WO2009143622A1

WO2009143622A1 - Methods of stratifying, prognosing and diagnosing schizophrenia, mutant nucleic acid molecules and polypeptides

Info

Publication number: WO2009143622A1
Application number: PCT/CA2009/000735
Authority: WO
Inventors: Guy A. Rouleau; Pierre Drapeau; Martine Gauthier; Claude Marineau
Original assignee: CHUM
Current assignee: CHUM
Priority date: 2008-05-29
Filing date: 2009-05-29
Publication date: 2009-12-03
Anticipated expiration: 2010-11-29
Also published as: EP2297325A4; EP2297325A1

Abstract

A method for diagnosing the presence of schizophrenia or predicting the risk of developing schizophrenia in a human subject, comprising detecting the presence or absence of a defect in the SHANK3 gene encoding a SHANK3 polypeptide, in a nucleic acid sample of the subject, whereby the detection of the defect is indicative that the subject has schizophrenia or is at risk of developing schizophrenia.

Description

TITLE OF THE INVENTION

METHODS OF STRATIFYING, PROGNOSING AND DIAGNOSING SCHIZOPHRENIA, MUTANT NUCLEIC ACID MOLECULES AND POLYPEPTIDES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] NA

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] N. A.

FIELD OF THE INVENTION

[0003] The present invention relates to the identification of a gene associated with schizophrenia. The present invention also relates to methods of stratifying, diagnosing and prognosing schizophrenia and to methods of screening for compounds useful in the treatment of schizophrenia.

BACKGROUND OF THE INVENTION

[0004] Schizophrenia is a chronic psychiatric disorder characterized by a profound disruption in cognition, behaviour and emotion that affects up to 1% of the worldwide population. Today, there is a poor understanding of the genes involved in schizophrenia which severely hampered the development of improved antipsychotic drugs.

[0005] Schizophrenia is thought to be due to polygenic inheritance¹ though a fraction of the cases could result from variably penetrant de novo mutations. The possibility that schizophrenia could be caused by de novo mutations was first proposed over a half century ago⁴. However, only recently has this proposition been seriously considered, based on studies demonstrating that the heritability of schizophrenia is 6-8 times higher in monozygotic versus dizygotic twins⁵⁶, as well as studies showing a significantly increased risk of schizophrenia with increasing paternal age⁷, reduced rates of marriage, fertility, and reproduction in schizophrenia patients compared to controls⁸⁹. Despite this low reproductive fitness and the extremely variable environmental factors, the incidence of schizophrenia is maintained at ~1% worldwide, suggesting that schizophrenia may be due, at least in part, to de novo mutations of key genes.

[0006] The SHANK3 gene maps to a region of chromosome 22q previously associated with large deletions causing mental retardation or other developmental brain abnormalities¹² and recently SHANK3 mutations were described in autistic patients²³. SHANK3 is a scaffolding protein abundant in the post-synaptic density of excitatory synapses on dendritic spines¹⁰. [0007] In view of the fact that little is known about the genes altered in schizophrenic subjects and of the small number of efficient therapeutic and diagnostic treatments available, there remains a need to identify new genes associated with schizophrenia and to provide new diagnostic methods and therapeutic targets for the treatment of schizophrenia.

[0008] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0009] The present invention reports the identification of rare cfe novo and inherited mutations in two schizophrenia cohorts in the gene encoding the synaptic scaffolding protein SHANK3 and shows that some of these mutations lead to loss of function in a biological model. As mutations in SHANK3 were previously reported in autism²³, these findings support a role for SHANK3 in the etiology of schizophrenia, define a molecular genetic link between schizophrenia and autism, and emphasize the importance of rare cfe novo mutations in schizophrenia.

[0010] More specifically, in accordance with the present invention, there is provided a method for diagnosing the presence of schizophrenia or predicting the risk of developing schizophrenia in a human subject, comprising detecting the presence or absence of a defect in a gene encoding a polypeptide comprising the sequence of Figure 5 (SEQ ID NO:2), in a nucleic acid sample of the subject, whereby the detection of the defect is indicative that the subject has or is at risk of developing schizophrenia.

[0011] In a specific embodiment of the method, said sample comprises DNA. In another specific embodiment, said sample comprises RNA. In another specific embodiment, the defect is a missense, nonsense or splice site mutation.

[0012] In another embodiment, the defect comprises a mutation in a gene causing a modification in gene or protein expression or in protein function. In a particular embodiment, said modification in gene or protein expression is a diminution of gene or protein expression. In another particular embodiment, the modification in protein function is a reduction in the biological activity of the SHANK3 protein. In another embodiment, the defect comprises a mutation in a gene causing a complete or partial deletion of one or more of the Homer- and Cortactin-binding sites and the sterile alpha motif (SAM) domain.

[0013] In another specific embodiment, the defect comprises a mutation in the gene resulting in a mutant polypeptide in which at least one amino acid residue of Figure 5 (SEQ ID NO: 2) is substituted with another amino acid residue, and wherein the at least one amino acid residue is selected from the group consisting of an alanine residue at position 224; an arginine residue at position 536; an arginine residue at position 1117; a proline residue at position 1134; an histidine residue at position 493, a serine residue at position 952, an alanine residue at position 1160 and a valine residue at position 1333. In another specific embodiment, the defect comprises a mutation in the gene resulting in a mutant polypeptide in which amino acid residue 224 of Figure 5 (SEQ ID NO: 2) is substituted with a threonine residue, in which amino acid residue 536 of Figure 5 (SEQ ID NO: 2) is substituted with a tryptophan; in which amino acid residue 1117 of Figure 5 (SEQ ID NO: 2) is substituted with a stop codon; in which amino acid residue 1134 of Figure 5 (SEQ ID NO: 2) is substituted with a histidine residue; in which amino acid residue 493 of Figure 5 (SEQ ID NO: 2) is substituted with a glutamine residue, in which amino acid residue 952 of Figure 5 (SEQ ID NO: 2) is substituted with a threonine residue, or in which amino acid residue 1333 is substituted with a glycine residue.

[0014] In another specific embodiment, the defect comprises a mutation in the SHANK3 cDNA, wherein the cDNA is as set forth in Figure 4 (SEQ ID NO: 1), and wherein the mutation is selected from the group consisting of a substitution of a cytosine at position c.3349 (R1117X) with another nucleotide, a substitution of a cytosine at position c.1606 (R536W) with another nucleotide, a substitution of a cytosine at position c.3401 (P1134H) with another nucleotide, a substitution of a guanine at position c.670 (A224T) with another nucleotide, and a substitution of a thymine at position c.3998 (V1333G) with another nucleotide (the positions are given with respect to the first nucleotide in the ATG initiator as nucleotide no.1). In another specific embodiment, the defect comprises a mutation in the SHANK3 gene, wherein the gene is as set forth in Figure 4 (SEQ ID NO: 1), and the mutation is selected from the group consisting of a substitution of a cytosine at position c.3349 with a thymine (R1117X), a substitution of a cytosine at position c.1606 with a thymine (R536W), a substitution of a cytosine at position c.3401 with an adenine (P1134H), a substitution of guanine at position c.670 with an adenine (A224T), and a substitution of thymine at position c.3998 with a guanine (V1333G) (the positions are given with respect to the first nucleotide in the ATG initiator as nucleotide no.1).

[0015] The present invention further provides a method of diagnosing schizophrenia or susceptibility to suffer from schizophrenia in a subject comprising determining in a biological sample from said subject the presence or absence of a mutation in a SHANK3 nucleic acid or a mutation which shows linkage disequilibrium therewith, wherein the identification of a mutation in said SHANK3 in at least one allele of said subject is indicative that the subject has or is at risk of developing schizophrenia, and wherein said mutation in said SHANK3 nucleic acid is selected from the group consisting of:

a) a mutation corresponding to a mutation at amino acid position 224 of a SHANK3 protein; b) a mutation corresponding to a mutation at amino acid position 536 of a SHANK3 protein; c) a mutation corresponding to a mutation at amino acid position 1134 of a SHANK3 protein; d) a mutation corresponding to a mutation at amino acid position 1333 of a SHANK3 protein; and e) a mutation corresponding to a mutation at amino acid position 1117 of a SHANK3 protein.

[0016] In accordance with another aspect of the present invention, there is provided a method of detecting the presence or absence of a mutation in a SHANK3 gene, said method comprising the steps of: a) analyzing a nucleic acid test sample containing the gene; b) comparing the results of said analysis of said sample of step a) with the results of an analysis of a control nucleic acid sample containing a wild type SHANK3 gene, wherein the wild type SHANK3 gene comprises the sequence of Figure 4 (SEQ ID NO: 1); and c) determining the presence or absence of at least one defect in the SHANK3 gene of the test sample.

[0017] In another specific embodiment of the method, the nucleic acid sample is amplified prior to analysis. In another specific embodiment, the defect is a mutation in the coding region of the SHANK3 gene. In another specific embodiment, the mutation is a missense, nonsense or splice site mutation.

[0018] In another specific embodiment, the analysis is selected from the group consisting of: sequence analysis; fragment polymorphism assays; hybridization assays and computer based data analysis.

[0019] In accordance with another aspect of the present invention, there is provided a SHANK3 polypeptide comprising a mutation in one or more amino acids selected from the group consisting of: arginine at position 215 (R215); alanine at position 224 (A224); isoleucine at position 245 (I245); arginine at position 536 (R536); alanine at position 721 (A721); proline at position 1134 (P1134); arginine at position 1298 (R1298); alanine at position 1324 (A1324); valine at position 1333 (V1333); isoleucine at position 1546; proline at position 1654; and arginine at position 1117 (R1117). In a particular embodiment, the SHANK3 polypeptide of the present invention comprises a mutation in one or more amino acids selected from the group consisting of: alanine at position 224 (A224); arginine at position 536 (R536); proline at position 1134 (P1134); valine at position 1333 (V1333); and arginine at position 1117 (R1117).

[0020] In accordance with another aspect of the present invention, there is provided a purified antibody that binds specifically to the polypeptide of the present invention.

[0021] In accordance with another aspect of the present invention, there is provided a method of determining whether a biological sample contains the SHANK3 polypeptide of the present invention, comprising contacting the sample with a purified ligand that specifically binds to the polypeptide, and determining whether the ligand specifically binds to the polypeptide, the binding being an indication that the sample contains the polypeptide. [0022] In a specific embodiment, the ligand is a purified antibody.

