(12) Patent Application Publication (10) Pub. No.: US 2017/0009296A1 Glessner Et Al

US 20170009296A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0009296A1 Glessner et al. (43) Pub. Date: Jan. 12, 2017

(54) ASSOCIATION OF RARE RECURRENT (60) Provisional application No. 61/376.498, filed on Aug. GENETIC VARATIONS TO 24, 2010, provisional application No. 61/466,657, ATTENTION-DEFICIT, HYPERACTIVITY filed on Mar. 23, 2011. DISORDER (ADHD) AND METHODS OF USE THEREOF FOR THE DAGNOSS AND TREATMENT OF THE SAME Publication Classification (71) Applicant: The Children's Hospital of Philadelphia, Philadelphia, PA (US) (51) Int. Cl. CI2O I/68 (2006.01) (72) Inventors: Joseph Glessner, Mullica Hill, NJ A63L/454 (2006.01) (US); Josephine Elia, Penllyn, PA (52) U.S. Cl. (US); Hakon Hakonarson, Malvern, CPC ...... CI2O I/6883 (2013.01); A61 K3I/454 PA (US) (2013.01); C12O 2600/156 (2013.01); C12O (21) Appl. No.: 15/063,482 2600/16 (2013.01) (22) Filed: Mar. 7, 2016 Related U.S. Application Data (57) ABSTRACT (63) Continuation of application No. 13/776,662, filed on Feb. 25, 2013, now abandoned, which is a continu ation-in-part of application No. PCT/US 11/48993, Compositions and methods for the detection and treatment filed on Aug. 24, 2011. of ADHD are provided. Patent Application Publication Jan. 12, 2017. Sheet 1 of 18 US 2017/000929.6 A1

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ASSOCATION OF RARE RECURRENT diagnosis and provide avenues for the development of GENETIC VARATIONS TO therapeutic agents having efficacy for the treatment of ATTENTION-DEFICIT, HYPERACTIVITY ADHD. DISORDER (ADHD) AND METHODS OF USE THEREOF FOR THE DAGNOSIS AND SUMMARY OF THE INVENTION TREATMENT OF THE SAME 0006. In accordance with the present invention, methods 0001. This application is a continuation of U.S. applica are provided for the diagnosis and treatment of ADHD. An tion Ser. No. 13/776,662 filed Feb. 25, 2013, which is a exemplary method entails detecting the presence of at least continuation in part of PCT/US2011/048993 filed Aug. 24, one CNV in a target polynucleotide wherein if said CNV(s) 2011 which in turn claims priority to U.S. Provisional is/are present, said patient has an increased risk for devel Applications 61/376.498 and 61/466,657 filed Aug. 24, 2010 oping ADHD. and Mar. 23, 2011 respectively, the entire contents of each 0007. In one aspect of the present invention, a method for being incorporated by reference as though set forth in full. detecting a propensity for developing attention deficit hyper activity disorder (ADHD) in a patient in need thereof is provided. An exemplary method entails detecting the pres FIELD OF THE INVENTION ence of at least 1, 2, 3, 4, 5, 6, 10, 20, 30 or all of the SNP 0002 This invention relates to the fields of genetics and containing nucleic acid in a target polynucleotide, said SNP the diagnosis of attention deficit hyperactivity disorder being informative of the presence of an ADHD associated (ADHD). More specifically, the invention provides compo copy number variation (CNV), wherein if said SNP is sitions and methods useful for the diagnosis and treatment of present, said patient has an increased risk for developing ADHD. ADHD, wherein said SNP containing nucleic acid is pro vided in Table 13. 0008. In another embodiment of the invention a method BACKGROUND OF THE INVENTION for identifying agents which alter neuronal signaling and/or 0003. Several publications and patent documents are morphology is provided. Such a method comprises provid cited through the specification in order to describe the state ing cells expressing at least one nucleic acid comprising the of the art to which this invention pertains. Each of these ADHD associated CNVs of the invention, (step a); provid citations is incorporated herein by reference as though set ing cells which express the cognate wild type sequences forth in full. which lack the CNV (step b); contacting the cells from each sample with a test agent and analyzing whether said agent 0004 Attention Deficit Hyperactivity Disorder (ADHD) alters neuronal signaling and/or morphology of cells of step is a common neuropsychiatric disorder with heritability a) relative to those of step b), thereby identifying agents estimates ranging from 30 to 90% (Derks, et al. 2008; Wood, which alter neuronal signaling and morphology. In a pre et al. 2008; Haberstic, et al. 2008). Most neurodevelopmen ferred embodiment the test agent modulates metabotropic tal disorders have been resistant to the genome wide asso glutamate receptor (mGluR) gene activity. In another ciation (GWA) approach, although recent progress has been embodiment the test agent is selected from a group consist made in autism (Glessner, et al. 2009: Derks, et al. 2008: ing of an mGluR positive allosteric modulators (PAM) (e.g., Wod, et al. 2008; Haberstic, et al. 2008: Wang, et al. 2009). mGluR5 PAM, mGluR7 PAM), an mGluR negative allos GWA studies have been reported in ADHD utilizing a cohort teric modulator (NAM) (e.g., mGluR2/3 NAM), and a of 958 parent-child trios recruited through the International tachykinin-3/neurokinin-3 receptor (TAC3/NK3R) antago Multicentre ADHD Genetics (IMAGE) study. Results of nist. In another embodiment, the test agent is selected from these studies did not report any association at genome-wide the group consisting of ADX63365, ADX50938, significance level (Franke, et al. 2009; Neale, et al. 2008). ADX71149, ADX48621, AMN082, 1-(hetero)aryl-3-amino Using quantitative measures of ADHD, Lasky-Su and col pyrrolidine derivatives (e.g. those provided in U.S. Patent leagues