WO2012106404A2 - Diagnosis and treatment of neurological disorders through vipr2 and vpac2r - Google Patents

Abstract

This invention provides diagnostic and therapeutic compositions and methods to detect and treat neurological disorders, such as schizophrenia, autism, and other related disorders through modulating vasointestinal peptide receptor 2 gene (VIPR2) expression or correcting pathogenic mutations of the VIPR2 gene, and through modulating the VIPR2 encoding vasointestinal peptide 2 receptor (VPAC2R) activity in the cycHc-AMP signaling with selective VPAC2R agonists, VPAC2R antagonists, and VIP or peptide derivatives or analogs thereof.

Description

DIAGNOSIS AND TREATMENT OF NEUROLOGICAL DISORDERS THROUGH VIPR2 AND VPAC2R

CROSS REFERENCE TO RELATED APPLICATIONS

[00011 This application claims a priority of U.S. Provisional Application Serial

No. 61/438,453, entitled "Diagnosis and treatment of schizophrenia and related disorders with selective agonists and antagonists of VPAC2R," filed February I, 201 1, the entire application is incorporated by reference herewith.

FIELD OF THE INVENTION

[0002] This invention provides diagnostic and therapeutic methods to detect and treat neurological disorders, such as schizophrenia, autism, and other related disorders.

BACKGROUND OF THE INVENTION

[0003] Rare copy number variants (CNVs) play a prominent role in the etiology of schizophrenia and other neuropsychiatric disorders' . Substantial risk for schizophrenia is conferred by large (>500 kb) CNVs at several loci, including microdeletions at lq21.1 ^z, 3q29 15q I3.3 ² and 22q l 1.2 - and microduplication at 16pl 1.2 However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood.

[00041 A majority of the rare CNVs that have been implicated in schizophrenia involve large (> 500 Kb) genomic regions where local segmental duplication architecture promotes frequent and nearly identical rearrangements by non-allelic homologous recombination (NAHR). Because of the high structural mutation rates at these loci, the strong phenotypic effects of the causal variants, and the excellent power of most array platforms to detect such large CNVs, these genomic hotspots were the first to be detected in studies of CNVs in schizophrenia. Since most of the genome lacks the duplication architecture of the NAHR hotspots described above and because a variety of mutational mechanisms can give rise to structural rearrangements, causal variants in other regions of the genome may consist of CNVs that are individually more rare and smaller (< 500 kb) than those arising at NAHR hotspots. For example, microdeletions of the gene Neurexin-1 (NRXN1), which are highly enriched in autism and schizophrenia ^&z, consist of overlapping deletions with non-recurrent breakpoints. NRXN 1 deletions are not flanked by segmental duplications, and may occur by different mutational mechanisms such as non-homologous end joining (NHEJ) or DNA replication-mediated rearrangement. |0005| VIPR2 encodes the vasoactive intestinal peptide (VIP) receptor VPAC2, a

G protein-coupled receptor that is expressed in variety of tissues including, in the brain, the suprachiasmatic nucleus, hippocampus, amygdala, and hypothalamus ^~. VPAC2 binds VIP ^u, activates cyclic-AMP signaling and PKA, regulates synaptic transmission in the hippocampus ^{i &} and promotes the proliferation of neural progenitor cells in the dentate gyrus '^. Genetic studies in mouse have established that VIP signaling plays a role in learning and memory— . VPAC2 also plays a role in sustaining normal circadian oscillations in the SCN ^{Ά Ά}, and VIPR2-null "^~ and VlPR2-overexpression ^ mice exhibit abnormal rhythms of rest and activity.

[0006] VIPR2 is known to play a role in the cardiovascular and gastrointestinal system, and it is this application that has driven early efforts to develop selective agonists , and antagonists of VPAC2R. Peptide derivatives ²⁶— and small molecules— have been identified that are selective VPAC2 agonists or antagonists

SUMMARY OF THE INVENTION

[0007] This invention provides diagnostic and therapeutic methods and compositions to detect and treat neurological disorders, such as schizophrenia, autism, and other related disorders through modulating vasointestinal peptide receptor 2 gene (VIPR2) expression or correcting pathogenic mutations of the VIPR2 gene, and/or through modulating the VIPR2 encoding vasointestinal peptide 2 receptor (VPAC2R) activity in the cyclic-AMP signaling with selective VPAC2R agonists, VPAC2R antagonists, and/or VIP or peptide derivatives thereof.

[0008] In certain embodiments, the present invention provides novel generic r_ methods for the; identification of patients that carry mutations in VIPR2, and further detection of mutations in DNA that impact the function of VIPR2. These methods include, but are not limited to, microarray, PCR and mass spectrometry based methods. The present invention further provides that the patients carrying the mutations in VIPR2 can benefit from treatment with selective agonists or antagonists of VPAC2R, and/or VIP or peptide derivatives.

[0009] The present invention further provides diat mutations in DNA that modify the expression of the V1PR2 gene are pathogenic. The pathogenic mutations include, but arc not limited to, copy number variants (CNVs), which can be detected using multiple methods that have been developed. These methods include, but are not limited to, ,. microarray analysis and computational methods for analysis of microarray data, PCR and Mass Spectrometry based detection. The present invention further provides that other mutations of the VIPR2 genomic region, including CNVs, indels and single nucleotide variants can be detected by DNA sequencing.

[0010] The present invention also provides that ovcrcxpression of the

Vasointestinal Peptide Receptor 2 (V1PR2) gene confers a high risk of a neurological : disorder, such as schizophrenia and autism. As a direct implication of this finding, specific compounds that selectively modulate the activity of the VPAC2R receptor that is encoded by VIPR2, including selective agonists, antagonists, VIP, and/or VIP peptide derivatives, are provided as treatments for these and related disorders. More specifically, the present invention provides the first conclusive genetic link between VIPR2 and disorders of the brain, and for the first time the pathogenic mechanism of V1PR2 including alterations in the_cexpression of the VIPR2 gene and alterations in cyclic-AMP signaling by the VPAC2 receptor.

{00111 In certain embodiments, the present invention provides the application of selective antagonists and agonists in the treatment of neurological disorders, including, but not limited to schizophrenia, autism, and related disorders. In certain embodiments, the present invention provides that treatment effective amounts of antagonists of VPAC2R, .:; such as compounds I and 2 published by Chu et al. peptide derivatives of VIP published by Moreno et al.— , such as selective antagonists PG-96-238 and PG-99-465, and partial agonist PG-97-277 and PG-97-278, modulate the activity of VIPR2, consequently modulate the disease process.

[0012| In one embodiment, the present invention provides that a synthetic peptide and a selective agonist, BAY-55-9837, modulates the pathogenic activity of VIPR2 in human cells. The present invention demonstrates that VIP and BAY 55-9837, a peptide derivative of VIP that selectively binds to VPAC2, elicit a high level of cyclic-AMP accumulation in lymophoblastoid cell lines of patients with pathogenic mutations of V1PR2. The cAMP accumulation observed in patients is significantly higher than in controls, indicating that the pathogenic mechanism of VIPR2 mutations is modulated by compounds active against the VPAC2 receptor. Therefore, the present invention provides that the pathogenic action of VIPR2 can be corrected by treatment with these and related compounds or peptides. A patient, in which human cells are overexpressing VIPR2 can be treated with a selective antagonist of VPAC2. Conversely a patient in which human cells are underexpressing VIPR2 can be treated with VIP or another selective agonist of ,^■ VPAC2, such as«AY 55-9837.

|0013| The present invention further provides a novel approach to using genetic testing to guide the selection of appropriate drugs for modulation of VPAC2R activity and consequently to treat neurological disorders, such as schizophrenia and autism. The present invention provides that copy number of the V1PR2 is directly correlated with VPAC2R activity, and thus a genetic test can be used for gene copy number to infer the ^■' effect of mutations on gene function. Furthermore, based on the VIPR2 expression and VPAC2R activity, a therapeutic strategy is provided whereby molecular testing of human cells can be effectively used to guide the selection of appropriate drugs for individual patients.

[0014] In certain embodiments, overexpression or underexpression of VIPR2 is detected through genetic analysis of DNA and RNA from human cells. Changes in VPAC2 activity are detected through analysis of cyclic-AMP accumulation in human cells. The efficacy of drug candidates is evaluated by monitoring DNA and RNA while treatment of human cells with drug candidates in culture. Human cells include, but are not limited to, blood, lymphoblastoid cell lines, fibroblasts, induced pluripotent stem cells (iPSCs) or cell types derived from iPSCs.

|0015| In certain embodiments, the novel approach to personalized treatment of schizophrenia and/or autism consists of applying the diagnostic and therapeutic inventions described above to the analysis of DNA and human cells from patients. Genetic analysis of DNA, biochemical analysis of V1PR2 expression, biochemical analysis of VPAC2 activity in human cells are used to infer the pathogenic mechanism (underexpression or overpression of VIPR2). Having determined the pathogenic mechanism, appropriate drugs are selected based on their ability to rescue VPAC2 activity in human cells from the patient. Appropriate drugs are selected based on their ability to rescue animal behavior in VIPR2 genetic mouse models, including a VIPR2 knockout and a VIPR2 expression mouse.

|0016] The present invention therefore provides that treatment of patients with compounds or peptides active against VPAC2 can be significantly improved using the diagnostic and personalized medicine of the present inventions provided herein. Selective antagonists or agonists of VPAC2 that are known to or later discovered to modulate the disease process appropriately in the in vitro and in vivo systems constitute new antipsychotic drugs for the treatment of schizophrenia, autism and related disorders.

; [0017] A. research tool model for identifying a therapeutic candidate and/or drug leads for treatment, diagnosis, prognosis or prevention of schizophrenia or autism in a mammal is also provided by the present invention. The present invention provides molecular assays of human cells for testing the efficacy of drug leads or candidates in vitro, in certain embodiments, the present invention provides appropriate genetic mouse models of VIPR2 for testing the efficacy of drug leads in vivo.