[0023] In accordance with another aspect of the present invention, there is provided an isolated nucleic acid molecule comprising the sequence of Figure 4 (SEQ ID NO:1) or the complement thereof comprising a mutation in one or more nucleotides selected from the group consisting of: a substitution of a cytosine at position c.3349 (R1117X) with another nucleotide, a substitution of a cytosine at position c.1606 (R536W) with another nucleotide, a substitution of a cytosine at position c.3401 (P1134H) with another nucleotide, a substitution of a guanine at position c.670 (A224T) with another nucleotide, and a substitution of a thymine at position c.3998 (V1333G) with another nucleotide. In another specific embodiment, the defect comprises a mutation in the SHANK3 gene, wherein the gene is as set forth in Figure 4 (SEQ ID NO: 1), and the mutation is selected from the group consisting of a substitution of a cytosine at position c.3349 with a thymine (R1117X), a substitution of a cytosine at position c.1606 with a thymine (R536W), a substitution of a cytosine at position c.3401 with an adenine (P1134H), a substitution of guanine at position c.670 with an adenine (A224T), and a substitution of thymine at position c.3998 with a guanine (V1333G). (the positions are given with respect to the first nucleotide in the ATG initiator as nucleotide no.1)

[0024] In accordance with another aspect of the present invention, there is provided an isolated nucleic acid molecule encoding a polypeptide comprising a mutation in one or more amino acids selected from the group consisting of: arginine at position 215 (R215); alanine at position 224 (A224); isoleucine at position 245 (I245); arginine at position 536 (R536); alanine at position 721 (A721); proline at position 1134 (P1134); arginine at position 1298 (R1298); alanine at position 1324 (A1324); valine at position 1333 (V1333); isoleucine at position 1546; praline at position 1654; and arginine at position 1117 (R1117). In a particular embodiment, the isolated nucleic acid molecule encodes a polypeptide comprising a mutation in one or more amino acids selected from the group consisting of: alanine at position 224 (A224); arginine at position 536 (R536); proline at position 1134 (P1134); valine at position 1333 (V1333); and arginine at position 1117 (R1117).

[0025] In accordance with another aspect of the present invention, there is provided a vector comprising the nucleic acid molecule of the present invention. In accordance with another aspect of the present invention, there is provided a recombinant host cell comprising the vector of the present invention.

[0026] In specific embodiments of the methods of the present invention, the subject is pre-diagnosed as suffering from schizophrenia or as being a likely candidate for developing schizophrenia. In another specific embodiment, the likely candidate has at least one family member (parent, sister, brother, uncle, aunt, cousin, daughter, son, grand-parent, grand-child, etc.) suffering from a mental illness. In yet another specific embodiment, the likely candidate has at least one family member suffering from schizophrenia.

[0027] The present invention further provides a method for determining the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the method comprising determining in a biological sample from said subject:

(a) the level of expression of a SHANK3 nucleic acid or encoded polypeptide;

(b) the level of activity of a SHANK3 protein;

(c) the presence or absence of a functional mutation in SHANK3 nucleic acid or encoded protein; or

(d) any combination of (a) to (c), wherein a difference in said level relative to a corresponding control level or the presence of a functional mutation in said SHANK3 nucleic acid or encoded protein is indicative has or is at risk of developing schizophrenia.

[0028] The present invention also concern a method of determining the existence of an association between a SHANK3 polymorphism and schizophrenia, comprising the steps of: (i) genotyping at least one polymorphism in a SHANK3 gene or encoded polypeptide, in a population having schizophrenia; (ii) genotyping said polymorphism in a control population; and, (iii) determining whether a statistically significant association exists between schizophrenia and said polymorphism.

[0029] Also provided is a kit or package for detecting the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the kit comprising means for determining in a biological sample from said subject:

(a) the level of expression of SHANK3 nucleic acid (e.g., SEQ ID NO:1) or encoded polypeptide (e.g., SEQ ID NO:2);

(b) the level of activity of SHANK3 protein;

(c) the presence or absence of a mutation in SHANK3 nucleic acid or encoded protein that modifies its activity and/or expression; or

(d) any combination of (a) to (d);

together with instructions for correlating said level or said mutation with the presence of schizophrenia or a predisposition to suffer from schizophrenia.

[0030] The present invention further provides a method of identifying a compound for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia, said method comprising determining whether: (a) the level of expression of a SHANK3 nucleic acid (e.g., SEQ ID N0:1) or encoded polypeptide (e.g., SEQ ID N0:2);

(b) the level of SHANK3 activity; or

(c) a combination of (a) and (b); is increased in the presence of a test compound relative to in the absence of said test compound; wherein said increase is indicative that said test compound can be used for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia.

[0031] In an embodiment, said shank 3 activity is determined by assessing whether said test compound is able to rescue or compensate (totally or partially) the developmental and/or behavioral effect of SHANK3 knock down in zebra fish. In an embodiment, said developmental effect is a reduction in size of the head, eyes and/or trunk of the zebrafish. In an embodiment, said behavioral effect is a reduction in the capacity of the zebrafish to swim in response to touch Jn an embodiment said SHANK3 activity is the level of neuronal differentiation or neurite outgrowth. In an embodiment, said shank 3 activity that is assessed is an increase in the level of somatic sprouting of neurites compared to control neurons.

[0032] The present invention further relates to a method of identifying or characterizing a compound for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia, said method comprising:

(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a SHANK3 gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and

(b) determining whether reporter gene expression or reporter protein activity is increased in the presence of said test compound; said increase in reporter gene expression or reporter protein activity being an indication that said test compound may be used for decreasing susceptibility schizophrenia or for preventing or treating schizophrenia

[0033] In an embodiment, the above mentioned SHANK3 gene or SHANK3 nucleic acid in the above screening methods comprises encodes a SHANK3 polypeptide comprising a mutation in one or more amino acids selected from the group consisting of: arginine at position 215 (R215); alanine at position 224 (A224); isoleucine at position 245 (I245); arginine at position 536 (R536); alanine at position 721 (A721); proline at position 1134 (P1134); arginine at position 1298 (R1298); alanine at position 1324 (A1324); valine at position 1333 (V1333); isoleucine at position 1546; proline at position 1654; and arginine at position 1117 (R1117). In a particular embodiment, the SHANK3 polypeptide of the present invention comprises a mutation in one or more amino acids selected from the group consisting of: alanine at position 224 (A224); arginine at position 536 (R536); proline at position 1134 (P1134); valine at position 1333 (V1333); and arginine at position 1117 (R1117). In yet another embodiment, the mutation is selected from: a tryptophane at position 536 (R536); a histidine at position 1134 (P1134); and stop codon at position 1117 (R1117), which result in a truncated protein.

[0034] The present invention further provides a method of diagnosing schizophrenia or susceptibility to suffer from schizophrenia in a subject comprising determining in a biological sample from said subject the presence or absence of a mutation in a SHANK3 nucleic acid or a mutation which shows linkage disequilibrium therewith, wherein the identification of a mutation in said SHANK3 in at least one allele of said subject is indicative that the subject has or is at risk of developing schizophrenia, and wherein said mutation in said SHANK3 nucleic acid is selected from the group consisting of:

[0035] In a particular embodiment, the above-mentioned Shank3 polypeptide in the above-mentioned methods and kits comprises a mutation at a histidine residue at position 493, a serine residue at position 952 or an alanine residue at position 1160. In an embodiment, the histidine residue at position 493 is substituted with a glutamine residue, the serine residue at position 952 is substituted with a threonine residue.

[0036] In another specific embodiment, the mutation or defect comprises a mutation in the SHANK3 gene, wherein the gene is as set forth in Figure 4 (SEQ ID NO: 1), and wherein the mutation is selected from the group consisting of a substitution of a cytosine at position c.1479 (H493) with another nucleotide, a substitution of a guanine at position c.2856 (S952) with another nucleotid, or a substitution of a thymine at position c.3482 (A1160) with another nucleotide, (the positions are given with respect to the first nucleotide in the ATG initiator as nucleotide no.1).

[0037] In another specific embodiment, the defect comprises a mutation in the SHANK3 gene, wherein the gene is as set forth in Figure 4 (SEQ ID NO: 1), and the mutation is selected from the group consisting of a substitution of a cytosine at position c.1479 with a guanine (H493), a substitution of a guanine at position c.2856 with a cytosine (S952), or a substitution of a thymine at position c.3482 with an cytosine (A1160)(the positions are given with respect to the first nucleotide in the ATG initiator as nucleotide no.1).

[0038] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In the appended drawings:

[0040] Figure 1 shows families with de novo and potentially deleterious transmitted mutation in the

SHANK3 gene, a) Pedigree of family SCZ1 showing segregation of the R1117X nonsense mutation in three affected brothers. The proband is represented by the arrow, b) Pedigree of family SCZ2 showing segregation of the R536W missense mutation in the proband but not in his non-affected brother, c) Pedigree of family SCZ4 showing the segregation of the P1134H missense mutation transmitted by the father who is diagnosed with mood and obsessive compulsive disorder.

[0041] Figure 2 shows SHANK3 variants, a) Localization on the linear protein structure of SHANK3 of the de novo mutations found in families with schizophrenia. ANK: ankyrin repeats, SH3: Src homology 3 domain, PDZ: post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DIgA), and zonula occludens-1 protein (zo-1) domain, SAM: sterile alpha motif domain, b) Alignment of SHANK3 orthologous peptide sequences near the R536W and P1134H missenses (SEQ ID NOs: 80-101, indicated by asterisk (^*)) showing amino acid conservation of the R536 and P1134 residues in 10 species. GenBank accession numbers: chimp, AC145340 (SEQ ID NOs: 3 and 76); Rhesus monkey, AANU01101358 (SEQ ID NOs:4 and 77); dog, XPJ48271 (SEQ ID NO:5); rat, P_067708 (SEQ ID NO:12); Opossum , XP_001366729 (SEQ ID NO:6); Lizard, AAWZ01039630 (SEQ ID NOs:7 and 78); Xenopus, CX494866 (SEQ ID NOs: 8 and 79); Zebrafish zs3.1, (SEQ ID NOs:9 and 102) and zs3.2 , (SEQ ID NOs: 10 and 15), human (SEQ ID NOs 1, 2 and 80, 81) c) Western blot analysis using HA antibody of HEK293T cells lysate transfected with empty vector (Control), HA-Shank3 (WT), R1117X and R536W. Shank3 wt and R536W have a similar size (200 kDa), whereas the nonsense R1117X results in a truncated protein (123 kDa).

[0042] Figure 3 shows the validation of SHANK3 mutations in zebrafish. Knockdown (KD) of either of the zebrafish SHANK3 genes (zs3.1 (SEQ ID NO:102), zs3.2 (SEQ ID NO:10)) using selective AMOs resulted in severe morphological (A) and behavioural deficits (B, representative images taken from high speed video films) compared to wild type (WT). Partial rescue was observed with co-injection of AMOs and rat SHANK3 mRNA. The pie charts depict the proportion (% of totals) of normal (control-like, white), severely affected (no swimming, black) and mildly affected embryos (slow swimming, gray) in each group. The results with co-injection of rat SHANK3 WT or mutated (R1117X, P1134H or R536W) mRNAs are summarized in bar graphs (C). The asterisks denote significant (p<0.001) differences from the KD group.

[0043] Figure 4 shows the nucleotide sequence of the SHANK3 cDNA (SEQ ID NO: 1). The initiator codon (ATG) is shown in bold and the terminator codon is shown in bold and underlined. Exon 10 is underlined.

[0044] Figure 5 shows the amino acid sequence of SHANK3 protein (SEQ ID NO: 2).

[0045] Figure 6 shows the effect of SHANK3 mutants on differentiation of hippocampal neurons.

