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  • C07—ORGANIC CHEMISTRY
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/136—Screening for pharmacological compounds
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/30—Psychoses; Psychiatry
  • G01N2800/302—Schizophrenia

Definitions

• 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.
• 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.
• Schizophrenia is thought to be due to polygenic inheritance 1 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 ago 4 .
• This proposition was seriously considered, based on studies demonstrating that the heritability of schizophrenia is 6-8 times higher in monozygotic versus dizygotic twins 56 , as well as studies showing a significantly increased risk of schizophrenia with increasing paternal age 7 , reduced rates of marriage, fertility, and reproduction in schizophrenia patients compared to controls 89 .
• 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.
• SHANK3 The SHANK3 gene maps to a region of chromosome 22q previously associated with large deletions causing mental retardation or other developmental brain abnormalities 12 and recently SHANK3 mutations were described in autistic patients 23 .
• SHANK3 is a scaffolding protein abundant in the post-synaptic density of excitatory synapses on dendritic spines 10 .
• [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.
• 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.
• mutations in SHANK3 were previously reported in autism 23 , 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.
• 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.
• said sample comprises DNA.
• said sample comprises RNA.
• the defect is a missense, nonsense or splice site mutation.
• the defect comprises a mutation in a gene causing a modification in gene or protein expression or in protein function.
• said modification in gene or protein expression is a diminution of gene or protein expression.
• the modification in protein function is a reduction in the biological activity of the SHANK3 protein.
• 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.
• SAM sterile alpha motif
• 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.
• 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.
• 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).
• 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).
• 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 method of detecting the presence or absence of a mutation in a SHANK3 gene 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.
• the nucleic acid sample is amplified prior to analysis.
• the defect is a mutation in the coding region of the SHANK3 gene.
• the mutation is a missense, nonsense or splice site mutation.
• the analysis is selected from the group consisting of: sequence analysis; fragment polymorphism assays; hybridization assays and computer based data analysis.
• 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).
• 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).
• 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.
• the ligand is a purified antibody.
• 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.
• 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)
• 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).
• 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).
• a vector comprising the nucleic acid molecule of the present invention.
• a recombinant host cell comprising the vector of the present invention.
• the subject is pre-diagnosed as suffering from schizophrenia or as being a likely candidate for developing schizophrenia.
• 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.
• the likely candidate has at least one family member suffering from schizophrenia.
• 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:
• 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.
• kits or package for detecting the presence of schizophrenia or a predisposition to suffer from schizophrenia in a subject comprising means for determining in a biological sample from said subject:
• SHANK3 nucleic acid e.g., SEQ ID NO:1
• encoded polypeptide e.g., SEQ ID NO:2
• 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);
• 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.
• 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.
• said developmental effect is a reduction in size of the head, eyes and/or trunk of the zebrafish.
• said behavioral effect is a reduction in the capacity of the zebrafish to swim in response to touch Jn
• said SHANK3 activity is the level of neuronal differentiation or neurite outgrowth.
• said shank 3 activity that is assessed is an increase in the level of somatic sprouting of neurites compared to control neurons.
• 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:
• 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).
• 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).
• 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.
• 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:
• 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.
• the histidine residue at position 493 is substituted with a glutamine residue
• the serine residue at position 952 is substituted with a threonine residue.
• 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).
• 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).
• 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
• 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
• FIG. 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.
• 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.
• Figure 5 shows the amino acid sequence of SHANK3 protein (SEQ ID NO: 2).
• 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 ( * );
• Figure 7 shows PCR primer (SEQ ID NOs:16-61) pairs used for screening of the SHANK3 gene.
• Figure 8 shows PCR primer (SEQ ID NOs:62-75) pairs used to determine the parental origin of the cfe novo mutations.
• Figure 9 shows the segregation of microsatellite markers in pedigrees showing SHANK3 cfe novo and potentially deleterious mutations.