^■ |0018| These and other inventions and variations will be apparent to one of skilled in the art in view of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1. Duplications and triplications of 7q36.3 result in increased VIPR2 transcription and cyclic-AMP signaling, (a) Quantitative PCR results of VIPR2 mRNA: from lymphoblastoid cell lines. Two to four subjects were tested for each of four genotypes (subtelomeric duplication, VIPR2 duplication, exon 3/4 triplication, and normal diploid copy number as control). Results arc expressed as the mean fold-change of CNV carriers relative to the mean of control samples, (b-c) Cyclic AMP accumulation was measured in the same cell lines in response to VIP ( H)OnM) and the VPAC2 agonist BAY 55-9837 (l OOnM). Results arc expressed as fold-change over forskolin/IBMX alone, (d) No significant differences were observed in cAMP response to another GPCR agonist, Prostaglandin E2 (PGE2, 1 μΜ), demonstrating that the effects are specific to VPAC2. For subjects, error bars represent standard error of the mean computed across replicates. Differences between the groups of 9 duplication carriers and 4 controls were tested using unpaired two sample t-tests. [0020] Figure 2. Gel Electrophoresis of Quantitative PGR (qPCR) Products from

Lymphoblastoid Cell Line-Derived RNAs Analyzed.

[0021] Figure 3. Structure of VPAC2 antagonist hits compounds 1 and 2. The full name of compound 1 is (2R,4S)-2-benzyl-4-hydroxy-N-((ls,2R)-2-hydroxy-2,3-dihydro- lH-inden-l-yl)5-(4-nitrophenylsulfonamido)pcntanamide. The full name of compound 2 - is (2R,4S)-2-benzyl-5-(4<ert-but>dphenylsulfonainido)-4-hydro-N-((lS_J2R)-2-hydroxy- 2,3-dihydro-lH-inden-I -yl)pentan amide.

[0022] Figure 4. Peptide sequence alignment of peptide derivative of VIP. PG 96-

238 (SEQ ID NO: I), PG-97-277 (SEQ ID NO:2), PG-97-278 (SEQ ID NO:3), and PG-99-465 (SEQ ID NO:4). All the peptides were carboxyl-terminally amidated. The residues boxed are different from those of VIP (AcH: acetyl-histidine; Nle: Norleucine; yrH: myristoyl-histidine: the bracket between ²¹ and D²⁵ indicates the presence of a lactam bridge between the ε aminoacid residue of the lysine 21 and the β carboxyl residue of aspartate 25).

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a novel approach for diagnosis and personalized treatment of neurological disorders, such as schizophrenia, autism, or other related neurological disorders by detecting one or more pathogenic mutations or an abnormal gene expression level of VIPR2 in the patient, then treating the patient with an effective amount of a modulator that is able to correct the pathogenic effect of the mutations, modulating the VIPR2 gene expression, resulting in modulating the activity of VPAC2R in the cyclic AMP signaling functions. The present invention also provides a novel approach for diagnosis and personalized treatment of neurological disorders, such as schizophrenia, autism, or other related neurological disorders by modulating VPAC2R activity directly with an effective amount of selective VPAC2R agonists, VPAC2R antagonists, and/or VIP or peptide derivatives or analogs thereof. Furthermore, a research tool model for identifying a therapeutic candidate and/or drug leads for treatment, diagnosis, prognosis or prevention of schizophrenia, autism, or other related neurological disorders is also provided by the present invention. |0024| In certain embodiments, the present invention provides a method and composition for treatment of a neurological disorder comprising administering to a patient in need a treatment effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically active agent selected from the group consisting of a selective antagonist and or agonist of vasoactive intestinal peptide 2 ,. receptor (VPAQ2R), a modulator of VPAC2R or VPAC2R gene (aka VIPR2), and a vasoactive intestinal peptide (VIP) derivative or analog.

10025) As used herein, the term ^: VPAC2R" or "VPAC2" is used to refer to and in conjunction with the particular receptor for a vasoactive intestinal peptide (VIP) and putuitary adenylate cyclase-activating peptide (PACAP) that encoded by VIPR2 gene (for review see Dickson & Finlayson 2009, Pharmacol Ther 121: 294-316). The term - "selective antagonist and/or agonist of vasoactive intestinal peptide 2 receptor (VPAC2R)" refers to an antagonist or agonist that selectively inhibits or activates the VPAC2 receptor with increased selectivity for the VPAC2 receptor compared to other known receptors. The degree of selectivity is determined by a ratio of VPAC2 receptor binding affinity to VPAC I receptor binding affinity and by a ratio of VPAC2 receptor binding affinity to PAC 1 receptor binding affinity. Preferably, the agonists of the present invention have a selectivity ratio where the affinity for the VPAC2 receptor is at least 50 times greater than for the VPACl and/or for PACl receptors. More preferably, the affinity is at least 100 times greater for VPAC2 than for VPACl and/or for PACl . Even more preferably, the affinity is at least 200 times greater for VPAC2 than for VPACl and/or for PACl . Still more preferably, the affinity is at least 500 times greater for VPAC2 than for VPACl and/or for PACl. Yet more preferably, the affinity is at least 1000 times greater for VPAC2 than for VPAC 1 and or for PAC 1.

r.

|0026| Compounds and/or peptides that are selective for VPAC2 receptor were initially identified by modifying vasoactive intestinal peptide (VIP) and /or PACAP (See e.g.,, WOO 1/23420, WO04/006839, which are hereby incorporated by reference). As used herein, the selective antagonist or agonist of VPAC2 receptor includes, but is not limited to, now known or later developed peptide and/or chemical compounds that show selectively high binding affinity to the VPAC2 as compared to the binding affinity to the other known receptors, such as VPACl and PAC l. Exemplary selective VPAC2 receptor antagonists and agonists are disclosed in literature, such as Chu et al. (2009, Molecular Pharmacology, 77( 1): 95-101 ) (disclosing compounds 1 and 2) and US Patent Publication No. 2009/0118167 to Bokvist et al. (disclosing selective VPAC2 receptor peptide agonists), the entire contents of these publications are incorporated by reference herewith. In certain embodiments of the present invention, the VPAC2 receptor antagonists are compound 1 and compound 2 with the chemical structures shown in Figure 3, and ,. derivatives and analogs thereof.

|0027J As used herein, the term "VIP" refers to naturally occurred vasoactive intestinal peptide (VIP) having a single amino acid sequence containing 28 naturally occurred amino acids. The term "naturally occurring amino acid" as used herein means the twenty amino acids coded for by the human genetic code (i.e. the twenty standard amino acids). These twenty amino acids are: Alanine, Arginine, Asparagine, Aspartic :^■ Acid, Cysteine, ^s Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and, Valine. The term "VIP derivative or analog" refers to peptides or compounds that differ from naturally occurred VIP amino acid sequence by substitution, addition or removal of one or more amino acid. The amino acid used for substitution or addition can be either naturally occurring amino acids as defined above, or non-naturally occurring amino acids, which include both synthetic amino acids and those modified by the body. These include D-amino acids, arginine-like amino acids (e.g., homoarginine), and other amino acids having an extra methylene in the side chain ("homo" amino acids), and modified amino acids (e g norlcucine, lysine (isopropyl)-- wherein the side chain amine of lysine is modified by an isopropyl group). Also included are amino acids such as ornithine and amino isobutyric acid.

- [0028] As used herein, the VIP derivatives and/or analogs include, but are not limited to, those that are widely known in the art or later developed. Exemplar)' known VIP derivatives and/or analogs are natural VIP related peptides, such as hclodermin and helospectin which are isolated from the salivary excretions of the Gila Monster - (Heloderma Suspectum); the VIP peptide analogs disclosed in W091/06565 comprising a combination of the amino acid sequence of VIP with a part of the amino acid sequence of helodermin; and linear or cyclic VIP analogies (see e.g., US Patent No. 5,677,419; EP 0536,741 ; US Patent Nos. 6,080,837; 6,316,593; WO 97/29126, each incorporated by reference) including a particular cyclic VIP analogue RO 15-1392 that shows to be a selective VPAC2 receptor agonist (Bolin et al. 1997, J. Pharmacol. Exp. Ther. 281(2):,. 629-633). In certain embodiments of the present invention, the VIP derivatives or ' analogues are selective VPAC2 receptor peptide antagonist or partial agonist developed from the cyclic VIP analogues as starting point, such selective VPAC2 receptor peptide antagonists or partial agonists include, but arc not limited to, PG 96-238, PG 99-465, PG 97-277, and PG 97-278 (see e.g., Moreno et al., 2000, Peptides, 21 : 1543-49). The amino acid sequences of these selective VPAC2 receptor peptide antagonist or partial agonist are shown in Figure 4. |0029| As used herein, the term "modulator" refers to any compounds and/or peptides, naturally occurring or synthetically synthesized, that provide any effect on the VPAC2 protein expression or the activity of the VPAC2 receptor or the expression or the activity of at least one protein involved in VPAC2 receptor induced c-AMP signaling pathway. The term "modulator" also refers to any compounds and/or peptides, naturally occurring or synthetically synthesized, that provide any effect on the expression, the activity, and/or correction of pathogenic mutations of the VIPR2 gene encoding the VPAC2 receptor protein. The "effect" as used herein refers to optionally a partial or complete stimulation, but preferably a partial or complete inhibition. Thus, the modulator as used herein preferably inhibits the expression or the activity of VPAC2 receptor and/or at least one proteins involved in the VPAC2 receptor induced c-AMP signaling pathway, or inhibits die VIPR2 gene expression or corrects one or more pathogenic mutations, ^" including but not limited to, copy number variants (CNVs), duplications, indels, or single nucleotide variants of V1PR2 gene. As used herein, the term "VIPR2 gene expression" is intended to mean the amount of mRNA of VIPR2 expressed, and the term "VPAC2 protein expression" is intended to mean the amount of VPAC2 protein expressed. [0030] In certain embodiments, the modulator is a VPAC2R or VPAC2 signaling pathway inhibitor. The term "inhibitor" refers to a chemical compound or substance which eliminates or substantially reduces the biological activity of the VPAC2R or VPAC2 signaling pathway proteins. The term "substantially" signifies a reduction of at least 25%, preferably of at least 35%,' even more preferably of at least 50%, and more preferably at least 70% or 90%. The modulator as used herein may be of any type. They may be of natural origin or may have been produced by chemical synthesis.