recently reported nominal evidence from a PBAT Application Publication No. 2008/0300266), LY341495, analysis of tagging SNPs located at CDH13 (rs6565113) and GSK1144814, and SB223412. Methods of treating ADHD GFOD1 (rs552655) (Lasky-Su, et al. 2008). A SNP in strong patients via administration of test agents identified using the linkage disequilibrium with rs6565113 impacting CDH13 methods described herein in patients in need thereof are also was also reported in a GWA study of an independent sample encompassed by the present invention. The invention also of ADHD adults (Lesch, et al. 2008). The applicants provides at least one isolated ADHD related SNP-containing reported previously on copy number variation (CNV) loci nucleic acid selected from the group listed in Table 13. In observed in the first 335 ADHD cases we recruited (Elia, et one embodiment, a multiplex SNP panel containing all of al. 2009). While none of the CNV loci detected in that study the informative SNPs from Table 13 is provided. Such SNP met criteria for significance, it is noteworthy that one family containing nucleic acids which indicate the presence of was observed to have a GRM5 deletion impacting all three ADHD associated CNV(s) may optionally be contained in a affected children, inherited from their affected father. A Suitable expression vector for expression in neuronal cells. GRM7 deletion in one family with ADHD was additionally Alternatively, they may be immobilized on a solid support. detected (Elia, et al. 2009). CNVs of metabotropic glutamate In yet another alternative, the panel may be provided in receptors (mGluR) in addition to the discovery of the NK3 silico. gene in ADHD have Suggested new therapeutic approaches 0009. According to yet another aspect of the present to the treatment of ADHD. invention, there is provided a method of treating ADHD in 0005. The development of improved accurate diagnostic a patient determined to have at least one prescribed single tests for this disorder based on associated genetic alterations nucleotide polymorphism indicative of the presence of an is highly desirable. Such tests would facilitate conclusive ADHD-associated copy number variation, as described US 2017/000929.6 A1 Jan. 12, 2017 herein below, by administering to the patient a therapeuti assays using Roche Universal probe were designed to Vali cally effective amount of at least one member of the pirac date every candidate CNV with a completely independent etam family of nootropic agents. This method provides a test test (representative series shown for each locus in case and and treat paradigm, whereby a patient’s genetic profile is control pairs). Error bars denote the standard deviation of used to personalize treatment with therapeutics targeted quadruplicate runs. Del, deletion; Dup, duplication. towards specific neurophysiological defects found in indi (0017 FIG. 8. Examples of CNV observance based on viduals exhibiting ADHD. Such a test and treat model may B-allele frequency (BAF) and Log R Ratio (LRR). benefit up to 50% of patients with ADHD with greater (0018 FIG. 9. An illustration of the deletion directly efficacy and fewer side effects than non-personalized treat impacting GRM5, exclusive to ADHD cases and replicated ment. Thus, any of the patients exhibiting an alteration in in IMAGE and PUWMa. Four CHOP ADHD case hemizy glutaminergic signaling can be tested for the presence of gous deletions in GRM5 replicated by 2 deletions and 3 Such a genetic alteration and then treated with the appropri larger deletions found in IMAGE and 1 PUWMa deletion. ate pharmaceutical Such as the agents listed above. SNP coverage of the Illumina 550k, Perlegen 600k, Illumina 1M, and Affymetrix 5.0 arrays are shown as vertical blue BRIEF DESCRIPTION OF THE DRAWINGS lines. 0010 FIGS. 1A-B. Agraphical distribution of CNV calls (0019 FIG. 10. A display of GRM receptor gene interac per individual cases (1A) compared to controls (1B). tion networks impacted in ADHD. GRM receptor genes are 0011 FIG. 2. A graphical display of the Normalized SNP shown as large diamond-shaped nodes while other interact Level Perlegen 600K Data. The X axis shows base pair ing genes within 2 degrees if interaction are shown as position in Megabases on chromosome 11. Raw SNP Level smaller circular nodes. Nodes are colored to represent Data Showing GRM5 Deletion in five samples from IMAGE enrichment of CNVs: dark red are deletions enriched in Perlegen 600K Data Normalized by Adapted PennCNV cases, light red are deletions enriched in controls, dark green Affy Protocol. Genotype data termed B-allele frequency are duplications enriched in cases, light green are duplica (BAF) and intensity data termed Log R Ratio (LRR) plotted. tions enriched in controls, and grey are diploid and devoid 0012 FIGS. 3A-F. Graphs of the full SNP-Level data: of CNVs. Blue thick dashed lines highlight edges connected 3A-C) Normalized Perlegen 600K data, 3D-E) Normalized to at least one GRM gene while grey thin dotted lines Illumina 1M PUWMa data, and 3F) Normalized Affymetrix represent all other gene interactions. Highly connected mod 5.0 IMAGE II data. ules enriched for significant functional annotations are high 0013 FIG. 4. Agraphical display of the IMAGE Perlegen lighted by blue shaded ellipses. 