WO2016205348A1 - Methods of diagnosing and treating autism - Google Patents

Abstract

Methods for diagnosing and treating autism spectral disorders are encompassed. In one embodiment, a patient is diagnosed as having autism spectral disorder if at least one CNV in an mGluR network gene is found in a patient sample. Patients with at least one mGluR network gene CNV are effectively treated with (+) - 5-oxo-Dprolinepiperidinamide monohydrate (NS-105) (also known as NFC-1 or fasoracetam).

Description

METHODS OF DIAGNOSING AND TREATING AUTISM

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to United States Nonprovisional Patent Application No. 14/740, 230, filed June 15, 2015, as well as to United States

Provisional Patent Applications 62/215,628, 62/215,633, 62/215,636, and

62/ 215,673, all of which were filed September 8, 2015. All of the above -listed applications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[002] Methods for diagnosing and treating autism spectrum disorders are provided.

BACKGROUND OF THE INVENTION

[003] Autism spectrum disorder (ASD) is a range of complex

neurodevelopmental disorders characterized by mild to severe social impairments, communication difficulties, and restrictive or repetitive behaviors. ASD, previously known as pervasive developmental disorders (PDD), is an umbrella term that includes various conditions that used to be diagnosed separately such as autistic disorder (or classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD- NOS), and social (pragmatic) communication disorder (SCD). (Vieux et al, "Autism Spectrum Disorder: DSM-5 Changes, Epidemiology & Outcomes," The Carlat Report, Vol. 11, Issue 6, June 2013.)

[004] ASD occurs in all racial, ethnic, and socioeconomic groups. (Durkin et al., "Socioeconomic inequality in the prevalence of autism spectrum disorder:

evidence from a U.S. cross-sectional study," PLOS One, 5(7), July 2010.) In 2013, ASD was estimated to occur in 2% of children between the ages of 6 and 17, a significant increase from the estimate of 1.16% in 2007. (see, e.g., Blumberg et. al., National Health Statistics Reports, No. 65, March 20, 2013) While it is not known how many adults suffer from ASD, a British study reported that 1.8% adult men and 0.2% adult women have ASD. (Brugha et al, "Epidemiology of autism spectrum disorders in adults in the community in England," Arch. Gen. Psych. 68: 459-66, 2011.)

[005] With the increased incidence of ASD comes increased costs to society. The CDC estimates that it costs between $17,000 -$21,000 more per year to care for a child diagnosed with ASD as compared to a child not diagnosed with ASD. Medical costs alone for children diagnosed with ASD can be elevated by $4,100-$6,200 per year, with behavioral interventions for children diagnosed with ASD costing $40,000 -$60,000 per year. It is estimated that total societal costs of caring for children diagnosed with ASD were over $9 billion in 2011.

[006] Some risk factors and patterns have emerged to assist in diagnosing ASD, and various treatment regimes are often used on a trial and error basis. While there have been improvements in diagnosis and treatment of ASD, there remains a need for consistent diagnosis and treatment of ASD using a genetic approach. SUMMARY OF THE INVENTION

[007] In accordance with the description, a method for treating autism spectrum disorder (ASD) in patients having at least one CNV in a GRM/ mGluR network gene, as shown in Figures 15A-D, comprising administering a therapeutically effective amount of fasoracetam (NS-105 or NFC-1), or member of the piracetam family of nootropic agents, is encompassed. CNVs in GRM/mGluR network genes are sensitive and specific biomarkers for diagnosing ASD. The inventors have identified drug candidates that specifically activate mGluRs, restoring normal neurophysiology in ASD patients having at least one CNV in any of the

GRM/ mGluR network genes shown in Figures 15A-D.

[008] In some embodiments, a CNV in at least one of the GRM/ mGluR network genes recited in Tables 16-18 indicates a diagnosis of ASD. These genes include ACATl, ACAT2, ACCNl, ACCN2, ACPI, ACTB, ACTR2, ADA, ADCYl, ADD1, ADD2, ADORA1, ADRA1B, ADRA2A, ADRA2C, ADRB2, ADRBKl, ALDOA, ANXA2, APP, APTX, AQP1, ARHGAP24, ARL15, ARRBl, ARRB2, ATXN7L3, BDKRBl, BDKRB2, BTBD2, BTG2, C17orf44, Clorfll6, C7orf25, CA8, CACNA1B, CACYBP, CALB2, CALM1, CALM2, CALM3, CAMK1,

CAMK2B, CAMK4, CCNBl, CDC42, CENTGl, CHGB, CHP, CHRM2, CHRM3, CIC, CMPK, CNP, CNR1, CNTN4, COPB2, CRHRl, CTNNA2, CYCS, DCN, DHCR7, DISCI, DLST, DPP6, DRD2, DRD3, DSTN, DYNLL1, ECHS1, EGFR, EIF3S3, ERBB2, F2R, F2RL2, F2RL3, F3, FKBP3, FPR1, FSCN1, FURIN, FYN, GAPDH, GLP1R, GLP2R, GNA15, GNAI1, GNAI2, GNAI3, GNAOl, GNAQ, GNB2L1, GOTl, GPIBA, GPR26, GRB2, GRB7, GRIAl, GRIKl, GRIK3, GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, GRM8, GSN, HBXIP, HD,

HNRPA3, HOMER1, HOMER3, HRPT2, HSP90AB1, HTR2A, IL8RB, IMPDH2, IQGAP2, ITGB1, ITGB7, ITPR1, KIAA0090, KIAA1683, LAMA4, LARP7, LRP2BP, LRRC59, LTA, LYAR, LYN, MAP4, MAPK1, MAPT, MARK4, MC4R, MGC11082, MRPL14, MRPS16, MTHFD1, MTNR1A, MTNR1B, MX1, MYC, MY06, NANS, NARGl, NCKl, NEGRI, NFKBIA, NLN, NMI, NPY2R, NUDC, OPRDl, PAFAH1B3, PCBP1, PCBP3, PCDHA4, PCID1, PCMT1, PDCD5, PDE1B, PDE1C, PDE6G, PGM1, PHKB, PHKG2, PICKl, PIK3CA, PIK3R1, PLA2G7, PLCB1, PLCB3, PLCG2, PPIH, PPP2R1A, PRDXl, PRKCA, PRLHR, PRMTl, PRPSAPl, PSATl, PSENl, PSMAl, PSMCl, PSMDl, PSMDll, PSMDl 3, PSMD6, PSME1, PTHR2, PXN, PYGL, PYGM, QRICH2, RAB2, RALA,

RANBP1, RAP2A, RCC1, RCC2, RGS12, RGS2, RHOA, RIF1, RPA2, RPLP2, RPN2, RPS14, RRMl, RUVBL2, RYRl, RYR2, S100A6, SACS, SARS, SCTR, SDC3, SELE, SERPINB9, SET, SETD4, SF3B14, SGTB, SHANK1, SHBG, SIAH1, SLC2A1, SLC6A3, SLC7A10, SNCA, SNRPB2, SOCS6, SOCS7, SORD, SRC, STAU1, STRAP, STX12, SYK, TBCA, TBXA2R, TCP1, TEAD3, TFAM, TGM2, TJP1, TK1, TLR10, TMEM4, TNIK, TPI1, TRAF2, TRMT112, TUBA1, TUBA1A, TUBA1B, TUBA2, TUBB, TUBG1, TXN, TXNDC4, TXNL2, TYMS, UBQLN4, UCHL1, USP24, VHL, VTPR1, YWHAQ, and ZAP70.

[009] Moreover, patients with at least one CNV in a GRM/ mGluR network gene will see improvement in ASD upon administration of a therapeutically effective amount of a member of the piracetam family of nootropic agents, as described in F. Gualtieri et al., Curr. Phann. Des., 8: 125-38 (2002). In one embodiment, the treating agent is a pyroglutamide. Details regarding the preparation and formulation of pyroglutamides, which may be used in the practice of this invention, are provided in U.S. Pat. No. 5,102,882 to Kimura et al. In one embodiment the nootropic agent for the treatment of ASD in patients determined to have one or more of the CNVs indicative as set forth in Figures 15A-D, is (+)-5-oxo-Dprolinepiperidinamide monohydrate (fasoracetam; NS-105).

[0010] In one embodiment, the patient has been diagnosed as having pervasive developmental disorder, or one or more conditions selected from autistic disorder (classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD- NOS), or social (pragmatic) communication disorder (SCD).

[0011] The CNV may be a duplication or deletion.

[0012] The ASD may be syndromic or non- syndromic.

[0013] In one embodiment, the ASD may be in a patient having 22qll.2 Deletion or Duplication Syndrome, Fetal Valproate Syndrome or Thalidomide Embryopathy.

[0014] In one embodiment, the patient has at least two CNVs in a gene selected from the group consisting of ACAT1, ACAT2, ACCN1, ACCN2, ACPI, ACTB, ACTR2, ADA, ADCYl, ADDl, ADD2, ADORAl, ADRAIB, ADRA2A, ADRA2C, ADRB2, ADRBKl, ALDOA, ANXA2, APP, APTX, AQP1, ARHGAP24, ARL15, ARRB1, ARRB2, ATXN7L3, BDKRB1, BDKRB2, BTBD2, BTG2, C17orf44, Clorfll6, C7orf25, CA8, CACNAIB, CACYBP, CALB2, CALMl, CALM2, CALM3, CAMK1, CAMK2B, CAMK4, CCNB1, CDC42, CENTG1, CHGB, CHP, CHRM2, CHRM3, CIC, CMPK, CNP, CNR1, CNTN4, COPB2, CRHR1, CTNNA2, CYCS, DCN, DHCR7, DISCI, DLST, DPP6, DRD2, DRD3, DSTN, DYNLL1, ECHS1, EGFR, EIF3S3, ERBB2, F2R, F2RL2, F2RL3, F3, FKBP3, FPR1, FSCN1, FURIN, FYN, GAPDH, GLP1R, GLP2R, GNA15,

GNAI1, GNAI2, GNAI3, GNAOl, GNAQ, GNB2L1, GOT1, GP1BA, GPR26, GRB2, GRB7, GRIA1, GRIK1, GRIK3, GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, GRM8, GSN, HBXIP, HD, HNRPA3, HOMER1, HOMER3, HRPT2, HSP90AB1, HTR2A, IL8RB, IMPDH2, IQGAP2, ITGBl, ITGB7, ITPRl, KIAA0090, KIAA1683, LAMA4, LARP7, LRP2BP, LRRC59, LTA, LYAR, LYN, MAP4, MAPK1, MAPT, MARK4, MC4R, MGC11082, MRPL14, MRPS16,

MTHFD1, MTNR1A, MTNR1B, MX1, MYC, MY06, NANS, NARG1, NCK1, NEGRI, NFKBIA, NLN, NMI, NPY2R, NUDC, OPRDl, PAFAH1B3, PCBP1, PCBP3, PCDHA4, PCID1, PCMTl, PDCD5, PDE1B, PDE1C, PDE6G, PGM1, PHKB, PHKG2, PICKl, PIK3CA, PIK3R1, PLA2G7, PLCB1, PLCB3, PLCG2, PPIH, PPP2R1A, PRDXl, PRKCA, PRLHR, PRMT1, PRPSAP1, PSAT1, PSEN1, PSMA1, PSMC1, PSMD1, PSMD11, PSMD13, PSMD6, PSME1, PTHR2, PXN, PYGL, PYGM, QRICH2, RAB2, RALA, RANBPl, RAP2A, RCCl, RCC2, RGS12, RGS2, RHOA, RIFl, RPA2, RPLP2, RPN2, RPS14, RRMl, RUVBL2, RYRl, RYR2, S100A6, SACS, SARS, SCTR, SDC3, SELE, SERPINB9, SET, SETD4, SF3B14, SGTB, SHANKl, SHBG, SIAHl, SLC2A1, SLC6A3, SLC7A10, SNCA, SNRPB2, SOCS6, SOCS7, SORD, SRC, STAU1, STRAP, STX12, SYK, TBCA, TBXA2R, TCPl, TEAD3, TFAM, TGM2, TJPl, TKl, TLR10, TMEM4, TNIK, TPIl, TRAF2, TRMT112, TUBA1, TUBA 1 A, TUBA1B, TUBA2, TUBB, TUBG1, TXN,

TXNDC4, TXNL2, TYMS, UBQLN4, UCHL1, USP24, VHL, VIPR1, YWHAQ, and ZAP70.

[0015] In one embodiment, the CNVs are detected in any type of biological sample taken from a human patient, including but not limited to, body fluids

(including blood, urine, serum, gastric lavage), any type of cell (such as brain cells, white blood cells, mononuclear cells) or body tissue.

[0016] Methods for determining whether a patient has a GRM/ mGluR network gene CNV include, for example, analyzing the biological sample on a SNP- array, taking into account both LOG-R ratio (intensity data) and BAF (B allele frequency), which assesses allele states. SNP array platforms are commercially available from, for example, Illumina, Affymetrix, and Agilent.

[0017] Other methods for determining whether a patient has a GRM/ mGluR network gene CNV include, for example, CGH (comparative genomic hybridization), which utilizes intensity data for evaluation of CNVs; whole exome or whole genome sequencing (or targeted sequencing); utilizing specific FISH probes; qPCR; droplet PCR; and taqman probes. Alternatively, certain companies that use proprietary methods for determining the presence or absence of CNVs may be provided samples to be tested for presence of CNVs in GRM/ mGluR network genes in accordance with the methods of the invention.

[0018] Some embodiments include methods of treating autism in a subject comprising administering an effective amount of a nonselective activator of

metabotropic glutamate receptors (mGluRs) to a subject, thereby improving at least one symptom of autism. Some embodiments include treating autism in a subject comprising administering an effective amount of a nonselective activator of

metabotropic glutamate receptors (mGluRs) to a subject that has at least one genetic alteration in an mGluR network gene (e.g. as shown in Figures 16- 18), thereby improving at least one symptom of autism. Some embodiments include methods of treating autism in a subject comprising obtaining results from a genetic screen that determines whether a subject has a genetic alteration in an mGluR network gene, and, if the results show that the subject has at least one genetic alteration in an mGluR network gene, treating the subject by administering an effective amount of a nonselective activator of mGluRs. In some such embodiments, the genetic alteration is a copy number variation (CNV) or single nucleotide variation (SNV). In some such embodiments, the nonselective activator of mGluRs is fasoracetam, such as

fasoracetam monohydrate (NS-105 or NFC-1).