Transfected hippocampal neurons were identified by GFP expression (a). Overexpression of WT Shank3 in neurons leads to an increase in primary neurite outgrowth from somata (b). Overexpression of R536W (c), similarly stimulated neurite outgrowth. In contrast expression of the R1117X truncating mutation (d) failed to do so. In panel (e) the data is quantified in a bar histogram along with SDs for each bar. Neurite outgrowth significantly different from control levels (PO.001) is indicated with an asterisk (^*);

[0046] Figure 7 shows PCR primer (SEQ ID NOs:16-61) pairs used for screening of the SHANK3 gene.

Exons 22 and 23 were not tested since they are not expressed in isoforms found in brain tissue²¹²;

[0047] Figure 8 shows PCR primer (SEQ ID NOs:62-75) pairs used to determine the parental origin of the cfe novo mutations.

[0048] Figure 9 shows the segregation of microsatellite markers in pedigrees showing SHANK3 cfe novo and potentially deleterious mutations.

[0049] Figure 10 shows the prediction of functional effect of detected missense and nonsense variants in the SHANK3 gene in schizophrenia patients and controls using PolyPhen™, SIFT™, and SNAP™ programs. ^* Observed in controls in Durand et al. 2007².

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

DIAGNOSIS METHODS AND KITS

[0050] The present invention provides diagnostic and prognostic methods based on the identification of a defect in a SHANK3 nucleic acid of the present invention in a subject. Within the context of the present invention, the term "diagnosis" includes the detection, monitoring, dosing, comparison, etc. of at least one SHANK3 defect, at various stages, including early, pre-symptomatic stages, and late stages, in adults, children and pre-birth. Prognosis typically includes the assessment of a predisposition or risk to develop schizophrenia and the characterization of a subject to define most appropriate treatment (pharmacogenetics).

[0051] A particular object of this invention resides in a method of detecting the presence of or predisposition to schizophrenia in a subject, the method comprising (i) detecting the presence of a defect in a SHANK3 gene locus of the present invention in a sample from the subject, the presence of said defect being indicative that the subject has or is at risk of developing schizophrenia.

[0052] A particular object of this invention resides in a method of detecting the presence of or a predisposition to schizophrenia in a subject, the method comprising (i) detecting the presence of a defect in a SHANK3 mRNA or gene of the present invention in a sample from the subject, the presence of said defect being indicative that the subject has or is at risk of developing schizophrenia.

[0053] An additional particular object of this invention resides in a method of detecting the presence of or a predisposition to schizophrenia in a subject, the method comprising (i) detecting the presence of a defect in a SHANK3 polypeptide of the present invention in a sample from the subject, the presence of said defect being indicative that the subject has or is at risk of developing schizophrenia.

[0054] A defect in the SHANK3 gene may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertion(s) in the coding and/or non-coding region of the locus, alone or in various combination(s) which modifies the normal level of expression or activity of the protein or nucleic acid encoding same. The detection of the presence of an altered SHANK3 gene or an altered SHANK3 mRNA sequence according to the present invention can be performed by sequencing all or part of the gene, polypeptide or RNA, by selective hybridization or by selective amplification, for instance.

[0055] In a related aspect, the present invention provides a method of detecting the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the method comprising (i) detecting the presence of an altered SHANK3 RNA and/or polypeptide expression in a sample from the subject, the presence of said altered SHANK3 RNA and/or polypeptide expression being indicative that the subject has or is at risk of developing schizophrenia .

[0056] Defective SHANK3 RNA expression includes the presence of an increased or decreased quantity of RNA as compared to the amount of RNA expressed in cells of healthy individuals not suffering from schizophrenia and the presence of an altered tissue distribution of RNA. This may be detected by various techniques known in the art, including by selective hybridization or selective amplification of all or part of said RNA, for instance.

[0057] Similarly, defective SHANK3 polypeptide expression includes the presence of decreased or increased quantity of polypeptide, the presence of an altered tissue distribution, etc. These may be detected by various techniques known in the art, including by binding to specific ligands (such as SHANK specific antibodies), for instance.

[0058] A further object of the present invention resides in a method of detecting the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the method comprising detecting the presence of a reduced SHANK3 protein activity in a sample from the subject, the presence of said reduced activity being indicative that the subject has or is at risk of developing schizophrenia.

[0059] In another aspect, the present invention provides a method for determining the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the method comprising determining in a biological sample from said subject:

(a) the level of expression of a SHANK3 nucleic acid or encoded polypeptide;

(b) the level of activity of a SHANK3 protein;

(d) any combination of (a) to (c),

wherein a difference in said level relative to a corresponding control level or the presence of a functional mutation in said SHANK3 nucleic acid or encoded protein is indicative that the subject has or is at risk of developing schizophrenia .

[0060] In another aspect, the present invention is concerned with a method of detecting a functional mutation in SHANK3 nucleic acid or encoded protein in a subject's sample, the presence of a defect in a SHANK3 gene being indicative that the subject has or is at risk of developing schizophrenia. The functional mutation in said SHANK3 gene or encoded protein reduces, modifies or abolishes the expression and/or activity of said encoded protein. In one or more embodiments, the identification of one or more functional mutations enables the adaptation of a prophylactic or treatment regimen.

[0061] An object of the present invention resides in a method of genotyping at least one polymorphism of a SHANK3 gene of the present invention, comprising determining the presence of a polymorphism in at least one allele of said SHANK3 gene in a sample from the subject. In an embodiment, the identity of the polymorphism is determined by performing a hybridization assay, a sequencing assay, a microsequencing assay or an allele-specific amplification assay.

[0062] The present invention also relates to a method of determining the existence of an association between a SHANK3 polymorphism and schizophrenia, comprising the steps of: (i) genotyping at least one polymorphism of SHANK3 in a population having schizophrenia; (ii) genotyping said polymorphism in a control population (i.e., in subjects not suffering from schizophrenia and not likely to develop schizophrenia); and, (iii) determining whether a statistically significant association exists between schizophrenia and said polymorphism.

[0063] As indicated above, various techniques known in the art may be used to detect or quantify altered genes or RNA expressions or sequences, including sequencing, hybridization, amplification and/or binding to specific ligands (such as SHANK3 specific antibodies). Other suitable methods include allele-specific oligonucleotide (ASO), allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single- stranded conformation polymorphism (SSCP), PFGE (pulse field gel electrophoresis), fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, heteroduplex analysis, RNase protection, chemical mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA), Restriction fragment length polymorphism (RFLP), etc.

[0064] Some of these approaches (e.g., SSCP, heteroduplex analysis, fragment analysis, high performance DNA sequencing) are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments may then be sequenced to confirm the defect. Some others are based on specific hybridization between nucleic acids from the subject and a probe specific for wild type or altered gene or RNA. The probe may be in suspension or immobilized on a substrate. The probe is typically labeled to facilitate detection of hybrids. By "specific hybridization" is intended a hybridization under stringent conditions.

[0065] Some of these approaches are particularly suited for assessing a polypeptide sequence or its expression level, such as Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time- of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, and assays based on a property of the protein including, but not limited to, DNA binding, ligand binding, or interaction with other protein partners. The latter requires the use of a ligand specific for the polypeptide, more preferably of a specific antibody.

[0066] Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Preferred techniques use allele-specific PCR or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers to initiate the reaction. Review concerning various genotyping techniques known in the art is provided by Nedelcheva Kristensen in Biotechniques (2001, Vol. 30: 318-332).

[0067] The term "quantifying" or "quantitating" when used in the context of quantifying transcription levels of a gene can refer to absolute or to relative quantification. Absolute quantification may be accomplished by inclusion of known concentration(s) of one or more target nucleic acids and referencing the hybridization intensity of unknowns with the known target nucleic acids (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of hybridization signals between two or more genes, or between two or more treatments to quantify the changes in hybridization intensity and, by implication, transcription level.

[0068] Methods for normalizing the level of expression of a gene are well known in the art. For example, the expression level of a gene of the present invention can be normalized on the basis of the relative ratio of the mRNA level of this gene to the mRNA level of a housekeeping gene or the relative ratio of the protein level of the protein encoded by this gene to the protein level of the housekeeping protein, so that variations in the sample extraction efficiency among cells or tissues are reduced in the evaluation of the gene expression level. A "housekeeping gene" is a gene the expression of which is substantially the same from sample to sample or from tissue to tissue, or one that is relatively refractory to change in response to external stimuli. A housekeeping gene can be any RNA molecule other than that encoded by the gene of interest that will allow normalization of sample RNA or any other marker that can be used to normalize for the amount of total RNA added to each reaction. For example, the GAPDH gene, the G6PD gene, the actin gene, ribosomal RNA, 36B4 RNA, PGK1 , RPLPO, or the like, may be used as a housekeeping gene.

[0069] Methods for calibrating the level of expression of a gene are well known in the art. For example, the expression of a gene can be calibrated using reference samples, which are commercially available. Examples of reference samples include, but are not limited to: Stratagene® QPCR Human Reference Total RNA, Clontech™ Universal Reference Total RNA, and XpressRef™ Universal Reference Total RNA.

[0070] A "reference" or "control" level may be determined, for example, by measuring the level of expression of a SHANK3 nucleic acid or encoded polypeptide, or the level of SHANK3 activity, in a corresponding biological sample obtained from one or more healthy subject(s) (i.e., not suffering from schizophrenia or unlikely to suffer from schizophrenia, not diagnosed with schizophrenia or related disorders). When such a control level is used, a lower or decreased level of SHANK3 measured in a biological sample from a subject (i.e., test sample) is indicative that said subject is suffering from or is at risk of developing schizophrenia, whereas a substantially similar level is indicative that said subject does not have or is not at risk of developing schizophrenia.

[0071] Alternatively, a "reference" level may be determined, for example, by measuring the level of expression of a SHANK3 nucleic acid or encoded polypeptide, or the level of SHANK3 activity, in a biological sample obtained from one or more subject(s) known to be suffering from or susceptible to schizophrenia. When such a reference level is used, a substantially similar level measured in a biological sample from a subject (i.e., test sample) is indicative that said subject has or is at risk of developing schizophrenia, whereas a higher or increased level is indicative that said subject does not have or is at not at risk of developing schizophrenia.

[0072] As used herein, a substantially similar level refers to a difference in the level of expression or activity between the level determined in a biological sample of a given subject (i.e., test sample) and the reference level which is 15% or less; in a further embodiment, 10% or less; in a further embodiment, 5% or less.

[0073] As used herein, a "higher" or "increased" level refers to a level of expression or activity in a biological sample of a given subject (i.e., test sample) which is at least 20% higher, in an embodiment at least 30% higher, in a further embodiment at least 40% higher; in a further embodiment at least 50% higher, in a further embodiment at least 100% higher (i.e., 2-fold), in a further embodiment at least 200% higher (i.e., 3-fold), in a further embodiment at least 300% higher (i.e., 4-fold), relative to the reference level.