600K Independent Validation data. Fluorescent probe-based 0020 FIG. 11. A schematic overview showing the inter qPCR assays using Roche Universal probe were designed to action of GRM receptors impacted in ADHD with modules validate every candidate CNV with a completely indepen of genes enriched for functional significance. GRM receptor dent test (11 of the 14 IMAGE samples with replicating genes are shown as diamonds colored either green or red to CNV calls for the loci reported were available for validation represent duplications and deletions respectively enriched in and all validated in comparison with control pairs; the other cases. Boxes highlight functional modules defined by the 3 loci were visually validated). Error bars denote the stan network of interacting genes that are significantly enriched dard deviation of quadruplicate runs. Del, deletion; Dup, for GO annotations. Functional modules describe significant duplication. functional annotations and are labeled with the cluster name 0014 FIG. 5. An illustration of the Eigenstrat Principle and the number of component genes in parenthesis. Func Components Analysis. Cases and Controls were simultane tional annotations that may be particularly pertinent to ously analyzed to minimize population Substructure in case ADHD underlying pathophysiology are bolded. Edges of the control CNV association. Samples deviating from the Cau network connect GRM receptor genes to functional mod casian cluster shown were removed. The genomic inflation ules: solid lines indicate membership of the GRM interact factor (GIF) within Plink was at an acceptable level (GIF=1. ing gene in the functional module, and dotted lines indicate 02409). We also checked pairwise population concordance a first-degree relationship between GRM receptor genes and to check for and filter out cryptic relatedness which could at least one component gene of a functional module. give rise to rare CNVs specific to ultra-stratified subpopu 0021 FIG. 12. A CNV peninsula false positive associa lations of Europe. tion example. An example from chr 2 is shown (location 0015 FIGS. 6A-B. An example of the SNP-based statis 51,777,616-51,784,033). All significant CNVRs are tics applied and the resulting highest significance region reviewed for CNV peninsulas indicating uncertainty in Called. Examples from chr 3 are shown: 6A) 1,327.963-2, boundary truncation. 376,095 and 6B) 1,847,000-1,862.261. Complex CNV over DETAILED DESCRIPTION OF THE lap is simplified by producing SNP-based statistics. As seen INVENTION in plots for cases deleted and controls deleted, each SNP has a specific number of CNVs. The cases and controls are (0022. Attention-Deficit, Hyperactivity Disorder (ADHD) compared with a Fisher's exact test and the negative log p is a common, heritable neuropsychiatric disorder of value is shown in the third plot. Regions of significance unknown etiology. Recently, we reported an enrichment of ranging within a power often are reported and the region of rare variants in genes involved in learning, behavior, Syn highest significance (local minimum p-value) within 1 MB aptic transmission and central nervous system development is reported. The IMAGE cases deleted plot shows only one in autism', suggesting that rare inherited structural variants case sample #11939 since the remaining red regions 3' are could also play a role in the etiology of ADHD, a related parents. neuropsychiatric disorder. 0016 FIG. 7. CHOP Illumina Human Hap550 Indepen 0023 To follow up on those studies, we performed a dent Validation using qPCR. Fluorescent probe-based qPCR whole-genome CNV study in a cohort of 1,013 ADHD cases US 2017/000929.6 A1 Jan. 12, 2017

and 4,105 healthy children of European ancestry who were tibility to genetic disorders is known to be associated not genotyped with 550,000 SNP markers. Positive findings only with single nucleotide polymorphisms (SNP), but also were evaluated in multiple independent cohorts, totaling with structural and other genetic variations, including 2,493 ADHD cases and 9.222 controls of European ancestry, CNVs. A CNV represents a copy number change involving with respective case-control cohorts genotyped on matched a DNA fragment that is ~1 kilobases (kb) or larger (Feuk et platforms. al. 2006a). CNVs described herein do not include those 0024 Our results identified several CNVs impacting variants that arise from the insertion/deletion of transposable metabotropic glutamate receptor genes which were signifi elements (e.g., -6-kb Kpnl repeats) to minimize the com cantly enriched across all independent cohorts (P=2.1 x 10 plexity of future CNV analyses. The term CNV therefore 9). Among them, deletions in GRM5 (glutamate receptor, encompasses previously introduced terms such as large metabotropic 5) occurred in ten cases across three indepen scale copy number variants (LCVs: Iafrate et al. 2004), copy dent cohorts and in only one control subject (P=1.36x10). number polymorphisms (CNPs; Sebat et al. 2004), and In addition, deletions in GRM7 occurred in six cases and intermediate-sized variants (ISVs; Tuzun et al. 2005), but GRM8 in eight cases, both with a control frequency of Zero. not retroposon insertions. The terminology "duplication GRM1 was duplicated in eight cases, a frequency notably containing CNV is also used herein below consistent with enriched above controls. Observed variants were experimen the CNV definition provided. tally validated using quantitative PCR. Subsequent gene 0030 “ADHD-associated SNP or “ADHD-associated network analysis demonstrated that genes interacting with specific marker” or ADHD-associated informational GRM receptors are significantly enriched for CNVs in cases sequence molecule' is a SNP or marker sequence which is compared to controls (P-4.38x10"), collectively impact associated with an increased or decreased risk of developing ing ~10% of ADHD cases in this study. Furthermore, we ADHD not found normal patients who do not have this found that GRMs serve as critical hubs that coordinate disease. Such markers may include but are not limited to highly connected modules of interacting genes, many of nucleic acids, proteins encoded thereby, or other Small which harbor CNVs and are enriched for synaptic and molecules. Thus, the phrase “ADHD-associated SNP con neuronal biological functions. taining nucleic acid' is encompassed by the above descrip 0025. The following definitions are provided to facilitate tion. an understanding of the present invention. 