[0019] In methods comprising fasoracetam administration, the fasoracetam may be administered at a dose of 50 mg, lOOmg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg, wherein the dose is administered once, twice, or three times daily. For example, fasoracetam may be administered at a dose of 50-400 mg, 100-400 mg, or 200-400 mg, wherein the dose is administered once, twice, or three times daily. For example, the fasoracetam is administered at a dose of 200-400 mg, such as 200 mg, 300 mg, or 400 mg, wherein the dose is administered twice daily.

[0020] In some of the above embodiments, the subject has a CNV in at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mGluR network genes. For example, the subject may have a CNV in an mGluR network gene, which is found by obtaining a nucleic acid- comprising sample from the subject and subjecting the sample to a screen that assesses CNVs in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or a l of Tier 1 mGluR network genes. In other cases, the subject may have a CNV in an mGluR network gene that is determined by obtaining a nucleic acid-comprising sample from the subject and subjecting the sample to a screen that assesses CNVs in at least 50, at least 100, at least 150, at least 175, or all of Tier 2 mGluR network genes. In yet other cases the subject may have a CNV in an mGluR network gene that is determined by obtaining a nucleic acid sample from the subject and subjecting the sample to a screen that assesses CNVs in at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all of Tier 3 mGluR network genes. In some embodiments, the screen does not assess CNVs in one or more of GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, or GRM8. In some embodiments, the subject does not have a CNV in one or more of GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, or GRM8.

[0021] In some of the above embodiments, the subject is a pediatric subject, for instance between the ages of 5 and 17, 5 and 8, 8 and 17, 8 and 12, or 12 and 17. In other embodiments the subject is an adult. [0022] In some embodiments above, the nonselective activator of mGluRs is administered in combination with another pharmaceutical or non-pharmaceutical therapy.

[0023] In some of the above embodiments, the subject has been diagnosed with autism spectrum disorder, pervasive developmental disorder, or one or more conditions selected from autistic disorder (classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD-NOS), and social (pragmatic) communication disorder (SCD). In some cases, the autism is syndromic ASD. In some cases, the patient has ASD and 22qll.2 Deletion or Duplication Syndrome, Fetal Valproate Syndrome or Thalidomide Embryopathy. In some embodiments, the patient does not have any of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression. In other cases, the autism patient also has one or more of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression.

[0024] Also encompassed herein are methods for diagnosing autism in a subject comprising isolating a nucleic acid comprising sample from a subject, analy2ing the sample for the presence or absence of a genetic alteration in at least one mGluR network genes, and diagnosing autism if the subject has at least one genetic alteration in a mGluR network gene. Further encompassed are methods for diagnosing autism in a subject comprising isolating a nucleic acid comprising sample from a subject, isolating nucleic acid from the sample, analy2ing the nucleic acid for the presence or absence of a genetic alteration in at least one mGluR network genes, and diagnosing autism if the subject has at least one genetic alteration in a mGluR network gene. Additional embodiments include methods for identifying a subject as having autism comprising obtaining a sample from a patient, optionally isolating nucleic acid from the sample, optionally amplifying the nucleic acid, and analy2ing the nucleic acid in the sample for the presence or absence of a genetic alteration, such as a CNV, in at least one mGluR network gene, wherein the subj ect is identified as having autism if at least one genetic alteration, such as a CNV, in an mGluR network gene is detected. Further encompassed are methods for diagnosing autism in a subject comprising analy2ing genetic

information about one or more mGluR network genes, comparing the subject's information to a control subject that does not have autism, and diagnosing autism if the genetic information suggests that the subject has at least one genetic alteration in a mGluRnetworkgene. Also encompassed are methods of confirming a diagnosis of autism in a subject comprising: obtaining a nucleic acid-comprising sample from a subject diagnosed with autism by a method that comprises detecting or analy2ing genetic alterations in mGluR network genes; optionally amplifying the nucleic acid in the sample; and determining whether the subject has at least one genetic alteration, such as a CNV, in an mGluR network gene; and confirming a diagnosis of autism if the subject has at least one genetic alteration in an mGluRnetworkgene. In any of those methods, the analysis for the presence or absence of at least one genetic alteration in an mGluR network gene can comprise microarrays, whole genome sequencing, exome sequencing, targeted sequencing, FISH, comparative genomic hybridization, genome mapping, or other methods using next-generation sequencing, Sanger sequencing, PCR, or TaqMan technologies.

[0025] In some embodiments, the subject has CNVs in at least two mGluR network genes. In some embodiments, the method comprises detecting CNVs in mGluR network genes by subjecting the sample to a screen that assesses CNVs in at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mGluR network genes. In some embodiments, CNVs in mGluR network genes are determined by subjecting the sample to a screen that assesses CNVs in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or all of Tier 1 mGluR network genes. In some embodiments, CNVs in mGluR network genes are determined by subjecting the sample to a screen that assesses CNVs in at least 50, at least 100, at least 150, at least 175, or all of Tier 2 mGluR network genes. In some embodiments, CNVs in mGluR network genes are determined by subjecting the sample to a screen that assesses CNVs in at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all of Tier 3 mGluR network genes.

[0026] In some of the above embodiments, the subject is a pediatric subject, such as a subject between the ages of 5 and 17, 5 and 8, 8 and 17, 8 and 12, or 12 and 17. In other embodiments, the subject is an adult subject.

[0027] In some embodiments, the subject is not assessed for genetic alterations or CNVs in one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, andGRM8.

[0028] In some embodiments, the method for determining the presence or absence of at least one mGluR network gene genetic alteration comprises microarrays, whole genome sequencing, exome sequencing, targeted sequencing,FISH,comparative genomic hybridization, genome mapping, or other methods using next-generation sequencing, Sanger sequencing, PCR, or TaqMan technologies.

[0029] In some of the above embodiments, the subject has been diagnosed with autism spectrum disorder, pervasive developmental disorder, or one or more conditions selected from autistic disorder (classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD-NOS), and social (pragmatic) communication disorder (SCD). In some cases, the autism is syndromic ASD. In some cases, the patient has ASD and 22qll.2 Deletion or Duplication Syndrome, Fetal Valproate Syndrome or Thalidomide Embryopathy. In some embodiments, the patient does not have any of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression. In other cases, the autism patient also has one or more of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression. In some cases, the patient has both autism and ADHD.

[0030] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. [0031] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0032] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 provides the design of a clinical study. In this two-stage design, 2076 cases vs 4754 controls were used in the discovery cohort (Stage 1), and 1159 cases vs 2546 controls were used for a replication cohort (Stage 2). All samples used passed minimal quality control metrics, but the default quality calls of PennCNV were used to discriminate the discovery cohort (best quality) from the replication cohort (lesser quality.)

[0034] Figure 2 is a graph showing certain classes of gene pathways, which are disrupted by the ASD-related CNVRs disclosed herein. All genes with exons disrupted by replicated CNVRs were submitted to Ingenuity to ascertain significance of pathway enrichment.

[0035] Figures 3A-F are a schematic of a first-degree interactome of the GABAR-A family highlighting copy number defects enriched in cases vs controls.

[0036] Figure 4 shows a test and treat model for targeting therapeutics to specific pathways defective in disease. The generic test and treat model is shown in black where a molecular diagnostic is used to genetically define a population with defective pathways that are likely to benefit from a targeted intervention. Examples of trastu2umab as a targeted intervention for HER2 specific breast cancer is shown as well as an extrapolation of behavioral programs and novel therapeutics that are being developed to target ASDs due to defective GABAR-A pathways.

[0037] Figure 5 shows certain identified genetic marker CNVRs by GWAS predictive of ASDs in European-derived or African-derived populations. The

Manhattan plots show the -log 10 transformed P value of association for each CNVR along the genome. Adjacent chromosomes are shown. The regions discovered in Europeans (P 0.0001) that replicated in Africans (P<=0.001) are highlighted with black arrows labeled by chromosome band. GWAS of 4,634 cases vs 4,726 controls in Europeans is shown on top and GWAS of 312 cases vs 4,173 controls in Africans is shown below.

[0038] Figures 6A-D show a representative enrichment of certain CNVRs across mGluR network of genes. Nodes of the network are labeled with their gene names, with red and green representing deletions and duplications respectively, while grey nodes lack CNV data. Dark and light colors represent enrichment in cases and controls respectively. The genes defining the network are showed as diamonds, while all other genes are shown as circles. Blue lines indicate evidence of interaction.

[0039] Figure 7 shows a representative graph of enrichment of optimal CNVRs across the CALM1 network. The first degree directed interaction network defined by CALM1 is shown. [0040] Figure 8 shows a diagram of representative study design. Results from this study show that children with ASD and gene changes in mGluR network

(mGluR+ASD) were more likely to have Syndromic ASD as compared to children with ASD without abnormalities of mGluR network genes (mGluR-ASD). P<0.0001. See US Patent application no. 14/ 292,480 incorporated herein by reference as though set forth in full.

[0041] Figure 9 shows significant CNVRs in the mGluR network. The table shows the 10 most significant CNVRs for 189 genes with data in the GFIN for the GRM gene family across a European-derived population, as well as the most significant CNVRs harbored by the GRM mGluR receptors themselves.

[0042] Figure 10 shows significant CNVRs across genes in the MXD network in European-derived populations. Where large CNVs span multiple genes, the component gene implicated within the MXD gene family interaction network is bolded.

[0043] Figure 11 shows significant CNVRs across genes in the CALM1 network in European-derived populations.

[0044] Figure 12 shows CNVRs distinguishing cases from controls significant across both European-derived populations (P≤ 0.0001 by Fisher's exact test) and African-derived populations (P≤0.001). CNVR=copy-number variable region;

OR=odds ratio. For each CNVR, the table lists the type (del or dup), the closest gene impacted, the chromosomal band, the approximate size of the defect (Kb), the number of contributing SNPs, the numbers of affected cases and controls, as well as P-value and odds ratio (OR) from Fisher's exact test for across all populations, and subsets of European-derived and African-derived populations. *Genes with an asterisk (*) harbor CNVRs that disrupt their exons of directly, while those without the asterisk are located in the genomic region around the intergenic CNVRs.

[0045] Figures 13A-B show significant gene family interaction networks (GFINs) by network permutation testing (Pperm 0.05) enriched for CNV defects across at least 5% of cases. The table lists the name and size of gene family tested, the number and frequency of network genes enriched in the second degree gene interaction network, the number and frequency of cases harboring defects across the network, the number and frequency of controls harboring defects across the network, the significance of association by Fisher's exact test, the enrichment of CNV defects in cases, and the significance of that enrichment by 1,000 random network

permutations.

[0046] Figure 14 shows significant individual gene interaction networks ranked by permutation testing. Figure 14 lists the name and gene family member tested, the number and frequency of network genes enriched, the number and frequency of cases harbouring defects, the number and frequency of controls harbouring defects, and the significance of association by Fisher's exact test, the odds ratio of the effect size, and the significance of association by random permutation of network while controlling for number of genes tested. Among other highly ranked first degree gene interaction networks were the nuclear receptor co-repressor 1 (NCOR1; Pfisher <1.11Ε-06, ennchment=13.37, Pperm <0.004) and BCL2- associated athanogene 1 (BAGl; Pfisher≤2.18E— 04, enrichment= 15.40, Pperm ≤0.014) networks. NCOR1 is a transcriptional co-regulatory protein that appears to assist nuclear receptors in the downregulation of DNA expression through recruitment of histone deacetylases to DNA promoter regions; it is a principal regulator in neural stem cells. The oncogene BCL2 is a membrane protein that blocks the apoptosis pathway, and BAGl forms a BCL2-associated athanogene and represents a link between growth factor receptors and antiapoptotic mechanisms. The BAGl gene has been implicated in age-related neurodegenerative diseases, including Akheimer's disease.

[0047] Figures 15A-D show the raw data described in Examples 1-4. Column A lists the GRM/ mGluR network genes that are enriched in patients with ASD. Column B indicates whether the CNV is a duplication or deletion and the genetic start and stop locus. The odds ratio recited in column J recites "none" for any gene where no CNV was found in the control group. In column K, enrichment, the word "case" is indicative of enrichment in CNVs in the associated gene in ASD patients as compared to controls. Finding at least one CNV in any of the genes listed in Figures 15A-D, Column A, indicates a diagnosis of ASD and implicates treatment with NS- 105.

[0048] Figures 16A-D show the mGluR network genes included in the Tier 1 gene set. These genes have 2 degrees of protein-protein interaction with mGluR genes (GRMl-8) based on the Cytoscape Human Interactome, which is software for integrating biomolecular interaction networks with high-throughput data (as described in Shannon P (2003) Genome Research 13:2498-2504). The Tier 1 gene set includes 76 genes. The exact location for each gene in Tier 1 is listed in both the Human

Genome version 18 (hgl8) and Human Genome version 19 (hgl9). In addition, the exact gene location plus 500 kilobase (i.e., the range from 500 kilobase before and 500 kilobase after the gene of interest) is listed for hgl9. The start single nucleotide polymorphism (StartSNP) (i.e., the SNP located 500 kilobases before the gene of interest) and the EndSNP (i.e., the SNP located 500 kilobases after the gene of interest) are also listed. Genes of the mGluRs themselves are noted as "GRM." The expanded regions (i.e., 500kg up and down stream) frequently harbor regulatory elements and if impacted by a CNV, can have the same impact on the gene expression and function as a CNV residing in the gene sequence itself.