[0074] As used herein, a "lower" or "decreased" level refers to a level of expression or activity in a biological sample of a given subject (i.e., test sample) which is at least 20% lower, in an embodiment at least 30% lower, in a further embodiment at least 40% lower; in a further embodiment at least 50% lower, in a further embodiment at least 100% lower (i.e., 2-fold), in a further embodiment at least 200% lower (i.e., 3-fold), in a further embodiment at least 300% lower (i.e., 4-fold), relative to the reference level.

[0075] The term "linkage disequilibrium" refers to any degree of non-random genetic association between one or more allele(s) of two different polymorphic DNA sequences, that is due to the physical proximity of the two loci. Linkage disequilibrium is present when two DNA segments that are very close to each other on a given chromosome will tend to remain unseparated for several generations with the consequence that alleles of a DNA polymorphism (or marker) in one segment will show a non-random association with the alleles of a different DNA polymorphism (or marker) located in the other DNA segment nearby. Hence, testing of a marker in linkage disequilibrium with the polymorphisms of the present invention at a SHANK3 gene (indirect testing), will give almost the same information as testing for the SHAN3 polymorphisms (mutations) directly. This situation is encountered throughout the human genome when two DNA polymorphisms that are very close to each other are studied. Various degrees of linkage disequilibrium can be encountered between two genetic markers so that some are more closely associated than others. Linkage disequilibrium and the use thereof in inheritance studies is well known in the art to which the present invention pertains as exemplified by publications such as Risch and Merikangas, Science 273: 1516-1517 (1996); Maniatis, Methods MoI Biol. 376: 109-21 (2007) and Borecki et al., Adv Genet 60: 51-74 (2008).Thus, in an embodiment, the methods of the present invention which comprising determining the presence of a defect in a SHANK3 gene identified herein may (instead of directly assessing the presence of a specific mutation) assess the presence of a marker (e.g., polymorphism) which shows linkage desiquilibrium with a SHANK 3 mutation of the present invention.

[0076] The present invention also provides a kit or package for detecting the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the kit comprising means for determining in a biological sample from said subject:

(a) the level of expression of SHANK3 nucleic acid or encoded polypeptide; (b) the level of activity of SHANK3 protein;

(d) any combination of (a) to (d),

[0077] The present invention also provides diagnostic and prognostic kits comprising primers, probes and/or antibodies for detecting in a sample from a subject the presence of a defect in a SHANK3 gene sequence, RNA sequence (SEQ ID NO:1) or expression level, encoded protein sequence (SEQ ID NO:2), expression level or protein activity. Optionally, said diagnostic kits further comprise buffers and reagents for detecting said defect as well as instructions for using said diagnostic kits.

[0078] In the above described methods and kits the terminology "sample", "biological sample", clinical sample" and the like is meant to include any tissue or material derived from a living or dead human (or from another animal) which may contain the SHANK3 target nucleic acid or protein. Non limiting examples of samples include any tissue or material that may contain cells expressing or containing the SHANK3 nucleic acid or protein such as blood or fraction thereof, biopsies, bronchial aspiration, feces, cerebrospinal fluid, skin, sputum, saliva, urine, or coughing samples from test patients (suspected schizophrenic patients and control patients) or other body fluids or tissue that might be tested for SHANK3 expression, activity or sequence. In another embodiment, the biological sample of the present invention is a crude sample (i.e., unpurified). In another embodiment, the biological sample is semi-purified or substantially purified (e.g., a nucleic acid extract or protein extract). The biological sample may be treated to physically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents, and the like which are used to prepare the sample for analysis. Biological samples to be tested include but should not be limited to samples from mammalian (e.g., human) or any other sources. Of course, human samples are preferred biological samples in accordance with the present invention. In one particularly preferred embodiment, the clinical sample from the patient is not obtained through an invasive method. Numerous clinical textbooks and articles exist and are well known in the art concerning means of obtaining clinical samples and treatment thereof (in some conditions and for some applications) prior to use in the molecular diagnosis or for other uses.

[0079] A mammal, for purposes of treatment, prevention, diagnosis or prognosis, refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, horses, cats, cows. etc. Preferably, the mammal is human. SCREENING ASSAYS:

[0080] In another aspect, the invention relates to the use of SHANK3 as a target in screening assays that may be used to identify compounds useful for the prevention or treatment of schizophrenia. In an embodiment, the present invention further provides a method of identifying a compound for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia, said method comprising determining whether:

(a) the level of expression of a SHANK3 nucleic acid or encoded polypeptide;

(b) the level of SHANK3 activity; or

(c) a combination of (a) and (b);

is increased in the presence of a test compound relative to in the absence of said test compound; wherein said increase is indicative that said test compound can be used for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia. In an embodiment, the above-mentioned method is an in vitro method.

[0081] In an embodiment, the SHANK3 nucleic acid or polypeptide is a wild type SHANK3 nucleic acid or encoded polypeptide found in a control subject. In another embodiment, the SHANK3 nucleic acid or polypeptide is a SHANK3 nucleic acid or polypeptide having a defect which reduces its expression or activity. In a particular embodiment, the SHANK3 nucleic acid encodes a polypeptide comprising a mutation in one or more amino acids selected from the group consisting of: alanine at position 224 (A224); arginine at position 536 (R536); proline at position 1134 (P1134); valine at position 1333 (V1333); and arginine at position 1117 (R1117).

[0082] In another specific embodiment, the defect comprises a mutation in the nucleic acid resulting in a mutant polypeptide in which amino acid residue 224 of a SHANK3 polypeptide is substituted with a threonine residue, in which amino acid residue 536 of a SHANK3 polypeptide is substituted with a tryptophan; in which amino acid residue 1117 of a SHANK3 polypeptide is substituted with a stop codon; in which amino acid residue 1134 of a SHANK3 polypeptide is substituted with a histidine residue; or in which amino acid residue 1333 of a SHANK3 polypeptide is substituted with a glycine residue. In another embodiment, the defect comprises a mutation causing a complete or partial deletion of one or more of the Homer- and Cortactin-binding sites and of the sterile alpha motif (SAM) domain. In yet a further embodiment, the defect comprises a mutation which deletes at least exon 10 from said nucleic acid resulting in a smaller SHANK3 polypeptide.

[0083] In another embodiment of the invention, a reporter assay-based method of selecting agents which modulate SHANK3 expression is provided. The method includes providing a cell comprising a nucleic acid sequence comprising a SHANK3 transcriptional regulatory sequence operably-linked to a suitable reporter gene. The cell is then exposed to the agent suspected of affecting SHANK3 expression (e.g., a test/candidate compound) and the transcription efficiency is measured by the activity of the reporter gene. The activity can then be compared to the activity of the reporter gene in cells unexposed to the agent in question. Suitable reporter genes include but are not limited to beta (β)-D-galactosidase, luciferase, chloramphenicol acetyltransferase and green fluorescent protein (GFP).

[0084] Accordingly, the present invention further provides a method of identifying or characterizing a compound for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia, said method comprising: (a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a SHANK3 gene (e.g., a promoter region naturally associated with a SHANK3 gene), operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is increased in the presence of said test compound; said increase in reporter gene expression or reporter protein activity being an indication that said test compound may be used for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia. In an embodiment, the above-mentioned method is an in vitro method.

[0085] In an embodiment, the promoter region normally associated with a SHANK3 gene is a wild type promoter region found in a control subject. In another embodiment, the promoter region normally associated with a SHANK3 gene is a promoter region having a mutation found in a subject suffering from schizophrenia which decreases the expression of the SHANK3 nucleic acid and polypeptide when compared to a promoter region found in an unaffected subject.

[0086] In an embodiment, the above-mentioned SHANK3 activity is determined by assessing whether said test compound is able to rescue or compensate (totally or partially) the developmental and/or behavioral effect of SHANK3 knock down in zebra fish. In an embodiment, said developmental effect is a reduction in size of the head, eyes and/or trunk of the zebrafish. In an embodiment, said behavioral effect is a reduction in the capacity of the zebrafish to swim in response to touch .In an embodiment said SHANK3 activity is the level of neuronal differentiation or neurite outgrowth. In an embodiment, said shank 3 activity that is assessed is an increase in the level of somatic sprouting of neurites compared to control neurons.

[0087] The above-noted assays may be applied to a single test compound or to a plurality or "library" of such compounds (e.g., a combinatorial library). Any such compounds may be utilized as lead compounds and further modified to improve their therapeutic, prophylactic and/or pharmacological properties for the prevention and treatment of parasite infection or associated disease.

[0088] Such assay systems may comprise a variety of means to enable and optimize useful assay conditions. Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal SHANK3 activity and stability (e.g., protease inhibitors), and temperature control means for optimal SHANK3 activity and/or stability. A variety of such detection means may be used, including but not limited to one or a combination of the following: radiolabelling (e.g., P³², C¹⁴, H³), antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.

[0089] The assay may be carried out in vitro utilizing a source of SHANK3 which may comprise naturally isolated or recombinantly produced SHANK3, in preparations ranging from crude to pure. Recombinant SHANK3 may be produced in a number of prokaryotic or eukaryotic expression systems. Such assays may be performed in an array format. In certain embodiments, one or a plurality of the assay steps are automated.

[0090] A homolog, variant and/or fragment of SHANK3 which retains totally or partially its activity may also be used in the methods of the present invention. Homologs include protein sequences, which are substantially identical to the amino acid sequence of a SHANK3, sharing significant structural and functional homology with a SHANK3 (e.g., comprising polymorphisms found in healthy individuals but not in schizophrenic subjects). Variants include, but are not limited to, proteins or peptides, which differ from SHANK3 by any modifications, and/or amino acid substitutions, deletions or additions (e.g., fusion with another polypeptide). Modifications can occur anywhere including the polypeptide backbone, (i.e., the amino acid sequence), the amino acid side chains and the amino or carboxy termini. Such substitutions, deletions or additions may involve one or more amino acids. Fragments include a fragment or a portion of a SHANK3 or a fragment or a portion of a homolog or variant of a SHANK3 which retains SHANK3 activity.

[0091] As used herein, the designation "functional derivative" denotes, in the context of a functional derivative of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural; e.g., SHANK3 function or structure) that is substantially similar to that of the original sequence. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid generally has chemico physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include "fragments", "segments", "variants", "analogs" or "chemical derivatives" of the subject matter of the present invention. The genetic code, the chemico-physical characteristics of amino acids and teachings relating to conservative vs. non- conservative mutations are well-known in the art. Non-limiting examples of textbooks teaching such information are Stryer, Biochemistry, 3rd ed.; and Lehninger, Biochemistry, 3rd ed. The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology, all these methods are well known in the art.