0031. The term "solid matrix” as used herein refers to any format, Such as beads, microparticles, a microarray, the I. DEFINITIONS surface of a microtitration well or a test tube, a dipstick or 0026. For purposes of the present invention, “a” or “an a filter. The material of the matrix may be polystyrene, entity refers to one or more of that entity; for example, “a cellulose, latex, nitrocellulose, nylon, polyacrylamide, dex cDNA refers to one or more cDNA or at least one cDNA. tran or agarose. As such, the terms 'a' or “an,” “one or more' and “at least 0032. The phrase “consisting essentially of when refer one can be used interchangeably herein. It is also noted that ring to a particular nucleotide or amino acid means a the terms “comprising,” “including, and “having can be sequence having the properties of a given SEQID NO. For used interchangeably. Furthermore, a compound “selected example, when used in reference to an amino acid sequence, from the group consisting of refers to one or more of the the phrase includes the sequence per se and molecular compounds in the list that follows, including mixtures (i.e. modifications that would not affect the functional and novel combinations) of two or more of the compounds. According characteristics of the sequence. to the present invention, an isolated, or biologically pure 0033. The phrase “partial informative CNV is used molecule is a compound that has been removed from its herein to refer to a nucleic acid that hybridizes to sequences natural milieu. As such, “isolated' and “biologically pure' comprising a duplication on a chromosome however, the do not necessarily reflect the extent to which the compound partial informative CNV may not be identical to the dupli has been purified. An isolated compound of the present cation, rather, the CNV may correspond to only a portion of invention can be obtained from its natural source, can be the duplication, but yet is still informative for the same. produced using laboratory synthetic techniques or can be 0034 “Target nucleic acid” as used herein refers to a produced by any such chemical synthetic route. previously defined region of a nucleic acid present in a 0027. The term “genetic alteration” as used herein refers complex nucleic acid mixture wherein the defined wild-type to a change from the wild-type or reference sequence of one region contains at least one known nucleotide variation or more nucleic acid molecules. Genetic alterations include which may or may not be associated with ADHD but is without limitation, base pair substitutions, additions and informative of the risk of ADHD. The nucleic acid molecule deletions of at least one nucleotide from a nucleic acid may be isolated from a natural source by cDNA cloning or molecule of known sequence. subtractive hybridization or synthesized manually. The 0028. A “single nucleotide polymorphism (SNP) refers nucleic acid molecule may be synthesized manually by the to a change in which a single base in the DNA differs from triester synthetic method or by using an automated DNA the usual base at that position. These single base changes are synthesizer. called SNPs or "snips.” Millions of SNP's have been cata 0035. With regard to nucleic acids used in the invention, loged in the human genome. Some SNPs such as that which the term "isolated nucleic acid' is sometimes employed. causes sickle cell are responsible for disease. Other SNPs are This term, when applied to DNA, refers to a DNA molecule normal variations in the genome. that is separated from sequences with which it is immedi 0029. A “copy number variation (CNV)" refers to the ately contiguous (in the 5' and 3' directions) in the naturally number of copies of a particular gene or segment thereof in occurring genome of the organism from which it was the genome of an individual. CNVs represent a major derived. For example, the "isolated nucleic acid may com genetic component of human phenotypic diversity. Suscep prise a DNA molecule inserted into a vector, such as a US 2017/000929.6 A1 Jan. 12, 2017 plasmid or virus vector, or integrated into the genomic DNA and cytosine, a "complement of this nucleic acid molecule of a prokaryote or eukaryote. An "isolated nucleic acid would be a molecule containing adenine in the place of molecule' may also comprise a cDNA molecule. An isolated thymine, thymine in the place of adenine, cytosine in the nucleic acid molecule inserted into a vector is also some place of guanine, and guanine in the place of cytosine. times referred to herein as a recombinant nucleic acid Because the complement can contain a nucleic acid molecule. sequence that forms optimal interactions with the parent 0036. With respect to RNA molecules, the term “isolated nucleic acid molecule. Such a complement can bind with nucleic acid primarily refers to an RNA molecule encoded high affinity to its parent molecule. by an isolated DNA molecule as defined above. Alterna 0040. With respect to single stranded nucleic acids, par tively, the term may refer to an