Methods of diagnosing and treating conduct disorder

Abstract

To provide methods for diagnosing and treating conduct disorder.SOLUTION: Diagnosis and treatment may be based upon an assessment of genetic alterations in metabotropic glutamate receptor (mGluR) network genes, and the treatment is performed with nonselective activators of mGluRs such as fasoracetam.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference to related applications This application claims priority to four U.S. provisional patent applications, including U.S. Provisional Patent Application Nos. 62 / 215628, 62 / 215633, 62 / 215636, and 62 / 215673, each filed on September 8, 2015, each of which is incorporated herein by reference in its entirety.

[0002] This application relates to the treatment of conduct disorder with non-selective activators of metabotropic glutamate receptors (mGluRs), and to the diagnosis and treatment of conduct disorder in subjects with genetic alterations, e.g., copy number variations (CNVs), in one or more mGluR network genes. [Background technology]

[0003] Conduct disorder encompasses a group of behavioral and emotional problems in children that make it extremely difficult to follow rules and behave in a socially acceptable manner. Conduct disorder is characterized by a repetitive pattern of behavior that violates the rights of others or breaks societal rules. Children and adolescents with conduct disorder exhibit behaviors such as aggression toward people and animals, destruction of property, cheating, lying, or stealing, and / or severe violation of age-appropriate rules. Factors that may contribute to the development of conduct disorder include brain injury, child abuse, genetic vulnerability, school failure, and traumatic life experiences (see American Academy of Child & Adolescent Psychiatry: Conduct Disorders (2013) and Ercan ES et al., Child Adolesc Psychiatry Ment Health. 5(1):10 (2011)).

[0004] The estimated lifetime prevalence of conduct disorder in the United States is 9.5%, with more men affected than women (see Nock MK et al., Psychol. Med. 36(5):699-710 (2006)). Conduct disorder symptoms, such as aggression and antisocial behavior, are the leading problems presented by children and adolescents in the United States leading to psychiatric referrals. Many children and adolescents with conduct disorder also have comorbid conditions such as mood disorders, anxiety, post-traumatic stress disorder (PTSD), substance abuse, attention-deficit hyperactivity disorder (ADHD), or learning disabilities. Untreated, children and adolescents with conduct disorder are more likely to exhibit antisocial behavior in adulthood and to continue to have relationship and workplace difficulties in adulthood. Childhood or adolescent conduct disorder is associated with an increased risk of related psychiatric disorders, legal problems, and premature mortality.

[0005] Currently, conduct disorder is diagnosed using one or more rating scales, such as the Conduct Disorder Rating Scale (CDRS) as described in Waschbusch DA Assessment 14(1):65-74 (2007), which has been validated for patients aged 5 to 12 years and has proven suitable for clinical studies of conduct disorder. Alternatively, conduct disorder can be assessed using the Aberrant Behavior Checklist (ABC) (see Sansone SM J Autism Dev Disord 42(7):1377-1392 (2012)). The ABC is widely used, has shown sensitivity in clinical trials of medications to treat behavioral problems, and can be used with patients with intellectual disabilities.

[0006] Behavioral and psychological therapy is usually required to help conduct disorder patients express and manage anger (see American Academy of Child & Adolescent Psychiatry: Conduct Disorders (2013)). Medications can also help conduct disorder patients manage depression, impulse control, and ADHD symptoms. In both children and adolescents, antipsychotics can be used to attempt to control aggressive symptoms of conduct disorder, but their effectiveness has not been proven (Toteja N, Int J Neuropsychopharmacol 17(7):1095-105 (2014)). While the use of antipsychotics in children and adolescents with conduct disorder can reduce aggression, their use also raises concerns about side effects, such as changes in prolactin levels (see James AC Adv Psychiatr Treat 16:63-75 (2010) and Ercan 2011). Currently, pharmacological treatment options for children and adolescents with conduct disorder are limited, and these treatments are only considered after behavioral treatment and family interventions have failed. Summary of the Invention

[0007] The present inventors have studied the genotypes of over 800 patients diagnosed with conduct disorder and found that these patients have a significantly higher frequency of genetic alterations in one or more metabotropic glutamate receptor (mGluR) network genes than healthy controls. The frequency of genetic alterations in mGluR network genes was also substantially higher in this conduct disorder population than in a control population without other neuropsychological disorders.

[0008] Thus, provided herein are methods for treating conduct disorder in a subject, the methods comprising administering to the subject an effective amount of a non-selective activator of metabotropic glutamate receptors (mGluRs), thereby treating the conduct disorder. In some embodiments, the subject has at least one genetic alteration, e.g., a copy number variation (CNV) or a single nucleotide variation (SNV), in an mGluR network gene. Also provided herein are methods for treating conduct disorder, the methods comprising administering an effective amount of a non-selective activator of metabotropic glutamate receptors (mGluRs) to a subject having at least one genetic alteration, e.g., a CNV, in an mGluR network gene, thereby treating the conduct disorder. In some embodiments, the subject has a CNV in an mGluR network gene, the CNV is a duplication or deletion.

[0009] Also provided is a method of treating conduct disorder in a subject, the method comprising obtaining results from a genetic screen to determine whether the subject has a genetic alteration in an mGluR network gene, and if the results indicate that the subject has at least one genetic alteration in an mGluR network gene, treating the subject by administering an effective amount of a non-selective activator of mGluR.

[0010] In some embodiments, a method for treating conduct disorder is provided, comprising administering an effective amount of a non-selective activator of metabotropic glutamate receptors (mGluRs) to a subject having at least one symptom of conduct disorder, thereby treating the conduct disorder. In some embodiments, a method for treating conduct disorder is provided, comprising administering an effective amount of a non-selective activator of metabotropic glutamate receptors (mGluRs) to a subject having at least one symptom of conduct disorder and at least one genetic alteration in an mGluR network gene, thereby treating the conduct disorder. One skilled in the art can identify symptoms of conduct disorder, for example, by referring to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) (2013). Symptoms include persistent, repetitive patterns of behavior that violate the fundamental rights of others or major societal norms.

[0011] In some embodiments of the above methods, the non-selective mGluR activator is fasoracetam, e.g., fasoracetam monohydrate (NS-105 or NFC-1). In some embodiments, fasoracetam is administered at a dose of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg, administered once, twice, or three times daily. In some embodiments, fasoracetam is administered at a dose of 50-400 mg, 100-400 mg, or 200-400 mg, administered once, twice, or three times daily. In some embodiments, fasoracetam is administered at a dose of 200-400 mg, e.g., 200 mg, 300 mg, or 400 mg, administered twice daily.

[0012] In some embodiments, the method includes determining whether the subject has a genetic alteration, e.g., a CNV, in an mGluR network gene based on the results of the screening. In some embodiments of the above methods, the subject has a CNV in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mGluR network genes. In some embodiments, the CNV in an mGluR network gene is determined by obtaining a nucleic acid-containing sample from the subject and subjecting the sample to a screen that evaluates CNV in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or all Tier 1 mGluR network genes. In some embodiments, the CNV in an mGluR network gene is determined by obtaining a nucleic acid-containing sample from the subject and subjecting the sample to a screen that evaluates CNV in at least 50, at least 100, at least 150, at least 175, or all Tier 2 mGluR network genes. In some embodiments, CNVs in mGluR network genes are determined by obtaining a nucleic acid sample from a subject and subjecting the sample to a screen that evaluates CNVs in at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all Tier 3 mGluR network genes. In some embodiments, the screen does not evaluate CNVs in one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, or GRM8. In certain aspects, the subject does not have a CNV in one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, or GRM8.

[0013] In some embodiments of the above methods, the subject has antisocial personality disorder. In some embodiments, the subject is a child or adolescent subject, for example, a child or adolescent subject between the ages of 5 and 17, between 5 and 8, between 8 and 17, between 8 and 12, between 12 and 18, between 13 and 18, or between 12 and 17. In other embodiments, the subject is an adult.

[0014] In some embodiments of the above methods, the non-selective activator of mGluRs is administered in combination with another drug, e.g., an antipsychotic agent, therapy, or non-drug therapy, which may include brain stimulation, e.g., vagus nerve stimulation, repetitive transcranial magnetic stimulation, magnetoconvulsive therapy, or deep brain stimulation.

[0015] In some embodiments, symptoms of inattention, hyperactivity, and / or impulsivity are reduced in the subject. In some embodiments, symptoms of aggression or antisocial behavior are reduced. In some embodiments, the method reduces other behavioral symptoms, such as mood disorders or sleep disorders. In some embodiments, the method also includes assessing symptoms, such as aggression or antisocial behavior and inattention, hyperactivity, and / or impulsivity, during or after administration, e.g., to determine whether one or more of these symptoms have been reduced in the subject. In some methods, such assessment can be based on the Corners Global Rating Scale. In some embodiments, the method further includes obtaining a clinical global impression of the subject's severity or improvement during or after administration. In some embodiments, the method can improve the subject's clinical global improvement score. Such symptoms can be reduced, for example, after one week of treatment with the activator, e.g., after two weeks of treatment, three weeks of treatment, or four weeks of treatment.

[0016] Also provided herein is a method for diagnosing conduct disorder in a subject, the method comprising isolating a nucleic acid-containing sample from the subject, analyzing the sample for the presence or absence of a genetic alteration in at least one mGluR network gene, and diagnosing the subject with a conduct disorder if the subject has at least one genetic alteration in an mGluR network gene. Also provided is a method for diagnosing conduct disorder in a subject, the method comprising isolating a nucleic acid-containing sample from the subject, isolating nucleic acid from the sample, analyzing the nucleic acid for the presence or absence of a genetic alteration in at least one mGluR network gene, and diagnosing the subject with a conduct disorder if the subject has at least one genetic alteration in an mGluR network gene. Also provided is a method for identifying a subject as having a conduct disorder, comprising obtaining a sample from a patient, optionally isolating nucleic acid from the sample, optionally amplifying the nucleic acid, and analyzing the nucleic acid in the sample for the presence or absence of at least one mGluR network genetic alteration, e.g., a CNV, wherein the subject is identified as having a conduct disorder if at least one genetic alteration, e.g., a CNV, in an mGluR network gene is detected.Also provided is a method for diagnosing conduct disorder in a subject, comprising analyzing genetic information for about one or more mGluR network genes, comparing the subject's information with control subjects without conduct disorder, and diagnosing the subject as having a conduct disorder if the genetic information suggests that the subject has at least one genetic alteration in an mGluR network gene.