|0031| In certain embodiments, the modulator may be an inhibitory anti-VPAC2R or anti-VPAC2R-signalling pathway antibody, including a monoclonal antibody. The modulator may also be a polypeptide, an antisense D A or RNA polynucleotide, an si- RNA, or a PNA (Peptide nucleic acid, polypeptide chain substituted with purine and pyrimidine bases, the spatial structure of which mimics that of the DNA and allows hybridization thereto). The modulator may also be an aptamer. The aptamer is a class of molecules representing, in terms of molecular recognition, an alternative to antibodies. They are oligonucleotide sequences having the ability to recognize virtually all classes of target molecules with high affinity and specificity. Such ligands can be isolated by systematic evolution of ligands by exponential enrichment (SELEX) of a random sequence library as described by Tuerk and Gold (1990, Science 249: 505-510). The random sequence library can be obtained by combinatorial chemical DNA synthesis. In this library, each member is an optionally chemically modified linear oligomer of a single sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena (1999, Clinical Chemistry 45(9): 1628-1650).

[0032] As used herein, the term "pharmacologically active agent," "therapeutic agent," "active agent," or "drug" is used interchangeably to refer to a chemical material or compound that induces a desired pharmacological, physiological effect, and include agents that are therapeutically or prophylactically effective. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives and analogs of those active agents specifically mentioned herein, including but are not limited to, salts, esters, amides, prodrugs, active metabolites, inclusion complexes, analogs, and the like. When the terms "pharmacologically active agent," "active agent," and "drug" are used, it is to be understood that applicants intend to include the active agent per sc as well as pharmaceutically. acceptable, pharmacological ly active salts, esters, amides, prodrugs, active metabolites, inclusion complexes, analogs, etc., which are collectively referred to herein as "pharmaceutically acceptable derivatives".

[0033| The pharmaceutically active agents as used herein may also refer to any oligonucleotides (antisensc oligonucleotide agents), polynucleotides (e.g. therapeutic DNA), ribozymes, dsRNAs, siRNA, RNAi, gene therapy vectors, and/or vaccines for :; therapeutic use.^; The term "antisense oligonucleotide agent" refers to short synthetic segments of DNA or RNA, usually referred to as oligonucleotides, which are designed to be complementary to a sequence of a specific mRNA to inhibit the translation of the targeted mRNA by binding to a unique sequence segment on the mRNA. Antisense oligonucleotides are often developed and used in the antisense technology. The term "antisense technology" refers to a drug-discovery and development technique that involves design and use of synthetic oligonucleotides complementary to a target mRNA to inhibit production of specific disease-causing proteins. Virtually all diseases are associated with inadequate or over-production of proteins. Traditional small molecule drugs are designed to interact with disease-causing proteins and inhibit their function. Γη contrast, antisense technology permits design of drugs, called antisense oligonucleotides, which intervene at the genetic level and inhibit the production of disease-associated proteins. Antisense oligonucleotide agents are developed based on genetic information. [00341 As an alternative to antisense oligonucleotide agents, ribozymes or double stranded RNA (dsRNA), RNA interference (RNAi), and/or small interfering RNA (siRNA), can also be used herewith as pharmaceutically active agents. As used herein, the term "ribozyme" refers to a catalytic RNA-based enzyme with ribonuclease activity that is capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which it has a complementary region. Ribozymes can be used to catal tically cleave target mRNA transcripts to thereby inhibit translation of target mRNA. The term "dsRNA," as used herein, refers to RNA hybrids comprising two strands of RNA. The dsRNAs can be linear or circular in structure. The dsRNA may comprise ribonucleotides, ribonucleotide analogs, such as 2'-0-methyl ribosyl residues, or combinations thereof. The term "RNAi" refers to RNA interfernce or post-transcriptional gene silencing (PTGS). The term "siRNA" refers to small dsRNA molecules (e.g., 21-23 nucleotides) that arc the mediators ■ _;: of the RNAi effects. RNAi is induced by the introduction of long dsRNA (up to 1 -2 kb) produced by in vitro transcription, and has been successfully used to reduce gene expression in variety of organisms. In mammalian cells, RNAi uses siRNA (e.g. 22 nucleotides long) to bind to the RNA-induced silencing complex (RISC), which then binds to any matching mRNA sequence to degrade target mRNA, thus, silences the gene.

[0035| As used herein, the pharmaceutically active agents also include any vectors/virus useti for gene therapy. The term "gene therapy" refers to a technique for correcting defective genes responsible for disease development. Such techniques may include inserting a normal gene into a nonspecific location within the genome to replace a nonfunctional gene; swapping an abnormal gene for a normal gene through homologous recombinations, reparing an abnormal gene to resume its normal function through selective reverse mutation; and altering or regulating gene expression and/or functions of a particular gene. _c In most gene therapy, a normal gene is inserted into the genome to replace an abnormal or disease-causing gene. As used herein, a term "vector/virus" refers to a carrier molecule that carries and delivers the "normal" therapeutic gene to the patient's target cells. Because viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner, mostcommon vectors for gene therapy are viruses that have been genetically altered to carry the normal human DNA. As used herein, the viruses/vectors for gene therapy include retroviruses, adenoviruses, adeno- associated viruses, and herpes simplex viruses. The term "retrovirus" refers to a class of viruses that can create double-stranded DNA copies of their RNA genomes, which can be further integrated into the chromosomes of host cells, for example, Human immunodeficiency virus (HIV) is a retrovirus. The term "adenovirus" refers to a class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in human, for instance, the virus that cause the common cold is an adenovirus. : The term "adenci-associated vims" refers to a class of small, single-strandcd DNA viruses that can insert their genetic material at a specific site on chromosome 19. The term "herpes simplex viruses" refers to a class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type I is a common human pathogen that causes cold sores. [0036] As used herein, a term "treatment effective amount" refers to the quantity that results in a desired therapeutic and/or prophylactic effect without causing unacceptable side effects when administered to a patient in need of the invention ■^: pharmaceutical composition. A "desired therapeutic effect" includes one or more of the following: 1 ) an amelioration of the symptom(s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment. For example, an "treatment effective amount" of a VPAC2 receptor antagonist for the treatment of schizophrenia is the quantity „ that would result, in greater control of symptoms associated with schizophrenia dian in the absence of treatment.

[0037] A " treatment effective amount" as used herein will also depend on the type and severity of the disease and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The dose of the invention composition administering to a patient in need will depend on a number of factors, among which are included, without limitation, the patient's sex. weight and age, the type and or severity of the disease, die route of administration and bioavailability, the pharmacokinetic profile of the agent, the potency, the formulation, and other factors within the particular knowledge of the patient and physician. Thus, it is not necessary to specify' an exact effective amount herein. However, an appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Furthermore, the exact effective amount of an active agent incorporated into a composition or dosage form of the present invention is not critical, so long as the concentration is within a range sufficient to permit ready application of the solution or formulation so as to deliver an amount of the active agent that is within a therapeutically effective range.

[0038) As used herein, solutions are homogeneous mixtures prepared by dissolving one or more chemical substances (solute) in another liquid such that the molecules of the dissolved substance are dispersed among those of the solvent. The solution may contain other pharmaceutically acceptable chemicals to buffer, stabilize or preserve the solute. Commonly used examples of solvents used in preparing solutions are saline, water, ethanol, propylene glycol or any other pharmaceutically acceptable vehicle. [0039) A typical dose range for the therapeutic composition of the present invention will range from about ^g per day to about 5000 μ§ per day. Preferably, the dose ranges from about 1 per day to about 2500 μg per day, more preferably from about 1 g per day to about 1000 ug per day. Even more preferably, the dose ranges from about 5 ug per day to about 100 |ag per day. Needless to say, a person skilled in the art will take care of select the suitable amount of the invention composition, such that a desired therapeutic and/or prophylactic effect is obtained without causing unacceptable side effects when administered to a patient in need of the invention pharmaceutical composition. ^~ |0040] As used herein, the term "pharmaceutically acceptable carrier" refers to carrier materials suitable for administration of a pharmaceutically active agent. Carriers useful herein include any such materials known in the art, which are nontoxic and do not interact with other components of the composition in a deleterious manner. Various additives, known to those skilled in the art, may be included in the composition of the present invention. For example, solvents, including relatively small amounts of alcohol, may be used t¾ solubilize certain drug substances. Other optional additives include opacifiers, antioxidants, fragrance, colorant, gelling agents, thickening agents, stabilizers, surfactants and the like. Other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, i.e., to inhibit growth of microbes such as yeasts and molds. Suitable antimicrobial agents are typically selected from the group consisting of the methyl and propyl esters of p-hydroxybenzoic acid (i.e., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, and combinations thereof.

|0041| The pharmaceutically active agent may be administered through any desirable route, such as oral and/or any parental administration, if desired, in the form of a salt, ester, amide, prodrug, derivative, or the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically. Salts, esters, amides, prodrugs and other derivatives of the active agents may be prepared using standard procedures known to those skilled in the ar^ of synthetic organic chemistry and described, for example, by March's Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 5th Ed. (Wiley- Interscience, 2001). Preparation of salts, ester, amides, prodrug, etc. is known to those skilled in the art or described in the pertinent literature. [0042) The invention composition is preferably administered to a human.