RNA molecule that has been ticularly oligonucleotides, the term “specifically hybridiz sufficiently separated from RNA molecules with which it ing refers to the association between two single-stranded would be associated in its natural State (i.e., in cells or nucleotide molecules of Sufficiently complementary tissues), such that it exists in a “substantially pure' form. sequence to permit such hybridization under pre-determined 0037. By the use of the term “enriched” in reference to conditions generally used in the art (sometimes termed nucleic acid it is meant that the specific DNA or RNA “substantially complementary'). In particular, the term sequence constitutes a significantly higher fraction (2-5 fold) refers to hybridization of an oligonucleotide with a substan of the total DNA or RNA present in the cells or solution of tially complementary sequence contained within a single interest than in normal cells or in the cells from which the stranded DNA or RNA molecule of the invention, to the sequence was taken. This could be caused by a person by substantial exclusion of hybridization of the oligonucleotide preferential reduction in the amount of other DNA or RNA with single-stranded nucleic acids of non-complementary present, or by a preferential increase in the amount of the sequence. For example, specific hybridization can refer to a specific DNA or RNA sequence, or by a combination of the sequence which hybridizes to any ADHD specific marker two. However, it should be noted that "enriched' does not gene or nucleic acid, but does not hybridize to other nucleo imply that there are no other DNA or RNA sequences tides. Also polynucleotide which “specifically hybridizes' present, just that the relative amount of the sequence of may hybridize only to a neurospecific specific marker, Such interest has been significantly increased. as an ADHD-specific marker shown in the Tables contained 0038. It is also advantageous for some purposes that a herein. Appropriate conditions enabling specific hybridiza nucleotide sequence be in purified form. The term “purified tion of single stranded nucleic acid molecules of varying in reference to nucleic acid does not require absolute purity complementarity are well known in the art. (such as a homogeneous preparation); instead, it represents 0041. For instance, one common formula for calculating an indication that the sequence is relatively purer than in the the stringency conditions required to achieve hybridization natural environment (compared to the natural level, this between nucleic acid molecules of a specified sequence level should be at least 2-5 fold greater, e.g., in terms of homology is set forth below (Sambrook et al., Molecular mg/ml). Individual clones isolated from a cDNA library may Cloning, Cold Spring Harbor Laboratory (1989): be purified to electrophoretic homogeneity. The claimed T=81.5"C+16.6 Log Na++0.41 (% G+C)-0.63(% DNA molecules obtained from these clones can be obtained formamide)-600, #bp in duplex directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably 0042. As an illustration of the above formula, using obtained via manipulation of a partially purified naturally Na+=0.368 and 50% formamide, with GC content of occurring Substance (messenger RNA). The construction of 42% and an average probe size of 200 bases, the T is 57"C. a cDNA library from mRNA involves the creation of a The T of a DNA duplex decreases by 1-1.5"C with every synthetic substance (cDNA) and pure individual cDNA 1% decrease in homology. Thus, targets with greater than clones can be isolated from the synthetic library by clonal about 75% sequence identity would be observed using a selection of the cells carrying the cDNA library. Thus, the hybridization temperature of 42"C. process which includes the construction of a cDNA library 0043. The stringency of the hybridization and wash from mRNA and isolation of distinct cDNA clones yields an depend primarily on the salt concentration and temperature approximately 10-fold purification of the native message. of the solutions. In general, to maximize the rate of anneal Thus, purification of at least one order of magnitude, pref ing of the probe with its target, the hybridization is usually erably two or three orders, and more preferably four or five carried out at salt and temperature conditions that are 20-25° orders of magnitude is expressly contemplated. Thus the C. below the calculated T of the hybrid. Wash conditions term 'substantially pure' refers to a preparation comprising should be as stringent as possible for the degree of identity at least 50-60% by weight the compound of interest (e.g., of the probe for the target. In general, wash conditions are nucleic acid, oligonucleotide, etc.). More preferably, the selected to be approximately 12-20°C. below the T of the preparation comprises at least 75% by weight, and most hybrid. In regards to the nucleic acids of the current inven preferably 90-99% by weight, the compound of interest. tion, a moderate Stringency hybridization is defined as Purity is measured by methods appropriate for the com hybridization in 6xSSC, 5xDenhardt’s solution, 0.5% SDS pound of interest. and 100 g/ml denatured salmon sperm DNA at 42°C., and 0039. The term “complementary” describes two nucleo washed in 2XSSC and 0.5% SDS at 55° C. for 15 minutes. tides that can form multiple favorable interactions with one A high Stringency hybridization is defined as hybridization another. For example, adenine is complementary to thymine in 6xSSC, 5xDenhardt’s solution, 0.5% SDS and 100 g/ml as they can form two hydrogen bonds. Similarly, guanine denatured salmon sperm DNA at 42°C., and washed in and cytosine are complementary since they can form three 1xSSC and 0.5% SDS at 65° C. for 15 minutes. A very high hydrogen bonds. Thus if a nucleic acid sequence contains stringency hybridization is defined as hybridization in the following sequence of bases, thymine, adenine, guanine 6xSSC, 5xDenhardt’s solution, 0.5% SDS and 100 g/ml US 2017/000929.6 A1 Jan. 12, 2017