[0017] Also provided herein is a method of confirming a diagnosis of conduct disorder in a subject, the method comprising obtaining a nucleic acid-containing sample from a subject diagnosed with conduct disorder by a method that does not include detecting or analyzing a genetic alteration in an mGluR network gene, optionally amplifying nucleic acid in the sample, and determining whether the subject has at least one genetic alteration, e.g., a CNV, in an mGluR network gene, and confirming the diagnosis of conduct disorder if the subject has at least one genetic alteration in an mGluR network gene.

[0018] In any of the above methods, analyzing the mGluR network genes for the presence or absence of at least one genetic alteration can include the use of 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 technology.

[0019] In some embodiments, the subject has CNV in one, two, or more mGluR network genes. In some embodiments, the method comprises detecting CNV in mGluR network genes by subjecting the sample to a screen that assesses CNV in at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mGluR network genes. In some embodiments, CNV in mGluR network genes is determined by subjecting the sample to a screen that assesses CNV in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or all Tier 1 mGluR network genes. In some embodiments, CNV in mGluR network genes is determined by subjecting the sample to a screen that assesses CNV in at least 50, at least 100, at least 150, at least 175, or all Tier 2 mGluR network genes. In some embodiments, the CNV of mGluR network genes is determined by subjecting the sample to a screen that evaluates the CNV of at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all Tier 3 mGluR network genes.

[0020] In some embodiments of the above methods, the subject has antisocial personality disorder. In some embodiments, the subject is a child or adolescent subject, e.g., between the ages of 5 and 17, between the ages of 5 and 8, between the ages of 8 and 17, between the ages of 8 and 12, between the ages of 12 and 18, between the ages of 13 and 18, or between the ages of 12 and 17. In other embodiments, the subject is an adult subject.

[0021] In some embodiments, screening methods for determining the presence or absence of at least one mGluR network gene genetic alteration include the use of 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 technology.

[0022] In some embodiments, the subject is not evaluated for genetic alterations or CNVs in one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, and GRM8. In some embodiments, the subject does not have a CNV in one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, and GRM8. In some embodiments, the subject does not have a CNV in any of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, and GRM8.

[0023] In any of the methods and embodiments described in the preceding paragraphs of this Summary of the Invention, the subject may have a conduct disorder and one or more co-morbid conditions, such as attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), autism, a mood disorder, Tourette's syndrome, an anxiety disorder, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior, self-injurious dermatology, a movement disorder, a developmental disorder, or depression. In other cases, the subject does not have one or more of ADHD, ODD, autism, a mood disorder, Tourette's syndrome, an anxiety disorder, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior, self-injurious dermatology, a movement disorder, a developmental disorder, or depression. In still other cases, the subject does not have any of ADHD, ODD, autism, a mood disorder, Tourette's syndrome, an anxiety disorder, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior, self-injurious dermatology, a movement disorder, a developmental disorder, or depression. In some embodiments, the subject does not have any of OCD, phobias, and depression.

[0024] In some embodiments, the subject has a co-morbid symptom of anxiety, and optionally the method reduces the anxiety symptoms. In some embodiments, the subject has OCD, and optionally the method reduces the OCD symptoms. In some embodiments, the subject has a co-morbid symptom of self-injurious dermatopathy, such as excessive skin picking, and optionally the method reduces those symptoms. In some embodiments, the subject has one or more co-morbid developmental disorders, and optionally the method reduces the severity of symptoms related to the developmental disorder.

[0025] In any of the methods herein, the subject may have ADHD and conduct disorder. In any of the methods herein, the subject may have one or more of the following phenotypic changes after at least one week, e.g., at least two weeks, at least three weeks, or at least four weeks of treatment with the activator: (a) the subject has anger control difficulties and anger control symptoms are improved; (b) the subject has disruptive behaviors and these disruptive behaviors are reduced; (c) the subject's CGI-I is reduced by at least 1 or at least 2; after 1, 2, 3, or 4 weeks of treatment, the subject's CGI-I score is 1 or 2; (d) after 1, 2, 3, or 4 weeks of treatment, the subject's CGI-S score is 1; or the subject's ADHD rating scale score is reduced by at least 25%, e.g., at least 30%, at least 35%, or less. (e) the subject has symptoms of inattention and the inattention symptom is reduced; the subject has symptoms of hyperactivity and the hyperactivity symptom is reduced; (f) the subject has symptoms of impulsivity and the impulsivity symptom is reduced; (g) the subject has symptoms of ODD, e.g., anger and irritability, argumentative and defiant attitudes, and / or vindictiveness and the ODD symptom is reduced; (h) the subject has symptoms of anxiety and the anxiety symptom is reduced; (i) the subject has symptoms of Tourette's syndrome and the Tourette's syndrome symptom is reduced; (j) the subject has symptoms of autism and the autism symptom is reduced; and (k) the subject has symptoms of movement disorder and the movement disorder symptom is reduced.

[0026] In one embodiment, a method for diagnosing an mGluR-related disorder is provided, wherein a subject is diagnosed with an mGluR-related disorder if at least one genetic alteration in an mGluR network gene is detected. [Brief explanation of the drawings]

[0027] [Figure 1]Figure 1 shows the number of samples from conduct disorder, depression, obsessive-compulsive disorder (OCD), and phobia patients included in a genotyping study designed to assess genetic alterations in mGluR network genes. Only fully genotyped patient samples were included in this data analysis. [Figure 2] Figure 1 shows the mGluR network genes included in the Tier 1 gene set. These genes have a protein-protein interaction degree of 2 with mGluR genes (GRM1-8) based on Cytoscape Human Interactome, a software for integrating biomolecular interaction networks with high-throughput data (described in Shannon P (2003) Genome Research 13:2498-2504). The Tier 1 gene set contains 76 genes. The exact locus for each gene in Tier 1 is listed in both Human Genome version 18 (hg18) and Human Genome version 19 (hg19). In addition, hg19 lists the exact gene locus +500 kb (i.e., ranging from 500 kb before to 500 kb after the gene of interest). The first single nucleotide polymorphism (StartSNP) (i.e., a SNP located 500 kb before the gene of interest) and the EndSNP (i.e., a SNP located 500 kb after the gene of interest) are also listed. The mGluR gene itself is designated "GRM." The expansion region (i.e., upstream and downstream of the 500 kb) often carries regulatory elements that, if affected by CNVs, can have the same effect on gene expression as CNVs present in the gene sequence itself. [Figure 3]This figure shows the mGluR network genes included in the Tier 2 gene set. These genes have a protein-protein interaction degree of 2 with mGluR genes (GRM1-8) based on Cytoscape Human Interactome, but do not include genes from Tier 1. The Tier 2 gene set includes 197 genes. The exact locus for each Tier 2 gene is listed in both Human Genome version 18 (hg18) and Human Genome version 19 (hg19). In addition, hg19 lists the exact gene locus +500 kb (i.e., ranging from 500 kb before to 500 kb after the gene of interest). The first single nucleotide polymorphism (StartSNP) (i.e., a SNP located 500 kb before the gene of interest) and EndSNP (i.e., a SNP located 500 kb after the gene of interest) are also listed in hg19. [Figure 4] This figure shows the genes in the Tier 3 gene set. Based on Cytoscape Human Interactome, genes with a protein-protein interaction level of 2 with mGluR genes are included. Genes included in Tier 1 and 2 are excluded from Tier 3. The Tier 3 gene set includes 599 genes. The exact locus for each Tier 3 gene is listed in both Human Genome Version 18 (hg18) and Human Genome Version 19 (hg19). In addition, hg19 lists the exact gene locus +500 kb (i.e., the range from 500 kb before to 500 kb after the gene of interest). The Start SNP (i.e., the SNP located 500 kb before the gene of interest) and End SNP (i.e., the SNP located 500 kb after the gene of interest) are also listed in hg19. [Figure 5]Figure 1 shows the number of copy number variation (CNV) calls involving Tier genes (mGluR network genes) in samples from 861 fully genotyped conduct disorder patients, 95 fully genotyped depression patients, 150 fully genotyped OCD patients, and 55 fully genotyped phobia patients. Note that several patients had more than one CNV call involving an mGluR network gene. [Figure 6] FIG. 1 shows the percentage of fully genotyped conduct disorder, depression, OCD or phobia patients who had CNVs within the Tier 1, Tier 1+2, or Tier 1+2+3 mGluR network gene sets. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition In addition to the definitions contained in this subsection, further definitions of terms are scattered throughout this document.

[0029] In the present invention, unless the context clearly dictates otherwise, "a" or "an" means "at least one" or "one or more," etc. The term "or" means "and / or," unless otherwise indicated. However, in the case of multiple dependent claims, the use of the term "or" refers back to more than one preceding claim and exclusively as an alternative to that claim.

[0030] "mGluR," or metabotropic glutamate receptor, refers to one of eight glutamate receptors expressed in neural tissue, named mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7, and mGluR8. Their genes are abbreviated as GRM1 through GMRM8. mGluR proteins are G protein-coupled receptors. They are typically classified into three subgroups: Group I receptors, including mGluR1 and mGluR5, are classified as slow excitatory receptors; Group II includes mGluR2 and mGluR3; and Group III includes mGluR4, mGluR6, mGluR7, and mGluR8. Groups II and III are classified as slow inhibitory receptors. mGluRs are distinct from ionotropic GluRs or iGluRs, which are ionotropic glutamate receptors and are classified as fast excitatory receptors.

[0031] "mGluR network genes," as used herein, include not only the mGluRm genes GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, and GRM8, but also each of the other genes listed in Figures 2-4 herein, as well as regions of DNA that regulate the genes listed in Figures 2-4. Additionally, "mGluR network proteins" are proteins encoded by mGluR network genes.

[0032] The mGluR network genes are classified into three subsets: Tier 1, Tier 2, and Tier 3 (see Figures 2-4). The Tier 1 mGluR network genes shown in Figure 2 contain 76 genes, including several GRM genes themselves as well as numerous other genes. The Tier 2 mGluR network genes shown in Figure 3 contain 197 genes and do not include Tier 1 genes.

[0033] Both Tier 1 and Tier 2 are included in the "primary mGluR network." The "primary network" of mGluR genes totals 276 genes and also includes the genes 4-Sep, LOC642393, and LOC653098. Evaluation of the 4-Sep, LOC642393, and LOC653098 genes is difficult with current technology. Therefore, although they are not included in Tier 1 or Tier 2, they are included in the primary network of genes of the present invention. Tier 1 and 2 genes differ in that alterations in Tier 1 genes have been documented in previous genotyping studies of subjects suffering from psychiatric disorders.