[0049] Figures 17A-I show the mGluR network genes included in the Tier 2 gene set. These genes have 2 degrees of protein-protein interaction with mGluR genes (GRM1-8) based on the Cytoscape Human Interactome but exclude genes from Tier 1. The Tier 2 gene set includes 197 genes. The exact location for each gene in Tier 2 is listed in both the Human Genome version 18 (hgl 8) and Human Genome version 19 (hgl 9). In addition, the exact gene location plus 500 kilobase (i.e., the range from500 kilobase before and 500 kilobase after the gene of interest) is listed for hgl 9. The start single nucleotide polymorphism (StartSNP) (i.e., the SNP located 500 kilobases before the gene of interest) and the EndSNP (i.e., the SNP located 500 kilobases after the gene of interest) in hgl 9 are also listed. [0050] Figures 18A-Y shows genes within the Tier 3 gene set. Genes with reciprocal gene querying with 2 degrees of protein-protein interaction with mGluR genes based on Cytoscape Human Interactome are included. Genes contained within Tiers 1 and 2 are excluded from Tier 3. The Tier 3 gene set includes 599 genes. The exact location for each gene in Tier 3 is listed in both the Human Genome version 18 (hgl8) and Human Genome version 19 (hgl9). In addition, the exact gene location plus 500 kilobase (i.e., the range from 500 kilobase before and 500 kilobase after the gene of interest) is listed for hgl9. The StartSNP (i.e., the SNP located 500 kilobases before the gene of interest) and the EndSNP (i.e., the SNP located 500 kilobases after the gene of interest) in hgl9 are also listed.

DESCRIPTION OF PARTICULAR EMBODIMENTS

Definitions

[0051] In this invention, "a" or "an" means "at least one" or "one or more," etc., unless clearly indicated otherwise by context. The term "or" means "and/ or" unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term "or" refers back to more than one preceding claim in the alternative only.

[0052] "Autism" and "Autism Spectrum Disorder" (ASD) are used

interchangeably herein. As noted earlier, autism or ASD comprises a range of complex neurodevelopmental disorders characterized by symptoms such as mild to severe social impairments, communication difficulties, and restrictive or repetitive behaviors. ASD, previously known as pervasive developmental disorders (PDD), is an umbrella term that includes various conditions that used to be diagnosed separately such as autistic disorder (or classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD-NOS), and social (pragmatic) communication disorder (SCD).

[0053] An "mGluR" or metabotropic glutamate receptor refers to one of eight glutamate receptors expressed in neural tissue named mGluRl, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7, and mGluR8. Their genes are abbreviated GRMl to GRM8. The mGluR proteins are G-protein-coupled receptors. They are typically placed into three sub-groups, Group I receptors including mGluRl and mGluR5 are classed as slow excitatory receptors. Group II includes mGluR2 and mGluR3. Group III includes mGluR4, mGluR6, mGluR7, and mGluR8. Groups II and III are classed as slow inhibitory receptors. The mGluRs are distinguished from the ionotropic GluRs or iGluRs, which are ion channel-associated glutamate receptors and are classed as fast excitatory receptors.

[0054] An "mGluR network gene," for purposes of this invention, comprises not only the mGluR genes GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, and GRM8, but also each of the other genes listed herein in Figs. 16-18 as well as the regions of DNA that regulate the genes listed in Figs 16-18. In addition, "mGluR network proteins" are the proteins encoded by the mGluR network genes.

[0055] The mGluR network genes are grouped into three subsets: Tier 1, Tier2, and Tier 3. (See Figs. 16-18.) Tier 1 mGluR network genes, shown in Figs. 16A-D, comprise 76 genes, including some GRM genes themselves as well as a number of other genes. The Tier 2 mGluR network genes, shown in Figs. 17A-I, comprise 197 genes, and exclude the Tier 1 genes.

[0056] Tiers 1 and 2 together are included in the "primary mGluR network." The "primary network" of mGluR genes also includes the genes 4-Sep, LOC642393, and LOC653098, for a total of 276 genes. There are presently technical difficulties in assessing the 4-Sep, LOC642393, and LOC653098 genes. Thus, they are not included in the genes of Tiers 1 and 2, although they are included in the primary network of genes of the present invention. The genes of Tier 1 and Tier 2 differ in that alterations in Tier 1 genes had been documented in previous genotyping studies of subjects suffering from mental disorders.

[0057] Tier 3 mGluR network genes, shown in Figs. 18A-Y, comprise 599 genes that are in the distal part of the mGluR network based on the merged human interactome provided by the Cytoscape Software (Shannon P et al. (2003) Genome Research 13:2498- 2504), and exclude the Tier 1 and Tier 2 genes. The Tier 3 genes are thus part of the "distal mGluR network." In addition to the Tier 3 genes, the genes LOC285147, LOC147004, and LOC93444 are included in the "distal mGluR network," although they were not assessed in the present study and are not included in Tier 3 due to technical difficulties in assessing genetic alterations in these genes.

[0058] A "genetic alteration" as used herein means any alteration in the DNA of a gene, or in the DNA regulating a gene, that results in a gene product that is

functionally changed as compared to a gene product produced from a non-altered DNA. A functional change maybe differing expression levels (up-regulation or down- regulation) or loss or change in one or more biological activities, for example. Agenetic alteration includes without limitation, copy number variations (CNVs), single nucleotide variants (SNVs), also called single nucleotide polymorphisms (SNPs) herein, frame shift mutations, or any other base pair substitutions, insertions, and deletions or duplications.

[0059] A "copy number variation (CNV)" refers to the number of copies of a particular gene in the genotype of an individual. CNVs represent a major genetic component of human phenotypic diversity. Susceptibility to genetic disorders is known to be associated not only with single nucleotide polymorphisms (SNP), but also with structural and other genetic variations, including CNVs. A CNV represents a copy number change involving a DNA fragment that is -1 kilobases (kb) or larger (Feuk et al. 2006a). CNVs described herein do not include those variants that arise from the insertion/deletion of transposable elements (e.g., .about.6-kb Kpnl repeats) to minimize the complexity of future CNV analyses. The term CNV therefore encompasses previously introduced terms such as large-scale copy number variants (LCVs; Iafrate et al. 2004), copy number polymorphisms (CNPs; Sebat et al. 2004), and intermediate-sized variants (ISVs; Tuzun et al. 2005), but not retroposon insertions.

[0060] A "CNV deletion" or "deletion CNV" or similar terms refer to a CNV in which a gene, DNA segment regulating a gene, or gene segment is deleted. A "CNV duplication" or "duplication CNV" or similar terms refer to a CNV in which a gene, DNA segment regulating a gene, or gene segment is present in at least two, and possibly more than two, copies in comparison with the single copy found in a normal reference genome.

[0061] A "single nucleotide polymorphism (SNP)" refers to a change in which a single base in the DNA differs from the usual base at that position. These single base changes are called SNPs or "snips." Millions of SNP's have been cataloged in the human genome. Some SNPs such as that which causes sickle cell are responsible for disease. Other SNPs are normal variations in the genome.

[0062] A "sample" refers to a sample from a subject that may be tested, for example, for presence of a CNV in one or more mGluR network proteins, as described herein. The sample may comprise cells, and it may comprise body fluids, such as blood, serum, plasma, cerebral spinal fluid, urine, saliva, tears, pleural fluid, and the like.

[0063] The terms "subject" and "patient" are used interchangeably to refer to a human. The terms "pediatric subject" or "pediatric patient" are used interchangeably to refer to a human less than 18 years of age. An "adult patient" refers to a human 18 years of age or older.

[0064] "Target nucleic acid" as used herein refers to a previously defined region of a nucleic acid present in a complex nucleic acid mixture wherein the defined wild-type region contains at least one known nucleotide variation, which may or may not be associated with autism. The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridi2ation or synfhesi2ed manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer. [0065] With regard to nucleic acids used in the invention, the term "isolated nucleic acid" is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived. For example, the "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote. An "isolated nucleic acid molecule" may also comprise a cDNA molecule. An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a recombinant nucleic acid molecule.

[0066] With respect to RNA molecules, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure" form.

[0067] By the use of the term "enriched" in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that "enriched" does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.

[0068] It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term "purified" in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/ ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10. sup. -6-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. [0069] The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest.

[0070] The term "complementary" describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is

complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a "complement" of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.

[0071] With respect to single stranded nucleic acids, particularly

oligonucleotides, the term "specifically hybridi2ing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridi2ation under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridi2ation of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence. For example, specific hybridization can refer to a sequence which hybridizes to any autism specific marker gene or nucleic acid, but does not hybridize to other nucleotides. Also polynucleotide which "specifically hybridizes" may hybridize only to a neurospecific specific marker, such an autism-specific marker shown in the Table contained herein. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.

[0072] For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989):

T_m=81.5 degrees C+16.6 Log [Na+]+0.41(% G+C)-0.63(% formamide)-

600/ #bp in duplex

[0073] As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57 degrees C. The T_m of a DNA duplex decreases by 1-1.5 degrees C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 degrees C.

[0074] The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridi2ation is usually carried out at salt and temperature conditions that are 20-25 degrees C below the calculated T_m of the hybrid.

[0075] Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20 degrees C below the T_m of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 micro-gram/ml denatured salmon sperm DNA at 42 degree C, and washed in 2xSSC and 0.5% SDS at 55 degree C for 15 minutes. A high stringency hybridization is defined as hybridization in 6xSSC, 5x Denhardt's solution, 0.5% SDS and 100 micro-gram/ml denatured salmon sperm DNA at 42 degree C, and washed in IxSSC and 0.5% SDS at 65 degree C for 15 minutes. A very high stringency hybridization is defined as hybridization in 6xSSC, 5x Denhardt's solution, 0.5% SDS and 100 micro-gram/ml denatured salmon sperm DNA at 42 degree C, and washed in O.lxSSC and 0.5% SDS at 65 degrees C for 15 minutes.

[0076] The term "oligonucleotide," as used herein is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide. Oligonucleotides, which include probes and primers, can be any length from 3 nucleotides to the full length of the nucleic acid molecule, and explicitly include every possible number of contiguous nucleic acids from 3 through the full length of the polynucleotide. Preferably, oligonucleotides are at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length.

[0077] The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction en2yme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences

complementary to the probe. A probe may be either single-stranded or double- stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.

[0078] The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction en2yme digestion, or produced

synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase en2yme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic

applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar en2yme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template -primer complex for the synthesis of the extension product.

Probes and primers having the appropriate sequence homology which specifically hybridized to CNV containing nucleic acids are useful in the detecting the presence of such nucleic acids in biological samples.

[0079] Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein.

[0080] The term "vector" relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.

[0081] Those skilled in the art will recognize that a nucleic acid vector can contain nucleic acid elements other than the promoter element and the autism specific marker gene nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance en2ymes or amino acid metabolic en2ymes, and nucleic acid sequences encoding secretion signals, locali2ation signals, or signals useful for polypeptide purification.

[0082] As used herein, the terms "reporter," "reporter system", "reporter gene," or "reporter gene product" shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.

Methods of Using Autism-Associated C Vs for Diagnosing a Propensity for the Development of Autism and Autistic Spectrum Disorders

[0083] Autism-related-CNV containing nucleic acids, including but not limited to those listed in the Tables and Figures provided herein, for example in Figures 15- 18, may be used for a variety of purposes in accordance with the present invention. Autism-associated CNV/SNP containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/ or expression of autism specific markers. Methods in which autism specific marker nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).

[0084] Further, assays for detecting autism-associated CNVs/SNPs may be conducted on any type of biological sample, including but not limited to body fluids (including blood, urine, serum, gastric lavage), any type of cell (such as brain cells, white blood cells, mononuclear cells) or body tissue. Such detection methods can include for example, southern and northern blotting, RFLP, direct sequencing and PCR amplification followed by hybridization of amplified products to a microarray comprising reference nucleic acid sequences.

[0085] Autism-associated CNV/SNP containing nucleic acids, vectors expressing the same, autism CNV/ SNP containing marker proteins and anti- Autism specific marker antibodies of the invention can be used to detect autism associated CNVs/SNPs in body tissue, cells, or fluid, and alter autism SNP containing marker protein expression for purposes of assessing the genetic and protein interactions involved in the development of autism.

[0086] In some embodiments for screening for autism-associated CNVs, the autism-associated CNV containing nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the templates as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are becoming increasingly important in the art. Alternatively, new detection technologies can overcome this limitation and enable analysis of small samples containing as little as 1 microgram of total RNA. Using Resonance Light Scattering (RLS) technology, as opposed to traditional fluorescence techniques, multiple reads can detect low quantities of mRNAs using biotin labeled hybridi2ed targets and anti-biotin antibodies. Another alternative to PCR amplification involves planar wave guide technology (PWG) to increase signal-to-noise ratios and reduce background interference. Both techniques are commercially available from Qiagen Inc. (USA).

[0087] Thus any of the aforementioned techniques may be used to detect or quantify autism-associated CNV marker expression and accordingly, diagnose autism or an autism spectrum disorder.

Methods of Diagnosing Autism

[0088] In some embodiments, the invention comprises a method of diagnosing autism (including autism spectrum disorder) in a subject comprising analy2ing the genetic information of the subject to determine whether the subject has a genetic variation in at least one mGluR network gene, and diagnosing the subject as having autism if a genetic variation is found. In some embodiments, the subject has autism but does not have ADHD, oppositional defiant disorder (ODD), conduct disorder, Tourette's syndrome (TS), anxiety disorder, phobia, or depression. In some embodiments, the subject has autism and also one or more of ADHD, conduct disorder, TS, anxiety disorder, phobia, and depression. In some embodiments, the subject has both autism and ADHD.

[0089] In other embodiments, the invention encompasses confirming a diagnosis of autism in a subject. As used herein, "confirming a diagnosis of autism" means diagnosing a subject who has already been diagnosed with autism. In some embodiments, the method of confirming a diagnosis of autism comprises analy2ing the genetic information of a subject that has been diagnosed as having autism by a method that does not comprise analy2ing mGluR network genes, to determine whether the subject has a genetic variation in at least one mGluR network gene, and confirming the diagnosis of autism if a genetic variation in at least one mGluR network gene is found. In some embodiments, a screen for the presence of mGluR network gene variations is one of two or more tests or evaluations that are performed to confirm a diagnosis in a subject. In some embodiments, the subject has autism but does not have ADHD, ODD, conduct disorder, TS,anxiety disorder, phobia, or depression. In some embodiments, the subject has autism and also one or more of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, and depression. In some embodiments the subject has both autism and ADHD.

[0090] In other embodiments, the invention comprises confirming a diagnosis of autism in a subject who does not have ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression, comprising analy2ing the genetic information of a subject that has been diagnosed as having autism by a method that does not comprise analy2ing mGluR network genes, to determine whether the subject has a genetic variation in at least one mGluR network gene, and confirming the diagnosis of autism if a genetic variation in at least one mGluR network gene is found.