^"' However, the invention composition can also be administered to any mammals including animal, e.g., companion animals (e.g.. dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

|0043| In certain embodiments, a subject of the invention relates to an in vitro method for diagnosing, determining an individual's susceptibility to developing, or monitoring the progression of a neurological disorder, such as schizophrenia or autism, in a patient, which comprises the steps of a) obtaining a biological sample from said patient; b) detecting one or more pathogenic mutation, such as copy number variants (CNVs), duplications, indels, or single nucleotide variants, or an expression level of V1PR2 gene in said biological sample; and c) diagnosing said patient being at risk for said neurological disorder wherein one or more pathogenic mutation or overexpressed level of VIPR2 gene is detected in said biological sample of said patient. The present invention also contemplates an in vitro method for diagnosing or monitoring the progression of a neurological disorder in a patient comprising comparing the expression or activity of VPAC2R and/or at least one of the VPAC2R signaling pathway proteins in a biological sample from a patient, relative to a biological sample from a control individual.

|0044| The expression of the proteins can be determined by assaying the VPAC2R

c

and/or VPAC2R signaling pathway proteins according to one of the methods such as Western blotting, immunohistochemistry, analysis by mass spectrometry (Maldi-TOF and LC/MS analysis), radioimmunoassay (RIA) and ELISA or any other method known to those skilled in the art. Another method, in particular for measuring the expression of VIPR2 or VPAC2R signaling pathway gene, is to measure the amount of the corresponding mRNA by any method such as RT-PCR, ribonuclease protection assay, ^■: northern blotting; hybridisation-based microarray technologies or any other method known to those skilled in the art. Those skilled in the art are familiar with techniques for the quantitative or semi-quantitative detection of the mRNA and mutations of a gene of interest. Assaying the activity of VPAC2R or VPAC2R signaling pathway can also be envisaged. |0045j In the context of a diagnosis, the "control" individual is a "healthy" individual. In the context of monitoring the progression of a neurological disorder, such as schizophrenia or autism, the "control individual" refers to the same individual at a different time, which preferably corresponds to the beginning of the treatment (TO). This ^'·^■" measurement of the difference in expression or in activity of VPAC2R or at least one of the VPAC2R signaling pathway proteins, or in expression or mutation(s) of die VIPR2 gene or in activity of VIPR2 or at least one of VPAC2R signaling pathway genes, makes it possible in particular to monitor the efficacy of a treatment, in particular a treatment with a pharmaceutically active agent, as envisaged above, or another treatment against the neurological disorder. Such monitoring can reassure the patient as to the well-founded _ grounds or the need for continuing this treatment.

[0046| In these in vitro diagnostic or prognostic methods, the biological sample tested may be any sample of biological fluid or a tissue sample obtained by invasive or non-invasive methods. In certain embodiments, the biological sample is a brain tissue or cell obtained from a patient in need.

|0047| In certain embodiments, the present invention also provides an in vitro or in vivo method or a research tool model for identifying compounds which arc therapeutic candidates for treatment, diagnosis, prognosis or prevention of a neurological disorder, such as schizophrenia or autism, in a patient, comprising comprises combining the therapeutic candidate with a VIPR2 gene or other genes encoding at least one of the VPAC2R signaling pathway proteins, and observing or determining modulation of VIPR2 gene expression or correction of pathogenic mutations, such as copy number variants (CNVs), duplications, indels, or single nucleotide variants of the VIPR2 gene, said modulation indicating the usefulness of the therapeutic candidate for treatment, diagnosis, prognosis or prevention of a neurological disorder, such as schizophrenia or autism. In other embodiments, the present invention also provides an in vitro or in vivo method or a research tool model for identifying compounds which are therapeutic candidates for treatment, diagnosis, prognosis or prevention of a neurological disorder, such as : schizophrenia or autism, in a patient, comprising comprises combining the therapeutic candidate with VPAC2R or at least one of VPAC2R signaling pathway proteins, and observing or determining modulation of VPAC2R expression and/or activity in cyclic- AMP signaling, said modulation indicating the usefulness of the therapeutic candidate for treatment, diagnosis, prognosis or prevention of a neurological disorder, such as schizophrenia or autism.

|0048j The therapeutic candidates are compounds of any type. They may be of natural origin or may have been produced by chemical synthesis. They may be a library of structurally defined chemical compounds, of non-characterized compounds or substances, or a mixture of compounds. Various techniques can be used to test these compounds and .. to identify the compounds of therapeutic interest which are antagonist or agonist of VPAC2R, modulators of VPAC2R or VIPR2, or VIP derivatives or analogs thereof. [0049] An in vivo screening method can be carried out in any laboratory animal, for example a rodent. In certain embodiments, the screening method comprises administering the test compound to the animal, then optionally sacrificing the animal by euthanasia and taking a suitable biological sample before evaluating the expression and/or mutations of the 'VIPR2 gene, or expression or activity of its encoding V AC2R or at least one of the VPAC2R signaling pathway proteins, by any method described herein.

|0050] The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein. However before the present peptides, compounds, compositions, and methods are disclosed and described, it is to be understood that this ^" invention is not limited to specific nucleic acids, specific peptides or proteins, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is also to be understood that as used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.

[0051] Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0052] It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. These and many other variations and embodiments of the invention will be apparent to one of skill in the art upon a review of the appended description and examples.

EXAMPLES

Materials and Methods

1. Data Collection a) Primary Cohort

10053] The initial discovery data set was composed of 1,761 unrelated subjects analyzed on the NimbleGcn HD2 Array-CGH platform. The unfiltcred sample consisted of 913 patients and 848 controls ascertained at ten sites. Ascertainment of these samples for family-based studies is described in previous publications ^{24: i}. Microarray - hybridizations using the NimbleGen HD2 platform were performed at the service laboratory of Roche NimbleGen according to the manufacturer's specifications. Samples were filtered from the dataset based on data quality measures described below. Five duplicate samples from Trinity College Dublin also present in the ISC dataset were removed from the primary dataset. The final discovery data set consisted of 906 unrelated patients and 742 controls. b) Secondary Cohort

|0054| The secondary cohort consisted of Affymetrix Genome-Wide Human SNP

Array 6.0 data from the Genetic Association Information Network (GAIN) supported case-control studies, "Genome-Wide Association Study of Schizophrenia" (phs000021.v2.p l ), and the "Molecular Genetics of Schizophrenia - nonGAIN Sample" (phs000167.v l .pl). Both of these studies are subsets of the larger "Molecular Genetics of Schizophrenia Genome-wide Association" study, so the datasets were combined and referred to the merged cohort as "MGS" herewith. In total, the combined MGS sample consisted of 4, 195 unrelated cases and 3,804 controls. A further 429 controls assayed on the Affymetrix 6.0 by the Bipolar Genome Study (BiGS) were combined with the MGS ^" cohort to increase the number of controls. Genotyping of the MGS and BiGS cohorts was performed at the Broad Institute Center for Genotyping and Analysis. Further details concerning the MGS collection sites, principal investigators and case-control inclusion criteria are available through dbGap. Further information on the BiGs Collaboration is also described elsewhere— .

|0055J Several NIMH controls overlapped between the primary and secondary cohorts. Therefore, overlapping samples were eliminated from the dataset by retaining the experiment with the greatest probe density. Sample and experiment redundancy were also minimized in the secondary cohort by prioritizing studies in order of GAIN-MGS > nonGAIN-MGS > BiGS. In total, 4,097 cases and 3,508 controls assayed on the Affymetrix 6.0 platform were included in stage two of the analysis. CNV data from the International Schizophrenia Consortium (ISC) ⁴¹ were further combined with the - processed data to increase the final size of the secondary cohort to 7,488 cases and 6,689 controls. Because sample identifiers were not available for CNVs in the ISC dataset, each CNV in the ISC call set was treated as coming from an independent sample. This approximation is reasonable given that the mean rare CNV burden in this datxiset was less than one CNV per sample— .

2. Intensity Data Processing

10056] With the exception of data collected by ISC, all processing of microarray data was performed at Cold Spring Harbor Laboratory. Different methods were used for dual color intensity data from NimbleGen HD2 and single color intensity data from the Affymetrix Genome-Wide Human SNP 6.0 genotyping array. a) NimbleGen HD2

- 10057] NimbleGen HD2 dual color intensity data were normalized in a two step process: (1) a spatial normalization of probes was performed to adjust for regional differences in intensities across the surface of the array, and (2) the Cy5 and Cy3 intensities were adjusted to a fitting curve by invariant set normalization.

[0058] Spatial normalization was performed using "an R module provided by Kyle

Munn (Rochc-NimbleGen Inc). Invariant set normalization of intensity data involves selection of a probe set with minimal variability between the ranked test and reference autosomal probe intensities as described in Li et al The test intensities of this invariant autosomal probe set are then adjusted to the reference distribution. Based on these adjustments, a fitting curve is established to which all other intensities are shifted, preserving the variability in the data. The intensities of X and Y chromosomes were then extrapolated to the fitting curve. The process is repeated while exchanging the test and

- reference to simulate a dye swap experiment. The log₂ ratio for probe is then estimated using the geometric mean of normalized and raw intensity data of test (Tst) and reference (Rfit) as follows:

' NormalizedTst. Raw Tst_t

\og₂(Ratio,)

RawRfo_i NormalizedRfii_; J

- b) Affymetrix Genome-Wide Human SNP Array 6.0

|0059| A two-step normalization method was developed to process Affymetrix

SNP Array single color intensity data (Affymetrix 500K, Affymetrix 5.0 & Affymetrix 6.0). First, all arrays were normalized by invariant set normalization to a single reference array. To obtain the single reference array, the median-most array was selected based on autosomal intensities; then the correlation matrix was built for the adjacent 100

s.