denatured salmon sperm DNA at 42°C., and washed in the initiation of synthesis by a polymerase or similar 0.1XSSC and 0.5% SDS at 65° C. for 15 minutes. enzyme. It is not required that the primer sequence represent 0044) The term "oligonucleotide,” as used herein is an exact complement of the desired template. For example, defined as a nucleic acid molecule comprised of two or more a non-complementary nucleotide sequence may be attached ribo- or deoxyribonucleotides, preferably more than three. to the 5' end of an otherwise complementary primer. Alter The exact size of the oligonucleotide will depend on various natively, non-complementary bases may be interspersed factors and on the particular application and use of the within the oligonucleotide primer sequence, provided that oligonucleotide. Oligonucleotides, which include probes the primer sequence has sufficient complementarity with the and primers, can be any length from 3 nucleotides to the full sequence of the desired template strand to functionally length of the nucleic acid molecule, and explicitly include provide a template-primer complex for the synthesis of the every possible number of contiguous nucleic acids from 3 extension product. through the full length of the polynucleotide. Preferably, 0047 Polymerase chain reaction (PCR) has been oligonucleotides are at least about 10 nucleotides in length, described in U.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965, more preferably at least 15 nucleotides in length, more 188, the entire disclosures of which are incorporated by preferably at least about 20 nucleotides in length. reference herein. 0045. The term “probe' as used herein refers to an 0048. The term “vector relates to a single or double oligonucleotide, polynucleotide or nucleic acid, either RNA Stranded circular nucleic acid molecule that can be infected, or DNA, whether occurring naturally as in a purified restric transfected or transformed into cells and replicate indepen tion enzyme digest or produced synthetically, which is dently or within the host cell genome. A circular double capable of annealing with or specifically hybridizing to a Stranded nucleic acid molecule can be cut and thereby nucleic acid with sequences complementary to the probe. A linearized upon treatment with restriction enzymes. An probe may be either single-stranded or double-stranded. The assortment of vectors, restriction enzymes, and the knowl exact length of the probe will depend upon many factors, edge of the nucleotide sequences that are targeted by restric including temperature, Source of probe and use of the tion enzymes are readily available to those skilled in the art, method. For example, for diagnostic applications, depending and include any replicon, such as a plasmid, cosmid, bac on the complexity of the target sequence, the oligonucleotide mid, phage or virus, to which another genetic sequence or probe typically contains 15-25 or more nucleotides, element (either DNA or RNA) may be attached so as to bring although it may contain fewer nucleotides. The probes about the replication of the attached sequence or element. A herein are selected to be complementary to different strands nucleic acid molecule of the invention can be inserted into of a particular target nucleic acid sequence. This means that a vector by cutting the vector with restriction enzymes and the probes must be sufficiently complementary so as to be ligating the two pieces together. able to “specifically hybridize' or anneal with their respec tive target strands under a set of pre-determined conditions. 0049 Many techniques are available to those skilled in Therefore, the probe sequence need not reflect the exact the art to facilitate transformation, transfection, or transduc complementary sequence of the target. For example, a tion of the expression construct into a prokaryotic or eukary non-complementary nucleotide fragment may be attached to otic organism. The terms “transformation”, “transfection'. the 5' or 3' end of the probe, with the remainder of the probe and “transduction” refer to methods of inserting a nucleic sequence being complementary to the target Strand. Alter acid and/or expression construct into a cell or host organism. natively, non-complementary bases or longer sequences can These methods involve a variety of techniques, such as be interspersed into the probe, provided that the probe treating the cells with high concentrations of salt, an electric sequence has sufficient complementarity with the sequence field, or detergent, to render the host cell outer membrane or of the target nucleic acid to anneal therewith specifically. wall permeable to nucleic acid molecules of interest, micro 0046. The term “primer' as used herein refers to an injection, PEG-fusion, and the like. oligonucleotide, either RNA or DNA, either single-stranded 0050. The term “promoter element” describes a nucleo or double-stranded, either derived from a biological system, tide sequence that is incorporated into a vector that, once generated by restriction enzyme digestion, or produced inside an appropriate cell, can facilitate transcription factor synthetically