[0091] In one embodiment, autism is diagnosed and/ or confirmed if at least one CNV, SNV, frameshift mutation, or any other base pair substitution, insertion, deletion or duplication in an mGluR network gene is detected. In other embodiments, autism is diagnosed and/ or confirmed if at least one CNV, SNV, frameshift mutation, or any other base pair substitution, insertion, or deletion in a Tier 1 mGluR network gene is detected. In another embodiment, autism is diagnosed and/ or confirmed if at least one CNV, SNV, frameshift mutation, or any other base pair substitution, insertion, or deletion in a Tier 2 mGluR network gene is detected. In still other embodiments, autism is diagnosed and/ or confirmed if at least one CNV, SNV, frameshift mutation, or any other base pair substitution, insertion, or deletion in a Tier 3 mGluR network gene is detected.

[0092] A diagnosis or confirmation of diagnosis of autism may be based or confirmed on finding a genetic alteration in a Tier 1, Tier 2, and/ or Tier 3 mGluR network gene. The genetic alteration may be a CNV. The CNV may be a duplication or deletion of a region of DNA that contains some or all of the DNA encoding and controlling/ regulating an mGluR network gene. In another embodiment, the diagnosis or confirmation of diagnosis of autism is made in a patient who does not have ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression. In some embodiments, the diagnosis or confirmation of diagnosis of autism is made in a patient who has autism as well as one or more of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, and depression.

[0093] In some embodiments, the diagnosis or confirmation of diagnosis of autism is based on a finding that the copy number of an mGluR network gene deviates from the normal diploid state. In some embodiments, the diagnosis or confirmation of diagnosis of autism is based on a copy number of 2ero or one, which indicates a CNV deletion. In some embodiments, the diagnosis or confirmation of diagnosis of autism is based on a copy number of three or greater, which indicates a CNV duplication. In another embodiment, the diagnosis or confirmation of diagnosis of autism is made in a patient who does not have ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression by the presence of a copy number of 2ero or one, by a copy number of three or greater, or by any deviation from the diploid state.

[0094] In one embodiment, a more severe form of autism is diagnosed if at least two CNVs in mGluR network genes are detected. In one embodiment, a more severe form of autism in a patient who does not have ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression is diagnosed if at least two CNVs in mGluR network genes are detected.

[0095] In one embodiment, a method of diagnosing and/ or confirming autism comprises: obtaining a nucleic acid-containing sample from a subject; optionally amplifying the nucleic acid; optionally labeling the nucleic acid sample; applying the nucleic acid to a solid support that comprises one or more nucleic acids of mGluR network genes, wherein the nucleic acids optionally comprise SNVs of mGluR network genes; removing any unbound nucleic acid sample; and detecting any nucleic acid that has bound to the nucleic acid on the solid support, wherein the subject is diagnosed or confirmed as having autism if bound nucleic acids are detected. In one embodiment the method further comprises comparing any bound nucleic acids to a standard or control and diagnosing or confirming autism if the analysis finds that the test sample is different from the control or standard. In another embodiment of this method, the patient with autism does not have ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression. In another embodiment, the autism patient also has one or more of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression.

[0096] In some diagnostic, confirming, and treatment method of the invention, the subject may have autism but not any of ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression. In other diagnostic, confirming, and treatment methods of the invention, the subject may have autism and one or more additional disorders such as ADHD, conduct disorder, TS, anxiety disorder, phobia, or depression. In some methods, the subject has both autism and ADHD.

Methods of Treating Autism

[0097] Encompassed herein are methods of treating autism in a subject comprising administering an effective amount of a nonselective mGluR activator. The term "treatment," as used herein, includes any administration or application of a therapeutic for a disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving the symptoms of the disease, or preventing occurrence or reoccurrence of the disease or symptoms of the disease.

[0098] The mGluR proteins are typically placed into three sub-groups, group I receptors including mGluRl and mGluR5 are classed as slow excitatory receptors.

[0099] Group II includes mGluR2 and mGluR3. Group III includes mGluR4, mGluR6, mGluR7, and mGluR8. Groups II and III are classed as slow inhibitory receptors.

[00100] The mGluRs are distinguished from the ionotropic GluRs or iGluRs, which are ion channel-associatedglutamate receptors andare classed as fast excitatory receptors.

[00101] A "nonselective activator ofmGluRs" refers to a molecule that activates mGluRs from more than one of the group I, II, and III categories. Thus, a nonselective activator of mGluRs may provide for a general stimulation of the mGluR networks. This is in contrast to specific mGluR activators that may only significantly activate a single mGluR, such as mGluR5, for example. Nonselective mGluRactivators include, for example, nonselective mGluR agonists.

[00102] In some embodiments, the nonselective mGluR activator is fasoracetam. Fasoracetam is a nootropic (i.e., cognitive-enhancing) drug that can stimulate both group I and group II/III mGluRs in in vitro studies. (See Hirouchi M, et al. (2000) European journal of Pharmacology 387:9—17.) Fasoracetam may stimulate adenylate cyclase activity through activation of group I mGluRs, while it may also inhibit adenylate cyclase activity by stimulating group II and III mGluRs. (Oka M, et al (1997) Brain Research 754:121—130.) Fasoracetam has been observed to be highly bioavailable (79%-97%) with a half-life of 5-6.5 hours in prior human studies (see Majl kh AG, et al. (2010) Drugs 70(3):287-312). Fasoracetam is a member of the racetam family of chemicals that share a five-carbon oxopyrrolidone ring. The

structure of fasoracetam is:

[00103] The term "fasoracetam" as used herein encompasses

pharmaceutically acceptable hydrates and any solid state, amorphous, or crystalline forms of the fasoracetam molecule. For example, the term fasoracetam herein includes forms such as NFC-1: fasoracetam monohydrate. In addition to NFC-1, fasoracetam is alsoknownasC-NS-105,NS105,andLAM-105.

[00104] NFC-1 has been previously studied in Phase I-III clinical trials in dementia-related cognitive impairment but did not show sufficient efficacy in dementia in Phase III trials. These trials demonstrated that NFC-1 was generally safe and well tolerated for those indications. Phase III data indicated that NFC-1 showed beneficial effects on psychiatric symptoms in cerebral infarct patients and adult dementia patients with cerebrovascular diseases.

[00105] In each of the method of treatment embodiments, a

metabotropic glutamate receptor positive allosteric modulator, a metabotropic glutamate receptor negative allosteric modulator, or a tachykinin-3/ neurokinin-3 receptor (TACR- 3/NK3R) antagonist may be administered alone or in combination with a nonselective activator of mGluRs to a subject, for example, having an alteration in an mGluR network gene. In some embodiments, the treatment agent comprises ADX63365, ADX50938, ADX71149, AMN082, a l-(hetero)aryl-3-amino- pyrrolidine derivative, LY341495, ADX48621, GSK1144814, or SB223412.

[00106] Also encompassed herein are methods of treating autism comprising administering fasoracetam to a subject that has a genetic alteration in at least one mGluR network gene. In some embodiments, this subject has autism but does not have ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression, while in other embodiments, the subject has autism as well as at least one of ADHD, ODD, conduct disorder, TS,anxiety disorder, phobia, or depression. In some embodiments, the subject has both autism and ADHD.

[00107] In some embodiments, the treatment methods comprise identifying or diagnosing a subject as having a genetic alteration in at least one mGluR network gene, and administering a nonselective mGluR activator such as fasoracetam to the identified or diagnosed subject. In some of the embodiments, the subject has autism, but does not have ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression. In other embodiments, the subject has autism, as well as one or more neuropsychological disorders such as ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, and depression.

[00108] In one embodiment, the nonselective mGluR activator, such as fasoracetam, is administered to a subject that has autism and has been confirmed as having at least one genetic alteration in an mGluR network gene. The genetic alteration may be in a Tier 1 mGluR network gene. The genetic alteration may be in a Tier 2 mGluR network gene. The genetic alteration may be in a Tier 3 mGluR network gene. The genetic alteration may be more than one genetic alteration, and the more than one alteration may be in one of Tiers 1, 2, or 3, or in any combination of Tiers.

[00109] Some embodiments include a method of treating autism comprising obtaining genetic information about a subject's mGluR network genes, and administering a nonselective mGluR activator, such as fasoracetam, if the subject has at least one genetic alteration, such as a CNV, in an mGluR network gene. Other embodiments encompass a method of treating autism comprising obtaining genetic information about a subject's mGluR network genes, and administering a nonselective mGluR activator, such as fasoracetam, if the subject has least one genetic alteration, such as a CNV, in a Tier 1 mGluR network gene. Other embodiments include a method of treating autism comprising obtaining genetic information about a subject's mGluR network genes, and administering a nonselective mGluR activator, such as fasoracetam, if the subject has at least one genetic alteration, such as a CNV, in a Tier 2 mGluR network gene. Still other embodiments encompass a method of treating autism comprising obtaining genetic information about a subject's mGluR network genes, and administering a nonselective mGluR activator, such as fasoracetam, if the subject has at least one genetic alteration, such as a CNV, in a Tier 3 mGluR network gene. Subjects having more than one CNV in any one Tier, or in a combination of any of the three Tiers, maybe treated by administering a nonselective mGluR activator, such as fasoracetam. In some embodiments, subjects having autism, but not having ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, or depression, may be treated. In other method of treatment embodiments of the invention, the subject has one or more neuropsychological disorders such as ADHD, ODD, conduct disorder, TS, anxiety disorder, phobia, and depression in addition to autism.

Dosing

[00110] In some embodiments, fasoracetam may be administered as fasoracetam monohydrate (NFC-1). In some embodiments, fasoracetam may be administered by mouth (i.e., per os). In some embodiments, fasoracetam may be administered as capsules. In some embodiments, fasoracetam capsules may contain 50, 60, 70, 80, 90, 100, 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg of fasoracetam monohydrate. In some embodiments, fasoracetam may be dosed once daily or twice daily. In some embodiments, the daily dose of fasoracetam maybe 50 mg once-daily, 100 mg once-daily, 200 mg once-daily, 400 mg once-daily, 50 mgtwice-daily, 100 mgtwice-daily, 200 mgtwice-daily, or 400 mg twice- daily. In some embodiments, fasoracetam dosing may be adjusted using a series of dose escalations. In some embodiments, pharmacokinetic data on drug level or clinical response is used to determine changes in dosing. In some embodiments, dose escalation of fasoracetam is not used. In some embodiments, subjects are treated at a dose of fasoracetam expected to be clinically efficacious without a dose-escalation protocol. Combination Therapies

[00111] In some embodiments, fasoracetam is used in combination with other agents for the treatment of autism.

Kits and Articles of Manufacture

[00112] Any of the aforementioned products can be incorporated into a kit which may contain a autism-associated CNV/ SNP specific marker polynucleotide or one or more such markers immobilized on a Gene Chip, an oligonucleotide, a polypeptide, a peptide, an antibody, a label, marker, or reporter, a pharmaceutically acceptable carrier, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.

[00113] Vectors and probes comprising any of the autism-associated

CNV/SNP specific marker polynucleotides or the Tier 1, 2, and/ or 3 genes described in Figures 15-18 are encompassed.

[00114] Host cells expressing the autism-associated CNVs/ SNPs of the present invention or functional fragments thereof are encompassed.

[00115] The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

EXAMPLES

Example 1

[00116] A large genome-wide association study (GWAS) of structural variants that disrupt gene family protein interaction networks in patients with autism was performed. Multiple defective networks in the ASDs were found, most notably rare copy-number variants (CNVs) in the metabotropic glutamate receptor (mGluR) signaling pathway in 5.8% of patients with the ASDs. Defective mGluR signaling was found in both ADHD and schizophrenia, two common neurop sychiatric disorders that are highly coincident with the ASDs. Furthermore, other attractive candidates were found such as the MAX dimerization protein (MXD) network that is implicated in cancer, and a Calmodulin 1 (CALM1) gene interaction network that is active in neuronal tissues. The numerous defective gene family interactions found to underlie autism present many novel translational opportunities to explore for therapeutic interventions.

[00117] To identify and comprehensively characterize defective genetic networks underlying the ASDs, a large-scale genome association study for copy- number variation (CNVs) enriched in patients with autism was performed. By combining the affected cases from previously published large ASD studies with more recently recruited cases from the Children's Hospital of Philadelphia, one of the largest searches for rare pathogenic CNVs in ASDs to date was executed. In sum, 6,742 genotyped samples from patients with the ASDs were compared with those from 12,544 neurologically normal controls recruited at The Children's Hospital of Philadelphia (CHOP).

[00118] These cases were each screened by neurodevelopmental specialists to exclude patients with known syndromic causes for autism. Genotyping was performed for the vast majority of the ASD cases as well as all the controls. After cleaning the data to remove sample duplicates and performing standard QC for CNVs, the continental ancestry of 5,627 affected cases and 9,644 disease-free controls was inferred using a training set defined by populations from HapMap 3 and the Human Genome Diversity Panel (Table 1). Using this QC criteria, it was estimated that the sensitivity and specificity of calling CNVs is -70% and 100%, respectively, across 121 different genomic regions assayed by PCR. Across all ethnicities, there was an increased burden of CNVs in cases versus controls, a statistically significantly difference (P≤ 0.001) in the larger European (63.3 versus 54.5 Kb, respectively) and African-derived (70.4 versus 48.0 Kb, respectively) populations.

[00119] Table 1: Distribution of CNVs across samples and estimated ancestry.

Continental ancestry Case Control Total

Europe

Number of samples 4,602 4,722 9,324

:_.C V burden (Kb) 63.3 54.5

Africa

Number of samples 312 4,189 4,481

:_.C V burden (Kb) 70.4 48.0

America

Number of samples 485 276 761

CNV burden (Kb) 59.1 58.4

Asia

Number of samples 201 350 551

CN burden (Kb) 56.1 54.1

Other

Number of samples 27 127 154

CNV burden (Kb) 51 .5 49.4 All Ethnicities

Number of samples 5,627 9,844 15,271

!CN burden (Kb) 63.0 51 .7

[00120] Referring to Table 1, CNV= copy-number variation. The table shows the distribution of cases, controls and CNV coverage across estimated continental ancestry. For groups of cases and controls across estimated ancestries, the table lists the numbers of subjects that passed quality control and their group-wise CNV burden, defined as the average span of CNVs in Kb for each group.