^" experiments (50 lower and 50 higher than the median-most). The single array most highly correlated with all others was selected as the common reference for Invariant Set Normalization based on the sum of Pearson's correlation coefficients. The reference array itself was not normalized. Second, for each experiment the ratio of intensities in comparison to a^r"virtual reference genome" (VRG) was calculated. The VRG for each "test" is a composite of multiple individuals from the study population selected to minimize the overall variance in probe ratios. The VRG consisted of the median of the 10 most highly-correlated experiments in the dataset based on the Pearson correlation of 5% of probes on the array. This defined probe set was comprised of every 20^th autosomal SNP probe and the nearest adjacent copy number (CN) probe. Separate virtual reference genomes were computed for males and females in order to have a sex-matched VRG.

c) GC Correction of log₂ Ratios

|0060| The final step of data processing involved the correction of genomic waves effects in Iog₂ ratios due to regional correlations with GC content based on the fitted linear regression model proposed by Diskin el al ^Μ.

3. Copy Number Variant Discovery

|0061) In this study, two segmentation algorithms were used to discover copy number variants in the GC-corrected NimblcGcn HD2 and Aff metrix 6.0 ratio data: H MSeg ⁴⁵ and Genome Alteration Detection Analysis (GADA) . CNVs detected by both algorithms were used for downstream data processing and analysis. CNVs detected by only one algorithm were excluded. In addition, CNVs of the same type (i.e., deletion or duplication) that were separated by <3 probes were merged into one contiguous segment. The proximal and distal boundaries of overlapping and adjacent CNVs were defined by the minimal chromosomal start position and the maximal chromosomal end position of the CNVs. All CNV coordinates are based on the human genome build NCBI36/hgl8.

4. Data Filtering

[0062] Data filtering criteria were designed to minimize the false discovery rates of rare CNVs in the primary and secondary analysis data sets. In this section the filtering of CNVs and experiments in the NimbleGen HD2 and Affymetrix data sets were described. Further details describing the filtering criteria of rare CNVs are outlined below. a) NimbleGen HD2

- [0063| Filtering of NimbleGen HD2 experiments from analysis was based on the experiment quality and filtered CNV properties. Experiments with autosomal median absolute deviations (aMAD) >0.23, with >400 filtered autosomal CNVs and with >30Mb in combined CNV length (~1% of the human genome) were removed. In addition, any sample with aneuploidy of autosomal chromosomes was excluded, as were experiments with conflicting gender and empirically derived X and Y median probe ratios. ^"" [0064] CNVs that overlapped regions of the genome prone to somatic cell rearrangements were removed from the primary data set. In particular, CNVs intersecting or overlapping T-ccll receptor regions (chr7:38,245J05-38,365/141; chr7: 141,647,285- 142,221,100; chr9:33,608,462-33,652,656; chrl4: 21,159,896-22,090,937) and abParts (chr2:88,937,989-89,411,302; chr2:88,966,183-89,377,035; chr2:89,589,457-89,897,555; chrl4: 105,065,301-106,352,275; chr22:20,715,572-21,595,082) were excluded. CNVs ■^•..■with median probe ratios (seg. median) between 0.83 and 1.15 were also removed as were CNVs smaller than 100 kb or larger than 10Mb. b) Affymetrix 6.0

[00651 CNV and experiment filtering criteria similar to those used to process the primary NimbleGen HD2 dataset were implemented for analysis of CNVs in the „ secondary data .set, taking into account the non-uniformity of Affymetrix 6.0 probe distribution across the human genome. Probable somatic T-ccll receptor and abPart rearrangements were removed and CNVs were filtered out based on segment median thresholds. Noisy experiments with Median Absolute Deviation (MAD) >0.2 and chromosomal aneuploidies were eliminated from further analysis.

5. Rare CNV Formulation

[0066| Within each cohort, rare CNV frequencies were determined in the combined set of cases and controls (separately within each ethnic group). CNV frequencies were estimated based on 50% reciprocal overlap between CNV calls of the same type. CNVs with frequency > 1% were removed.

[0067] Rare CNV calls that passed the 1% frequency filter were further filtered based on the confidence score (CS). Thresholds for CS were adjusted for various size classes of CNVs and for each platform separately as shown in Table 5. As the CS score of a CNV call, the P-value derived from the outlier detection genotyping method, median Z- score outlier detection ( eZOD)— was used. Appropriate CS thresholds for each size class and platform were determined by examining patterns of endelian inheritance in a set of mother-father-child trios (of confirmed parentage). CS was adjusted to achieve a 5% rate of Mendelian inconsistency for rare CNVs (i.e., 5% of CNVs called in the child were not inherited from a parent). This process maintained an error rate of 5% across a range of CN V sizes.

6. CNV Analysis

[0068] An unbiased genome-wide search for regions associated with schizophrenia was performed using differences in regional CNV burdens in cases and controls, a two- stage approach was employed: first, the regions of interest (ROI) was defined in the primary dataset as regions of the genome in which CNVs were present in 2 or more cases and absent from controls; and second, statistical significance of these regions was estimated using a secondary dataset (combined MGS/ISC). P-value was estimated as - follows: all CNVs overlapping an ROI were piled up, and the CNV breakpoints that fell within the ROI were used to partition the ROI into a series of non-overlapping segments or bins, as schematically shown in Figure 4. Based on the CNV counts within a segment, association was quantified using the Exact Conditional test, with ancestry and study as covariates. The segment with the lowest one-sided p-value was the peak of association within an ROI. Because segments in different ROls are driven by the underlying genetic . architecture, their numbers and sizes varied widely.

[0069] Furthermore, numbers of CNVs in nearby segments are highly correlated.

To address these issues, a permutation -based ?-value correction scheme was applied in which the observed one-sided />-value of the association peak is compared to the distribution of minimal one-sided /rvalues of any segment within the ROI, computed based on data with case/control labels shuffled at the sample level. This empirical / value was reported as the -value for the ROI. In these experiments, -value was estimated by running 100,000 permutations and ^-values of the 8 nominally significant regions were further refined by running 200,000 permutations. For each ROI both the region and peak ORs estimated by the use of Exact Conditional test were reported. In order to avoid -r infinite OR estimates, which occur when the number of controls across all strata is equal to zero, 0.5 was add to all cells in the contingency table (Haldane correction). Within the permutation-based procedure, no correction of the counts was performed, because a mixture of corrected and uncorrected minimal 7-values, which may occur within an ROI, would interfere with accurate estimation of the null distribution.

Results

._^■· *

1. Experimental Validation of 7q36.3 CNVs a) Sequenom MassArray

[0070] Absolute copy number (ACN) detection method by Sequenom MassArray combines real time competitive PCR (rPCR) with MassEXTEND procedures and matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The CNV assay involves spiking genomic DNA template with genomic DNA from a single reference chimpanzee (Pan troglodytes) that was purchased from Southwest National Primate Research Center (San Antonio, Texas, USA). The genotype assay targets a specific single nucleotide difference between human and chimpanzee, and relative copy number is determined based the ratio of peak areas of the human and chimp alleles.

~ [0071] CNV Assay Design: two CNV assays for each of the two segments

(proximal and distal) of the 7q36.3 region were designed. These four assays were used to validate all CNV calls, with the exception of a single duplication in control sample D0024922 that did not overlap either the proximal or the distal segment. For this CNV a third pair of assays was developed. In addition, similar assays for four copy number invariant regions of the genome were developed. All assays were designed to be carried out in a single multiplex reaction. [0072] Sequence differences between human and chimpanzee were identified within CNV target regions by aligning the respective genomic DNA using UCSC Blat. Nucleotide differences between human and chimpanzee sequences were then categorized based on the position of difference in the alignment., alleles in human and chimpanzee, and .. direction of aligument. All aligned human-chimpanzee loci were processed to identify the location of any variant bases (single nucleotide polymorphisms and insertions/deletions) within a given distance of the specified human-chimpanzee loci. All known human and chimpanzee SNPs and indels were excluded in₍CNV assay design. Single base nucleotide extension (SBE) assays were then designed to target the non-conserved nucleotides using Sequenom Assay Design v3.1 software.

- (0073j PCR: PCR was performed on Biorad thermocyclcrs in a 5 μΐ reaction using 15ng of genomic human DNA (/wDNA). 15ng of P. troglodytes DNA (p/DNA), 0.5pmol of each primer in multiplex, 250μ dNTPs. 0.5μ1 of 10X 20 mM MgCl PCR Buffer and 0.5units of Taq polymerase (Roche, USA). The PCR cycling parameters were 95°C for 15 min, followed by 45 cycles of 94°C for 30s, 56°C for 30s and 72°C for 1 min, followed by 72°C for 3 min. Following PCR, 5μ1 PCR product was treated with 0.17μ1 of 10X SAP .. (shrimp alkalinej>hosphate) buffer and 0.30μ1 of SAP enzyme and incubated at 37°C for 40 min followed by heat denaturation of enzyme at 85°C for 5 min.

|0074) Single Base Primer Extension. Primer extension was performed on MJ thermocyclers in 9μ1 reactions using the 7μ1 SAP-treated PCR product, 7-14pmol of each primer in multiplex, 0.20μ1 of iplex buffer, 0.20μ1 of iplex termination mix and 0.05μ1 of iplex enzyme (Sequenom, San Diego, CA, USA). The cycling parameters were 94°C for ·· 30s, followed by 40 cycles of 94°C for 5s and a nested 5 cycles of 52°C for 5s and 80°C for 5s, followed by 72°C for 3 min. |0075| Quantification of absolute copy number. The absolute copy number for target regions was determined using the method described by Williams et al. (2008) ^w. All copy number measurements from each sample were normalized against copy number measurements from the assays targeting the invariant (normal diploid copy) regions of the

- genome in the same sample to control for any sample to sample (i.e., well to well) loading variation. Data were analyzed using Splus 8.0. b) Fine mapping of 7q36.3 Duplications in MGS subjects using the Nimblegen HD2 array

(0076) Duplications of 7q36.3 detected by the Affymctrix 6.0 array in the MGS sample were mapped at higher resolution using the NimbleGen HD2 platform. Hybridizations were performed as described above. Based on the initial and fine mapping results most duplications have unique boundaries, suggesting that each is a result of a different mutation event. Furthermore, the NimbleGen HD2 data revealed additional structural complexity, including triplicated regions in samples 03C23091 and 00C02873 that were not readily apparent from the original Affymetrix 6.0 scans. . c) Fluorescence in situ hybridization (FISH)

[0077| Cytogenetic confirmation was obtained for two samples with duplication of

VIPR2 (05C43079 and 05C48386). Subtelomeric probes were obtained for chromosome 7p and 7q (Abbott Molecular). The genomic coordinates of the TelVysion 7q probe were Chr7: 158,551,912-158,650,925 (NCBI36/hgl8), which overlaps exons 1-4 of VIPR2. Hybridizations were performed according to manufacturer's protocols.

|00781 In images from fluorescence in sihi hybridization (FISH) using a subtelomeric 7q probe, three distinct red signals can be seen in interphase cells, confirming a heterozygous duplication. In metaphase cells, all signals localize to the subtelomeric region of 7q, confirming that the duplications lie adjacent to each other in the 7q36.3 region.