[0034] The Tier 3 mGluR network genes shown in Figure 4 include 599 genes in the distal part of the mGluR network based on the merged human interactome provided by Cytoscape software (Shannon P et al. (2003) Genome Research 13:2498-2504), excluding Tier 1 and Tier 2 genes. Therefore, Tier 3 genes are part of the "distal mGluR network." In addition to Tier 3 genes, genes LOC285147, LOC147004, and LOC93444 are also included in the "distal mGluR network." However, due to technical challenges in assessing genetic alterations in these genes, they were not evaluated in this study and are not included in Tier 3.

[0035] As used herein, "genetic alteration" refers to any alteration in the DNA of a gene or in the DNA regulating a gene that can result in, for example, a functionally altered gene product compared to the gene product produced from the unaltered DNA. A functional alteration can be, for example, a different expression level (up-regulation or down-regulation), or a loss or alteration of one or more biological activities. Genetic alterations include, but are not limited to, copy number variations (CNVs), single nucleotide variations (SNVs) (also referred to herein as single nucleotide polymorphisms (SNPs)), frameshift mutations, or any other base pair substitutions, insertions, and deletions or duplications.

[0036] "Copy number variation" or "CNV" refers to the duplication or deletion of a DNA segment relative to a reference genome, where the DNA segment encompasses a gene, genes, a segment of a gene, or a DNA region regulating a gene. In some embodiments, CNV is determined based on variation from the normal diploid state. In some embodiments, CNV refers to copy number changes involving DNA fragments that are 1 kilobase (kb) or larger. CNVs as described herein do not include variants resulting from insertion / deletion of transposable elements (e.g., 6 kb KpnI repeats). Thus, the term CNV encompasses terms such as large 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 does not encompass retrotransposon insertions.

[0037] A "CNV deletion" or "deletion CNV" or similar terms refers to a CNV in which a gene, a DNA segment regulating a gene, or a gene segment is deleted. A "CNV duplication" or "duplication CNV" or similar terms refers to a CNV in which a gene, a DNA segment regulating a gene, or a gene segment is present in at least two copies, and possibly more than two copies, compared to the single copy found in a normal reference genome.

[0038] "Sample" refers to a sample from a subject that can be tested, for example, for the presence of a CNV in one or more mGluR network gene proteins, as described herein. A sample can include cells or a bodily fluid, such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, tears, pleural fluid, etc.

[0039] "Conduct disorder" is defined in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) (2013) as a persistent, repetitive pattern of behavior that violates the fundamental rights of others or major societal norms. Conduct disorder is typically diagnosed in patients before adulthood (i.e., before age 18). A diagnosis of conduct disorder can be based on significant impairment in social, academic, or occupational functioning. Currently, conduct disorder is diagnosed using one or more rating scales, such as the CDRS (Waschbusch 2007) or the ABC (Sansone 2012). Patients with conduct disorder may also have inattention, hyperactivity, anxiety, mood, and sleep disorders.

[0040] "Antisocial personality disorder" is defined by the DSM-5 (2013) as a pervasive pattern of disregard for and violation of the rights of others in individuals who are at least 18 years of age, but whose onset of symptoms occurred before age 15. Accordingly, antisocial personality disorder may be diagnosed later in adulthood in individuals who exhibited symptoms consistent with conduct disorder during childhood or adolescence. Patients with antisocial personality disorder have previously been diagnosed with conduct disorder during childhood or adolescence. As used herein, conduct disorder patients include subjects over the age of 18 who have previously been diagnosed with conduct disorder. As used herein, conduct disorder patients also include subjects over the age of 18 who were not diagnosed with conduct disorder as children or adolescents, but who exhibited symptoms consistent with conduct disorder during childhood or adolescence. Accordingly, antisocial personality disorder is included within the definition of conduct disorder within this application.

[0041] 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 under the age of 18. An "adult patient" refers to a human aged 18 years or older. An "adolescent patient" or "adolescent subject" is a subject typically between about 12 and 18 years of age, e.g., between 12 and 17 years of age, or between 13 and 18 years of age.

[0042] How to Diagnose Conduct Disorder In some embodiments, the present invention includes a method of diagnosing conduct disorder in a subject, comprising analyzing the subject's genetic information to determine whether the subject has a genetic variation in at least one mGluR network gene, and diagnosing the subject as having a conduct disorder if a genetic variation is found. In some embodiments, the subject has conduct disorder but does not have ADHD, oppositional defiant disorder (ODD), autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression. In some embodiments, the subject has conduct disorder and also has one or more of ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression.

[0043] As used herein, "developmental disorders" include, for example, those classified under International Classification of Diseases, Ninth Revision (World Health Organization) codes 299.80, 299.90, 315.2, 315.39, 315.4, 315.5, 315.8, and 315.9, which may affect behaviors such as learning, coordination, and speech. "Self-injurious dermatopathy," also known as excoriation disorder or skin picking disorder, is a disorder involving excessive picking of one's own skin, resulting in damage, and includes picking at intact skin as well as real or imagined skin defects, such as moles, freckles, or scars.

[0044] In other embodiments, the present invention encompasses confirming a diagnosis of an anxiety disorder in a subject. As used herein, "confirming a diagnosis of conduct disorder" refers to diagnosing a subject who has already been diagnosed with a conduct disorder. In one embodiment, a method of confirming a diagnosis of conduct disorder comprises analyzing genetic information of a subject diagnosed with conduct disorder by a method that does not involve analyzing mGluR network genes to determine whether the subject has a genetic variation in at least one mGluR network gene, and confirming the diagnosis of conduct disorder if a genetic variation in at least one mGluR network gene is found. In some embodiments, the subject has a conduct disorder but does not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression. In some embodiments, the subject has a conduct disorder and also has one or more of ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, and depression.

[0045] In another embodiment, the invention comprises confirming a diagnosis of conduct disorder in a subject who does not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, movement disorders, developmental disorders, and depression, comprising analyzing genetic information of a subject diagnosed with conduct disorder by a method that does not include analyzing mGluR network genes to determine whether the subject has a genetic variation in at least one mGluR network gene, and confirming the diagnosis of conduct disorder if a genetic variation in at least one mGluR network gene is found.

[0046] In one embodiment, conduct disorder is diagnosed and / or confirmed when 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 another embodiment, conduct disorder is diagnosed and / or confirmed when at least one CNV, SNV, frameshift mutation, or any other base pair substitution, insertion, deletion, or duplication in a Tier 1 mGluR network gene is detected. In another embodiment, conduct disorder is diagnosed and / or confirmed when at least one CNV, SNV, frameshift mutation, or any other base pair substitution, insertion, duplication, or deletion in a Tier 2 mGluR network gene is detected. In yet another embodiment, conduct disorder is diagnosed and / or confirmed when at least one CNV, SNV, frameshift mutation, or any other base pair substitution, insertion, duplication, or deletion in a Tier 3 mGluR network gene is detected.

[0047] A diagnosis or confirmation of conduct disorder can be based on or confirmed by finding a genetic alteration in Tier 1, Tier 2, and / or Tier 3 mGluR network genes. The genetic alteration can be a CNV. A CNV can be a duplication or deletion of a DNA region containing some or all of the DNA encoding and regulating an mGluR network gene. In another embodiment, a diagnosis or confirmation of a diagnosis of conduct disorder is made in a patient who does not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression. In some embodiments, a diagnosis or confirmation of a diagnosis of conduct disorder is made in a subject who does have ADHD, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, and / or depression.

[0048] In some embodiments, a diagnosis or confirmation of a diagnosis of a conduct disorder is based on finding that the copy number of mGluR network genes deviates from the normal diploid state. In some embodiments, a diagnosis or confirmation of a diagnosis of a conduct disorder is based on a copy number of 0 or 1, indicating a CNV deletion. In some embodiments, a diagnosis or confirmation of a diagnosis of a conduct disorder is based on a copy number of 3 or more, indicating a CNV duplication. In some embodiments, a diagnosis or confirmation of a diagnosis of an anxiety disorder is made by the presence of a copy number of 0 or 1, or a copy number of 3 or more, or any deviation from the diploid state in patients without ADHD, ODD, autism, mood disorder, anxiety disorder, Tourette's syndrome, phobia, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, movement disorder, developmental disorder, and depression.

[0049] In other embodiments, a diagnosis of conduct disorder is made or confirmed by the presence of a copy number of 0 or 1, or a copy number of 3 or more, or any deviation from the diploid state in a patient with ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, an obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression. In another embodiment, a diagnosis of conduct disorder is made or confirmed by the presence of a copy number of 0 or 1, or a copy number of 3 or more, or any deviation from the diploid state in a patient without ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, an obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression.

[0050] In some embodiments, more severe forms of conduct disorder are diagnosed when at least two CNVs in mGluR network genes are detected. In one embodiment, more severe forms of conduct disorder are diagnosed in patients without ADHD, anxiety disorders, Tourette's syndrome, phobias, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, movement disorders, developmental disorders, and depression when at least two CNVs in mGluR network genes are detected.

[0051] In one embodiment, the method for diagnosing and / or confirming conduct disorder comprises: obtaining a nucleic acid-containing sample from a subject; optionally amplifying said nucleic acid; optionally labeling said nucleic acid sample; applying one or more nucleic acids of an mGluR network gene to a solid support, the nucleic acid optionally comprising an SNV of an mGluR network gene; Removing any unbound nucleic acid sample; and and detecting any nucleic acid bound to the nucleic acid on the solid support, wherein if bound nucleic acid is detected, the patient is diagnosed or confirmed as having a conduct disorder. In one embodiment, the method further comprises comparing any bound nucleic acid to a standard or control, and diagnosing or confirming a conduct disorder if analysis reveals that the test sample is different from the control or standard. In another embodiment of this method, the patient with a conduct disorder does not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression. In some embodiments, the patient with a conduct disorder has one or more of ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression.

[0052] In each of the diagnostic, confirmatory, and therapeutic methods of the invention, the subject may have a conduct disorder but may not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression. In other diagnostic, confirmatory, and therapeutic methods of the invention, the subject has a conduct disorder and one or more additional disorders, such as ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression.

[0053] How to Treat Conduct Disorder

[0013] Also encompassed herein are methods for treating conduct disorder in a subject, comprising administering an effective amount of a non-selective mGluR activator. As used herein, the term "treatment" includes any administration or application of a therapy for a disease or disorder in a subject, including suppressing the disease, preventing its onset, alleviating symptoms of the disease, or preventing the occurrence or recurrence of the disease or symptoms of the disease. In some embodiments, conduct disorder is said to be treated if there is an improvement in antisocial behavior, a reduction in episodes of violating the rights of others, or a reduction in conflicts with the law.