[00121] Referring to Table 1, *Statistically significant (P< 0.01 by

PLINK permutation test) differences in CNV burden are marked with an asterisk(*). Example 2

[00122] A search was performed for pan-ethnic CNV regions (CNVRs) discovered in the European-derived data set (4,602 cases versus 4,722 controls; P≤ 0.0001 by Fisher's exact test) and replicated in an independent ASD data set of African ancestry (312 cases versus 4,169 controls; P≤ 0.001 by Fisher's exact test) with subsequent measurement of overall significance across the entire multi-ethnic discovery cohort (5,627 cases versus 9,644 controls) for maximal power (Fig. 5, Fig. 10).

[00123] Figure 5 shows the significance of CNVRs by GWAS of ASDs in European-derived or African-derived populations. The Manhattan plots show the — loglO transformed P-value of association for each CNVR along the genome.

Adjacent chromosomes are shown in alternating red and blue colors. The regions discovered in Europeans (P≤ 0.0001) that replicated in Africans (P≤ 0.001) are highlighted with black arrows labeled by chromosome band. GWAS of 4,634 cases versus 4,726 controls in Europeans is shown on top and GWAS of 312 cases versus 4,173 controls in Africans is shown below.

[00124] On the basis of these selection criteria, two large well-known

ASD risk loci emerged that harbored multiple duplications in the Prader

Willi/ Angelman syndrome (15qll— 13) critical region, and multiple deletions were detected in the DiGeorge syndrome (22ql 1) critical region, albeit notably smaller than the 22ql 1 deletion syndrome. A third locus harboring deletions in poly ADP- ribose polymerase family 8 (PARP8) on chromosome 5ql 1 was also discovered. PARP8 has previously been identified as associated with the ASDs in a Dutch population but it has not previously been described for its pan ethnic distribution across European-derived and African-derived populations.

Example 3

[00125] The genetic interaction networks derived from gene families with members localized to the Prader Willi/ Angelman syndrome (15ql 1-13) critical region, the DiGeorge syndrome (22qll) critical region, and the novel PARP8 (5qll) region were examined using a method previously applied to ADHD; however, hardly any of the most significant genes harboring significant CNVRs clustered within gene families. Consequently, the search for gene family interaction networks (GFINs) was broadened to search the entire genome for GFINs with CNVs enriched in autism. For every gene family, a GFIN was defined as the genetic interaction network spawned by its multiple duplicated members. Standard HUGO gene names were used to define 1,732 GFINs across which were searched for enrichment of network defects associated with the ASDs. However, because there is an a priori excess of CNV burden in ASD cases over disease-free controls (Table 1), larger GFINs are expected to display significant enrichment of case defects by virtue solely of their increased size and complexity. Therefore, for each GFIN, a network permutation test of case enrichment was used across 1,000 random sets of networked genes to control for the GFIN size and complexity. With this approach, network defects associated with the ASDs were identified by minimizing statistical artifact derived from any a priori excessive CNV burden in cases over controls, as well as other unknown biases that may be inherent in the human interactome data that were mined.

[00126] Out of 1,732 GFINs, the network permutation test was used to rank 1,557 GFINs with defined CNVs for enrichment of genetic defects in the ASDs. Among the top GFINs (Figures 13A-B) was the metabotropic glutamate receptor (mGluR) pathway defined by the GRM family of genes that impacts glutamatergic neurotransmission. The GRM family contains eight members, all of which were defined in the human interactome to cumulatively spawn a GFIN of 279 genes (Figs. 6A-D). Across this GFIN for the GRM family of genes, we found CNV defects in 5.8% of European-derived ASD cases (265/4,602) versus only 3% of ethnically matched controls (153/4,722), a 1.8-fold enrichment of frequency (PFisher 2.40E-09). By 1,000 random network permutations, we found this excess of enrichment across cases in the mGluR pathway to also be statistically significant (Pperm≤ 0.05). In addition, 69.2% (124/ 181) of the informative genes within our mGluR network showed an excess of CNVs among cases. However, the component genes that harbor the most significant CNVRs contributing to this overall network significance reveal that the duplicated mGluR genes themselves (GRM1, GRM3, GRM4, GRM5, GRM6, GRM7 and GRM8) fail to achieve significance individually, although there is a trend for an excess of CNV defects across a specific subset of mGluR receptors (GRM1, GRM3, GRM5, GRM7, GRM8) that is unique to cases (Fig. 9).

[00127] Figures 6A-D shows enrichment of optimal CNVRs across mGluR network of genes. Nodes of the network are labeled with their gene names, with red and green representing deletions and duplications, respectively, while grey nodes lack CNV data. Dark and light colors represent enrichment in cases and controls, respectively. The genes defining the network are shown as diamonds, while all other genes are shown as circles. Blue lines indicate evidence of interaction.

Example 4

[00128] Many large studies of CNVs implicate genes within the glutamatergic signaling pathway in the etiology of the ASDs, and SNP and CNV duplications of GRM8 have been reported in association with the ASDs before in humans. Moreover, a recent functional study demonstrated that in mouse models of tuberous sclerosis and fragile X, two different forms of syndromic autism, the autistic phenotype was ameliorated by modulation of GRM5 in opposite directions for each syndrome, which suggests that GRM5 functional activity is central in defining the axis of synaptopathophysiology in syndromic autism. The GRM network findings herein implicate rare defects in mGluR signalling also contribute to the ASDs outside of fragile X and tuberous sclerosis, and functional mGluR synaptopathophysiology may be initiated from many do2ens if not hundreds of defective genes within the mGluR pathway that may account for as much as 6% of the endophenotypes of the ASDs (Figures 13A-B).

[00129] In addition, the importance of mGluRs in ADHD, a highly coincident neuropsychiatric disorder within the autism spectrum, has been

demonstrated. However, in contrast to ADHD where defects within the mGluR receptors themselves (GRMs) were among the most significant copy-number defects contributing to the overall network significance, it was found that in the ASDs defects of component GRMs contributed only modestly to the overall significance of the mGluR pathway. Nonetheless, the defects within GRMl, GRM3, GRM5, GRM7 and GRM8 that were identified as unique to cases and thus enriched are the same GRMs that were identified as being pathogenic in ADHD and may impact glutamatergic signaling.

[00130] Among the most highly ranked GFINs by permutation testing, the MAX dimerization protein (MXD) GFIN (PFisher <3.83E-23,

enrichment=2.53, Pperm≤0.042) was the most enriched. The MXD family of genes encode proteins that interact with MYC/MAX network of basic heli -loop-helix leucine 2ipper (bHLHZ) transcription factors that regulate cell proliferation, differentiation and apoptosis (MIM 600021); MXD genes are important candidate tumour suppressor genes as the MXD-MYC-MAX network is dysregulated in various types of cancer. Interestingly an epidemiological link between autism and specific types of cancer has been reported, and anticancer therapeutics were recently shown to modulate ASD phenotypes in the mouse through regulation of synaptic NLGN protein levels. Within the component genes contributing to the MXD GFIN significance, duplications in PARP10 (P≤4.06E-11, OR=2.04) and UBE3A

(1.50E— 06, OR=inf) are the most significantly enriched (Figure 12). It is notable that PARP8 was found to be significant across ethnicities as described earlier (Figure 12), and the importance of structural defects in UBE3A in the ASDs was previously described.

[00131] Other notable significant GFINs uncovered were POU class 5 homeobox (POU5F) GIFN (PFisher≤2.96E-17, ennchment=2.3, Pperm <0.008, and the SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily c (SMARCC) GFIN (PFisher <1.22E-09, ennchment=1.9, Pperm ≤0.035). The POU5F family of genes encodes for transcription factors containing a POU homeodomain, and their role has been demonstrated in embryonic

development, especially during early embryogenesis, and it is necessary for embryonic stem cell pluripotency. Component genes of the SMARCC gene family are members of the SWI/ SNF family of proteins, whose members display helicase and ATPase activities and which are thought to regulate transcription of certain genes by altering the chromatin structure around those genes. Most interestingly, the KIAA family of genes ranked among the top GFINs (PFisher≤3.12E— 23, enrichment=1.6, Pperm ≤0.040). KIAA genes have been identified in the Ka2usa cDNA sequencing project and are predicted from novel large human cDNAs; however, they have no known function.

[00132] Some component members of gene families may contribute disproportionately to the significance of a GFIN because they are highly connected to interacting gene partners that are enriched for CNV defects in ASD. Therefore, the 1,732 gene families were decomposed into their 15,352 component duplicated genes of which 1,218 had defined networks with data to test for significance by genome-wide network permutation. The calmodulin 1 (CALMl) gene interaction network ranked highest by network permutation testing of case enrichment for CNV defects across 1,000 random gene networks (Figure 7, Figure 14) and represents a novel and attractive candidate gene for the ASDs. Across the CALMl network, CNV defects were found in 14/4,618 cases versus only 1/4726 controls (Pfisher ≤4.16E— 04, enrichment= 14.37, Pperm 0.002), and these defects were distributed such that 90% (9/ 10) of genes that harbored CNVs in the CALMl interactome were enriched in cases. Closer inspection of the most significant CNVR contributing to the CALMl network significance (Figure 11) revealed that no single gene was significant on its own; instead, with the exception of only one gene (PTH2R), each contributing CNVR tagged highly penetrant rare defects unique to cases. Calmodulin is the archetype of the family of calcium-modulated proteins of which nearly 20 members have been found. Calmodulin contains 149 amino acids that define four calcium- binding domains used for Ca2+-mediated coordination of a large number of en2ymes, ion channels and other proteins including kinases and phosphatases; its functions include roles in growth and cell cycle regulation as well as in signal transduction and the synthesis and release of neurotransmitters [MIM 114180].

[00133] Figure 7 shows the enrichment of optimal CNVRs across

CALMl network. The first degree-directed interaction network defined by CALMl is shown.

[00134] The comprehensive, unbiased analytical approach described herein has identified a diverse set of specific defective biological pathways that contribute to the underlying aetiology of the ASDs. Among GFINs robustly enriched for structural defects, the most enriched was that of the MXD family of genes that has been implicated in cancer pathogenesis, thereby providing concrete genetic defects to explore the reported coincidence of specific cancers with the ASDs. The most highly ranked component duplicated gene interaction network involves defects in CALMl and its multiple interacting partners that are important in regulating voltage-independent calcium-activated action potentials at the neuronal synapse. Moreover, there was significant enrichment for defects within the GFIN for GRM that defines the mGluR pathway. While specific mGluR gene family members have been shown to underlie syndromic ASDs, these findings suggest that rare defects in mGluR signaling also contribute to idiopathic autism across the entire GFIN for GRM genes.

[00135] Given the unmet need for better treatment for

neurodevelopmental diseases, the functionally diverse set of defective genetic interaction networks reported herein presents attractive genetic biomarkers to consider for targeted therapeutic intervention in ASDs and across the

neuropsychiatric disease spectrum.

Methods applicable to Examples 1-4

[00136] The majority of cases (5,049 of 6,742) and all controls (12,544) were geno typed with genome-wide coverage using the Infinium II platform across various iterations of the HumanHap BeadChip with 550 K, 610 K, 660 K and 1 M markers by the Center for Applied Genomics at The Children's Hospital of

Philadelphia (CHOP). There were 1,693 cases genotyped by the AGP consortium. All cases and ~\n50% of controls were re-used from previously published large ASD studies. All cases were diagnosed by ADI-R/ADOS and fulfilled standard criteria for ASDs. Duplicate samples were removed by selecting unique samples with the best quality (based on genotyping statistics used to QC samples) from clusters defined by single linkage clustering of all pairs of samples with high pairwise identity by state measures (IBS≥0.9) across 140 K non-correlated SNPs. Ethnicity of samples was inferred by a supervised k-means classification (k=3) of the first 10 eigenvectors estimated by principal component analysis across the same subset of 140 K non- correlated SNPs. We used HapMap 3 and the Human Genome Diversity Panel samples with known continental ancestry to train the k-means classifier implemented by the R Language for Statistical Computing. C V inference and association

[00137] CNVs with the PennCNV algorithm, which combines multiple values, including genotyping fluorescence intensity (Log R Ratio), population frequency of SNP minor alleles (B-allele frequency) and SNP spacing were called into a hidden Markov model. The term 'CNV represents individual CNV calls, whereas 'CNVR' refers to population-level variation shared across subjects. Quality control thresholds for sample inclusion in CNV analysis included a high call rate (call rate ≥95%) across SNPs, low s.d. of normali2ed intensity (s.d.≤0.3), low absolute genomic wave artifacts ( | GCWF | 0.02) and low numbers of CNVs called (#CNVs ≤100). Genome -wide differences in CNV burden, defined as the average span of CNVs, between cases and controls and estimates of significance were computed using PLINK. CNVRs were defined based on the genomic boundaries of individual CNVs, and the significance of the difference in CNVR frequency between cases and controls was evaluated at each CNVR using Fisher's exact test.

Gene family interaction networks definition and association

[00138] Previous work on ADHD was extended here to rank all GFINs by a network permutation test. Specifically, using merged human interactome data from three different yeast two hybrid generated data sets accessed through the Human Interactome Database, the directed second-degree gene interaction network was defined for all gene families just as was done for the sole metabotropic glutamate receptor gene family network in ADHD. Specifically, GFIN was used to refer to these gene family-derived interaction networks. In sum, 2,611 gene families were found with at least two members based on official HUGO gene nomenclature, and generated 1,732 GFINs using. For 1,557 GFINs with defined CNVs an odds ratio was calculated of cumulative network enrichment over all genes harboring CNVs within the network. Moreover, for each GFIN, its enrichment was quantified by a permutation test of 1,000 second-degree gene interaction networks derived from a random set of N genes, where N is the number of members of a given gene family. Because the CNVs focused on are so rare, there is relatively not enough power to achieve significance by permutation testing after correcting for multiple GFIN tests. However, all GFINs are reported in order of their nominal/ marginal significance. Experimental validation of CNVs

[00139] Significant CNVRs that were identified were validated using commercially available qPCR Taqman probes run on the ABI GeneAmp 9700 system from Life Technology. Figure 9 lists 251 reactions that were tested using 121 different genomic probes across 85 different samples for which DNA was available. For deletions,

NPV=1.00 and PPV=0.88. For duplications, sensitivity=0.68, specificity=0.99, NPV=0.94 and PPV=0.91.