[0092] As commonly known, a "mutation" is a detectable change in the genetic material which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position. Spontaneous mutations (inherited or cte novo) and experimentally induced mutations exist. The result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule. A mutant polypeptide can be encoded from this mutant nucleic acid molecule. A functional mutation is a mutation that modifies the normal biological activity of a gene or protein that it encodes. Functional mutations are generally found in the coding region of a gene but may be in the non-coding region (including splicing junction) and affect, for example the expression level of the polypeptide or its activity. A missense mutation (a type of nonsynonymous mutation), is a point mutation in which a nucleotide is changed, resulting in a codon that codes for a different amino acid. A non-sense mutation is a mutation (a change) in a base in the DNA that prematurely stops the translation (reading) of messenger RNA (mRNA) resulting in a polypeptide chain that ends prematurely and a protein product that is truncated (abbreviated) and incomplete and usually nonfunctional. As used herein, the term "missense mutation" or "non sense mutations" is meant to denote mutations which cause SHANK3 to be less functional (active) or inactive. For example, the missense or non-sense functional mutations of the present invention fail to rescue knock down of sz3.1 and sz3.2 in zebrafish.

[0093] "Homology" and "homologous" and "homolog" refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is "homologous" to or is a "homolog" of another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness). Two nucleic acids or amino acid sequences are considered "substantially identical" if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences. An "unrelated" or "non-homologous" sequence shares less than 50% identity, though preferably less than about 25 % identity and more preferably less than 10%.

[0094] Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% with a SHANK3 nucleic acid of the present invention. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. MoI. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. MoI. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W₁ T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0095] An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, more preferably highly stringent conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65⁰C, and washing in 0.2 x SSC/0.1% SDS at 42⁰C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68⁰C (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology -- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.

[0096] The assay may in an embodiment be performed using an appropriate host cell comprising

SHANK3 activity. Such a host cell may be prepared by the introduction of DNA encoding SHANK3 (e.g., comprising the nucleotide sequence set forth in Figure 4 (SEQ ID NO:1) or the coding sequence thereof, or a fragment/variant thereof having SHANK3 activity) into the host cell and providing conditions for the expression of SHANK3. Such host cells may be prokaryotic or eukaryotic, bacterial, yeast, amphibian or mammalian.

[0097] "Transcriptional regulatory sequence" or "transcriptional regulatory element" as used herein refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably linked. A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since enhancers generally function when separated from the promoters by several kilobases and intronic sequences which may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. As used herein, a transcriptionally regulatory element "normally" associated with for example a SHANK3 gene refers to such an element or a functional portion thereof derived from sequences operably-linked to for example a SHANK3 gene in its naturally-occurring state (i.e., as it occurs in a genome in nature). In another embodiment, the construct may comprise an in frame fusion of a suitable reporter gene within the open reading frame of a SHANK3 gene. The reporter gene may be chosen as such to facilitate the detection of its expression, e.g., by the detection of the activity of its gene product. Such a reporter construct may be introduced into a suitable system capable of exhibiting a change in the level of expression of the reporter gene in response to exposure to a suitable biological sample. Such an assay would also be adaptable to a possible large scale, high-throughput, automated format, and would allow more convenient detection due to the presence of its reporter component.

[0098] The above-described assay methods may further comprise determining whether any compounds so identified can be used for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia. ANTIBODIES:

[0099] The present invention also provides SHANK3 specific antibodies which are able to specifically bind and detect defects in a SHANK3 polypeptide and which are thus useful in the diagnostic and prognostic methods and kits of the present invention.

[00100] In a particular embodiment, the SHANK3 antibodies of the present invention enable the identification of a polymorphism present at amino acid positions R536; A224; P1134; V1333 or R1117 (the position are given with reference to the wild type SHANK3 polypeptide sequence of Figure 5). In an embodiment, the antibody of the present invention specifically binds to a SHANK3 polypeptide comprising a threonine at position 224; and/or a tryptophan at position 536; a histidine at position 1134; and/or a glycine at position 1333. In another embodiment, the SHANK3 antibody of the present invention specifically binds to a SHANK3 polypeptide comprising an alanine at position 224; and/or an arginine at position 536; and/or a proline at position 1134 and/or a valine at position 1333.

[00101] As used herein, the term "anti-SHANK3 antibody" or "immunologically specific anti-SHANK3 antibody" refers to an antibody that specifically binds to (interacts with) a SHANK3 protein and displays no substantial binding to naturally occurring proteins other than the ones sharing the same antigenic determinants as the SHANK3 protein. The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies™, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.

[00102] In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody A Laboratory Manual, CSH Laboratories). The term antibody encompasses herein polyclonal, monoclonal antibodies and antibody variants such as single-chain antibodies, humanized antibodies, chimeric antibodies and immunologically active fragments of antibodies (e.g., Fab and Fab' fragments) which inhibit or neutralize their respective interaction domains in Hyphen and/or are specific thereto. [00103] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injections of the relevant antigen with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or RIN=C=NR, where R and R1 are different alkyl groups.

[00104] Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermal^ at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with 1/5 to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

[00105] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al. Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Patent No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Patent Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293 (see also, e.g., Lindenbaum et al., 2004).

[00106] In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized (e.g., as hereinabove described) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.

[00107] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[00108] As used herein the term "purified" in the expression "purified polypeptide" means altered "by the hand of man" from its natural state (i.e. if it occurs in nature, it has been changed or removed from its original environment) or it has been synthesized in a non-natural environment (e.g., artificially synthesized). These terms do not require absolute purity (such as a homogeneous preparation) but instead represents an indication that it is relatively more pure than in the natural environment. For example, a protein/peptide naturally present in a living organism is not "purified", but the same protein separated (about 90-95% pure at least) from the coexisting materials of its natural state is "purified" as this term is employed herein.

[00109] Similarly, as used herein, the term "purified" in the expression "purified antibody" is simply meant to distinguish man-made antibody from an antibody that may naturally be produced by an animal against its own antigens. Hence, raw serum and hybridoma culture medium containing anti-SHANK3 antibody are "purified antibodies" within the meaning of the present invention.

EXAMPLE 1 Materials and methods

Subject collection

Cohort A

[00110] The SCZ samples included in this study were selected from over 1,000 SCZ families and 3000 DNA samples we collected from several large SCZ clinical genetic research centres in the world. These include: (1) Lynn E. DeLisi cohort of cases from USA and Europe (45 cases): Dr .DeLisi and her collaborators had identified and collected over 500 families with schizophrenia or schizoaffective disorder in at least two siblings over the last two decades. Diagnoses were made by using DSM-III-R criteria on the basis of structured interviews³⁷, review of medical records from all hospitalizations or other relevant treatment, and structured information obtained from at least one reliable family member about each individual. Two independent diagnoses (one made by L.E.D.) were made for each individual in the study. In cases of disagreement between the diagnosing clinicians, a third diagnostician was consulted, and final diagnoses were made by consensus after discussion. The sibling with earlier age of onset and more definite SCZ diagnosis were selected for the initial screening. (2) Ridha Joober cohort (103 cases): Dr Joober has collected over 300 SCZ families in Montreal in the past 10 years. The same clinical assessment procedures have been followed as in Dr DeLisi's study. In addition, extensive pharmacological data have been collected in this cohort. (3) Judith Rapoport cohort of childhood SCZ in USA (33 cases): Cases with childhood onset schizophrenia (COS) were recruited nationwide and assessed as previously described. Individuals in this cohort known to carry the VCFS deletion on chromosome 22q11 were excluded. To summarize briefly, all patients met DSM-IIIR/DSM-IV criteria for schizophrenia or psychosis not otherwise specified (NOS), had premorbid full-scale IQ scores of 70 or above and onset of psychotic symptoms by age 12 years. (4) Marie-Odile Krebs cohort in France (4 cases): All subjects were examined according to a standardized interview, the Diagnostic Interview for Genetic Studies (DIGS 3.0) (translated into French by Krebs and colleagues) was used. Family histories of psychiatric disorders were also collected using the Family Interview for Genetic Studies (FIGS). All DIGS and FIGS have been reviewed by two or more psychiatrists for a final consensus diagnosis based on DSM-IIlR or DSM-IV at each centre. Exclusion criteria for all subjects included neurologic hard signs, a history of head trauma and substance abuse or dependence. We obtained a SCZ cohort of 185 trios from the following population ancestries: 135 European, 35 non-European Caucasians, 5 African, and 10 mixed origins. The 285 controls (225 European, 58 non-European Caucasians, 1 African and 1 Asian) were recruited by advertisements in local newspapers; and the responding volunteers were interviewed using DIGS and clinical examinations. Only individuals without any neuropsychiatric symptoms or family history of neuropsychiatric problems, including any psychotic symptoms, were included as negative controls. The ethnicity was determined by self-reported ethnic origin of four grandparents. 95 were recruited by MO Krebs (INSERM) and 190 by R. Joober (Montreal). All samples were collected through informed consent following approval of each of the studies by the respective institutional ethics review committees. Genomic DNA was extracted from blood using a Puregene extraction kit (Gentra System, USA). In all cases, rare mutations were confirmed using blood derived DNA. Parents were not available for the schizophrenia negative controls.

Cohort B

[00111] The second cohort studied included subjects having parental as well as de now mutations.

Schizophrenic subjects were selected from over 1000 schizophrenia families (2000 affected individuals) ascertained for genetic studies of schizophrenia, for which DNA samples were available. In order to ensure accurate diagnoses all individuals were evaluated by experienced investigators using the Diagnostic Interview for Genetic Studies (DIGS) or Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS)³⁷³⁸, and multidimensional neurological, psychological, psychiatric, and pharmacological assessments at different centres. Family history for psychiatric disorders was also collected using the Family Interview for Genetic Studies (FIGS). All DIGS and FIGS have been reviewed by two or more psychiatrists for a final consensus diagnosis based on DSM-IIIR or DSM-IV at each centre. For all probands, specific inclusion criteria for the present study were : (1) The selected proband was definitely affected with schizophrenia only, not schizophreniform psychosis or bipolar depression with psychosis; (2) In families with multiple affected individuals, was selected the most severe schizophrenia case with early (<18 yrs) or childhood onset (<12 yrs), and/or additional neurodevelopmental problems, such as mental retardation, dyslexia and epilepsy, but not autistic disorder; for the childhood onset schizophrenia (COS) cohort, mental retardation and epilepsy were exclusionary (3) DNA from both parents was available, except for 8 proband samples, including 7 from Pakistan and one from Montreal, which were chosen from large multiplex families regardless of the availability of the parents' DNA; (4) Family history was well documented. Exclusion criteria included: (a) patients with psychotic symptoms mainly caused by alcohol, drug abuse, or other clinical diagnoses including major cytogenetic abnormalities; (b) patients with any of the four grandparents with Asian, African, Jewish, Arabic, Hispanic and American-Indian backgrounds were excluded. The final panel of the samples included 143 schizophrenia subjects from 5 different centres, i.e., 28 cases of COS from NIMH, USA²², 33 cases of adult onset schizophrenia from Paris, France²³, 12 cases of adult onset schizophrenia from Montreal, Canada²⁴, 63 cases of adult onset familial schizophrenia from New York, USA²⁵, and 7 cases of adult onset schizophrenia selected from each of seven large highly consanguineous pedigrees with ≥ 10 affected individuals with schizophrenia and schizoaffective disorders from Pakistan. Detailed description of ascertainment strategy, diagnostic instrument and criteria was reported by each centre in previous publications, except for the Pakistani samples. Unrelated, ethnically matched control individuals were used in this study. All samples were collected through informed consent, after approval of each of the studies by the respective institutional ethics review committees. Cohort B included 190 matched negative controls.