• Figure 10 shows the prediction of functional effect of detected missense and nonsense variants in the SHANK3 gene in schizophrenia patients and controls using PolyPhenTM, SIFTTM, and SNAPTM programs. * Observed in controls in Durand et al. 2007 2 .
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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 .
• 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.
• 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.
• 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.
• 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 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 .
• 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.
• the identification of one or more functional mutations enables the adaptation of a prophylactic or treatment regimen.
• 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.
• the identity of the polymorphism is determined by performing a hybridization assay, a sequencing assay, a microsequencing assay or an allele-specific amplification assay.
• 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.
• RNA expressions or sequences 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.
• ASO allele-specific oligonucleotide
• SSCP single- stranded conformation polymorphism
• Some of these approaches 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• Methods for calibrating the level of expression of a gene are well known in the art.
• the expression of a gene can be calibrated using reference samples, which are commercially available.
• reference samples include, but are not limited to: Stratagene® QPCR Human Reference Total RNA, ClontechTM Universal Reference Total RNA, and XpressRefTM Universal Reference Total RNA.
• 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).
• a control level i.e., 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.
• 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.
• a substantially similar level measured in a biological sample from a subject i.e., test sample
• a higher or increased level is indicative that said subject does not have or is at not at risk of developing schizophrenia.
• 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.
• 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.
• 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.
• 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.
• testing of a marker in linkage disequilibrium with the polymorphisms of the present invention at a SHANK3 gene 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.
• 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.
• a marker e.g., polymorphism
• 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:
• 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.
• said diagnostic kits further comprise buffers and reagents for detecting said defect as well as instructions for using said diagnostic kits.
• sample biological sample
• clinical sample 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.
• 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.
• the biological sample of the present invention is a crude sample (i.e., unpurified).
• 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.
• 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.
• 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.
• the mammal is human.
• 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.
• 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:
• the above-mentioned method is an in vitro method.
• 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.
• 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).
• 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.
• 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.
• the defect comprises a mutation which deletes at least exon 10 from said nucleic acid resulting in a smaller SHANK3 polypeptide.
• a reporter assay-based method of selecting agents which modulate SHANK3 expression 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 ( ⁇ )-D-galactosidase, luciferase, chloramphenicol acetyltransferase and green fluorescent protein (GFP).
• 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.
• the above-mentioned method is an in vitro method.
• the promoter region normally associated with a SHANK3 gene is a wild type promoter region found in a control subject.
• 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.
• 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.
• said developmental effect is a reduction in size of the head, eyes and/or trunk of the zebrafish.
• said behavioral effect is a reduction in the capacity of the zebrafish to swim in response to touch .
• said SHANK3 activity is the level of neuronal differentiation or neurite outgrowth.
• said shank 3 activity that is assessed is an increase in the level of somatic sprouting of neurites compared to control neurons.
• 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.
• 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., P 32 , C 14 , H 3 ), 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.
• radiolabelling e.g., P 32 , C 14 , H 3
• antibody-based detection e.g., fluorescence, chemiluminescence
• spectroscopic methods e.g., generation of a product with altered spectroscopic properties
• reporter enzymes or proteins e.g., horseradish peroxidase, green fluorescent protein
• specific binding reagents e.g., biotin/(stre
• 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.
• one or a plurality of the assay steps are automated.
• 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.
• 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.
• 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.
• the substituting amino acid 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.
• a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell.
• a mutation can be, for example, a detectable change in one or more deoxyribonucleotide.
• nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position.
• Spontaneous mutations inherited or cte novo
• 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.
• 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.
• missense mutation or “non sense mutations” is meant to denote mutations which cause SHANK3 to be less functional (active) or inactive.
• missense or non-sense functional mutations of the present invention fail to rescue knock down of sz3.1 and sz3.2 in zebrafish.
• 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).
• sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.
• 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%.
• 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.
• 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 W 1 T and X determine the sensitivity and speed of the alignment.