Conclusions

[00140] The data presented herein is evidence that ASD can be diagnosed after recognition of at least one CNV in an mGluR network gene selected from the group consisting of ACAT1, ACAT2, ACPI, ACTR2, ADCYl, ADDl, ADRA2C, ADRBKl, AGAP2, ALDOA, APTX, ARHGAP24, ARL15, BDKRB1, BDKRB2, ClorfU6, C4orf3, C7orf25, CA8, CACNA1B, CALB2, CALM1, CAMK2B, CHP, CHRM3, CNPY2, CNRl, COPB2, DCN, DHCR7, DISCI, DSTN, ECHS1, EGFR, ERBB2, ERP44, F2RL2, FKBP3, FURIN, GLP1R, GLP2R, GNA15, GNAI1, GNAI2, GNAI3, GNAOl, GNAQ, GNB2L1, GRB2, GRB7, GRIK1, GRM1, GRM3, GRM5, GRM7, GRM8, GSN, HNRNPA3, HOMER1, HOMER3, HTT, IMPDH2, IQGAP2, ITGB7, ITPR1, LAMA4, LRP2BP, LYAR, LYN, MAP4, MAPK1, MTHFD1, MTNR1A, MX1, MY06, NAA15, NCK1, NPY2R, PCBP1, PCBP3, PCDHA4, PDE1C, PICKl, PIK3CA, PLA2G7, PLCB1, PRDXl, PRKCA, PRPSAP1, PSMC1, PSMD1, PSMD13, PSME1, PXN, RAB2A, RANBPl, RAP2A, RCCl, RGS12, RPA2, RPN2, RUVBL2, RYR2, S100A6, SACS, SDC3, SERPINB9, SLC6A3, SNRPB2, SRC, STRAP, STX12, SYK, TCPl, TEAD3, TFAM, TJP1, TRAF2, TUBA1A, TUBA3C, TXN, UBQLN4, VHL, and YWHAQ (also shown in Figures 15A-D).

[00141] The CNVs in these genes are sensitive and specific biomarkers for selecting and treating ADHD due to defective mGluR pathways. Furthermore, the present inventors have identified drug candidates that specifically activate the mGluRs, potentially restoring normal neurophysiology in ASD patients with mutations in any of the mGluR network genes, as shown in Figures 15A-D.

[00142] For example, compounds which may be administered in implementing the test and treat paradigm described herein include the piracetam family of nootropic agents, as described in F. Gualtieri et al., Curr. Phann. Des., 8: 125-38 (2002). More preferably, the treating agent is a pyroglutamide. Details regarding the preparation and formulation of pyroglutamide s, which may be used in the practice of this invention, are provided in U.S. Pat. No. 5,102,882 to Kimura et al. A particularly preferred agent for the treatment of ASD in patients determined to have one or more of the CNVs indicative as set forth in Figures 15A-D, is (+)-5-oxo- Dprolinepiperidinamide monohydrate (NS-105).

Example 5

The Role of mGluR Copy Number Variation in Genetic and Environmental Forms of Syndromic Autism Spectrum Disorder

[00143] Abnormal signaling mediated through mGluR5 is involved in the pathophysiology of Autism Spectrum Disorder (ASD) in Fragile X Syndrome and Tuberous Sclerosis. However, the role of other mGluR associated network/ signaling genes in syndromic ASD is unknown. To determine whether copy number variants (CNVS) are enriched in syndromic ASD, microarrays were used to identify mGluR network CNVs in children with ASD. We set out to determine 1) whether rate of syndromic features vary between children with ASD with and without CNVs in mGluR network genes; and 2) whether "second hits" in mGluR network genes occur more often in children with ASD in children with 22qll.2 Deletion Syndrome (who all have haplo insufficiency of RANBP1, an mGluR network gene in the 22ql 1.2 region.

[00144] Individuals in our biorepository with parental report of ASD

(n=6,452) were screened for parental consent to access clinical evaluations in the Electronic Health Record at the Children's Hospital of Philadelphia (n=539). Our syndromic comparison cohort included children with 22qll.2 Deletion Syndrome with full access to past medical and neuropsychological evaluations (n=75), including those with diagnosis of ASD (n=25) and those with no concern for ASD (n=50).

[00145] Patient categorization (syndromic vs nonsyndromic) was done via blinded medical chart review in all mGluR positive and 100 randomly selected mGluR negative cases.

[00146] Our results, explained further herein show that 11.5% of ASD had mGluR CNV's vs. 3.2% in healthy controls (p<0.001). Syndromic ASD was more prevalent in children with mGluR CNVs (72% vs 16%, p<0.001). A

comparison cohort of children with 22qll.2 Deletion Syndrome (n=25 with ASD, n=50 without ASD), all haplo-insufficient for mGluR network gene RANBP1, was evaluated to determine whether "second hits" in mGluR network genes confer additional risk for ASD. 20% with 22qll.2DS+ASD had "second hits" in mGluR signaling genes vs 2% in 22qll.2DS-ASD (p<0.014). Conclusions: We propose that altered RANBPl expression may provide a mechanistic link between ASD in 22qll.2DS, Thalidomide Embryopathy and Fetal Valproate Syndrome, providing a link for seemingly unrelated genetic and environmental forms of ASD.

[00147] The results suggest that CNV's in mGluR network genes, previously implicated in altered neurological development in Fragile X Syndrome and Tuberous Sclerosis, may link many other genetic and environmental forms of Autism Spectrum Disorder. As discussed in the previous examples, Autism Spectrum

Disorder (ASD) occurs in approximately 1/88 individuals and is characterized by impairment in social communication and repetitive interests and activities. Approximately 20% of cases occur in the context of an identifiable syndrome.

Genetic syndromes with ASD are heterogeneous, including cytogenetically visible chromosomal alterations (e.g. Trisomy 21), microdeletion and microduplication syndromes (e.g. 22qll.2 deletion syndrome [22qll.2DS]; 22qll.2 duplication syndrome [22qll.2DupS]), and monogenic disorders (e.g. Fragile X Syndrome [FXS], Tuberous Sclerosis [TS]). In addition, prenatal exposure to thalidomide, valproic acid, misoprostol, ethanol and maternal rubella infection, have been associated with an elevated risk of ASD.

[00148] The mechanism for the development of ASD in most forms of idiopathic and syndromic forms of ASD remains elusive. Recently, signaling through metabotropic glutamate receptor 5 (mGluR5) has been implicated in the development of ASD in FXS and TS. In FXS, abnormal production of Fragile X Mental

Retardation Protein (FMRP) removes normal inhibition of signaling through the mGluR pathway. Tuberous Sclerosis leads to over inhibition of signaling. Auerbach and colleagues (2011) demonstrated abnormal synaptic learning and atypical behavior in mouse models of FXS and TS, and reversed these effects by breeding the two strains together— mice harboring both mutations had normal mGluR signaling, and learning and behavior that was indistinguishable from control mice. Other studies have demonstrated normalization of learning and behavior in Fragile X mice by administration of an mGluR5 antagonist. In addition to elucidating the mechanism for cognitive and behavioral differences in FXS and TS, these studies suggest a promising avenue for pharmacological treatment. To determine whether additional forms of syndromic ASD may share a similar mechanism (through disruption of the mGluR gene network), we analy2ed DNA from 539 children with ASD (not filtered for comorbid genetic syndrome) followed at the Children's Hospital of Philadelphia.

[00149] The following materials and methods are provided to facilitate the practice of Example 5.

[00150] Participants: Phenotypic data for patients with ASD as reported on parental health questionnaires from our biorepository (n= 6,452) were evaluated to identify patients who received clinical assessment at the Children's Hospital of Philadelphia and agreed to Electronic Health Record chart review. DNA from these cases (n=539) were selected for further phenotypic and genotypic analysis. Children were recruited for inclusion in the general Center for Applied Genomics

biorepository when they were getting blood drawn for another purpose at The Children's Hospital of Philadelphia, so there is an overrepresentation of children with at least one medical problem in this patient cohort. The parents of all patients gave consent for participation in the study, which was approved by the Institutional Review Board at the Children's Hospital of Philadelphia (IRB 06-004886).

[00151] Chart Review: Subject selection and randomi2ation process: All patients with an mGluR CNV (n=62) and 100 patients without mGluR CNV were randomly selected for chart review. This procedure was selected to ensure that all patients with mGluR CNV received detailed chart review with an adequately si2ed comparison cohort. A three-step process was done to ensure blinded chart review. The selection of the 162 charts was done by a geneticist with access to CNV data but without access to the Electronic Health Record (CK). Another author who had no access to CNV data nor the Electronic Health Record blinded and randomized the patient ID's (RTS). Finally, a physician with access to the Electronic Health Record but blinded to mGluR status (TLW) reviewed charts for documentation of ASD diagnosis and presence of other medical comorbidities.

[00152] ASD: Charts were reviewed to confirm a diagnosis of ASD and also to determine medical comorbidities for each patient. Diagnosis of ASD was confirmed in the chart, but as this was a retrospective chart review, gold-standard research instruments (e.g. Autism).

[00153] Medical Comorbidities: Structural birth defects, genetic testing and medical conditions were recorded for each patient. Cases were categorized as "Syndromic ASD" if they had ASD and presence of a medical condition or structural birth defect (e.g. cleft palate) that occurs in less than 1% of the general population. This criteria was established to define a subset of patients whose ASD and other medical problems would be highly unlikely to occur coincidentally— With a baseline rate of ASD at 1/88 and a medical condition that occurs in <1% of the general population, the compound likelihood of both occurring by chance would be approximately 0.001%. See FIG. 8.

[00154] Genotyping Arrays and CNV Calling: DNA from subjects with

ASD were each genotyped on the Human610-Quad or HumanHap550 SNP arrays from Illumina. For 22qll DS cohorts, subjects were typed either on Illumina SNP arrays (Human610-Quad vl.O or HumanHap550) or Affymetrix 6.0 SNP arrays. Clustering and SNP calling was performed using GenomeStudio (Illumina) to generate normalized intensity (i.e. Log-R ratio, or LRR) and B-allele frequencies (BAF). CNV calling was performed using the PennCNV algorithm [PMID:

17921354] following wavmess correction [PMID: 18784189]. In brief, PennCNV uses a hidden Markov model (HMM) that incorporates information from LRR, BAF, as well as features of the array (e.g. distance between neighboring SNPs) to detect CNVs.

[00155] CNV Quality Control: Samples with SNP arrays of poor quality were excluded from CNV calling, since typically the proportion of false positives increases considerably for these samples. Those samples where the genotyping call rate>96%, standard deviation of LRR (LRR sd)<0.4, GC-wave factor (GCWF) is between -0.2 and 0.2 after waviness correction, and total number of CNV calls for the sample <100 were included in analysis.

[00156] CNV Annotation: For syndromic ASD regions, genomic coordinates were those described by Betancur [PMID: 21129364]. The GRM/mGluR network generated by Cytoscape from the Human Interactome database was described by Elia et al. [PMID: 22138692] using UCSC Genome Browser definitions for gene coordinates (UCSC genes). This network from Cytoscape was used to define mGluR+ vs. mGluR-subsets. For 22qll DS cohort analysis, additional

GRM/ mGluR network genes were identified based on 1st degree interaction network of the eight GRM genes using the program Ingenuity Pathway Analysis (Ingenuity Systems Inc./ Qiagen; Redwood City, Calif.) as well as the genes encoding the group I mGluR signaling pathway described in Kelleher et al. [PMID: 22558107]. CNV calls were analy2ed for overlap to known syndromic regions and GRM network genes. All syndromic aberrations detected by clinical cytogenetic laboratory testing were confirmed on corresponding SNP arrays.

Results: mGluR Network Copy Number Variations (CNVs) are Prevalent in Syndromic ASD Compared to Nonsyndromic ASD

[00157] CNVs in the mGluR network genes were found in 74% of patients with syndromic ASD compared to 16% of patients with nonsyndromic ASD (p<0.001). Most of the mGluR CNVs in patients with syndromic ASD (75%) were included in larger clinically significant CNVs. As mGluR network genes are present in the 22qll.2 region (RANBPl) and on chromosome 21 (APP GRIKl MXl PCBP3 SETD4), patients with ASD in the presence of 22qll.2DS, 22qll.2DupS or Trisomy 21 accounted for 15 (33%) of the patients with Syndromic ASD+mGluR network changes. The remainder of observed cytogenetic changes had individual non- overlapping deletions or duplications. The analysis was repeated after exclusion of children with Trisomy 21, 22qll.2DS and 22qll.2DupS, (the syndromes in children in this study which have previously been associated with ASD). After their exclusion, the effect remained significant (p<0.001).

Autism Spectrum Disorder in 22qll.2 Deletion Syndrome is Associated with "Second Hit" in mGluR Pathway

[00158] As a comparison cohort, data from children with 22ql 1.2 DS with ASD (n=25) and without ASD (n=50) who had completed high density microarray evaluation (either Affymetrix 6.0, Illumina 500K, and Illumina 610Q) and clinical developmental assessments (as enrolled through a parallel study, approved by the Children's Hospital of Philadelphia Institutional Review Board, IRB 07-005352) were examined for the presence of a second mGluR network hit outside of the 22qll.2 region. "Second hits", deletions of an mGluR network gene outside of the 22qll.2 region, were found in 20% (5/ 25) of patients with ASD and only 2% (1/50) without ASD (p<0.014).

[00159] Prior studies have demonstrated that abnormal signaling (either too much or too little) through mGluR5 could be the basis for abnormal neural development (and possibly ASD) in FXS and TS. Our data suggest that derangement of the mGluR network may be responsible for increased rates of ASD seen in cytogenetically distinct forms of syndromic ASD. mGluR network genes are found in the 22qll.2 region as well as on Chromosome 21, which may be involved in the increased prevalence of ASD in both Down Syndrome and 22qll.2 DS. However, all patients with Trisomy 21 or 22qll.2 DS harbor the change in the mGluR network suggesting a second hit outside of the region may be necessary for expression of the ASD phenotype.