[00112] There is some overlap between patients from cohorts A and B.

Clinical cases

Family SCZ1

[00113] Of the 3 schizophrenia affected brothers, one was diagnosed with schizoaffective disorder with age of onset of 19 and the other two had a final diagnosis of an atypical chronic psychosis (PNOS) with ages of onset of 21 and 16 respectively. All 3 had evidence of mild mental retardation, but no formal testing was done. None had "schizophreniform psychosis". The mother was diagnosed with major depression and an unspecified neurological disorder for which she was administered carbamazepine. Father was well. No other psychiatric illness was present in the extended family on either side. The father's and mother's ages when the oldest affected son was born were 26 and 22, respectively.

Family SCZ2

[00114] This 23 year old girl was diagnosed with childhood onset schizophrenia with an age of onset of

11. At the time of evaluation in May and June 1995 she began to have periodic "episodes" consisting of agitation, disorganization, pressured speech and bizarre behavior (standing dazed in the shower) lasting several days and followed by episodes of withdrawal and loss of energy. She began having difficulty with school work, significant decline in overall functioning, and continued/worsening psychotic behaviors. Clinical status at last follow-up in 2005: she had not been rehospitalized but was living in a residential treatment centre which reported it was ill- equipped to manage her needs. Overall, she had limited/poor functioning. Medications at last follow-up in 2005 were Clozapine, and Olanzapine. The father's and mother's ages at the patient's birth were 38 and 39, respectively. There is no family history of schizophrenia. However, history of depression exists in both parents, both being on antidepressants as of 1999. The father and the mother had no Axis I or Il diagnosis within the affected definition of schizophrenia. The unaffected brother is healthy overall. Family SCZ4

[00115] This 13 year old boy at referral had a delay of several years in age-appropriate interactions with peers and delayed language. The Autism Screening Questionnaire score was 20. At age 8 he had a marked change in behaviors with oppositional behavior and deterioration in class work and behavior in school. He was confused about assignments and started to hear voices and talk inappropriately. He was diagnosed with undifferentiated type and chronic schizophrenia. The father of the proband has anxiety, mood disorder and obsessive compulsive disorder. The mother is considered normal.

DNA preparation

[00116] Genomic DNA was extracted from blood sample for each individual using Puregene™ extraction kit (Gentra System, USA). For certain individuals where blood DNA was limited, DNA isolated from a lymphoblastoid cell line derived from the individual was used for the screen. In all cases, rare mutations were confirmed using blood derived DNA to rule out variations having arisen during production or growth of the lymphoblastoid cell line.

Paternity testing

[00117] Paternity, maternity and unique genetic identification of each individual of all families (subject families and controls) was confirmed using at least 5 highly informative unlinked microsatellite markers. Families for which genotyping data were previously available (used in other genotyping projects, published and unpublished data) were genotyped using a panel of 5 highly informative microsatellite markers (D3S1043, D4S3351, D6S1043, D8S1179, and D15S659). Families not previously studied were genotyped using a panel of 9 additional markers (D1S533, D2S1327, D9S1118, D10S677, D11S1984, D12S1295, D14S587, D16S748, and D17S2196). PCR fragments were labelled by incorporation of radiolabeled -³⁵S-deoxyadenosine (³⁵SdATP) monophosphate, the fragments were separated on 5% denaturing polyacrylamide gels, and the gels were exposed to Hyperfilm MP film (Amersham). All fragments were amplified using a common PCR amplification protocol: 50 ng of DNA template were used with Taq polymerase (Qiagen) PCR initiation at 94⁰C for 5 min, denaturation at 94⁰C for 30 sec, 35 cycles at 55⁰C for 30 sec, elongation at 72⁰C for 30 sec, final elongation at 72⁰C for 10 min. Allele sizes were determined by comparison to an M13mp18 sequence ladder and numbered according to the Fondation Jean Dausset CEPH database (http://www.cephb.fr/). Microsatellite primer pair sequences were obtained from the Human Genome Database web site (http://www.gdb.org/). Primer pairs for markers used to determine the parental origin of the de novo mutations are described in Figure 8. PCR conditions were as described above except a cycling temperature of 55⁰C was used. Parentage was confirmed using the CERVUS™ (v3.0) program²⁶. Gene screening, variation analysis and bioinformatics

[00118] The coding region (exons) and the splice junctions of SHANK3 gene were sequenced in each proband. Exon numbering¹² and cDNA sequence³ were as previously described. Primers were designed using the Exon Primer program from the UCSC genome browser (Figure 7). PCR products were sequenced at the McGiII University and Genome Quebec Innovation Centre in Montreal, Canada (www.genomequebecplatforms.com/mcgill/) on a 3730XL™ DNA Analyzer System. In each case, variations were confirmed by re-amplifying the fragment and re-sequencing of the proband and both parents using reverse and forward primers. PolyPhred™ (version 6.11) and Mutation Surveyor™ (version 3.10, Soft Genetics Inc.) were used for mutation detection analysis. The PolyPhen²⁷, SIFT²⁸ and SNAP²⁹ programs were used to predict the overall severity of the missense mutations. In all instances default parameters were used for each program. Orthologous protein sequences were identified from GenBank by performing BLASTp or tBLASTn searches (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) using the mouse peptide sequence (NP_067398, SEQ ID N0:11)) on the following model organisms: chimp (AC145340, SEQ ID NO:76)), Rhesus monkey (AANU01101358, SEQ ID NO:77)), dog (XPJ48271 , SEQ ID NO:5)), rat (NP_067708, SEQ ID NO:12)), Opossum (XP.001366729, SEQ ID N0:6)), Lizard (AAWZ01039630, SEQ ID NO:78)), Xenopus (CX494866, SEQ ID NO:79)), Zebrafish zs3.1 (, SEQ ID NO:9)) and zs3.2 (, SEQ ID NO:15)).

Statistical methods for quantifying when a gene has an extreme number of cfe novo mutations with high relative risk

[00119] The probability that any individual harbours a de novo mutation in a gene depends on the length of that gene and the rate at which de novo mutations arise. The goal is to determine whether or not an observed de novo mutation (DNM) in a gene among controls is "too extreme" to have happened by chance. Specifically, we are interested in the cases when total number of DNMs m and the relative risk ©/(^-^) are both at least as large as what is observed. The cumulative probability of observing by chance values of (m_fn) that are more extreme in this direction than a given X₁ = (Mi₁ N₁) is thus sought. The data for each gene consists of a number of individuals with de novo mutations distributed among K cases and C controls. Consider a gene of coding length L, and let m and n be the numbers of individuals with (at least) one de novo mutation among the cases and controls, respectively. The data can be viewed in a 2 x 2 contingency table as shown below:

[00120] A gene is viewed as extreme when either the number of de novo mutations m + n or the relative risk ascribed to de novo mutations mC/nK combine to be surprisingly large. To quantify this level of surprise, we make the naive assumption that each nucleotide examined has an equal probability q of mutating in one generation. As each individual has two copies of the gene of interest, the probability that any one individual has at least one cte novo mutation in that gene is the probability p = 1 - (1 - q)^2L of observing at least one cfe novo mutation in 2L nucleotides. Under the null hypothesis that the relative risk ascribed to de novo mutations is one, the probability of observing m de novo cases and n de novo controls in a gene of length L is:

PKM, JV) =p*^+w(l - p)^C+z-^M-» φ Q

Thus, the probability of seeing a chance result at least as extreme as what was observed is the p-value

where 1_A is the indicator function of the event A.

[00121] For a per base mutation rate of 3 X 1(H for humans¹³, the number of cfe novo mutations at the

SHANK3 locus was significantly higher in the cases (p = 1.06 x 1(H) versus controls. Assuming a higher per base mutation rate of 1 x 1(F, as observed for CpG sites, the number of de novo mutations at the SHANK3 locus remained significant (p=0.024).

Test for excess of deleterious mutations at SHANK3 locus.

[00122] The McDonald-Kreitman (MK) test2 compares the ratio of synonymous and nonsynonymous polymorphisms within a species with the ratio of synonymous and non-synonymous fixations between species (in comparison with an outgroup species) within a single gene region; the expectation is that this ratio is the same under neutrality (Table 1 below). A Fisher's Exact test was performed. Even though the ratio of polymorphism is two times larger than that of substitutions, the two-tailed p-value (p= 0.22) suggests that there is no significant excess of deleterious mutations.

Molecular Cloning

[00123] Full-length (HA)-rat Shank3 in pRK5 was a gift from Paul F. Worley. The HA-rat Shank3-

Δ3'UTR was cloned into pcGlobin2³⁰ to synthesize the mRNA by using the mMESSAGE mMACHINE™ T7 kit (Applied Biosystems). The human R536W and R1117X mutations, corresponding to R535W and R1119X of the rat Shank3 protein, respectively, were generated by site-directed mutagenesis (Stratagene, La JoIIa, CA), and confirmed by sequencing. The human and rat Shank3 peptide sequences share 94,7 % identity. HEK293 Shank3 expression

[00124] HEK293 cells were transfected with a control plasmid or with plasmids encoding the HA-rat

Shank3 wt, R1117X and R536W using Lipofectamine 2000 (Invitrogen, CA). After cell lysis, the total protein extracts were subjected to SDS-polyacrylamide gel electrophoresis, transferred to Hybond-P™ PVDF membrane (GE Healthcare, UK) and immunoblotted with an anti-HA antibody (HA.C5, Abeam, MA).

Validation of SHANK3 mutations in zebrafish

[00125] Antisense morpholino oligonucleotides (AMO) (Gene Tools LLC, Philomath, OR) were used to knockdown both zebrafish Shank3 orthologous genes (zs3.1 or zs3.2) Two antisense morpholino oligonucleotides (AMO, Gene Tools LLC, Philomath, OR) were designed to target the initial codon of zs3.1 (5'- AGATCCTCCATAGGTTCGGAGCCAC-S¹, (SEQ ID NO:13)) and near a splicing junction of zs3.2 (5'- CTCCTCGCAAGACAAAGCCGAATCC-3', (SEQ ID NO: 14)). Full-length (HA)-rat SHANK3 pRK5 was a generous gift from Paul F. Worley. The HA-rat SHANK3-Δ3'UTR was cloned into pcGlobin2³⁰. The aa identity for the two zebrafish genes is 71% between one another and 63% (zs3.1, SEQ ID NO:9) and 65% (zs3.2 SEQ ID NO: 15) compared with the human sequence. The morpholinos were selective for either zebrafish homologue. The KD was dose-dependent, as expected. Further, the similarity of the phenotypes caused by KD of either gene (zs3.1 or zs3.2; data combined) and the partial rescue by rat Shank3 in both cases is consistent with a significant and specific KD. The KD is likely partial rather than complete and this would also be consistent with the heterozygous disease phenotype. The human R536W, R1117X, and P1134H mutations, corresponding to R535W, R1119X and P1136H of the rat SHANK3 protein, respectively, were generated by site-directed mutagenesis (Stratagene, La JoIIa, CA, see the "Molecular cloning section above). The AMOs were pressure- injected in one to four cell stage blastulae. After establishing the phenotype (0.75mM AMO), rescue experiments were performed in which both AMOs and rat SHANK3 wild type mRNA or mutated mRNA (100ng) were injected. 48 hours post fertilization transmitted light images were captured using a digital camera (Axio Cam HRC, Zeiss) mounted on a dissecting microscope (Stemi SV 11, Zeiss) and Axiovision™ 4.2 software and the response to touch was documented using high-speed (250 frames/sec) Photron Fastcam™ PCI video camera mounted on a Zeiss dissection microscope. Representative images from these films were used to reconstruct the movements of larvae in Figure 3a and b. The response to touch was documented at high-speed (250 frames/sec). Since no human SHANK3 cDNA was available for our analysis, and the human (SEQ OD NO:1) and rat SHANK3 (SEQ ID NO: 12) protein sequences are 95% identical, we considered the rat gene to be a reasonable substitute. The rat cDNA is what has been used primarily in functional studies of SHANK3.

