the kit include, microarrays, one or more buffers (e.g., block buffer, wash buffer, substrate buffer, etc), other reagents such as substrate (e.g., chromogen) which is chemically altered by an enzymatic label, epitope retrieval solution, control samples (positive and/or negative controls), control slide(s) etc. [00137] While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

[00138] All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention. Embodiments of inventive compositions and methods are illustrated in the following examples.

EXAMPLES

[00139] The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Example 1: Multiple rare SAPAP 3 missense variants in trichotillomania and OCD [00140] Mice deficient in the postsynaptic SAP90/PSD95-associated protein 3 (SAPAP3, also known as Dlgap3) develop an OCD-like phenotype, which includes compulsive grooming and increased anxiety. Interestingly, the phenotype of SAPAP3 knock-out mice can be rescued by administering selective serotonin reuptake inhibitors ⁴ . It was hypothesized that rare variants in the human orthologue SAPAP3 could contribute to disorders in the OCD spectrum. To test this, SAPAP3 were re-sequenced in three case populations, including 77 unrelated TTM probands collected at Duke University, 44 OCD with TTM probands from NIMH, and 44 OCD cases without TTM from NIMH ⁵ ' ⁶ . Controls were 48 OCD spectrum-negative subjects from NIMH ⁶ and a psychiatric comparison sample of 138 subjects screened for depression but not specifically for OCD from Duke University ⁷ . A board-certified psychiatrist saw all patients and controls and diagnoses met DSM-IV criteria. Samples were collected under approved institutional review board protocols. In 165 cases and 178 controls the complete coding region and flanking intronic sequence of SAPAP 3 was re-sequenced using standard capillary sequencing methods (Applied Biosystems, Foster City, CA).

[00141] Seven novel non-synonymous heterozygous variants, with all but Al 89V occurring only once (Table 1, Figure 1). Thus, in total, heterozygous SAPAP 3 variants were present in 4.2% of diagnosed TTM/OCD patients, but only in 1.1% of controls (two changes in Duke control samples, with one developing depression subsequent to entry into the study). The majority of changes presented missense mutations; one variant was an in- frame insertion of 5 amino acids, A148insGPAGA. In-silico analysis of the missense variants applying PMut and PolyPhen predicted two, or three, respectively, variants as of functional relevance (Table 1). The remaining polymorphisms were considered benign, including the two changes detected in controls. Further, 6 of the identified variants in the TTM/OCD subjects were genotyped in an additional sample of 281 OCD cases and in 751 general population controls ⁶ . R13C and P606T were found in one control each, while A189V was present in three controls. This indicates that these specific variants were not by themselves disease-causing abnormalities, but still leaves open the possibility that an aggregate of susceptibility variants may prove contributory to disease, as indicated for some other disorders including autism as well as OCD. The combined analyses of 2766 alleles showed that all changes were very rare, with minor allele frequencies between 0.00036 (T523K, K910R) and 0.002 (Al 89V). [00142] Available pedigrees from TTM/OCD mutation carriers were enriched for a diverse set of psychiatric conditions, including panic disorder, ADHD, depression, bipolar disorder, and substance abuse as well as OCD spectrum disorders (Figure 2 and Table 2). This situation is quite typical for psychiatric genetic studies and complicates allele segregation studies. Co-segregation of genotype and phenotype is also confounded by phenotypic penetrance rates, limited psychometric instruments, and assortative mating. Thus, it is considered herein, more significant to study the combined mutation load of the SAPAP3 comparing cases to controls. A significant case-control association was observed in this moderately sized sample (Fisher's one-sided exact test p=0.045). With generally still limited abilities to determine functional consequences of genetic variants, it was speculated that the predicted moderate functional consequences (Table 1) were not detrimental for protein function but rather increase susceptibility for OCD spectrum behavior, possibly through permissive or epistatic interactions with additional genetic and environmental factors. A recent study estimated that up to 70% of low-frequency missense alleles in humans have mildly deleterious effects ⁹ . The excess of rare mildly deleterious variants in any OCD risk gene could be promoted by an inefficient evolutionary selection against OCD risk alleles, which is supported by the high OCD spectrum frequency of 2-4% in the population, early disease onset, and normal reproductive fitness.

[00143] In summary, on the background of an intriguing SAPAP3-OCD mouse model the present data support a role for SAPAP 3 in trichotillomania and OCD. Expansion of this approach and modeling of rare genetic variants in SAPAP3 will be used to further test this hypothesis.

[00144] Table 1 : Identified rare variants in SAPAP3 and predicted functional relevance

Analyzed samples Prediction algorithms

Detected

121 nonsynonymous 44 Duke TTM and 48 130 variants NIMH OCD w/o PMut* PolyPhen* NIMH OCD w NIMH controls Duke controls

TTM

TTM

R13C Possibly

1 0 0 0 Pathological C.38OT daSAPAP3ng

A148insGPAGA c.441_442insGGGC O 1 0 0 N/A ^& N/A ^& CAGCAGGGGCA

T156M

O 0 0 1 Neutral Benign C.467OT

Al 89V

1 1 0 1 Neutral Benign c.566C>T

T523K Possibly

1 0 0 0 Pathological C.1569-70COAA daSAPAP3ng

P606T Possibly

O 1 0 0 Neutral c.l816C>A daSAPAP3ng

K910R

1 0 0 0 Neutral Benign c.2728A>G

Combined allele

7/330 TTM & OCD (2.1%) 2/356 Controls (0.56%) frequencies

Abbreviations: NIMH, National Institute of Mental Health; OCD, obsessive-compulsive disorder; TTM, trichotillomania. aPMut: mmb2.pcb.ub.es:8080/PMut; PolyPhen: genetics.bwh.harvard.edu/pph. bPrediction of the effects of in/dels is not possible with PMut and PolyPhen, but a functional effect is likely for a five amino acid insertion. [00145] Table 2: Summary of pedigree analysis illustrating the aggregation of psychiatric phenotypes in the studied families.

Cumulative index patient counts Criteria

6 out of 7 Female gender

5 out of 7 Early-onset TTM/ OCD

4 out of 7 Multiple psychiatric diagnoses

3 out of 7 Multiple sibs with reported symptoms of TTM/ OCD

4 out of 7 Parents with reported symptoms of TTM/ OCD

5 out of 7 Family members with reported symptoms of TTM/ OCD

6 out of 7 Family members with reported psychiatric disorders

[00146] Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

[00147] The Abstract of the disclosure will allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the following claims.

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