[0054] mGluR proteins are typically classified into three subgroups: Group I receptors, including mGluR1 and mGluR5, are classified as slow excitatory receptors; Group II includes mGluR2 and mGluR3; and Group III includes mGluR4, mGluR6, mGluR7, and mGluR8. Groups II and III are classified as slow inhibitory receptors. mGluRs are distinct from ionotropic GluRs or iGluRs, which are ionotropic glutamate receptors and are classified as fast excitatory receptors.

[0055] A "non-selective activator of mGluRs" refers to a molecule that activates mGluRs from more than one of the Group I, II, and III categories. Thus, a non-selective activator of mGluRs can provide a general stimulus to the mGluR network. This is in contrast to a specific mGluR activator that can only significantly activate a single mGluR, such as mGluR5. Non-selective mGluR activators include, for example, non-selective mGluR agonists.

[0056] In some embodiments, the non-selective mGluR activator is fasoracetam. Fasoracetam is a nootropic (i.e., cognitive-enhancing) drug that can stimulate both group I mGluRs and group II / III mGluRs in in vitro studies. (See Hirouchi M et al. (2000) European Journal of Pharmacology 387:9-17.) While fasoracetam can stimulate adenylate cyclase activity by activating group I mGluRs, it can 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 shown in previous human studies to be highly bioavailable (79%-97%) and have a half-life of 5-6.5 hours (see Malykh 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: The file is TIFF2025179063000001.tif21170.

[0057] The term "fasoracetam" as used herein encompasses pharma- ceutically acceptable hydrates and any solid, amorphous, or crystalline form of the fasoracetam molecule. For example, the term fasoracetam herein includes forms such as fasoracetam monohydrate NFC-1. In addition to NFC-1, fasoracetam is also known as C-NS-105, NS105, and LAM-105.

[0058] NFC-1 has previously been studied in Phase I-III clinical trials for dementia-related cognitive impairment, but Phase III trials did not demonstrate sufficient efficacy for dementia. These trials demonstrated that NFC-1 is generally safe and well-tolerated for these indications. Phase III data showed that NFC-1 has beneficial effects on psychiatric symptoms in patients with cerebral infarction and adult dementia patients with cerebrovascular disease.

[0059] In each method of treatment embodiment, 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 can be administered alone or in combination with, for example, a non-selective activator of mGluRs to a subject with alterations in mGluR network genes. In some embodiments, the therapeutic agent comprises ADX63365, ADX50938, ADX71149, AMN082, 1-(hetero)aryl-3-amino-pyrrolidine derivatives, LY341495, ADX48621, GSK1144814, or SB223412.

[0060] Also encompassed herein are methods for treating conduct disorder, comprising administering a non-selective mGluR activator, such as fasoracetam, to a subject having a genetic alteration in at least one mGluR network gene. In some embodiments, the subject has an anxiety disorder but does not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control problems, disruptive behavior, self-injurious dermatology, a movement disorder, a developmental disorder, or depression, while in other embodiments, the subject has conduct disorder and at least one of ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control problems, disruptive behavior, self-injurious dermatology, a movement disorder, a developmental disorder, or depression.

[0061] In some embodiments, a method of treatment comprises identifying or diagnosing a subject as having a genetic alteration in at least one mGluR network gene and administering a non-selective mGluR activator, such as fasoracetam, to the identified or diagnosed subject. In some embodiments, the subject has a conduct disorder but does not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression. In other embodiments, the subject has a conduct disorder and also has one or more neuropsychological disorders, such as ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, or depression.

[0062] In each of the treatment method embodiments of the present invention, non-selective mGluR activators can improve symptoms of inattention, hyperactivity, anxiety, mood, and sleep disturbances that may be seen in patients with conduct disorder. They can also reduce symptoms of aggression and / or antisocial behavior.

[0063] Some embodiments include methods of treating conduct disorder, comprising identifying or diagnosing a subject as having a genetic alteration in at least one mGluR network gene and administering to the identified or diagnosed subject an effective amount of a non-selective activator of mGluR, such as fasoracetam. In some embodiments, the subject has conduct disorder but does not have ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior, self-injurious dermatology, a movement disorder, a developmental disorder, or depression. In other embodiments, the subject has conduct disorder and one or more neuropsychological disorders, such as ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger control difficulties, disruptive behavior, self-injurious dermatology, a movement disorder, a developmental disorder, or depression.

[0064] In each treatment method embodiment of the invention, the subject may have a conduct disorder but may not have one or more neuropsychological disorders, such as ADHD, ODD, autism, mood disorders, anxiety disorders, Tourette's syndrome, phobias, and depression.

[0065] In one embodiment, an effective amount of a non-selective mGluR activator of mGluRs, such as fasoracetam, is administered to a subject with conduct disorder who has been confirmed to have 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 any combination of Tiers.

[0066] Some embodiments include methods of treating conduct disorder, comprising obtaining genetic information about mGluR network genes in a subject, and administering an effective amount of a non-selective mGluR activator, such as fasoracetam, if the subject has at least one genetic variation, e.g., a CNV, in an mGluR network gene. Other embodiments include methods of treating conduct disorder, comprising obtaining genetic information about mGluR network genes in a subject, and administering a non-selective mGluR activator, such as fasoracetam, if the subject has at least one genetic variation, e.g., a CNV, in a Tier 1 mGluR network gene. Other embodiments include methods of treating conduct disorder, comprising obtaining genetic information about mGluR network genes in a subject, and administering a non-selective mGluR activator, such as fasoracetam, if the subject has at least one genetic variation, e.g., a CNV, in a Tier 2 mGluR network gene. Yet other embodiments include methods of treating conduct disorder, comprising obtaining genetic information about mGluR network genes in a subject, and administering a non-selective mGluR activator, such as fasoracetam, if the subject has at least one genetic variation, e.g., a CNV, in a Tier 3 mGluR network gene. Subjects with more than one CNV in any one Tier, or a combination of any of the three Tiers, can be treated by administering a non-selective mGluR activator, such as fasoracetam. In some embodiments, subjects with conduct disorder but not ADHD, ODD, autism, mood disorders, anxiety disorders, Tourette's syndrome, phobias, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavior symptoms, self-injurious dermatopathy, movement disorders, developmental disorders, or depression can be treated. In other treatment method embodiments of the invention, the subject has one or more neuropsychological disorders in addition to a conduct disorder, such as ADHD, ODD, autism, a mood disorder, an anxiety disorder, Tourette's syndrome, a phobia, obsessive-compulsive disorder (OCD), anger management difficulties, disruptive behavioral symptoms, self-injurious dermatopathy, a movement disorder, a developmental disorder, and depression.

[0067] Methods for determining the presence or absence of genetic alterations Any biological sample, including but not limited to blood, urine, serum, gastric washings, central nervous system fluid, any cell type (e.g., brain cells, white blood cells, mononuclear cells), or body tissue, can be used to determine the presence or absence of mGluR network genetic alterations. Any biological sample from which DNA can be extracted can be used to determine the presence or absence of mGluR network genetic alterations. The sample can be freshly collected or previously collected for any use / purpose and stored until the time to test for genetic alterations. Previously purified DNA for different purposes can also be used.

[0068] A variety of methods are known for determining genetic alterations, including the following:

[0069] 1. Single Nucleotide Variant (SNV) / Single Nucleotide Polymorphism (SNP) Genotyping Determining whether a patient has a genetic alteration, e.g., a CNV, in an mGluR network gene can be performed by SNP / SNV genotyping. A "single nucleotide variant (SNV)," also referred to herein as a "single nucleotide polymorphism (SNP)," refers to a change in DNA in which a single base differs from the normal base at that position. Millions of SNVs in the human genome have been cataloged. Some SNVs are normal variations in the genome, while others are associated with disease. While certain SNVs may be associated with disease states or susceptibility, high-density SNV genotyping can be undertaken, in which sequencing information from SNVs is used to determine an individual's unique genetic makeup.

[0070] In SNV genotyping, SNVs can be determined by hybridizing complementary DNA probes to SNV sites. A wide range of platforms can be used with SNV genotyping tools to accommodate various sample throughputs, multiplexing capabilities, and chemistries. In high-density SNV arrays, hundreds of thousands of probes are arrayed on a small chip so that many SNVs can be interrogated simultaneously when target DNA is processed on the chip. Specific SNV alleles can be determined by determining the amount of hybridization of target DNA in a sample to probes (or redundant probes) on the array. The use of arrays for SNV genotyping enables large-scale interrogation of SNVs.

[0071] When analyzing CNV, after analyzing SNV, SNV data can be manipulated using computer programs to arrive at CNV data. In this case, PennCNV or similar programs can be used to detect signal patterns throughout the genome and identify continuous genetic markers related to copy number changes. (See Wang K et al. (June 2008) Cold Spring Harbour Protocol). PennCNV allows kilobase resolution detection of CNV (See Wang K et al. (Nov 2007) Genome Res.17(11):1665-74).

[0072] CNV analysis compares SNV genotyping data with the behavior of normal diploid DNA. The software uses SNV genotyping data to determine signal intensity data and the distribution of SNV allele ratios, and then uses these data to determine when there is a deviation from the normal diploid state of the DNA, indicating a CNV. This is done in part by using the log R ratio (LRR), a normalized measure of the total signal intensity for the two alleles of an SNV (Wang 2008). If the software detects a region of adjacent SNVs with an intensity (LRR) trending below zero, this indicates a CNV deletion. If the software detects a region of adjacent SNVs with an intensity (LRR) trending above zero, this indicates a CNV duplication. If no change in LRR is observed compared to the behavior of diploid DNA, the sequence is in a normal diploid state with no CNV present. The software also uses the B allele frequency (BAF), a normalized measure of the allele intensity ratio of two alleles, which changes when an allele is lost or gained, as well as when an allele is lost or gained, as well as when a CNV is lost or duplication. For example, CNV deletion is indicated by both a decrease in LRR value and the absence of heterozygotes in BAF value.In contrast, CNV duplication is indicated by both an increase in LRR value and the division of heterozygote genotype BAF cluster into two different clusters.The software automates the calculation of LRR and BAF to detect CNV deletion and duplication in whole genome SNV data.By simultaneously analyzing intensity data and genotype data, normal diploid state is accurately defined and CNV is determined.

[0073] Array platforms such as those from Illumina, Affymetrix, and Agilent can be used in SNV genotyping. Custom arrays can also be designed and used based on the data described herein.