Neuronal culture and transfection

[00126] Hippocampal neuron cultures were prepared from embryonic rats (E18) as described³¹. Briefly, dissociated neurons were co-transfected with GFP and wild type or mutant forms of Shank3 by electroporation using a Nucleofector Kit (Amaxa Inc.). Shank3 expression was detected in GFP-transfected cells with monoclonal antibodies (NeuroMabs, Davis, CA₁) and a secondary Alexa Fluor 555-labeled donkey anti-mouse IgGI antibody (Molecular Probes, Eugene, OR). F-Actin was visualized by Coumarin Labeled Phalloidin (Sigma- Aldrich). To quantify neurite numbers, neurites extending from the cell bodies (primary neurites) were quantified with Northern Eclipse Version 7.0 image analysis software (Empix Imaging, Mississauga, ON, Canada).

EXAMPLE 2

Identification of inherited and de novo mutations in SHANK3

[00127] Twenty-one out of twenty-two exons encoding the major brain-expressed isoform of SHANK3 were screened in both cohorts (A and B) of schizophrenia patients, using PCR amplification from genomic lymphoblastoid cell line DNA followed by direct sequencing. A total of 15 coding variants were identified, including 11 missense, 7 silent and 1 nonsense mutations. Each of these variants was genotyped in the respective proband's parents to determine inheritance mode. Two variants (R1117X and R536W) were not found in the parents and therefore constituted cfe novo mutations (Figure 1). Both mutations involved a C to T transversion at a CpG dinucleotide. Parentage of proband/parent trios and DNA authenticity in all family members were confirmed with >99.9% confidence using a panel of microsatellite markers (Figure 9). Each de novo mutation was confirmed in DNA isolated from blood. These two mutations nor any other protein-truncating SHANK 3 mutations were not detected in all ethnically matched unaffected controls (cohorts A and B).

[00128] The nonsense cfe novo mutation, R1117X, was detected in a proband and his two affected brothers (Fig. 1A, SCZ1). DNA from the proband's two affected brothers was genotyped for the R1117X mutation. Surprisingly, all 3 affected brothers shared the same R1117X nonsense mutation, suggesting the presence of germline mosaicism in one of the parents (Figure 1a). Haplotype analysis using markers near the SHANK3 gene (Figure 9A, PED 419 (SCZ1)) determined that all 3 brothers inherited the same paternal chromosome, but individuals II-2 and 11— 3 inherited different maternal chromosomes, suggesting that the nonsense mutation was inherited from the father. The proband is of European ancestry and has a diagnosis of schizoaffective disorder (age of onset 19 years), while the two brothers are diagnosed with atypical chronic psychosis (ages of onset of 21 and 16 years). All three had evidence of mild mental retardation. No other psychiatric illness was present in the extended family on either side. The R1117X mutation results in a truncated protein, as confirmed by expression analysis, lacking the Homer- and Cortactin-binding sites and the SAM (Sterile alpha motif) domain (Fig. 2A and C).

[00129] The missense cfe novo R536W mutation was identified in a 23-year-old girl, also of European ancestry, who was diagnosed as schizoaffective (age of onset 11 years). This patient had normal growth, non- dysmorphic features, speech impairment and poor academic and social performance. In order to exclude a possible diagnosis of autism, the Autism Screening Questionnaire³³ was completed and her score was = 1 (score > 15 = autism). The R536W mutation was absent from the healthy brother and unaffected parents' DNA (Figure 1b). It was not possible to determine whether the cfe novo R536W mutation had occurred in the father or the mother, or in the conceptus (Figure 9, PED56, SCZ2)). The R536 residue is located in close proximity to the SH3 domain and is perfectly conserved from mammals to fish (Fig. 2b), suggesting an important role for this residue in the function of the protein. The cfe novo R536W missense mutation is predicted to have a damaging effect on SHANK3 function (according to PolyPhen™, 1.89; SIFT™, O; and SNAP™, 4%²⁷'²^). Thus two αfe novo mutations in SHANK3 were identified in tow cohorts of unrelated schizophrenia patients. Each mutation was predicted to severely affect gene expression or protein function and at least one cfe novo mutation was paternally derived.

[00130] To evaluate whether SHANK3 is accumulating deleterious mutations in human populations, we performed a standard population genetic test³⁴ which asks whether there is an excess of potentially disruptive amino acid mutations accumulating at this locus more so than expected by chance. (Table 1, below). No excess of amino acid mutations segregating at this locus relative to a neutral expectation (2-tailed, p= 0.6796) were detected. Furthermore, over the entire coding locus (5196 nucleotides), only 3 fixed amino acid differences were observed between humans and chimps, relative to 16 silent substitutions which is similarly constrained compared to other brain expressed genes³⁵. This suggests that SHANK3 is not accumulating deleterious mutations or mutating at an unusual rate in the population at large and that the observations in the schizophrenia cohort are an exception.

[00131] Table 1: variations within the SHANK3 coding region. Substitutions were tabulated by comparing human and chimp (Pan troglodytes) orthhologous DNA sequence alignments.

[00132] Another mutation, the P1134H variant was found in one schizophrenia patient, but not in control subjects (cohort B). This mutation was chosen for further study because of its presence in a conserved proline- rich functional domain. The Pro1134 residue is conserved in all species examined (Figure 2b), and the substitution was predicted to be damaging (Figure 10). The P1134H missense was inherited from the proband's father, who is diagnosed with anxiety, mood disorder and obsessive compulsive disorder (Figure 1c and figure 9, SCZ 4). EXAMPLE 3 In vivo effect of SHANK3 mutations

Identification of transmitted mutations in sporadic cases

[00133] It was established herein that deleterious mutations in the SHANK3 gene cause schizophrenia. In fact, Applicant has discovered two different deleterious de novo mutations (not present in the parents, R1117X and R536W, Table 1) in individuals affected with schizophrenia. During our SHANK3 gene screening in a cohort of 185 sporadic schizophrenia patients (cohort A) 5 transmitted missenses (Table 2) not detected in controls were also identified (see Table 2 below).

Table 2. Transmitted mutations in sporadic cases

EXAMPLE4 In vivo effect of SHANK3 mutations

[00134] To determine whether the R1117X₁ R536W, and P1134H mutations had a detrimental effect on

SHANK3 function in vivo, their ability to rescue a SHANK3 knockdown phenotype in the zebrafish embryo was tested. This was tested by monitoring swimming activity that is due to a well-integrated synaptic drive³². The expression of either of the two zebrafish zSHANK3 orthologous genes (zs3.1 (SEQ ID NO:102 ) and zs3.2 (SEQ ID NO: 10 )) was knocked down by injecting selective antisense morpholino oligonucleotides (AMOs) into blastocysts. Knockdown of either gene (n=99 for zs3.1, n=92 for zs3.2) resulted in a reduction in size of the head, eyes and trunk (Figure 3a). In addition, the knockdown embryos were unable to swim in response to touch (Figure 3b and c, n=191). A small proportion (8%) showed milder deficits and could slowly swim in response to touch (Figure 3b and c). Because human SHANK3 cDNA was not readily available, the ability of rat SHANK3 to rescue the knockdown phenotype was tested and a partial rescue (Figure 3c, n=107) resulting in a significantly increased proportion of mild (78%) compared to severe (16%) phenotypes (p<0.001) was observed. In contrast, co-injection of rat SHANK3 mRNA bearing the R1117X mutation (n=43) or the P1134H mutation (n=58) failed to rescue the phenotype, whereas mRNA bearing the R536W mutation (n=39) partially rescued the phenotype (Figure 3b and c). Overexpression of wt (n=76) or any of the mutations (n=39 for R536W, n=35 for P1134H, n=24 for R1117X) was without effect (not shown). These results suggest that the R1117X and P1134H mutations had a dramatic loss-of-f unction and not a gain-of-function effect in vivo. The R536W had no obvious effect in the assay, though this does not rule out a pathogenic effect in schizophrenia. EXAMPLE 5 Effect of SHANK3 mutations on differentiation of rat hippocampal neurons

[00135] The consequences of Shank3 mutations on the overexpression of Shank3 in transfected rat hippocampal neurons was also examined. Expression of WT (Fig. 4B) or R536W (Fig. 4C) Shank3 resulted in increased somatic sprouting of neurites compared to control neurons (Fig. 4A; summarized in Fig. 4E), whereas the equivalent truncation mutation R1117X failed to promote sprouting (Fig. 4D). These results suggest that the R1117X mutation has a dramatic loss-of-function and not a gain-of-function effect in vivo. Although the R536W had no obvious effect in either of our assays, our genetic findings suggest it is likely to exert a pathogenic effect in humans in the form of SCZ.

[00136] SHANK3 is a scaffolding protein that promotes the formation and maturation of dendritic spines^{11 17}. Although the R536W missense mutation requires additional study, we predict that the R1117X and P1134H mutations reported here will result in a loss of SHANK3 function during synaptic development. Accordingly, individuals harbouring a mutated SHANK3 allele are expected to display immature or abnormal dendritic structure, as observed in the hippocampus and neocortex in some schizophrenia patients¹⁸. This would also be compatible with current hypotheses suggesting that synaptic dysfunction occurs in a significant fraction of schizophrenia cases. Finally, it is proposed that the de novo mutation mechanism described here for SCZ is plausible for other brain diseases which have so far resisted conventional genetic approaches.

[00137] The most likely explanation for the identical de novo nonsense mutation in three affected brothers in pedigree SCZ1 is germline mosaicism in the father, as described previously for a 300 kb deletion of the neurexin 1 gene in two autistic sisters¹⁶, for a cfe novo 1 Mb duplication at 17p12 in two affected autistic sibs¹⁶, and for a cfe novo 4.36 Mb deletion involving the SHANK3 gene in two autistic sibs (pedigree ASD3)³. The SCZ1 pedigree represents an example of why conventional genetic approaches have mostly failed to identify schizophrenia risk factors, since even a low frequency of de novo mutations would lead to significant allelic genetic heterogeneity and render association studies problematic. Given the monozygotic twin schizophrenia concordance rates of 65%-80% it is entirely plausible that a SHANK3 mutation carrier, such as the father of SCZ4 and possibly the father of SCZ3, would not have schizophrenia.