which, when placed in the proper environment, and/or polymerase binding and Subsequent transcription of is able to functionally act as an initiator of template portions of the vector DNA into mRNA. In one embodiment, dependent nucleic acid synthesis. When presented with an the promoter element of the present invention precedes the appropriate nucleic acid template, Suitable nucleoside 5' end of the ADHD specific marker nucleic acid molecule triphosphate precursors of nucleic acids, a polymerase such that the latter is transcribed into mRNA. Host cell enzyme, Suitable cofactors and conditions such as a Suitable machinery then translates mRNA into a polypeptide. temperature and pH, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a 0051. Those skilled in the art will recognize that a nucleic polymerase or similar activity to yield a primer extension acid vector can contain nucleic acid elements other than the product. The primer may vary in length depending on the promoter element and the ADHD specific marker nucleic particular conditions and requirement of the application. For acid molecule. These other nucleic acid elements include, example, in diagnostic applications, the oligonucleotide but are not limited to, origins of replication, ribosomal primer is typically 15-25 or more nucleotides in length. The binding sites, nucleic acid sequences encoding drug resis primer must be of sufficient complementarity to the desired tance enzymes or amino acid metabolic enzymes, and template to prime the synthesis of the desired extension nucleic acid sequences encoding secretion signals, localiza product, that is, to be able anneal with the desired template tion signals, or signals useful for polypeptide purification. strand in a manner sufficient to provide the 3' hydroxyl 0.052 A “replicon' is any genetic element, for example, moiety of the primer in appropriate juxtaposition for use in a plasmid, cosmid, bacmid, plastid, phage or virus, that is US 2017/000929.6 A1 Jan. 12, 2017

capable of replication largely under its own control. A nucleic acid molecule of the invention. Alternatively, this replicon may be either RNA or DNA and may be single or term may refer to a protein that has been sufficiently double stranded. separated from other proteins with which it would naturally 0053 An "expression operon” refers to a nucleic acid be associated, so as to exist in “substantially pure' form. segment that may possess transcriptional and translational “Isolated' is not meant to exclude artificial or synthetic control sequences, such as promoters, enhancers, transla mixtures with other compounds or materials, or the presence tional start signals (e.g., ATG or AUG codons), polyade of impurities that do not interfere with the fundamental nylation signals, terminators, and the like, and which facili activity, and that may be present, for example, due to tate the expression of a polypeptide coding sequence in a incomplete purification, addition of stabilizers, or com host cell or organism. pounding into, for example, immunogenic preparations or 0054 As used herein, the terms “reporter,” “reporter pharmaceutically acceptable preparations. system”, “reporter gene,” or “reporter gene product' shall 0060 A “specific binding pair comprises a specific mean an operative genetic system in which a nucleic acid binding member (sbm) and a binding partner (bp) which comprises a gene that encodes a product that when have a particular specificity for each other and which in expressed produces a reporter signal that is a readily mea normal conditions bind to each other in preference to other Surable, e.g., by biological assay, immunoassay, radio immu molecules. Examples of specific binding pairs are antigens noassay, or by colorimetric, fluorogenic, chemiluminescent and antibodies, ligands and receptors and complementary or other methods. The nucleic acid may be either RNA or nucleotide sequences. The skilled person is aware of many DNA, linear or circular, single or double Stranded, antisense other examples. Further, the term “specific binding pair is or sense polarity, and is operatively linked to the necessary also applicable where either or both of the specific binding control elements for the expression of the reporter gene member and the binding partner comprise a part of a large product. The required control elements will vary according molecule. In embodiments in which the specific binding pair to the nature of the reporter system and whether the reporter comprises nucleic acid sequences, they will be of a length to gene is in the form of DNA or RNA, but may include, but hybridize to each other under conditions of the assay, not be limited to. Such elements as promoters, enhancers, preferably greater than 10 nucleotides long, more preferably translational control sequences, poly A addition signals, greater than 15 or 20 nucleotides long. transcriptional termination signals and the like. 0061 “Sample' or “patient sample' or “biological 0055. The introduced nucleic acid may or may not be sample' generally refers to a sample which may be tested for integrated (covalently linked) into nucleic acid of the recipi a particular molecule, preferably an ADHD specific marker ent cell or organism. In bacterial, yeast, plant and mamma molecule, such as a marker described hereinbelow. Samples lian cells, for example, the introduced nucleic acid may be may include but are not limited to cells, body fluids, includ maintained as an episomal element or independent replicon ing blood, serum, plasma, cerebral spinal fluid, urine, saliva, Such as a plasmid. Alternatively, the