Autism Spectrum Disorder in 22qll.2 Deletion Syndrome, 22qll.2 Duplication Syndrome, Thalidomide Embryopathy and Fetal Valproate Syndrome

[00160] The 22qll.2 DS is the most common microdeletion syndrome in humans, occurring in 1 in 4,000 individuals. The typical deletion spans

approximately 3 Mb and includes approximately 45 genes, causing a variety of medical and behavioral disorders. ASD occurs in approximately 20%, and psychosis in 25%. The 22qll.2DupS results in the same types of birth defects and medical comorbidities seen in 22qll.2 DS, but at a lower rate (among over 60 patients in our clinical cohort). There are no cases of psychosis in 22qllDupS in the literature. sup.29 or our cohort. Among our cases with documentation of developmental evaluation after the age of 4, the prevalence of ASD is 27%, which is slightly higher than the rate in children with 22qll.2DS.

[00161] Thalidomide exposure during pregnancy causes a variety of birth defects that have all been reported in 22qll.2DS, including some that are extremely rare (e.g. phocomelia, radial ray defects). Miller and Stromland reported an elevated risk of ASD following exposure to thalidomide during early embryogenesis. This study included prospective evaluation by a psychiatrist was done for adults who had been exposed to thalidomide during pregnancy and evaluation by a physician to document birth defects and associated features. All cases of ASD following thalidomide exposure had ear anomalies, suggesting exposure between days 24-28 post-fertili2ation. Among individuals exposed at this time, there was a 27% rate of ASD. Replications of this study in additional cohorts of children have not been possible because the use of thalidomide in pregnant women was widely restricted in the 1960's; therefore, additional cases are not available. Though several mechanisms for the cause of many of the birth defects in thalidomide embryopathy have been proposed, animal studies of the teratogenic effects of thalidomide have been limited due to significant species differences. One of the reasons thalidomide was used widely in the 1960's was because of a lack of teratogenicity in animals at levels that are highly teratogenic in humans. This has resulted in significant limitation in the ability of researchers to determine the teratogenic mechanism of thalidomide, as studies have taken place in animals for which thalidomide is not particularly teratogenic, or using dosages which are much higher than that used in humans. Recent changes in legislation have allowed for a study to be completed in human embryonic stem cells— the first of its kind to use human cells and dosages which would have been analogous to that experienced by women taking thalidomide in the 1950's and 1960's.sup.30. This study, conducted by Meganathan and colleagues (2012) proposed that the teratogenic effects of thalidomide may be mediated through RANBPl. Valproic acid (VPA) is widely used as an anticonvulsant, mood stabilizer, and to prevent migraine headaches. Exposure to VPA during pregnancy causes an increased rate of several birth defects, all of which have been reported in 22qll.2DS, and most of which have been seen in Thalidomide Embryopathy. The comparison of all birth defects seen in 22qll.2 DS to the exposures syndromes was not made because 22qll.2 DS includes deletion of dozens of additional genes that we do not propose to be affected in Thalidomide Embryopathy or Fetal Valproate Syndrome. In addition to structural defects, children exposed to VPA in utero have an elevated risk of developing ASD. Rodent models of autism have used prenatal exposure to VPA to reproduce some of the neuroanatomic features of autism and abnormal behavior. Due to its action as a Histone Deacetylase Inhibitor, VPA affects expression of many genes. Based on homology, decreased expression of RanBPl mRNA is predicted in VPA-treated rats. Moreover, a recent study showed reversal of atypical behaviors in V A-exposed mice with treatment with an mGluR antagonist.

CONCLUSIONS

[00162] Derangement of genes in the mGluR network are found at a high rate in patients with different forms of Syndromic ASD, including 22qll.2DS, 22qll.2DupS, Trisomy 21 and a large number of other seemingly-unrelated chromosomal alterations. Moreover, among children with 22qll.2DS, the presence of a "second hit" in the mGluR network was identified in 20% of children with ASD, and only 2% of those without ASD (p<0.014). Significantly, four children, all with autism phenotype, had a small deletion in the vicinity of the RANBP1 gene. While the expression level of RANBP1 was not affected in one individual available for testing (data not shown), these atypical deletions could impact gene function with resulting dysregulation of the RANBPl protein.

[00163] Taken together, these data implicate dysregulation of the mGluR network as a likely permissive factor that increases the propensity to develop an ASD, including syndromic ASD, and ASD in individuals with 22qll.2 Deletion or Duplication Syndrome, Fetal Valproate Syndrome and Thalidomide Embryopathy. The striking increase in prevalence of ASD with a CNV affecting a second gene in the network suggests perturbations of mGluR signaling at multiple points is likely.

[00164] CNVs in the genes recited in Figures 15A-D are sensitive and specific biomarkers for selecting and treating ASD, syndromic ASD, and ASD in individuals with 22qll.2 Deletion or Duplication Syndrome, Fetal Valproate Syndrome and Thalidomide Embryopathy. Furthermore, the present inventors have identified drug candidates that specifically activate the mGluRs, potentially restoring normal neurophysiology in patients with mutations in any of the mGluR network genes, as shown in Figures 15A-D.

[00165] For example, compounds which may be administered in implementing the test and treat paradigm described herein include the piracetam family of nootropic agents, as described in F. Gualtieri et al., Curr. Phann. Des., 8: 125-38 (2002). More preferably, the treating agent is a pyroglutamide. Details regarding the preparation and formulation of pyroglutamides, which may be used in the practice of this invention, are provided in U.S. Pat. No. 5,102,882 to Kimura et al. A particularly preferred agent for the treatment of ASD in patients determined to have one or more of the CNVs indicative as set forth in Figures 15A-D, is (+)-5-oxo- Dprolinepiperidinamide monohydrate (NS-105).

[00166] Therefore, in one embodiment, methods for treating an ASD, syndromic ASD, and ASD in patients with 22qll.2 Deletion or Duplication

Syndrome, Fetal Valproate Syndrome or Thalidomide Embryopathy are encompassed comprising administering NS-105 to a patient having at least one CNV in an

GRM/ mGluR network gene selected from the group consisting of ACATl, ACAT2, ACPI, ACTR2, ADCY1, ADD1, ADRA2C, ADRBKl, AGAP2, ALDOA, APTX, ARHGAP24, ARL15, BDKRB1, BDKRB2, Clorfll6, C4orf3, C7orf25, CA8, CACNA1B, CALB2, CALM1, CAMK2B, CHP, CHRM3, CNPY2, CNR1, COPB2, DCN, DHCR7, DISCI, DSTN, ECHS1, EGFR, ERBB2, ERP44, F2RL2, FKBP3, FURIN, GLP1R, GLP2R, GNA15, GNAI1, GNAI2, GNAI3, GNAOl, GNAQ, GNB2L1, GRB2, GRB7, GRIK1, GRMl, GRM3, GRM5, GRM7, GRM8, GSN, HNRNPA3, HOMER1, HOMER3, HTT, IMPDH2, IQGAP2, ITGB7, ITPR1, LAMA4, LRP2BP, LYAR, LYN, MAP4, MAPK1, MTHFD1, MTNR1A, MX1, MY06, NAA15, NCK1, NPY2R, PCBP1, PCBP3, PCDHA4, PDE1C, PICKl, PIK3CA, PLA2G7, PLCB1, PRDXl, PRKCA, PRPSAP1, PSMC1, PSMD1, PSMD13, PSMEl, PXN, RAB2A, RANBPl, RAP2A, RCCl, RGS12, RPA2, RPN2, RUVBL2, RYR2, S100A6, SACS, SDC3, SERPINB9, SLC6A3, SNRPB2, SRC, STRAP, STX12, SYK, TCPl, TEAD3, TFAM, TJPl, TRAF2, TUBAIA, TUBA3C, TXN, UBQLN4, VHL, and YWHAQ.

Example 6

Treatment with Fasoracetam Monohydrate (NFC-1) of ADHD Patients with CNVs in mGluR Network Genes and Impact on Autism Symptoms

[00167] An open-label Phase lb clinical trial was conducted to investigate the safety, pharmacokinetics and efficacy of NFC-1 (fasoracetam monohydrate) in adolescent subjects between the ages of 12 and 17 previously diagnosed with ADHD who also had at least one genetic alteration in an mGluR network gene.

[00168] The study included 30 ADHD subjects who were between ages 12 and 17, of any ancestry or race, and of weight within the 5^th to 95^th percentile for their age, and otherwise judged to be in good medical health. Subjects were genotypedand included in the trial if they possess at least one genetic alteration in the form of at least one copy number variation (deletion or duplication) in an mGluR network gene that potentially disrupts the function of the gene. Seventeen of the 30 subjects have a CNV in a tier 1 mGluR network gene, while 7 subjects have a CNV in a tier 2 gene and 6 in a tier 3 gene. At enrollment, several trial subjects showed evidence of co-morbid phenotypes,includinga few subjects with symptoms related to autism.

[00169] Exclusion criteria comprised subjects suffering from a clinically significant illness, either mental or physical, that, in the investigator's opinion, might confound the results of the study or that might prevent them from completing the study, subjects that are pregnant or nursing, subjects that test positive for illicit drugs of that have a history of drug abuse, subjects that consume alcoholic beverages, or subjects for which the investigator is otherwise concerned regarding their compliance or suitability.

[00170] NFC-1 capsules of either 50 mg or 200 mg comprising fasoracetam monohydrate as active ingredient and placebo capsules comprising microcellulose were used for the study. The design of the trial was a phone screening (1 day) , enrollment phase (1 to 2 days) , a wash-out phase for subjects currently on ADHD medications (l-14days),pharmacokinetic (PK) assessment (2 days), followed byadose- escalation phase (35 days) and a follow-up phone visit approximately four weeks after the last dose, for a maximum of 127 days. All ADHD medications were discontinued during the wash-out phase prior to the study. The wash-out period for stimulants was 2-3 days and that for atomoxetine or noradrenergic agonists was 10-12 days. No new ADHD medications were started during the study. [00171] A dose-escalation phase of the trial ran over a 5-week period, after the initial wash-out period and the PK and initial safety assessments. During week 1, all subjects were administeredplacebo capsules twice daily. After one week of placebo treatment, patients were started on 50 mg bid NFC-1 for 1 week. If safety and responsiveness data from prior dose level of fasoracetam indicated it was appropriate, subjects were then escalated to the next higher dose (100, 200, or 400 mg). Subjects who showed tolerance to the 50 mg bid dose as well as response to the drug were to be maintained at that level for the remaining 3 weeks of the trial.

[00172] Subjects who showed tolerance but lack of response or partial response to the 50 mg bid dose were to be moved up to the next higher dose of 100 mg during the following week. Subj ects who showed tolerance at 100 mg but lack of response or partial response were to be moved up to the 200 mg dose the following week while those who showed both tolerance and response at 100 mg were to be kept at 100 mg bid for the remainder of the trial. Similarly, subjects moved up to the 200 mg dose who showed both tolerance and response were to be kept at 200 mg for the final week of the trial while those showing tolerance but lack of response or partial response were moved to a 400 mg dose for the final week. Of the 30 trial subjects, 3 received a maximum dose of 100 mg, 9 received a maximum dose of 200 mg, and the remaining 18 received a maximum dose of 400 mg.

[00173] Whilethis studywasnotspecificallydirectedatidentifying autism sufferers or monitoring their symptoms, 7 of the 30 ADHD subjects showed certain signs of autism, particularly lack of social interactions or engagement, that appeared to improve from the therapy with NFC-1. For example, two subjects showed increased social interactions after treatment. One of these two subjects, who had not communicated with a close relative for a long time, initiated contact with the relative. One subject went from not making eye contact to initiating conversation with clinicians. This same subject also had episodes of rapid repetitive movements, particularly when excited. These movements stopped while on the NFC-1 treatment and started again when the drug was discontinued. A teacher also commented during the trial that this subject also had increased social interactions. One subject went from not wanting to talk to peers to joining a school debating team. One subject was reported to be better able to relate to peers and to organize a peer get together. One subject was able to go to their locker at school without being fearful. And in a further subject, there was a decrease in self-talk. Thus, overall, in these seven subjects, social interactions appeared to improve while on NFC-1 medication.

EQUIVALENTS

[00174] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof. [00175] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/ -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:

1. A method of treating autism in a human patient comprising administering a therapeutically effective amount of fasoracetam (NS-105 or NFC-1) to the patient, wherein the patient has at least one CNV in a gene selected from the group consisting of ACAT1, ACAT2, ACCN1, ACCN2, ACPI, ACTB, ACTR2, ADA, ADCY1, ADD1, ADD2, ADORA1, ADRA1B, ADRA2A, ADRA2C, ADRB2, ADRBKl, ALDOA, ANXA2, APP, APTX, AQP1, ARHGAP24, ARL15, ARRB1, ARRB2, ATXN7L3, BDKRBl, BDKRB2, BTBD2, BTG2, C17orf44, Clorfll6, C7orf25, CA8, CACNA1B, CACYBP, CALB2, CALM1, CALM2, CALM3, CAMK1,

CAMK2B, CAMK4, CCNB1, CDC42, CENTG1, CHGB, CHP, CHRM2, CHRM3, CIC, CMPK, CNP, CNR1, CNTN4, COPB2, CRHR1, CTNNA2, CYCS, DCN, DHCR7, DISCI, DLST, DPP6, DRD2, DRD3, DSTN, DYNLL1, ECHS1, EGFR, EIF3S3, ERBB2, F2R, F2RL2, F2RL3, F3, FKBP3, FPR1, FSCN1, FURIN, FYN, GAPDH, GLP1R, GLP2R, GNA15, GNAI1, GNAI2, GNAI3, GNAOl, GNAQ, GNB2L1, GOTl, GPIBA, GPR26, GRB2, GRB7, GRIAl, GRIKl, GRIK3, GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, GRM8, GSN, HBXIP, HD,

HNRPA3, HOMER1, HOMER3, HRPT2, HSP90AB1, HTR2A, IL8RB, IMPDH2, IQGAP2, ITGB1, ITGB7, ITPR1, KIAA0090, KIAA1683, LAMA4, LARP7, LRP2BP, LRRC59, LTA, LYAR, LYN, MAP4, MAPK1, MAPT, MARK4, MC4R, MGC11082, MRPL14, MRPS16, MTHFD1, MTNR1A, MTNR1B, MX1, MYC, MY06, NANS, NARGl, NCKl, NEGRI, NFKBIA, NLN, NMI, NPY2R, NUDC, OPRDl, PAFAH1B3, PCBP1, PCBP3, PCDHA4, PCID1, PCMTl, PDCD5, PDE1B, PDE1C, PDE6G, PGM1, PHKB, PHKG2, PICKl, PIK3CA, PIK3R1, PLA2G7, PLCB1, PLCB3, PLCG2, PPIH, PPP2R1A, PRDXl, PRKCA, PRLHR, PRMTl, PRPSAPl, PSATl, PSENl, PSMAl, PSMCl, PSMDl, PSMDll, PSMDl 3, PSMD6, PSME1, PTHR2, PXN, PYGL, PYGM, QRICH2, RAB2, RALA,

RANBP1, RAP2A, RCC1, RCC2, RGS12, RGS2, RHOA, RIF1, RPA2, RPLP2, RPN2, RPS14, RRMl, RUVBL2, RYRl, RYR2, S100A6, SACS, SARS, SCTR, SDC3, SELE, SERPINB9, SET, SETD4, SF3B14, SGTB, SHANK1, SHBG, SIAH1, SLC2A1, SLC6A3, SLC7A10, SNCA, SNRPB2, SOCS6, SOCS7, SORD, SRC, STAU1, STRAP, STX12, SYK, TBCA, TBXA2R, TCP1, TEAD3, TFAM, TGM2, TJPl, TKl, TLR10, TMEM4, TNIK, TPIl, TRAF2, TRMT112, TUBAl, TUBAIA, TUBAlB, TUBA2, TUBB, TUBGl, TXN, TXNDC4, TXNL2, TYMS, UBQLN4, UCHL1, USP24, VTTL, VIPR1, YWHAQ, and ZAP70, thereby improving at least one symptom of autism in the patient.