[0074] 2. Comparative Genomic Hybridization Comparative genomic hybridization (CGH) is another method that can be used to assess genetic alterations, such as CNVs. CGH is a molecular cytogenetic method that uses competitive fluorescence in situ hybridization (FISH) to analyze genetic alterations, such as CNVs, in comparison to a reference sample. DNA is isolated from the patient and reference source and, after DNA denaturation, independently labeled with fluorescent molecules (i.e., fluorophores). Hybridization of the fluorophores to the resulting samples is compared along the length of each chromosome to identify chromosomal differences between the two sources. Color discrepancies indicate gain or loss of material within specific regions of the test sample, while color matches indicate no difference in genetic alterations, such as copy number, between the test and reference samples in a particular region.

[0075] 3. Whole Genome Sequencing, Whole Exome Sequencing, and Targeted Sequencing Whole genome sequencing, whole exome sequencing, or targeted sequencing can also be used to analyze genetic changes such as CNVs. Whole genome sequencing (also known as full-length genome sequencing, complete genome sequencing, or whole genome sequencing) involves sequencing the full-length genome of a species, including protein-coding and non-coding genes. In contrast, whole exome sequencing is the sequencing of only protein-coding genes in the genome (approximately 1% of the genome). Targeted sequencing involves sequencing only selected parts of the genome.

[0076] Those skilled in the art will be familiar with a wide range of techniques for performing whole genome, whole exome or targeted sequencing using purified DNA from a subject.Similar techniques can be used for different types of sequencing.The techniques used for whole genome sequencing include nanopore technology, fluorophore technology, DNA nanoball technology, and pyrosequencing (i.e., sequencing by synthesis).In particular, next-generation sequencing (NGS) involves the parallel sequencing of millions of small fragments of DNA, followed by the use of bioinformatics analysis to piece together the sequencing data from the fragments.

[0077] Whole exome sequencing does not require the sequencing of a large amount of DNA, as in whole genome sequencing, so a wide range of techniques can be used.Methods for whole exome sequencing include polymerase chain reaction, NGS, molecular inversion probe, hybrid capture using macroarray, in-solution capture, and classical Sanger sequencing.Targeted sequencing allows obtaining sequence data for specific genes instead of the whole genome, and can use any technique used in other types of sequencing, including specialized microarrays that contain materials for sequencing the target gene.

[0078] 4. Other methods for determining genetic alterations Proprietary methods, such as those from BioNano or OpGen, that use genome mapping technology can also be used to assess genetic alterations such as CNVs.

[0079] Genetic alterations such as CNVs can be assessed using standard molecular biology methods, such as quantitative polymerase chain reaction (PCR), droplet PCR, and TaqMan probes (i.e., hydrolysis probes designed to increase the specificity of quantitative PCR). Fluorescence in situ hybridization (FISH) probes may also be used to assess genetic alterations such as CNVs. Analysis of genetic alterations such as CNVs present in patients with anxiety disorders is not limited by the exact method used to determine genetic alterations such as CNVs.

[0080] Methods for diagnosing conduct disorder based on CNV data In some embodiments, the genetic variation is an SNV or CNV. The SNV or CNV associated with conduct disorder is found in an mGluR network gene, such as a gene listed in Tier 1, Tier 2, or Tier 3, as shown in Figures 2-4, or a set or panel of such genes.

[0081] In some embodiments, gene sets of mGluR network genes are used to analyze samples from patients with or suspected of having conduct disorder. In some embodiments, the presence of CNV duplications or deletions within these gene sets or panels is determined. In some embodiments, CNVs of the Tier 1 genes shown in Figure 2 are determined. In some embodiments, a panel of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or all 76 Tier 1 genes is evaluated for the presence of CNVs. Within any such gene panel, specific individual Tier 1 genes may be excluded from the analysis set. For example, any or all of GRM1-8 may be excluded from the panel.

[0082] In some embodiments, Tier 2 genes, such as those shown in Figure 3, are analyzed for the presence of genetic alterations, such as CNVs. Tier 2 genes are closely related to mGluRs but are not included in Tier 1. In some embodiments, Tier 2 genes are evaluated along with Tier 1 genes. In some embodiments, at least 100 Tier 2 genes are evaluated, while in some embodiments, at least 150 or 197 Tier 2 genes are evaluated. In some embodiments, individual specific Tier 2 genes may be excluded from the gene set for evaluation.

[0083] In some embodiments, 599 Tier 3 genes, such as those shown in Figure 4, are evaluated for the presence of genetic alterations, such as CNVs. In some embodiments, Tier 3 genes are evaluated along with Tier 1 and / or Tier 2 genes. In some embodiments, at least 100 Tier 3 genes are evaluated, while in some embodiments, at least 150, 200, 250, 300, 350, 400, 450, or 599 Tier 3 genes are evaluated. In some embodiments, individual specific Tier 3 genes may be excluded from the gene set for evaluation.

[0084] Administration methods and combination therapy In some embodiments, the agent that modulates mGluR signaling is fasoracetam or fasoracetam monohydrate (also known as C-NS-105, NFC1, NS105, or LAM-105).

[0085] dosage In some embodiments, fasoracetam can be administered as fasoracetam monohydrate (NFC-1). In some embodiments, fasoracetam can be administered orally (i.e., orally). In some embodiments, fasoracetam can be administered as a capsule. In some embodiments, fasoracetam capsules can 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 can be administered once daily or twice daily. In some embodiments, the daily dose of fasoracetam can be 50 mg once daily, 100 mg once daily, 200 mg once daily, 400 mg once daily, 50 mg twice daily, 100 mg twice daily, 200 mg twice daily, or 400 mg twice daily. In some embodiments, a series of dose escalations can be used to adjust fasoracetam dosing. In some embodiments, pharmacokinetic data on drug levels or clinical response are used to determine changes in dosing. In some embodiments, dose escalation of fasoracetam is not used. In some embodiments, subjects are treated with a fasoracetam dose expected to be clinically effective without a dose escalation protocol.

[0086] Combination therapy In some embodiments, fasoracetam is used in combination with another agent for the treatment of conduct disorder. The other agent used in combination with fasoracetam can be an antipsychotic agent (e.g., haloperidol, chlorpromazine, amisulpride, aripiprazole, asenapine, blonanserin, clozapine, iloperidone, lurasidone, melperone, olanzapine, paliperidone, quetiapine, risperidone, sertindole, sulpiride, ziprasidone, or zotepine).

[0087] In some embodiments, fasoracetam can be used in combination with non-pharmacological treatments, such as psychotherapy or brain stimulation therapy. In some embodiments, fasoracetam is used in combination with brain stimulation, which can be vagus nerve stimulation, repetitive transcranial magnetic stimulation, magnetoconvulsive therapy, deep brain stimulation, or any other therapy involving modulation of brain function by electricity, magnets, or implants.

[0088] manufactured goods In some embodiments, the invention includes articles of manufacture that can be used in the methods and treatments described herein. In one embodiment, the article of manufacture is a solid support or microarray for use in detecting genetic alterations in some or all of the mGluR network genes (i.e., Tiers 1-3) listed in Figures 1-3. (See also Tables 1-3 herein, which provide start and end positions for different mGluR network-associated SNPs. This information can be useful in creating microarrays.) In some embodiments, genes included in multiple Tiers are evaluated within the same solid support or microarray. In some embodiments, certain mGluR network genes are excluded. In some embodiments, GRM genes are excluded.

[0089] Thus, for example, in some embodiments assaying mGluR network genes to determine whether one or more of the genes have a genetic alteration, such as a CNV, a solid support or microarray, e.g., a chip, is used that contains suitable probes for determining the presence of genetic alterations in 10, 20, 30, 40, 50, 60, 70, or all Tier 1 genes. In some embodiments, the probes are labeled. In some embodiments, the probes are labeled with a non-naturally occurring moiety. In some embodiments, the solid support or microarray may also contain suitable probes for determining the presence of genetic alterations in at least 10, 20, 30, 50, 100, 150, or all Tier 2 genes. In some embodiments, it may further contain suitable probes for determining the presence of genetic alterations in at least 10, 20, 50, 100, 200, 300, 400, 500, or all Tier 3 genes. For example, such solid supports, microarrays or chips can be used to determine the presence of genetic alterations, e.g., CNVs or SNVs, in the Tier 1, Tier 1+2, or Tier 1+2+3 mGluR gene networks as part of a method for treating patients with ADHD or 22q deletions and / or duplications.

[0090] In some embodiments, the product is a probe set for mGluR network genes of interest from Tier 1, 2, and / or 3. In some embodiments, the probes are labeled. Similarly, probe sets can be manufactured to determine the presence of genetic alterations in 10, 20, 30, 40, 50, 60, 70, or all Tier 1 genes. In some embodiments, probes can be manufactured to determine the presence of genetic alterations in at least 10, 20, 30, 50, 100, 150, or all Tier 2 genes. In some embodiments, probes can further include those to determine the presence of genetic alterations in at least 10, 20, 50, 100, 200, 300, 400, 500, or all Tier 3 genes. These various probe sets can be used in methods to determine the presence of genetic alterations, e.g., CNVs or SNVs, in the Tier 1, Tier 1+2, or Tier 1+2+3 mGluR gene network as part of a method of treating patients with ADHD or 22q deletions and / or duplications. [Example]

[0091] Example 1. Enrichment of CNV calls involving mGluR network genes in samples from patients with conduct disorder We undertook a study of potential enrichment of mGluR network genes in patients with conduct disorder, phobia, depression, or obsessive-compulsive disorder. Given the important role of mGluRs and their signaling networks, copy number alterations of mGluR network genes may provide a new approach for the diagnosis or treatment of psychiatric disorders.

[0092] Previously, a large-scale genomic association study (as described in Elia et al., Nature Genetics, 44(1): 78-84 (2012)) of copy number variations abundant in ADHD patients was conducted. Elia's study included approximately 2,493 ADHD patients and approximately 9,222 controls, all of whom were of European descent and aged between 6 and 18 years. The study noted that the rate of CNVs involving mGluR network genes was 1.2% in the control group, and this rate increased to 11.3% in ADHD patients.

[0093] Samples for this study were selected based on ICP-9 codes for diagnoses from the electronic health records of children and adolescents treated at Children's Hospital of Philadelphia (CHOP). All subjects were evaluated by a child psychiatrist who entered a diagnosis of conduct disorder, depression, obsessive-compulsive disorder (OCD), or phobia. Figure 1 shows the number of patient samples analyzed in each of the conduct disorder, depression, OCD, and phobia groups. Selected patients in each group were fully genotyped as indicated in Figure 1. Data from fully genotyped patients were used for the analysis.