.. 2. Critical Evaluation of CNV Detection Sensitivity in Cases and Control Across the Primary and Secondary Cohorts

|0079| Sensitivity of CNV calls in the 7q36.3 region was examined to determine the possibility of a spurious association, which can arise, for instance, if there is a differential sensitivity to detect CNVs in control samples compared with the case samples. Sensitivity of the segmentation-based CNV calling methods was evaluated by (a) ^:: examining CNVs within the 7q36.3 region after relaxing our stringent filtering criteria, and by comparing segmentation calls in cases and controls to CNV genotype calls made in the (b) 7q36.3 region and (c) elsewhere in the genome using targeted genotyping algorithms with enhanced sensitivity. These efforts were focused only on the microarray datasets that were processed for this study (the primary cohort and MGS subset in the secondary cohort). Published CNV call sets generated by the ISC study of schizophrenia have undergone evaluation in earlier studies ^i!. a) CNV segmentation calls in the 7q36.3 region examined using a lower sensitivity threshold

[0080] After eliminating the primary filtering criterion, the CNV confidence score

^" (CS). no additional CNVs were detected in the 7q36.3 region; therefore, filtering based on confidence score does not account for the observed differences in CNV frequencies. Second, rare CNVs in the 7q36.3 region were examined after relaxing the minimum CNV size to 5 probes for imbleGen and 16 probes for the Aftymetrix arrays. Additional small CNVs were detected that did not negatively influence the observed association. The two- stage analysis of the 7q36.3 region was repeated using the unfiltered CNV call set, and the statistical signal- was slightly improved compared with the original result from the stringently filtered CNV calls (permutation P-value = 1.5* 10^"5, peak OR = 17.1 |3.3,oo|). This improved signal can be accounted for by the smaller target region and reduced multiple testing correction. The improved signal cannot be accounted for by the detection of additional small CNVs in cases, because only 3 smaller duplications overlapped the peak of association, one in cases and two in controls. - b) Concordance between segmentation calls at 7q36.3 region and CNV calls by a more sensitive outlier-detection based CNV detection method (MeZOD)

[0081] As a second approach to evaluating the sensitivity of the methods, a more sensitive outiicr-detection based method, Median Z-score Outlier Detection (MeZOD) was applied to the analysis of the two CNV target regions at 7q36. Outlier detection-based methods ⁷⁴ provide greater sensitivity for CNVs within defined target regions. If a significant number of CNVs overlapping the target region were undetected by the segmentation algorithms or filtered out of the call set, such variants can often be detected by MeZOD, leading to discordance between the segmentation calls and the MeZOD genotypes. When the distribution of z-scores for each target region was examined, punctate clusters of data points that are distinguishable from the overall distribution was observed. Perfect concordance between MeZOD genotypes and CNV calls were observed. Based on these results, it is unlikely that the segmentation-based CNV calling algorithms failed to detect CNVs that are > 100 kb in size within this region. c) Sensitivity to detect CNPs is comparable in cases and controls

(0082] Sensitivity at other loci throughout the genome was evaluated by examining sensitivity to detect a set of validated common CNPs (>100 kb in size) characterized as part of HapMap phase 3— . Sensitivity to detect common CNPs by the segmentation algorithms is equivalent to that of rare CNVs (because parameters of HMM- , based segmentation algorithms arc not adjusted based on prior knowledge of common CNVs). However, targeted genotyping methods have much greater sensitivity and accuracy. Therefore, concordance between the segmentation calls and a predefined set of common CNP genotypes were used as a measure of the segmentor sensitivity.

[0083] First, all common CNPs >100 kb were selected from HapMap 3, and these markers were compared with a list of common CNP markers that previously assembled for die NimbleGen HD2 platform, all CNPs that were present in both sets (total of 7) were selected. The seven CNPs were then genotyped by manual examination of clusters. Genotypes were compared with segmentation calls, and sensitivity was defined as the average fraction of genotyped "gains" or ^!ilosses" that were detected by segmentation per individual. By this approach, similar overall sensitivity between cases and controls were observed in the primary sample and in the MGS samples, with the possible exception that sensitivity was sh^'ghtly (but significantly) greater in African American controls compared with cases in the MGS study (case: control sensitivity ratio 0.96, P=0.001). d) Examining the impact of differential sensitivity of platforms used in different studies

[0084] The primary dataset consisted of NimbleGen 2.1M array data. The secondary dataset consisted of the MGS study (Affymetrix 6.0) and the ISC study of schizophrenia (which included data from Affymetrix 5.0 and 6.0 platforms). Combining data from multiple studies has the potential to create spurious associations. For instance, if data from two studies are combined and the study with the most sensitive platform had a large number of cases and a small number controls, a spurious enrichment of CNVs in cases could arise (if factors such as platform and study are not controlled for). There was no such a disparity in any of the 3 studies comprising our primary and secondary cohorts, which consisted of roughly equal numbers of cases and controls in each study. In the conditional exact-based permutation test for association, platform and study were included as factors. Second, heterogeneity was tested in the association across all 3 studies in the primary and secondary samples; no significant heterogeneity was observed across the 3 populations when tested using the Breslow-Day Tarone test (P = 0.19-0.86). Therefore, no _; evidence was found that the observed association can be attributed to differential sensitivity of platforms used across studies.

3. Quantitative PCR Measurement of V1PR2 mRNA

[0085| Levels of VIPR2 mRNA were measured in lymphoblastoid cell lines obtained from the MGS study collection at the NIMH genetics repository, including patients with duplications and triplications of 7q36.3 and control individuals with normal diploid copy number of this region. The samples and genotypes used in these experiments were as follows: controls: 04C347 1, 04C28567, 05C47472, 04C34070: duplication of VIPR2: 05C44574, 05C48386, 05C48694; Duplication of VIPR2 with a nested triplication of exons 3 and 4: 00C02204, 03C23250, 05C43079, 03C23091; duplication of upstream subtelomeric region: 05C51123, 05C46770. CNV genotypes were confirmed in cell lines by Sequenom assay.

|0086] Cell lines from two subjects were analyzed with each genotype. From each cell line, a series of two RNA preparations were made in order to provide two biological replicates of each subject. Each biological replicate was in turn analyzed with triplicate qPCR reactions. Results presented for each subject represent the mean and standard error of 6 replicates. Standard error and P-values for pooled results were computed across individuals (Figure 1).

[0087| Tissue Culture. EBV-transformcd lymphoblastoid cells from MGS patients and controls were obtained from the NIMH genetics initiative repository. Cell lines were cultured in Gibco's RPMI-1640 supplemented with 15% FBS and IX Penn-Strep at 37°C and 5% C0₂. [0088] Sample Preparation. Total RNA was prepared using Qiagen's RNEasy

Plus kit, and cD A was prepared using Quanta Qscript cDNA mix using lug RNA for each 20ul reaction.

(0089J Quantitative PCR (qPCR): Measurement of VIPR2 mRNA abundance was - carried out on mRNA from lymphoblastoid lines using the V1PR2 TaqMan® assay (Applied Biosystems Inc.). Assays were carried out by the Center for Aids Research (CFAR) core facility at UCSD. cDNA from 500ng of total RNA starting material was used for each qPCR reaction. Samples were done in triplicates using the VIPR2 and GAPDH primers provided by the manufacturer. qPCR cycling conditions were as follows: 50°C for 2min, 95°C for l Omin, then 45 cycles of 95°C for 15sec, 60°C for lmin. Ct values obtained were normalized with GAPDH Ct values. VIPR2 mRNA was detectable in all cell lines analyzed in this study (Figure 2).

[0090| Low but measurable levels of VIPR2 expression was observed in all lymphoblastoid cell lines in this study (the range of Ct values was 33-36). This is consistent with the low VIPR2 transcript abundance in the published transcriptome data from the cell lines of 60 Nigerian subjects by Pickrell et al. (21 reads/968 million) ^5l. This ,. is also consistent with very low level of VPAC2 receptor signalin that observed in control cell lines.

[0091] In cell lines from patients carrying duplications of terminal 7q, an approximately 3-fold increase in VIPR2 expression relative to controls was observed (Figure la).