[00138] Comparative genomic methods estimate that in any single conceptus there are -1-3 new deleterious mutations that lead to an altered amino acid per genome, which is on average 1 new mutation in 10,000 genes/zygote¹³'¹⁴'¹⁵. The significant excess of de novo mutations at SHANK3 found in schizophrenia subjects tested herein versus controls strongly suggests that disruption of this gene plays a role in the development of schizophrenia in some individuals. Deleterious SHANK3 mutations have previously been reported in mentally retarded and autistic individuals²¹², suggesting that the phenotypic spectrum of such mutations is rather broad. The present inventors report for the first time that mutations in the SHANK3 gene predispose to a schizophrenia phenotype. The subjects included in this study all had a strict diagnosis of schizophrenia and were ascertained as part of schizophrenia genetic studies. One of many possible explanations for this gene leading to several different phenotypes could be the type of causative mutation. In fact, 6 of the 7 de novo SHANK3 mutations described in autistic patients²³ were microdeletions encompassing the SHANK3 gene. The other mutation was a missense (Q321R) located in the ankyrin repeat domain of the protein.

[00139] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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Claims

CLAIMS:

1 A method for diagnosing the presence of schizophrenia or predicting the risk of developing schizophrenia in a human subject, comprising detecting the presence or absence of a defect in a SHANK3 gene encoding a polypeptide comprising a sequence as set forth in SEQ ID NO 2, in a nucleic acid sample of the subject, whereby the detection of the defect is indicative that the subject has or is at risk of developing schizophrenia

2 The method of claim 1 , wherein said sample compnses DNA

3 The method of claim 1 , wherein said sample compnses RNA

4 The method of claim 1, wherein the defect is a missense, nonsense or splice site mutation

5 The method of claim 1, wherein the defect is a nonsense or splice site mutation resulting in a truncated polypeptide lacking a Homer-binding site, a Cortactin-binding site and a sterile alpha motif domain

6 The method of claim 4, wherein the defect compnses a missence or nonsense mutation in the gene resulting in a mutant polypeptide in which at least one amino acid residue of SEQ ID NO 2 is substituted with another amino acid residue, and wherein the at least one amino acid residue is selected from the group consisting of alanine 224, arginine 536, proline 1134, valine 1333, histidine 493, serine 952, Alanine 1160, histidine 493 and arginine 1117

7 The method of claim 6, wherein the defect compnses a mutation in the gene resulting in a mutant polypeptide in which at least one amino acid residue of SEQ ID NO 2 is substituted with another amino acid residue, and wherein the at least one amino acid residue is selected from the group consisting of arginine 536, proline 1134, and arginine 1117

8 The method of claim 4, wherein the defect compnses a mutation in the gene resulting in a mutant polypeptide in which a) amino acid residue 224 of SEQ ID NO 2 is substituted with a threonine residue, or amino acid 536 of SEQ ID NO 2 is substituted with a tryptophan residue, or ammo acid 1134 of SEQ ID NO 2 is substituted with a histidine residue, or amino acid 1333 of SEQ ID NO 2 is substituted with a glycine residue, amino acid 493 is substituted with a glutamine residue , amino acid 952 is substituted with a threonine residue, or in which amino acid 1117 is substituted with a stop codon,, or b) a Homer binding site, a Cortactin binding site and/or a sterile alpha motif are absent

9 The method of claim 1, wherein the defect compnses a missense or nonsense mutation in the gene, wherein the corresponding cDNA is as set forth in SEQ ID NO 1, the mutation being selected from the group consisting of a substitution of a cytosine at position c.3349 (R1117) with another nucleotide, a substitution of a cytosine at position c.1606 (R536) with another nucleotide, a substitution of a cytosine at position c.3401 (P1134) with another nucleotide, a substitution of a guanine at position c.670 (A224) with another nucleotide, and a substitution of a thymine at position c.3998 (V1333) with another nucleotide, wherein said position are given with respect to the first nucleotide in the ATG initiator as nucleotide 1.

10. The method of claim 1 , wherein the defect comprises a mutation in the gene, wherein the corresponding cDNA is as set forth in SEQ ID NO: 1 , the mutation being selected from the group consisting of a substitution of a cytosine at position c.3349 with a thymine (R1117X), a substitution of a cytosine at position c.1606 with a thymine (R536W), a substitution of a cytosine at position c.3401 with an adenine (P1134H), a substitution of guanine at position c.670 with an adenine (A224T), and a substitution of thymine at position c.3998 with a guanine (V1333G).

11. A method of detecting the presence or absence of a mutation in a SHANK3 gene, said method comprising the steps of:

a) analyzing a nucleic acid test sample containing the gene;

b) comparing the results of said analysis of said sample of step a) with the results of an analysis of a control nucleic acid sample containing a wild type SHANK3 gene, wherein the wild type SHANK3 gene encodes a polypeptide comprising the sequence of Figure 5 (SEQ ID NO: X); and

c) determining the presence or absence of at least one defect in the SHANK3 gene of the test sample.

12. The method of any one of claims 1 to 11 , wherein the nucleic acid sample is amplified prior to analysis.

13. The method of any one of claims 1 to 12, wherein the defect is a mutation in the coding region of the SHANK3 gene.

14. The method of claim 11 , wherein the mutation is a missense, nonsense or splice site mutation.

15. The method of claim 11 , wherein the analysis is selected from the group consisting of sequence analysis, fragment polymorphism assays, hybridization assays and computer based data analysis.

16. A method of detecting the presence or absence of a mutation in a SHANK3 gene, said method comprising the steps of: a) analyzing a nucleic acid test sample containing the gene;

b) comparing the results of said analysis of said sample of step a) with the results of an analysis of a control nucleic acid sample containing a wild type SHANK3 gene, wherein the wild type SHANK3 gene comprises the sequence as set forth in SEQ ID NO: 1 ; and

17. The method of claim 16, wherein the nucleic acid test sample is amplified prior to analysis.

18. A method for determining the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the method comprising determining in a biological sample from said subject:

(a) the level of expression of a SHANK3 nucleic acid or encoded polypeptide;

(b) the level of activity of a SHANK3 protein;

(c) the presence or absence of a functional mutation in a SHANK3 nucleic acid or encoded protein; or

(d) any combination of (a) to (c), wherein a difference in said level relative to a corresponding control level or the presence of a functional mutation in said SHANK3 nucleic acid or encoded protein is indicative that said subject has or is at risk of developing schizophrenia.

19. A method of determining the existence of an association between a SHANK3 polymorphism and schizophrenia, comprising the steps of: (i) genotyping at least one polymorphism in a SHANK3 gene or encoded polypeptide, in a population having schizophrenia; (ii) genotyping said polymorphism in a control population; and, (iii) determining whether a statistically significant association exists between schizophrenia and said polymorphism.

20. A kit or package for detecting the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject, the kit comprising means for determining in a biological sample from said subject:

(a) the level of expression of SHANK3 nucleic acid or encoded polypeptide;

(b) the level of activity of SHANK3 protein;

(c) the presence or absence of a mutation in SHANK3 nucleic acid or encoded protein that modifies its activity and/or expression; or (d) any combination of (a) to (d);

together with instructions for correlating said level or said mutation with the presence of schizophrenia or a predisposition to suffer from schizophrenia, wherein a decrease in the expression of SHANK3 nucleic acid or encoded protein, a decrease in the activity of SHANK3 protein or the presence of a mutation in SHANK3 nucleic acid or encoded protein is indicative that said subject is suffering from schizophrenia or is predisposed to suffer from schizophrenia

21. A method of identifying a compound for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia, said method comprising determining whether:

(a) the level of expression of a SHANK3 nucleic acid or encoded polypeptide;

(b) the level of SHANK3 activity; or

22. A method of identifying or characterizing a compound for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia, said method comprising:

(b) determining whether reporter gene expression or reporter protein activity is increased in the presence of said test compound; said increase in reporter gene expression or reporter protein activity being an indication that said test compound may be used for decreasing susceptibility to schizophrenia or for preventing or treating schizophrenia

23. A purified SHANK3 polypeptide comprising at least one of: a valine at position 224, a tryptophan at position 536, a Histidine at position 1134, a glycine at position 1333, and a deletion at position 1117.

24. A purified SHANK3 polypeptide comprising a mutation in at least one of: an alanine at position 224, an arginine at position 536, a proline at position 1134, a valine at position 1333, an histidine at position 493, a serine at position 952, an alanine at position 1160 and an arginine at position 1117.

25. A purified antibody that binds specifically to the polypeptide of claim 23.

26. A method of determining whether a biological sample contains the polypeptide of claim 23, comprising contacting the sample with a purified ligand that specifically binds to the polypeptide, and determining whether the ligand specifically binds to the sample, the binding being an indication that the sample contains the polypeptide.

27. The method of claim 26, wherein the ligand is a purified antibody.

28. An isolated SHANK3 nucleic acid moloecule comprising a sequence as set forth in SEQ ID N0:1 comprising a substitution of a cytosine at position c.3349 (R1117) with another nucleotide, a substitution of a cytosine at position c.1606 (R536) with another nucleotide, a substitution of a cytosine at position C.3401 (P1134) with another nucleotide, a substitution of a guanine at position c.670 (A224) with another nucleotide, and a substitution of a thymine at position c.3998 (V1333) with another nucleotide.

29. A vector comprising the nucleic acid molecule of claim 28.

30. A recombinant host cell comprising the vector of claim 29.

31. An isolated nucleic acid molecule encoding a polypeptide comprising a valine at position 224; a tryptophan at position 536; a histidine at position 1134; a glycine at position 1333; a glutamine at position 493, a threonine at position 952, a deletion at position 1117 or a combination thereof.

32. A vector comprising the nucleic acid molecule of claim 31.

33. A recombinant host cell comprising the vector of claim 32.

34. A method of diagnosing schizophrenia or susceptibility to suffer from schizophrenia in a subject comprising determining in a biological sample from said subject the presence or absence of a mutation in a SHANK3 nucleic acid or a mutation which shows linkage disequilibrium therewith, wherein the identification of a mutation in said SHANK3 in at least one allele of said subject is indicative that the subject has or is at risk of developing schizophrenia, and wherein said mutation in said SHANK3 nucleic acid is selected from the group consisting of:

35. An isolated SHANK3 nucleic acid moloecule comprising a sequence as set forth in SEQ ID N0:1 comprising a substitution of a nucleotide at codon R1117 with another nucleotide, a substitution of a nucleotide at codon R536 with another nucleotide, a substitution of a nucleotide at codon P1134 with another nucleotide, a substitution of a nucleotide at codon A224 with another nucleotide, and a substitution of nucleotide at codon V1333 with another nucleotide, and a substitution of nucleotide at codon H493 with another nucleotide with another nucleotide, a substitution of nucleotide at codon S952 with another nucleotide, a substitution of nucleotide at codon A1160A, with another nucleotide, wherein said substitution results in a non sense or missense functional mutation.