[0094] CNVs were determined using single nucleotide variant (SNV) / single nucleotide polymorphism (SNP) genotype data. SNV genotyping provides an individual's genetic fingerprint by using a large number of SNV markers to generate high-density SNV genotyping data (see Wang K et al. (Nov 2007) Genome Res. 17(11):1665-74). HumanHap550 Gentyping BeadChip™ (Illumina) or Human610-Quad v1.0 BeadChip™ (Illumina) were used in this study. The same 520 SNVs were analyzed for both chips. Therefore, data from these two chips are interchangeable. Standard manufacturing protocols were used for all genotyping assays. Illumina readers were used for all experiments.

[0095] The SNV genotyping data from each fully genotyped patient sample was analyzed using PennCNV software to determine the signal intensity data and the distribution of SNV allele ratios.These data were then used to determine CNVs by simultaneous analysis of intensity and genotype data (as previously described in Wang 2008).Using this analysis, data showing regions of adjacent SNV loss will result in a CNV deletion call.Data showing regions of adjacent SNV gain will result in a CNV duplication call.A single individual may have multiple CNV deletions / duplications or no CNVs at all.

[0096] As previously discussed, three tiers of mGluR network genes were developed. Figures 2-4 show the genes included in the three gene sets: Tier 1 (76 genes) in Figure 2, Tier 2 (197 genes) in Figure 3, and Tier 3 (599 genes) in Figure 4. Note that these gene sets were non-inclusive, so a single gene was only included in a single tier.

[0097] Figure 5 shows data on the number of CNV calls in each mGluR gene tier for different patient groups. CNVs are either duplications or deletions. The data show that a higher number of CNV calls were found in each gene set of mGluR network genes in samples from conduct disorder patients compared to depression, OCD, and phobia patients.

[0098] The percentage of patients with CNV calls (either duplications or deletions) in each gene set of mGluR network genes is shown in Figure 6. These data indicate that a substantially higher percentage of conduct disorder patients had CNV calls in each of the mGluR network gene sets compared with healthy control patients or subjects with depression, phobia, or OCD. The patient-control frequency with CNVs in mGluR network genes has previously been estimated to be 1.2% in a large study of over 9,000 controls of European descent (see Elia). Based on the data in this study, the frequency of patients with CNVs for OCD, phobia, or depression was less than 1.6% for Tier 1 mutation-positive patients, whereas the frequency of patients with CNVs among conduct disorder patients was 11.3% for Tier 1 mutation-positive subjects, 16.59% for Tier 1 and 2 mutation-positive subjects, and 34.94% for Tier 1, 2, and 3 mutation-positive subjects. See Figure 6.

[0099] As shown in Figures 5-6, there was a significant enrichment of mGluR network genes in patients with conduct disorder. Therefore, diagnostics and treatments focusing on regulating the mGluR gene network may be particularly useful in patients with conduct disorder.

[0100] Example 2. Analysis of mGluR network genes contained within CNVs from conduct disorder patient samples Next, we analyzed genotyping data from 861 fully genotyped conduct disorder patients to identify genes associated with CNVs. The mGluR network genes contained within CNVs from this group of patients are presented as examples of genes within the mGluR network that are contained within CNVs either by duplication or deletion.

[0101] Table 1 shows representative CNV data from patients with conduct disorder in which Tier 1 mGluR network genes were located within or near the CNVs in the patient samples. CNVs can result in structural changes that affect the transcription of genes located outside but near the CNV. Therefore, mGluR network genes in Tier 1 of the Tiers located within 500,000 base pairs of the CNV were also included in the analysis. If an mGluR network gene is contained within a listed CNV, it is marked with a "distance from gene" value of 0. If an mGluR network gene is not contained within the CNV but is in close proximity to it, it is given a "distance from gene" value greater than 0.

[0102] Table 1 lists the chromosomes on which the CNVs are located, along with their start and end positions relative to Human Genome version 19 (hg19). The number of SNPs located within the CNV is noted as "Num SNPs," and the length of the CNV is noted in base pairs. The Start SNP and End SNP of the CNV are also given.

[0103] The column "State, CN" indicates the copy number resulting from the CNV. Normally, human DNA (i.e., without CNVs) would be diploid and would have a "State, CN" of 2. CNVs with a "State, CN" of 0 or 1 indicate copy number deletions. In contrast, CNVs with a "State, CN" of 3 or greater indicate copy number duplications.

[0104] Confidence values ​​indicate the relative confidence that a CNV call is correct. All CNVs included in this analysis had positive confidence values, indicating a high likelihood that the CNV call is correct. Values ​​of 15 or higher were observed for most CNVs, which is considered very high confidence in the CNV call based on qPCR and Taqman genotyping validation.

[0105] The "mGluR Gene" column in Table 1 lists the specific mGluR network genes within Tier 1 that are included in the listed CNVs. Table 1 is sorted to show all CNVs that include a given Tier 1 mGluR network gene. Some Tier 1 genes may be represented in multiple CNVs from different patients in this study, resulting in multiple rows for those specific mGluR network genes. Some Tier 1 genes may not be represented in CNVs from this particular patient population.

[0106] Table 2 shows data from specific CNVs that included Tier 1 or Tier 2 mGluR network genes. The structure of Table 2 is similar to that of Table 1. The "mGluR Gene" column lists the specific mGluR network genes within Tier 1 or Tier 2 that are included in the listed CNVs. Table 2 is sorted to show all CNVs that included a given Tier 1 or Tier 2 mGluR network gene. Some Tier 1 or Tier 2 genes may be represented in multiple CNVs from different patients in this study, resulting in multiple rows for those particular genes. Some Tier 1 or Tier 2 genes may not be represented in CNVs from this particular patient population.

[0107] Table 3 shows data from specific CNVs that included Tier 1, 2, or 3 mGluR network genes. The structure of Table 3 is similar to that of Tables 1 and 2. The "mGluR Gene" column lists the specific mGluR network genes within Tier 1, Tier 2, or Tier 3 that were included in the listed CNVs. Table 3 is sorted to show all CNVs that included a given Tier 1, 2, or 3 mGluR network gene. Some Tier 1, 2, or 3 genes may be represented in multiple CNVs from different patients in this study, resulting in multiple rows for those specific mGluR network genes. Some Tier 1, 2, or 3 genes may not be represented in CNVs from this particular patient population.

[0108] Taken together, the data in Tables 1-3 indicate that a wide variety of mGluR network genes contained within each Tier are present in CNVs from conduct disorder patients. When larger patient cohorts with the conduct disorder phenotype are genotyped, all genes within Tier-1, Tier-2, and Tier-3 show enrichment for CNVs in conduct disorder patients. TIFF2025179063000002.tif247170TIFF2025179063000003.tif249170TIFF2025179063000004.tif248170TIFF2025179063000005.tif248170TIFF2025179063000006.tif247170TIFF2025179063000007.tif248170TIFF2025179063000008.tif248170TIFF2025179063000009.tif249170TIFF2025179063000010.tif248170TIFF2025179063000011.tif248170TIFF2025179063000012.tif248170TIFF2025179063000013.tif249170TIFF2025179063000014.tif248170TIFF2025179063000015.tif248170TIFF2025179063000016.tif249170TIFF2025179063000017.tif251170TIFF2025179063000018.tif249170TIFF2025179063000019.tif250170TIFF2025179063000020.tif249170TIFF2025179063000021.tif249170TIFF2025179063000022.tif248170TIFF2025179063000023.tif249170TIFF2025179063000024.tif249170TIFF2025179063000025.tif248170TIFF2025179063000026.tif250170TIFF2025179063000027.tif248170TIFF2025179063000028.tif250170TIFF2025179063000029.tif249170TIFF2025179063000030.tif249170TIFF2025179063000031.tif248170TIFF2025179063000032.tif250170TIFF2025179063000033.tif250170TIFF2025179063000034.tif250170TIFF2025179063000035.tif250170TIFF2025179063000036.tif250170TIFF2025179063000037.tif249170TIFF2025179063000038.tif249170TIFF2025179063000039.tif249170TIFF2025179063000040.tif248170TIFF2025179063000041.tif248170TIFF2025179063000042.tif248170TIFF2025179063000043.tif249170TIFF2025179063000044.tif249170TIFF2025179063000045.tif250170TIFF2025179063000046.tif249170TIFF2025179063000047.tif249170TIFF2025179063000048.tif249170TIFF2025179063000049.tif250170TIFF2025179063000050.tif250170TIFF2025179063000051.tif251170TIFF2025179063000052.tif249170TIFF2025179063000053.tif248170TIFF2025179063000054.tif252170TIFF2025179063000055.tif253170TIFF2025179063000056.tif252170TIFF2025179063000057.tif251170TIFF2025179063000058.tif253170TIFF2025179063000059.tif252170TIFF2025179063000060.tif253170TIFF2025179063000061.tif253170TIFF2025179063000062.tif254170TIFF2025179063000063.tif251170TIFF2025179063000064.tif251170TIFF2025179063000065.tif251170TIFF2025179063000066.tif252170TIFF2025179063000067.tif252170TIFF2025179063000068.tif252170TIFF2025179063000069.tif253170TIFF2025179063000070.tif252170TIFF2025179063000071.tif251170TIFF2025179063000072.tif252170TIFF2025179063000073.tif251170TIFF2025179063000074.tif253170TIFF2025179063000075.tif252170TIFF2025179063000076.tif252170TIFF2025179063000077.tif252170TIFF2025179063000078.tif251170TIFF2025179063000079.tif252170TIFF2025179063000080.tif251170TIFF2025179063000081.tif251170TIFF2025179063000082.tif252170TIFF2025179063000083.tif252170TIFF2025179063000084.tif251170TIFF2025179063000085.tif251170TIFF2025179063000086.tif252170TIFF2025179063000087.tif251170TIFF2025179063000088.tif252170TIFF2025179063000089.tif252170TIFF2025179063000090.tif251170TIFF2025179063000091.tif251170TIFF2025179063000092.tif252170TIFF2025179063000093.tif251170TIFF2025179063000094.tif252170TIFF2025179063000095.tif251170TIFF2025179063000096.tif251170TIFF2025179063000097.tif252170TIFF2025179063000098.tif251170TIFF2025179063000099.tif251170TIFF2025179063000100.tif252170TIFF2025179063000101.tif253170TIFF2025179063000102.tif251170TIFF2025179063000103.tif251170TIFF2025179063000104.tif252170TIFF2025179063000105.tif252170TIFF2025179063000106.tif251170TIFF2025179063000107.tif251170TIFF2025179063000108.tif251170TIFF2025179063000109.tif251170TIFF2025179063000110.tif252170TIFF2025179063000111.tif251170TIFF2025179063000112.tif251170TIFF2025179063000113.tif250170TIFF2025179063000114.tif251170TIFF2025179063000115.tif251170TIFF2025179063000116.tif251170TIFF2025179063000117.tif251170TIFF2025179063000118.tif251170TIFF2025179063000119.tif251170TIFF2025179063000120.tif251170TIFF2025179063000121.tif252170TIFF2025179063000122.tif251170TIFF2025179063000123.tif251170TIFF2025179063000124.tif251170TIFF2025179063000125.tif251170TIFF2025179063000126.tif251170TIFF2025179063000127.tif251170TIFF2025179063000128.tif252170TIFF2025179063000129.tif251170TIFF2025179063000130.tif251170TIFF2025179063000131.tif250170TIFF2025179063000132.tif251170TIFF2025179063000133.tif251170TIFF2025179063000134.tif251170TIFF2025179063000135.tif251170TIFF2025179063000136.tif251170TIFF2025179063000137.tif251170TIFF2025179063000138.tif252170TIFF2025179063000139.tif251170TIFF2025179063000140.tif251170TIFF2025179063000141.tif251170TIFF2025179063000142.tif251170TIFF2025179063000143.tif251170TIFF2025179063000144.tif251170TIFF2025179063000145.tif251170TIFF2025179063000146.tif251170TIFF2025179063000147.tif251170TIFF2025179063000148.tif252170TIFF2025179063000149.tif252170TIFF2025179063000150.tif252170TIFF2025179063000151.tif251170TIFF2025179063000152.tif251170TIFF2025179063000153.tif251170TIFF2025179063000154.tif251170TIFF2025179063000155.tif251170TIFF2025179063000156.tif251170TIFF2025179063000157.tif251170TIFF2025179063000158.tif251170TIFF2025179063000159.tif251170TIFF2025179063000160.tif251170TIFF2025179063000161.tif251170TIFF2025179063000162.tif252170TIFF2025179063000163.tif251170TIFF2025179063000164.tif251170TIFF2025179063000165.tif252170TIFF2025179063000166.tif251170TIFF2025179063000167.tif251170TIFF2025179063000168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[0109] Example 3. Treatment with fasoracetam monohydrate (NFC-1) in ADHD patients with CNVs in mGluR network genes and the effect on conduct disorder An open-label, phase Ib clinical trial was conducted to investigate the safety, pharmacokinetics, and efficacy of NFC-1 (fasoracetam monohydrate) in adolescent subjects aged 12 to 17 years, previously diagnosed with ADHD, who also had at least one genetic alteration in an mGluR network gene.