4. Measurement of VIP-induced cAMP Signaling in Lymphoblastoid Cell Lines

[0092] Cyclic AMP accumulation was measured in lymphoblastoid cell lines (0.5 million per ml) prc-incubatcd for 20 minutes with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 200 μΜ), before the addition of the stimulatory agonists forskolin (10 μΜ) +/- VIP, [100 nM ], BAY 55-9837 (l OOnM) or prostaglandin E2 [PGE2, 1 μΜ] for 10 min. Reactions were terminated by pelleting the cells, aspiration -■^■■of the medium and addition of 100 μΐ of cold 7.5% (wt/vol) trichloroacetic acid (TCA). Cyclic AMP content in TCA extracts was determined by radioimmunoassay and normalized to the number of cells per well. Data are expressed as cA P accumulation in response to the GPCR agonists relative to the response to non-GPCR agonist forskolin (10uM) and IBMX (200 mM) alone. Results presented for each subject represent the mean and standard error of at least ten replicates. Standard error and -values for pooled results were computed across individuals (Figures lb-d).

[0093] In cell lines from patients carrying duplications of terminal 7q, a significant increase in cyclic-AMP accumulation in response to two different VPAC2 agonists relative to controls was observed (Figures lb,c). No difference in cyclic-AMP response was observed in response to a different GPCR agonist PGE2 (Figure Id). 5. Sequencing of Transcripts in Patients with Duplications of VIPR2 Exons 3 and 4

|0094| In four MGS patients, complex duplication/triplications consisting of a duplication of the entire gene and another duplication of exons 3 and 4 nested within it was observed. The exact structure of this haplotype is not known. If the nested duplication of exons 3 and 4 lies adjacent and in a direct orientation, then this rearrangement would result in a mutant transcript with tandem duplication of exons 3 and 4, which would result in a shift of the open reading frame. However, if the nested duplication lies outside of the gene or in an inverted orientation, then nested duplication would not have an obvious impact on either of the two copies of VIPR2 (thus, the duplication/triplication might be functionally equivalent to a tandem duplication). [0095) No transcript was identified with tandem duplication of exons 3 and 4 in mRNA from four subjects with duplication triplications. Using primers designed for exons 2 and 5^ VIPR2 cDNA products from the cell lines listed in Figure 1 were sequenced. The normal V1PR2 transcript was detected in all samples. No a larger product corresponding to the mutant transcript was detected in any sample. A truncated product was observed in one of the triplication carriers (05C43079). Sequencing of this transcript revealed an aberrantly spliced exon 3 and the creation of a premature translation termination site (data not shown). These results do not indicate that the duplication triplication of VIPR2 results in a mutant transcript. If such a transcript is ^~ produced in patients with complex rearrangements of VIPR2, it is probably degraded by nonsense-mediated decay. [0096] Clinical Description of 7q36.3 Duplication Carriers 02-0016 and 02-0135:

Both patients were from Maria Karayiorgou's family triad sample collected in the US ³². All patients in the sample participated in a formal diagnostic interview conducted by an experienced, licensed clinician specially trained in the use of the Diagnostic Interview for Genetic Studies (DIGS). Diagnostic criteria were applied as described in the Diagnostic and Statistical Manual 4 (DSM-IV). All diagnoses were confirmed by a senior licensed clinical psychologist.

|0097) Subject 02^0016 is male, age 44, of Norwegian descent. His diagnosis is schizoaffective disorder, depressed type, with onset at the age of 21. Family history: mother suffers from depression. Patient has never been married. He completed 4 years of college. He currently resides in a halfway house and attends a day treatment program. He is unemployed and receives disability. At age 16, he reported first feeling paranoid and having trouble in school. At age 21, he reported experiencing his first psychotic break and was hospitalized for 4 months. His symptoms primarily entailed paranoia. He also reported being depressed during his hospitalization. At age 22, he was hospitalized for a second time for suicidal thoughts, depression and feelings of .hopelessness. At age 26, he experienced a relapse with prominent paranoid delusions. The patient had a history of hyperactivity prior to the age of 16. Developmentally, he reported maturation lag (i.e., growing up more slowly than other children). He also reported motor and vocal tics. |0098| Subject 02-0135 is female, age 50, of Irish/German English/Scottish descent. Her diagnosis is schizoaffective disorder, bipolar type, with onset at the age of 22. Family history: None known. Patient has never been married and her education includes some college course work. She currently resides in a semi-independent group home and has been unemployed due to her psychiatric problems. Her primary symptoms are paranoia and irritability. She began having significant psychiatric problems when she was 21 while at college. She became aggressive and loud and started showing poor judgment. She has been hospitalized numerous times (at least 8) and her functioning has deteriorated steadily over the years. She has experienced at least 4 manic episodes, each lasting several months, with the first occurring when she was 25. The patient exhibited " pressured speech, irritable mood, and grandiose ideation during each manic episode. She also reported obsessions with patterns but did not meet diagnostic criteria for OCD. Developmentally, the patient has a history of delayed speech (did not speak words until the age of 3) and delayed puberty (did not menstruate until college). She lacked close friendships during childhood.

Discussion and Summary [0099) . In order to identify novel schizophrenia genes, copy number variation genome-wide was investigated using an approach that detects enrichment of multiple overlapping rare variants. Regions of interest were defined in a primary sample of 802 ^" patients and 742 controls as genomic segments containing CNVs in at least two cases and in no controls. This discovery step yielded 114 genomic regions of interest. In the secondary cohort of 7,488 patients and 6,689 controls, the association of these regions with schizophrenia were assessed. All CNVs overlapping each of the 1 14 regions of interest were collected, and CNV breakpoints falling within each region were used to partition the region into a series of non-overlapping segments or bins. Significance was --. tested within each bin by the exact conditional test, with ethnicity and study as covariates. The segment with the minimal p-value was defined as the peak of association within the region, and a permutation-based multiple testing correction scheme was applied in order to obtain the p-value for the region.

[00100] Of the 1 14 regions detected in the first step, four had statistically significant associations in the secondary sample after Bonferroni correction (a = 0.05/1 14 = 4.4x1ο^"4). - There were four "regions with significant p-values meeting this criterion and an ddditional four loci with nominally significant p-values (P<0.05) in the secondary cohort. Regions with significant associations were loss of copy number at 22q 1 1.2 (P < 5x10^"*, OR = 14.2), gain at 7q36.3 (P = 4.0x l0^"5, OR = 16.4), gain at 16pl 1.2 (P = l.OxlO^"4, OR = 16.1) and loss at 15ql3.3 (P = 1.6 l0^'4, OR = 14.9). No significant heterogeneity was observed for these genomic regions across studies (Breslow-Day-Tarone P = 0.42 - 0.83).

[00101) 15ql3.3, 16pl l .2 and 22q 11.2 are well-documented loci conferring increased risk for schizophrenia ^*. All are hotspots for non-allelic homologous recombination (NAHR), and all alleles contributing to the association consist of large deletions with similar breakpoints. By contrast, microduplications at 7q36.3 have not been previously implicated in neuropsychiatric disorders. The 7q36.3 region harbored CNVs that overlapped but differed in size and breakpoint positions. The peak of association is located in the subtelonieric region of 7q, upstream of the gene VIPR2. Also, ranking fifth among the associations genome-wide was another region, 125 kb proximal to the peak at 7q36.3 (P = 0.0007). Combining the two 7q36.3 regions into a single 362 kb region (chr7: 158,448,321-158,810,016), duplications were detected in 29 of 8,290 (0.35%) patients and 2 of 7,431 (0.03%) controls in this study. The p-valuc for the combined region in the combined sample was 5.7xl0^"7 and the OR was 14.1 [3.5. 123.9].

[001021 Sensitivity and specificity of CNV calls in the 7q36 region were examined to determine the possibility of a spurious association. No additional duplications >100 kb were detected after reducing the stringency of the CNV filtering criteria. Second, identical CNV calls were obtained using' a more sensitive targeted CNV calling algorithm, median Z-score Outlier Detection (McZOD)⁵. All but one of the duplications (control sample 06C52730) were confirmed using the Sequenom MASSarray genotyping platform with assays designed for the proximal region and for the distal region. Validated CNVs discovered in the MGS subjects were mapped at higher resolution using the NimbleGen HD2 platform and plots of probe intensity ratios from the HD2 array were also obtained. In addition, tandem duplications of the V1PR2 gene were confirmed in two patients by fluorescence in situ hybridization (FISH).

[00103] Unexpectedly, manual examination of probe ratios revealed additional structural complexity within some of the 7q36.3 CNVs. Copy number profiles in four patients (03C23250, 05C43079, 03C23091 and 00C02204) suggested triplications nested within duplications of the proximal region. In all four patients, a triplication overlapped with exons 3 and 4 of the gene VIPR2. A copy number of four was confirmed in these samples using the Sequenomc MASSarray CNV assay, and results for all samples were consistent with results in plots of probe intensity ratios for 16 CNVs detected in the primary and MGS datasets. VIPR2 transcripts were amplified from mRNA samples from the four triplication carriers. The normal VIPR2 transcript was detected in all samples, and a larger product corresponding to a transcript with duplicated exons was not observed.

[00104] Inheritance of the duplication at 7q36.3 could be evaluated in three families. In family 02-135, the duplication was confirmed in the proband, but not detected in either of the unaffected parents, and thus is apparently de novo. In family 02-01 , the duplication was detected in the proband and in a mother with a diagnosis of depression. In family LWJ02, the duplication was detected in the proband and in an unaffected mother. The proband's mother also had a son with a diagnosis of schizophrenia (LW-102-03) from a second marriage, but DNA was not available on this individual. Clinical psychiatric reports of patients 02-016 and 02-135 are provided below.

|00105| Variable expressivity is often characteristic of pathogenic CNVs^'A The spectrum of psychiatric phenotypes associated with 7q36.3 duplications by screening for these events were evaluated in individuals with bipolar disorder or autism spectrum disorder (ASD). Microarray data were available for 2,777 patients from the Bipolar Genome Study (BiGS), for 996 ASD patients from the Autism Genome Project Consortium (AGP), and from unpublished analyses of 1 14 patients with ASD using the NimbleGen HD2 platform. Microduplications of 7q36.3 (>100 kb in size) were detected in three of 1,1 10 (0.27%) of patients with ASD; compared with the controls described above, P=0.018. Microduplications at 7q36.3 were detected in two of 2,777 (0.07%) ^' patients with bipolar disorder; compared with the controls, P=0.23. These results provide evidence that the clinical phenotypes associated with 7q36.3 duplications may include pediatric neurodcvelopmental disorders such as autism, but do not include bipolar disorder. Larger chromosomal abnonnalities involving 7q have been reported in association with neurodevelopmental disorders, including deletions of 7q36-7qter ^a~ and duplications of 7q35-7qter—; a 550 kb duplication of 7qter (of unknown clinical .. relevance) has been reported in a patient with neurofibromatosis— .