• W word length
• B BLOSUM62 scoring matrix
• E expectation
• P(N) the smallest sum probability
• 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.
• 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 65 0 C, and washing in 0.2 x SSC/0.1% SDS at 42 0 C (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).
• 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 68 0 C (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).
• 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.
• 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.
• 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
• Such host cells may be prokaryotic or eukaryotic, bacterial, yeast, amphibian or mammalian.
• 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.
• a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
• operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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, PepBodiesTM, 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.
• 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.
• a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin,
• Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermal ⁇ at multiple sites.
• the antigen or conjugate e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice
• 3 volumes of Freund's complete adjuvant e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice
• the antigen or conjugate e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice
• the antigen or conjugate e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice
• the antigen or conjugate e.g., with 1/5 to 1/10 of the original amount used to immunize
• the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
• 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.
• 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).
• a mouse or other appropriate host animal such as a rat, hamster or monkey
• is immunized e.g., as hereinabove described
• 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.
• 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.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
• 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.
• purified antibody 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.
• raw serum and hybridoma culture medium containing anti-SHANK3 antibody are “purified antibodies” within the meaning of the present invention.
• 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 interviews 37 , 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.
• the 285 controls 225 European, 58 non-European Caucasians, 1 African and 1 Asian
• 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.
• 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.
• DIGS Diagnostic Interview for Genetic Studies
• K-SADS Kiddie Schedule for Affective Disorders and Schizophrenia
• 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, USA 22 , 33 cases of adult onset schizophrenia from Paris, France 23 , 12 cases of adult onset schizophrenia from Montreal, Canada 24 , 63 cases of adult onset familial schizophrenia from New York, USA 25 , 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.
• Genomic DNA was extracted from blood sample for each individual using PuregeneTM 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.
• PCR fragments were labelled by incorporation of radiolabeled - 35 S-deoxyadenosine ( 35 SdATP) 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 94 0 C for 5 min, denaturation at 94 0 C for 30 sec, 35 cycles at 55 0 C for 30 sec, elongation at 72 0 C for 30 sec, final elongation at 72 0 C for 10 min.
• Taq polymerase Qiagen
• PolyPhredTM version 6.11
• Mutation SurveyorTM version 3.10, Soft Genetics Inc.
• the PolyPhen 27 , SIFT 28 and SNAP 29 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)).
• 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.
• DNS de novo mutation
• the data for each gene consists of a number of individuals with de novo mutations distributed among K cases and C controls.
• 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:
• 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.
• the probability of observing m de novo cases and n de novo controls in a gene of length L is:
• 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).
• ⁇ 3'UTR was cloned into pcGlobin2 30 to synthesize the mRNA by using the mMESSAGE mMACHINETM 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 cells were transfected with a control plasmid or with plasmids encoding the HA-rat
• Antisense morpholino oligonucleotides (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-S 1 , (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- ⁇ 3'UTR was cloned into pcGlobin2 30 .
• 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.
• 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.
• Hippocampal neuron cultures were prepared from embryonic rats (E18) as described 31 . 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, CA 1 ) 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).
• 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).
• 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 PolyPhenTM, 1.89; SIFTTM, O; and SNAPTM, 4% 27 ' 2 ⁇ ).
• SHANK3 function accordinging to PolyPhenTM, 1.89; SIFTTM, O; and SNAPTM, 4% 27 ' 2 ⁇ .
• two ⁇ fe 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.
• Table 1 variations within the SHANK3 coding region. Substitutions were tabulated by comparing human and chimp (Pan troglodytes) orthhologous DNA sequence alignments.
• 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 drive 32 .
• AMOs selective antisense morpholino oligonucleotides
• Shank3 mutations 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).
• the R1117X mutation has a dramatic loss-of-function and not a gain-of-function effect in vivo.
• 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.
• SHANK3 is a scaffolding protein that promotes the formation and maturation of dendritic spines 11 17 .
• 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 patients 18 . 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.
• 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.

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