[0110] The study enrolled 30 ADHD subjects between 12 and 17 years of age, of any origin or race, with weights within the 5th and 95th percentiles for their age, and judged to be in otherwise good medical health. Subjects were genotyped and enrolled if they had 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 gene function. Seventeen of the 30 subjects had CNVs in tier 1 mGluR network genes, while seven subjects had CNVs in tier 2 genes and six had CNVs in tier 3 genes. At enrollment, several study subjects, including two with recurrent tics, showed evidence of comorbid phenotypes.

[0111] Exclusion criteria included subjects who, in the opinion of the investigator, suffered from any clinically significant mental or physical illness that might confound the results of the study or prevent completion of the study; subjects who were pregnant or breastfeeding; subjects with a history of drug abuse, subjects who tested positive for illicit drugs; subjects who consumed alcoholic beverages; or subjects who otherwise raised concerns with the investigator regarding their compliance or suitability.

[0112] The study included either 50 mg or 200 mg NFC-1 capsules containing fasoracetam monohydrate as the active ingredient, and placebo capsules containing microcellulose. The study design included telephone screening (day 1), a registration period (days 1 to 2), a washout period (days 1-14) for subjects currently receiving ADHD medication, a pharmacokinetic (PK) assessment (day 2), a dose-escalation period (day 35), and a follow-up visit approximately 4 weeks after the last dose, for a maximum of 127 days. All ADHD medications were discontinued during the pre-study washout period. The washout period for stimulants was 2-3 days, and the washout period for atomoxetine or noradrenergic agonists was 10-12 days. No new ADHD medications were initiated during the study.

[0113] After an initial washout period, and PK and initial safety assessments, the dose-escalation phase of the study lasted for 5 weeks. During the first week, all subjects received placebo capsules twice daily. After 1 week of placebo treatment, patients began 1 week of NFC-1 at 50 mg bid. If safety and response data from the previous dose level of fasoracetam were adequate, the subject's dose was increased to the next higher dose (100, 200, or 400 mg). Subjects who tolerated and responded to the 50 mg bid dose were to remain at that level for the remaining 3 weeks of the study.

[0114] Subjects who tolerated but showed a lack of response or partial response to the 50 mg bid dose were to be increased to the next higher 100 mg dose for subsequent weeks. Subjects who tolerated 100 mg but showed a lack of response or partial response were to be increased to the 200 mg dose for subsequent weeks, while subjects who tolerated and responded to 100 mg were to remain on 100 mg bid for the remainder of the study. Similarly, subjects who tolerated and responded to the 200 mg dose were to remain on 200 mg for the final week of the study, while subjects who tolerated but showed a lack of response or partial response were changed to the 400 mg dose for the final week. Of the 30 study subjects, 3 received the maximum dose of 100 mg, 9 received the maximum dose of 200 mg, and the remaining 18 received the maximum dose of 400 mg.

[0115] Although this study was not specifically aimed at conduct disorder, two study subjects met behavioral criteria for conduct disorder and scored 2 or 3 on at least two of the 14 Vanderbilt Inventory items 27-40, which assess conduct disorder symptoms. One of these two subjects did not complete the study. The other showed improvement in their conduct disorder symptoms based on a reduction in Vanderbilt items 27-40 from 16 to 12 between weeks 1 and 5.

[0116] Example 4. Treatment of ADHD patients with CNVs in mGluR network genes with fasoracetam monohydrate (NFC-1) and the effect on obsessive-compulsive symptoms Eight of the 30 ADHD subjects studied in the open-label Phase Ib clinical trial described in Example 3 had symptoms of obsessive-compulsive disorder (OCD). One of the tics subjects also had symptoms of OCD. In all eight subjects, OCD symptoms improved during treatment with NFC-1.

[0117] One subject with OCD also had a history of ear-scratching behavior (i.e., self-injurious dermatitis), which led to bleeding ulcers. The bleeding ulcers healed during treatment with NFC-1. This means that the subject's self-injurious dermatitis symptoms were reduced during treatment with NFC-1.

[0118] Example 5. Study of phenotypes associated with mGluR network CNVs A total of 1,000 ADHD patients aged 6-17 years were enrolled in the trial to examine phenotypes potentially associated with CNVs in Tier 1 or 2 mGluR network genes. Saliva samples were collected by the study site. Each DNA sample was then subjected to DNA extraction, gene sequencing, and DNA biobanking.

[0119] Gene sequencing results, along with medical history, were used to assess genotype (based on gene sequencing) and phenotype (based on interviews conducted by clinicians and the subject's parents / guardians). Subjects had ADHD as defined by the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-V).

[0120] A single clinician asked the parent or patient's legal guardian a series of questions regarding possible behavioral or health phenotypes. For each individual phenotype, the parent / guardian was asked, "Is this a current concern?" and a yes or no response was collected. The clinician determined the frequency of yes or no responses to generate phenotype data.

[0121] This study found that the prevalence of anger control as a current parental concern was 58.9% in ADHD subjects with Tier 1 or 2 mGluR network gene CNVs, but only 47.4% in ADHD subjects without such mGluR network gene CNVs. This difference was statistically significant (odds ratio of 1.59, P=0.003). This odds ratio, greater than 1, implies a higher prevalence of current anger control concerns in ADHD subjects with Tier 1 or 2 mGluR network gene CNVs compared with subjects without such CNVs.

[0122] The prevalence of disruptive behavior as a current patient concern was 57.1% in ADHD subjects with Tier 1 or 2 mGluR network gene CNVs and 43.9% in ADHD subjects without such mGluR network gene CNVs. This difference was also statistically significant (odds ratio of 1.70, P<0.001). This difference indicates a higher prevalence of parental disruptive behavior as a current concern among ADHD subjects who also have mGluR network gene mutations compared with those without mutations.

[0123] Example 6: Copy number variations of mGluR network genes in ADHD subjects with comorbid disorders Samples from 2707 pediatric subjects (mean age 10-10.5 years) known to have ADHD were genotyped using the 550 / 610 Illumina chip to determine whether they had one or more CNVs in Tier 1 or Tier 2 genes. The 2707 subjects included 759 females and 1778 males of African American or Caucasian ethnicity (1063 and 1483, respectively). 430 of the 2707 subjects (16.9%) had at least one CNV in an mGluR Tier 1 or Tier 2 gene.

[0124] The records of the 2707 subjects were also reviewed to determine whether they were diagnosed with comorbidities according to the World Health Organization's International Classification of Diseases, Ninth Revision (ICD-9). Of the 2707 subjects, 1902 (approximately 70%) had comorbidities, while 805 did not. Of these 1902 subjects with comorbidities, approximately 30% had more than one comorbidity, and approximately 20% had two or more, although a small percentage had more comorbidities.

[0125] The most prevalent comorbidities that were present in more than 100 subjects each are listed in Table 4. This table lists the comorbidities by ICD-9 code and provides the number of cases (column titled "N") among the 2707 subjects and the name of each comorbid condition or disorder. TIFF2025179063000174.tif144170

[0126] The comorbidities in Table 4 tend to cluster into a few distinct groups: anxiety, depression, or mood-related disorders; high-prevalence developmental disorders; low-prevalence developmental disorders; and autism and related disorders.

[0127] We then combined the phenotypic and comorbidity data to determine how many subjects with Tier 1 or 2 mGluR network gene CNVs also had comorbidities. We found that 316 of the subjects with such CNVs (approximately 18% of CNV-positive subjects or 12% of all subjects) also had at least one comorbidity, while 114 of the subjects without Tier 1 or 2 mGluR network gene CNVs (approximately 15% of CNV-negative subjects or 4% of all subjects) also had at least one comorbidity. This difference had a P value of 0.118. Thus, overall, comorbidities tended to be more prevalent in CNV-positive subjects than in CNV-negative subjects. When considering only subjects identified as Caucasian, there was a highly significant correlation between mGluR CNVs and ADHD comorbidity. Specifically, 218 of 1483 subjects had at least one CNV in a Tier 1 or 2 mGluR network gene, and of these 218 subjects, 169 also had a comorbidity, while 49 did not. This difference had a P-value of 0.004.