2. The method of claim 1, wherein the patient has been diagnosed with autism spectrum disorder, pervasive developmental disorder, or one or more conditions selected from autistic disorder (classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD-NOS), and social (pragmatic) communication disorder (SCD).

3. The method of claim 1 or claim 2, wherein the CNV is a duplication or deletion.

4. The method of claim 1 or 2, wherein the autism is syndromic ASD.

5. The method of claim 1 or 2, wherein the patient has ASD and 22qll.2

Deletion or Duplication Syndrome, Fetal Valproate Syndrome or Thalidomide Embryopathy.

6. The method of claim 1 or 2, wherein the patient has at least two CNVs in a gene selected from the group consisting of ACAT1, ACAT2, ACCN1, ACCN2, ACPI, ACTB, ACTR2, ADA, ADCY1, ADD1, ADD2, ADORA1, ADRAIB, ADRA2A, ADRA2C, ADRB2, ADRBKl, ALDOA, ANXA2, APP, APTX, AQPl, ARHGAP24, ARL15, ARRBl, ARRB2, ATXN7L3, BDKRBl, BDKRB2, BTBD2, BTG2, C17orf44, Clorfll6, C7orf25, CA8, CACNAIB, CACYBP, CALB2, CALMl, CALM2, CALM3, CAMK1, CAMK2B, CAMK4, CCNB1, CDC42, CENTG1, CHGB, CHP, CHRM2, CHRM3, CIC, CMPK, CNP, CNR1, CNTN4, COPB2, CRHR1, CTNNA2, CYCS, DCN, DHCR7, DISCI, DLST, DPP6, DRD2, DRD3, DSTN, DYNLL1, ECHS1, EGFR, EIF3S3, ERBB2, F2R, F2RL2, F2RL3, F3, FKBP3, FPR1, FSCN1, FURIN, FYN, GAPDH, GLP1R, GLP2R, GNA15,

GNAI1, GNAI2, GNAI3, GNAOl, GNAQ, GNB2L1, GOT1, GP1BA, GPR26, GRB2, GRB7, GRIA1, GRIK1, GRIK3, GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, GRM8, GSN, HBXIP, HD, HNRPA3, HOMER1, HOMER3, HRPT2, HSP90AB1, HTR2A, IL8RB, IMPDH2, IQGAP2, ITGBl, ITGB7, ITPRl, KIAA0090, KIAA1683, LAMA4, LARP7, LRP2BP, LRRC59, LTA, LYAR, LYN, MAP4, MAPK1, MAPT, MARK4, MC4R, MGC11082, MRPL14, MRPS16,

MTHFD1, MTNR1A, MTNR1B, MX1, MYC, MY06, NANS, NARG1, NCK1, NEGRI, NFKBIA, NLN, NMI, NPY2R, NUDC, OPRDl, PAFAH1B3, PCBP1, PCBP3, PCDHA4, PCID1, PCM 1, PDCD5, PDE1B, PDE1C, PDE6G, PGM1, PHKB, PHKG2, PICKl, PIK3CA, PIK3R1, PLA2G7, PLCB1, PLCB3, PLCG2, PPIH, PPP2R1A, PRDXl, PRKCA, PRLHR, PRMTl, PRPSAP1, PSAT1, PSEN1, PSMA1, PSMC1, PSMD1, PSMD11, PSMD13, PSMD6, PSME1, PTHR2, PXN, PYGL, PYGM, QRICH2, RAB2, RALA, RANBPl, RAP2A, RCCl, RCC2, RGS12, RGS2, RHOA, RIFl, RPA2, RPLP2, RPN2, RPS14, RRMl, RUVBL2, RYRl, RYR2, S100A6, SACS, SARS, SCTR, SDC3, SELE, SERPINB9, SET, SETD4, SF3B14, SGTB, SHANK1, SHBG, SIAH1, SLC2A1, SLC6A3, SLC7A10, SNCA, SNRPB2, SOCS6, SOCS7, SORD, SRC, STAU1, STRAP, STX12, SYK, TBCA, TBXA2R, TCPl, TEAD3, TFAM, TGM2, TJPl, TKl, TLR10, TMEM4, TNIK, TPIl, TRAF2, TRMT112, TUBA1, TUBA 1 A, TUBA1B, TUBA2, TUBB, TUBG1, TXN,

TXNDC4, TXNL2, TYMS, UBQLN4, UCHL1, USP24, VHL, VIPR1, YWHAQ, and ZAP70.

7. A method of treating autism in a subject comprising administering an effective amount of a nonselective activator of metabotropic glutamate receptors (mGluRs) to a subject, thereby improving at least one symptom of autism.

8. A method of treating autism in a subject comprising administering an effective amount of a nonselective activator of metabotropic glutamate receptors (mGluRs) to a subject that has at least one genetic alteration in an mGluR network gene, thereby improving at least one symptom of autism.

9. A method of treating autism in a subject comprising obtaining results from a genetic screen that determines whether a subject has a genetic alteration in an mGluR network gene, and, if the results show that the subject has at least one genetic alteration in an mGluR network gene, treating the subject by administering an effective amount of a nonselective activator of mGluRs.

10. The method of claim 8, wherein the genetic alteration is a copy number variation (CNV) or single nucleotide variation (SNV).

11. The method of any one of claims 7 to 10, wherein the nonselective activator of mGluRs is fasoracetam.

12. The method of claim 11 , wherein fasoracetam is fasoracetam monohydrate (NS- 105 or NFC-1).

13. The method of claim 11 or 12, wherein fasoracetam is administered at a dose of 50 mg, lOOmg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg, and wherein the dose is administered once, twice, or three times daily.

14. The method of claim 11 or 12, wherein fasoracetam is administered at a dose of 50-400 mg, 100-400 mg, or 200-400 mg, and wherein the dose is administered once, twice, or three times daily.

15. The method of claim 11 or 12, wherein the fasoracetam is administered at a dose of 200-400 mg, such as 200 mg, 300 mg, or 400 mg, and wherein the dose is

administered twice daily.

16. The method of any one of claims 7-15, wherein the subject has a CNV in at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mGluR network genes.

17. The method of any one of claims 10-16, wherein a CNV in an mGluR network gene is determined by obtaining a nucleic acid-comprising sample from the subject and subjecting the sample to a screen that assesses CNVs in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or all of Tier 1 mGluR network genes.

18. The method of any one of claims 10-17, wherein a CNV in an mGluR network gene is determined by obtaining a nucleic acid-comprising sample from the subject and subjecting the sample to a screen that assesses CNVs in at least 50, at least 100, atleast 150, at least 175, or all of Tier 2 mGluR network genes.

19. The method of any one of claims 10-18, wherein a CNV in an mGluR network gene is determined by obtaining a nucleic acid sample from the subject and subjecting the sample to a screen that assesses CNVs in at least 50, atleast 100, at least 200, at least 300, at least 400, at least 500, or all of Tier 3 mGluR network genes.

20. The method of claim 19, wherein the screen does not assess CNVs in one or more of GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, or GRM8.

21. The method of any one of claims 1-20, wherein the subject does not have a CNV in one or more of GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, or GRM8.

22. The method of any one of claims 1-21, wherein the subject is a pediatric subject.

23. The method of claim 22, wherein the pediatric subject is between the ages of 5 and 17, 5 and 8, 8 and 17, 8 and 12, or 12 and 17.

24. The method of any one of claims 1-21 wherein the subject is an adult.

25. The method of any one of claims 1-22, wherein the nonselective activator of mGluRs is administered in combination with another pharmaceutical or non- pharmaceutical therapy.

26. The method of any one of claims 7-25, wherein the patient has been diagnosed with autism spectrum disorder, pervasive developmental disorder, or one or more conditions selected from autistic disorder (classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD-NOS), and social (pragmatic) communication disorder (SCD).

27. The method of claim 26, wherein the autism is syndromic ASD.

28. The method of claim 26 or 27, wherein the patient has ASD and 22qll.2 Deletion or Duplication Syndrome, Fetal Valproate Syndrome or Thalidomide Embryopathy.

29. A method for diagnosing autism in a subject comprising isolating a nucleic acid comprising sample from a subject, analy2ing the sample for the presence or absence of a genetic alteration in at least one mGluR network genes, and diagnosing autism if the subject has at least one genetic alteration in a mGluR network gene.

30. A method for diagnosing autism in a subject comprising isolating a nucleic acid comprising sample from a subject, isolating nucleic acid from the sample, analy2ingthe nucleic acid for the presence or absence of a genetic alteration in at least one mGluR network genes, and diagnosing autism if the subject has at least one genetic alteration in a mGluR network gene.

31. A method for identifying a subject as having autism comprising obtaining a sample from a patient, optionally isolating nucleic acid from the sample, optionally amplifying the nucleic acid, and analy2ing the nucleic acid in the sample for the presence or absence of a genetic alteration, such as a CNV, in at least one mGluR network gene, wherein the subj ect is identified as having autism if at least one genetic alteration, such as a CNV, in an mGluR network gene is detected.

32. A method for diagnosing autism in a subject comprising analy2ing genetic information about one or more mGluR network genes, comparing the subject's information to a control subject that does not have autism, and diagnosing autism if the genetic information suggests that the subject has at least one genetic alteration in a mGluRnetworkgene.

33. A method of confirming a diagnosis of autism in a subject comprising: obtaining a nucleic acid-comprising sample from a subject diagnosed with autism by a method that comprises detecting or analy2ing genetic alterations in mGluR network genes;

optionally amplifying the nucleic acid in the sample;and determining whether the subject has at least one genetic alteration, such as a CNV, in an mGluR network gene; and confirming a diagnosis of autism if the subject has at least one genetic alteration in an mGluR network gene.

34. The method of any one of claims 29-33, wherein the analysis for the presence or absence of at least one genetic alteration in an mGluR network gene comprises microarrays, whole genome sequencing, exome sequencing, targeted sequencing, FISH, comparative genomic hybridi2ation, genome mapping, or other methods using next- generation sequencing, Sanger sequencing, PCR, or TaqMan technologies.

35. The method of any one of claims 29-34, wherein the subject has CNVs in at least two mGluR network genes.

36. The method of any one of claims 29-35, wherein the method comprises detecting CNVs in mGluR network genes by subjecting the sample to a screen that assesses CNVs in at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mGluR network genes.

37. The method of any one of claims 29-36, wherein CNVs in mGluR network genes are determined by subjecting the sample to a screen that assesses CNVs in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or all of Tier 1 mGluR network genes.

38. The method of any one of claims 29-37, wherein CNVs in mGluR network genes are determined by subjecting the sample to a screen that assesses CNVs in at least 50, at least 100, at least 150, at least 175, or all of Tier 2 mGluR network genes.

39. The method of any one of claims 29-38, wherein CNVs in mGluR network genes are determined by subjecting the sample to a screen that assesses CNVs in at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all of Tier 3 mGluR network genes.

40. The method of any one of claims 29-40, wherein the subject is a pediatric subject, such as a subject between the ages of 5 and 17, 5 and 8, 8 and 17, 8

and 12, or 12 and 17.

41. The method of any one of claims 29-40, wherein the subject is an adult subject.

42. The method of any one of claims 29-41, wherein the subject is not assessed for genetic alterations or CNVs in one or more of GRMl, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, and GRM8.

43. The method of any one of claims 29-42, wherein the method for determining the presence or absence of at least one mGluR network gene genetic alteration comprises microarrays, whole genome sequencing, exome sequencing, targeted sequencing, FISH, comparative genomic hybridization, genome mapping, or other methods using next-generation sequencing, Sanger sequencing, PCR, or TaqMan technologies.

44. The method of any one of claims 1-43, wherein the subject has autism as well as ADHD.

45. The method of any one of claims 1-43, wherein the subject does not have one or more of ADHD, schizophrenia, conduct disorder, TS, anxiety disorder, phobia, or depression.

46. The method of any one of claims 29-45, wherein the patient has autism spectrum disorder, pervasive developmental disorder, or one or more conditions selected from autistic disorder (classic autism), Asperger's syndrome, childhood disintegrative disorder, Rett's disorder, pervasive developmental disorder not otherwise specified (PDD-NOS), and social (pragmatic) communication disorder (SCD).

47. The method of claim 46, wherein the patient has syndromic ASD.

48. The method of claim 46 or 48, wherein the patient has ASD and 22qll.2 Deletion or Duplication Syndrome, Fetal Valproate Syndrome or Thalidomide Embryopathy.