1001061 These genetic data implicate the gene VIPR2. All variants contributing to the observed association at 7q36.3 overlap with this gene or lie within the gene-less subtelomeric region <89 kb from the transcriptional start site of VIPR2. V1PR2 encodes the vasoactive intestinal peptide (VIP) receptor VPAC2, a G protein-coupled receptor that is expressed in a variety of tissues including, in the brain, the suprachiasmatic nucleus, hippocampus, amygdala, and hypothalamus— . VPAC2 binds VIP— , activates cyclic- AMP signaling and PKA, regulates synaptic transmission in the hippocampus ^13J&, and promotes the proliferation of neural progenitor cells in the dentate gyrus— . Genetic studies in mouse have established that VIP signaling plays a role in learning and memory— . VPAC2 also plays a role in sustaining normal circadian oscillations in the SCN and VIPR2-nuil and VIPR2-overe.\pression ³¹ mice exhibit abnormal rhvthms of rest and activity.

[00107| Cyclic-AMP signaling has been implicated in schizophrenia Increases in VIPR2 transcription and VPAC2-mediated cAMP signaling would be a consequence of the microduplications at 7q36.3. Accordingly, VIPR2 mRNA and cAMP accumulation in response to VIP^' and BAY 55-9837, a VPAC2-selective agonist in lymphoblastoid cell lines from eight MGS study subjects were assessed: two with subtelomeric duplications, three with duplications of VIPR2, four with partial triplications, and four controls with normal copy number of the region. VIPR2 transcripts were present at low but measurable levels in all cell lines. VIPR2 mRNA levels were significantly increased in duplication carriers compared with controls (Figure la). Likewise, cAMP responses to VIP and BAY 55-9837 were significantly greater in lymphoblastoid lines from carriers as compared to controls (Figures lb and lc). In contrast, no group difference in cAMP accumulation in response to a different GPCR agonist, prostaglandin E2 were observed (Figure Id), thus confirming that the effect of 7q36 duplications on cAMP accumulation is mediated by VPAC2R.

[00108] Other VPC2R antagonists and agonists, such as compounds I and 2 published by C et al. ² (Figure 3), and peptide derivatives of VIP published by Moreno ct al.— , such as selective antagonists, PG-96-238 and PG-96-465, and a partial agonist, PG-97-278 (Figure 4), are also encompassed in the present invention for modulating VIPR2 expression and/or VPC2R activity, and subsequently for treating brain disorders.

[001091 In summary, a large two-stage genome-wide scan of rare CNVs was performed and the significant association of copy number gains at chromosome 7q36.3 with schizophrenia (P= 4.0x10 , OR = 16.14 [3.06, ∞\) was report herewith. Microduplications with variable breakpoints occurred within a 362 kb region and were detected in 29 of 8,290 (0.35%) patients versus two of 7,431 (0.03%) controls in the combined sample (p-value = 5.7x 10-7, odds ratio (OR) = 14.1 [3.5, 123.9]). All duplications overlapped or were located within 89 kb upstream of the vasoactive intestinal peptide receptor VIPR2. V1PR2 transcription and cyclic-AMP signaling were ^■- significandy increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered VIP signaling in the pathogenesis of schizophrenia and suggest VIPR2 as a potential target for the development of novel antipsychotic drugs.

{00110] The expression patterns that observed suggest that a variety of different genomic duplications can influence the transcription of VIPR2. Given that some risk variants are upstream of the gene and others are complex rearrangements that could potentially disrupt the duplicate copy, these results cannot be explained simply by an increase in gene dosage, but that duplications of 7q36 impact the regulation of VIPR2. Tandem duplication of regulatory sequences, for instance, could affect expression of the gene. Alternatively, the subtelomcric location of V1PR2 could be relevant to the mechanism. Intrinsic regulation of telomere structure and function often impacts the : transcriptional regulation of adjacent genes, a phenomenon known as telomere position effect (TPE) ^'²⁵. If VIPR2 is under such epigenetic regulation, any large tandem duplication of the subtelomeric region could potentially cause the gene to escape repression. [001111 i light of the emerging roles of VIPR2 in the brain, these results support that the pathogenesis of schizophrenia, in some patients, involves the dysregulation of ^" cellular processes such as adult neurogenesis and synaptic transmission and of the corresponding cognitive processes of learning and memory. Furthermore, in light of the brain expression patterns of VIPR2— , these results support the involvement of certain brain regions, such as hippocampus, amygdala and suprachiasmatic nucleus. The link between VIPR2 duplications and schizophrenia has significant applications for the development of molecular diagnostics and treatments for this disorder,

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100112) Genetic testing for duplications of the 7q36 region could enable the early detection of a subty pe of patients characterized by overexprcssion of VIPR2. Significant potential also exists for the development of therapeutics targeting this receptor. For instance, a selective antagonist of VPAC2R could have therapeutic potential in patients who carry duplications of the VIPR2 region. Peptide derivatives— and small molecules ²² have been identified that are selective VPAC2 inhibitors, and these pharmacological studies offer potential leads in the development of new drugs. While duplications of V1PR2 account for a small percentage of patients, the rapidly growing list of rare CNVs that are implicated in schizophrenia suggests that this psychiatric disorder is, in part, a

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Claims

-45- What is claimed is: .

1. A method for treatment of a neurological disorder comprising administering to a patient in need a treatment effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically active agent selected rom the group consisting. of a selective vasointestinal peptide 2 receptor (VPAC2R) antagonist, a selective VPAC2R agonist, a modulator of VPAC2R or at least one of VPAC2R signaling pathway proteins, a modulator of vasointestinal peptide receptor 2 (VIPR2) gene expression or one or more gene mutations, and a vasointestinal peptide (VIP) derivative or analog thereof.

2. The method of claim 1, wherein said neurological disorder is schizophrenia or autism.

3. The method of claim I , wherein said modulator decreases VIPR2 gene expression or corrects one or more pathogenic mutation of VIPR2 gene in said patient.

4. The method of claim 1 , wherein said pharmaceutically active agent is an antagonist ofVPAC2R.

5. The method of claim 1, wherein said pharmaceutically active agent is an agonist of VPAC2R.

6. The method of claim 1, wherein said pharmaceutically active agent is a VIP derivative.

7. A composition for treatment of a neurological disorder in a patient comprising a pharmaceuticall acceptable carrier and a pharmaceutically active agent selected from the group consisting of a selective vasointestinal peptide 2 receptor (VPAC2R) antagonist, a selective VPAC2R agonist, a modulator of VPAC2R or at least one VPAC2R signaling pathway proteins, a modulator of vasointestinal peptide receptor 2 (VIPR2) gene expression or one or more gene mutations, and a vasointestinal peptide (VIP) derivative or analog thereof.

8. The composition of claim 7, wherein said neurological disorder is schizophrenia or autism. -46-

9. The composition of claim 7, wherein said pharmaceutically active agent is an antagonist of VPAC2R.

10. The composition of claim 7, wherein said pharmaceutically active is an agonist of VPAC2R.

11. The composition of claim 7, wherein said pharmaceutically active agent is a VIP derivative.

12. The composition of claim 7, wherein said modulator decreases V1PR2 gene expression or corrects one or more pathogenic mutation of VIPR2 gene in said patient.

13. A method for diagnosis of a patient at risk for a neurological disorder comprising: a) obtaining a biological sample from said patient; b) detecting one or more pathogenic mutation or an expression level of VIPR2 gene in said biological sample; and

c) diagnosing said patient being at risk for said neurological disorder wherein one or more pathogenic mutation or overexpressed level of V1PR2 is detected in said biological sample of said patient.

14. The method of claim 13, wherein said pathogenic mutation comprises copy number variants (CNVs), duplications, indels, or single nucleotide variants of V1PR2.

15. The method claim 13, wherein said biological sample is brain tissue or cell.

16. A method for personalized treatment of a neurological disorder in a patient in need comprising: a) providing a genetic test for a gene copy number of VIPR2 to infer an effect of mutations on gene function in said patient; b) correlating said gene copy number of VIPR2 with VPAC2R activity; and -47- c) selecting a therapeutically effective dosage and pharmaceutically active agent that modulates VPAC2R activity and further corrects pathogenic mutations and gene expression of VIPR2.

17. The method of claim 16, wherein said pathogenic mutation comprises copy number variants CNVs), duplications, indels, or single nucleotide variants of V1PR2.

18. The method of claim 16, wherein said pharmaceutically active agent is selected from the group consisting of a selective vasointestinal peptide 2 receptor (VPAC2R) antagonist, a selective VPAC2R agonist, a modulator of VPAC2R or at least one VPAC2R signaling pathway proteins, a modulator of vasointestinal peptide receptor 2 (VIPR2) gene expression or one or more gene mutations, and a vasointcstinal peptide (VIP) derivative or analog thereof. ^s

19. A research tool model for identifying a therapeutic candidate for treatment, diagnosis, prognosis or prevention of a neurological disorder in a mammal comprising combining the therapeutic candidate with a VIPR2 gene, and observing modulation of VIPR2 gene expression or correction of padiogenic mutations of VIPR2 gene, or combining the therapeutic candidate with VPAC2R receptors, and observing modulation of VPACs activity in cyclic-AMP signaling.