Claims

1. A method for treating conduct disorder in a subject, comprising administering to the subject an effective amount of a non-selective activator of metabotropic glutamate receptors (mGluRs), thereby treating the conduct disorder.

2. 10. The method of claim 1, wherein the subject has at least one genetic alteration in an mGluR network gene.

3. 1. A method for treating conduct disorder in a subject, comprising administering an effective amount of a non-selective activator of metabotropic glutamate receptors (mGluRs) to a subject having at least one genetic alteration in an mGluR network gene, thereby treating the conduct disorder.

4. 1. A method of treating conduct disorder in a subject, comprising obtaining results from a genetic screen to determine whether the subject has a genetic alteration in an mGluR network gene, and if the results indicate that the subject has at least one genetic alteration in an mGluR network gene, treating the subject by administering an effective amount of a non-selective activator of mGluR.

5. 5. The method of claim 2, wherein the genetic alteration is a copy number variation (CNV) or a single nucleotide variation (SNV).

6. The method of claim 5 , wherein the genetic alteration is a CNV.

7. The method of claim 6, wherein the CNV is a deletion or duplication.

8. 8. The method of claim 1, wherein the non-selective activator of mGluR is fasoracetam.

9. 9. The method according to claim 8, wherein the fasoracetam is fasoracetam monohydrate (NS-105 or NFC-1).

10. 10. The method of claim 8 or claim 9, wherein fasoracetam is administered in a dose of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg or 400 mg, said dose being administered once, twice or three times daily.

11. 10. The method of claim 8 or claim 9, wherein fasoracetam is administered in a dose of 50-400 mg, 100-400 mg, or 200-400 mg, said dose being administered once, twice, or three times daily.

12. 10. The method of claim 8 or 9, wherein fasoracetam is administered in a dose of 200-400 mg, for example 200 mg, 300 mg or 400 mg, said dose being administered twice daily.

13. 13. The method of any one of claims 1 to 12, wherein the subject has CNVs in at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mGluR network genes.

14. CNVs in mGluR network genes (a) obtaining a nucleic acid-containing sample from said subject and subjecting said 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 Tier 1 mGluR network genes; or (b) Obtain a report describing the results of a genetic test that screened for CNVs in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or all Tier 1 mGluR network genes. The method of any one of claims 6 to 13, wherein the determination is made by:

15. CNVs in mGluR network genes (a) obtaining a nucleic acid-containing sample from said subject and subjecting said sample to a screen that assesses CNVs in at least 50, at least 100, at least 150, at least 175, or all Tier 2 mGluR network genes; or (b) obtaining a report describing the results of a genetic test that screened for CNVs in at least 50, at least 100, at least 150, at least 175, or all Tier 2 mGluR network genes; The method of any one of claims 6 to 13, wherein the determination is made by:

16. CNVs in mGluR network genes (a) obtaining a nucleic acid sample from said subject and subjecting said 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 Tier 3 mGluR network genes; or (b) obtaining a report describing the results of a genetic test that screened for CNVs in at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all Tier 3 mGluR network genes; The method of any one of claims 5 to 13, wherein the determination is made by:

17. 17. The method of any one of claims 5 to 16, wherein the screening does not evaluate CNVs of one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7 or GRM8.

18. 18. The method of any one of claims 1 to 17, wherein the subject does not have a CNV in one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, or GRM8.

19. 19. The method of any one of claims 1 to 18, wherein the patient being treated has antisocial personality disorder.

20. 20. The method of any one of claims 1 to 19, wherein the subject is a child or adolescent subject.

21. 21. The method of claim 20, wherein the child or adolescent subject is between 5 and 17 years of age, between 5 and 8 years of age, between 8 and 17 years of age, between 8 and 12 years of age, between 12 and 18 years of age, between 13 and 18 years of age, or between 12 and 17 years of age.

22. 20. The method of any one of claims 1 to 19, wherein the subject is an adult.

23. 23. The method of any one of claims 1 to 22, wherein the non-selective activator of mGluRs is administered in combination with another drug or non-drug therapy.

24. 24. The method of claim 23, wherein the non-drug therapy comprises brain stimulation, such as vagus nerve stimulation, repetitive transcranial magnetic stimulation, magnetoconvulsive therapy, or deep brain stimulation.

25. 25. The method of claim 23 or 24, wherein the activator is administered in combination with an antipsychotic agent.

26. 26. The method of any one of claims 1 to 25, wherein symptoms of inattention, hyperactivity and / or impulsivity are reduced in a subject after at least 1 week, e.g., at least 2 weeks, at least 3 weeks, or at least 4 weeks of treatment with the activator.

27. 27. The method of any one of claims 1 to 26, wherein symptoms of aggression or antisocial behavior are reduced in the subject after at least 1 week, e.g., at least 2 weeks, at least 3 weeks, or at least 4 weeks of treatment with the activator.

28. 1. A method for diagnosing conduct disorder in a subject, comprising: (a) isolating a sample comprising nucleic acid from a subject; (b) analyzing the sample for the presence or absence of a genetic alteration in at least one mGluR network gene; and (c) diagnosing the subject with conduct disorder if the subject has at least one genetic alteration in an mGluR network gene. A method comprising:

29. 1. A method for diagnosing conduct disorder in a subject, comprising: (a) obtaining results of a genetic test in which the subject is screened for the presence or absence of a genetic alteration in at least one mGluR network gene; and (b) diagnosing conduct disorder if the results indicate that the subject has at least one genetic alteration in an mGluR network gene. A method comprising:

30. 1. A method for identifying a subject as having a conduct disorder, comprising obtaining a sample from a patient, optionally isolating nucleic acid from said sample, optionally amplifying said nucleic acid, and analyzing said nucleic acid in said sample for the presence or absence of at least one mGluR network genetic alteration, e.g., CNV, wherein if at least one genetic alteration, e.g., CNV, in an mGluR network gene is detected, said subject is identified as having a conduct disorder.

31. 1. A method for diagnosing conduct disorder in a subject, comprising: analyzing genetic information for one or more mGluR network genes; comparing the subject's information with control subjects who do not have conduct disorder; and diagnosing the subject with conduct disorder if the genetic information suggests that the subject has at least one genetic alteration in an mGluR network gene.

32. 1. A method of confirming a diagnosis of conduct disorder in a subject, comprising: (a) obtaining a sample comprising nucleic acid from a subject diagnosed with conduct disorder by a method that does not involve detecting or analyzing genetic alterations in mGluR network genes; (b) optionally amplifying the nucleic acid in the sample; and (c) determining whether the subject has at least one genetic alteration, e.g., a CNV, in an mGluR network gene; (d) confirming a diagnosis of conduct disorder if the subject has at least one genetic alteration in an mGluR network gene. A method comprising:

33. 33. The method of any one of claims 28 to 32, wherein the subject has CNVs in at least two mGluR network genes.

34. 34. The method of any one of claims 28 to 33, comprising 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.

35. 35. The method of claim 34, wherein CNV in mGluR network genes is determined by subjecting the sample to a screen that assesses CNV in at least 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70 or all Tier 1 mGluR network genes.

36. 36. The method of any one of claims 28 to 35, wherein CNVs in mGluR network genes are determined by subjecting the sample to a screen that evaluates CNVs in at least 50, at least 100, at least 150, at least 175, or all Tier 2 mGluR network genes.

37. 37. The method of any one of claims 28 to 36, wherein CNVs in mGluR network genes are determined by subjecting the sample to a screen that evaluates CNVs in at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all Tier 3 mGluR network genes.

38. 38. The method of any one of claims 28 to 37, comprising detecting CNVs in mGluR network genes by subjecting the sample to a screen that evaluates CNVs in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, or all Tier 1 mGluR network genes, and at least 50, at least 100, at least 150, at least 175, or all Tier 2 mGluR network genes, and at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or all Tier 3 mGluR network genes.

39. 39. The method of any one of claims 28 to 38, wherein the subject is a child or adolescent subject.

40. 40. The method of claim 39, wherein the pediatric patient is between 5 and 17 years of age, between 5 and 8 years of age, between 8 and 17 years of age, between 8 and 12 years of age, between 12 and 18 years of age, between 13 and 18 years of age, or between 12 and 17 years of age.

41. 39. The method of any one of claims 28 to 38, wherein the subject is an adult subject.

42. 42. The method of any one of claims 28 to 41, wherein the subject is not evaluated for genetic alterations or CNVs in one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7 and GRM8.

43. 43. The method of any one of claims 28 to 42, wherein the method for determining the presence or absence of at least one mGluR network gene genetic alteration comprises the use of microarray, 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 technology.

44. 44. The method of any one of claims 28 to 43, wherein the subject is not evaluated for CNV of one or more of GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7 and GRM8.

45. 45. The method of any one of claims 1 to 44, wherein the subject has a conduct disorder and one or more of ADHD, ODD, anxiety disorder, Tourette's syndrome, phobia, or depression.

46. 45. The method of any one of claims 1 to 44, wherein the subject does not have one or more of ADHD, ODD, anxiety disorders, Tourette's syndrome, phobias, and depression.

47. 47. The method of claim 46, wherein the subject does not have any of ADHD, ODD, anxiety disorders, Tourette's syndrome, phobias, and depression.

48. The subject exhibits the following phenotypic changes after at least 1 week, e.g., at least 2 weeks, at least 3 weeks, or at least 4 weeks of treatment with the activator: (a) the subject has anger control difficulties, and anger control symptoms are improved; (b) the subject has disruptive behavior, and the disruptive behavior is reduced; (c) the subject's CGI-I is reduced by at least 1 or at least 2; the subject's CGI-I score is 1 or 2 after 1, 2, 3, or 4 weeks of treatment; (d) the subject's CGI-S score after 1, 2, 3, or 4 weeks of treatment is 1; the subject's ADHD rating scale score is reduced by at least 25%, e.g., at least 30%, at least 35%, or at least 40%; (e) the subject has a symptom of inattention and the symptom of inattention is reduced; the subject has a symptom of hyperactivity and the symptom of hyperactivity is reduced; (f) the subject has symptoms of impulsivity, and the impulsive symptoms are reduced; (g) the subject has symptoms of ODD, e.g., anger and irritability, argumentative and defiant behavior, and / or vindictiveness, and the ODD symptoms are reduced; (h) the subject has symptoms of anxiety, and the anxiety symptoms are reduced; (i) the subject has symptoms of Tourette's syndrome, and the symptoms of Tourette's syndrome are reduced; (j) the subject has symptoms of autism, and the autism symptoms are reduced; and (k) the subject has symptoms of a movement disorder, and the movement disorder symptoms are reduced.

48. The method of any one of claims 1 to 47, comprising one or more of:

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