introduced nucleic acid tears, pleural fluid and the like. may become integrated into the nucleic acid of the recipient 0062. The terms "agent” and “compound are used inter cell or organism and be stably maintained in that cell or changeably herein and denote a chemical compound, a organism and further passed on or inherited to progeny cells mixture of chemical compounds, a biological macromol or organisms of the recipient cell or organism. Finally, the ecule, or an extract made from biological materials such as introduced nucleic acid may exist in the recipient cell or host bacteria, plants, fungi, or animal (particularly mammalian) organism only transiently. cells or tissues. Biological macromolecules include siRNA, 0056. The term “selectable marker gene' refers to a gene shRNA, antisense oligonucleotides, peptides, peptide/DNA that when expressed confers a selectable phenotype. Such as complexes, and any nucleic acid based molecule which antibiotic resistance, on a transformed cell. exhibits the capacity to modulate the activity of the CNV or 0057 The term “operably linked' means that the regu SNP-containing nucleic acids described herein or their latory sequences necessary for expression of the coding encoded proteins. Agents and compounds may also be sequence are placed in the DNA molecule in the appropriate referred to as “test agents’ or “test compounds” which are positions relative to the coding sequence so as to effect evaluated for potential biological activity by inclusion in expression of the coding sequence. This same definition is screening assays described herein below. Sometimes applied to the arrangement of transcription units 0063. The term “modulate” as used herein refers to and other transcription control elements (e.g. enhancers) in increasing/promoting or decreasing/inhibiting a particular an expression vector. cellular, biological or signaling function associated with the 0058. The terms “recombinant organism”, or “transgenic normal activities of the CNV containing molecules organism' refer to organisms which have a new combination described herein or the proteins encoded thereby. For of genes or nucleic acid molecules. A new combination of example, the term modulate refers to the ability of a test genes or nucleic acid molecules can be introduced into an compound or test agent to interfere with signaling or activity organism using a wide array of nucleic acid manipulation of a gene or protein of the present invention. techniques available to those skilled in the art. The term "organism' relates to any living being comprised of a least II. METHODS OF USING ADHD-ASSOCIATED one cell. An organism can be as simple as one eukaryotic cell CNVS AND/OR SNPS FOR DIAGNOSING A or as complex as a mammal. Therefore, the phrase “a PROPENSITY FOR THE DEVELOPMENT OF recombinant organism' encompasses a recombinant cell, as ADHD well as eukaryotic and prokaryotic organism. 0064. The present invention provides methods of diag 0059. The term “isolated protein' or “isolated and puri nosing ADHD in a patient or methods for identifying a fied protein’ is sometimes used herein. This term refers patient having an increased risk of developing ADHD. primarily to a protein produced by expression of an isolated Diagnosis, as used herein, includes not only the initial US 2017/000929.6 A1 Jan. 12, 2017

identification of ADHD associated with the genetic altera 0070 Alternatively, new detection technologies can over tions described herein in a patient but confirmatory testing, come this limitation and enable analysis of Small samples or screening in patients who have previously been identified containing as little as 1 lug of total RNA. Using Resonance as having or likely to have ADHD. The methods include the Light Scattering (RLS) technology, as opposed to traditional steps of providing a biological sample from the patient, fluorescence techniques, multiple reads can detect low quan measuring the amount of particular sets, or any all of the tities of mRNAs using biotin labeled hybridized targets and ADHD associated markers (Table 13) present in the bio anti-biotin antibodies. Another alternative to PCR amplifi logical sample, preferably a tissue and/or blood plasma cation involves planar wave guide technology (PWG) to sample, and determining if the patient has a greater likeli increase signal-to-noise ratios and reduce background inter hood of ADHD based on the amount and/or type of ADHD marker expression level determined relative to those expres ference. Both techniques are commercially available from sion levels identified in patient cohorts of known outcome. Qiagen Inc. (USA). A patient has a greater likelihood of having ADHD when the 0071 Any of the aforementioned techniques may be used sample has a CNV marker expression profile associated with to detect or quantify ADHD-associated CNV or SNP marker patients previously diagnosed with ADHD. The composi expression and accordingly, diagnose ADHD. tions and methods of the invention are useful for the prognosis and diagnosis and management of ADHD III. KITS AND ARTICLES OF MANUFACTURE 0065. In another aspect, the patient sample may have been previously genotyped and thus the genetic expression 0072 Any of the aforementioned products can be incor profile in the sample may be available to the clinician. Accordingly, the method may entail storing reference porated into a kit which may contain a ADHD-associated ADHD associated marker sequence information in a data CNV or SNP specific marker polynucleotide or one or more base, i.e., those CNVs statistically associated with a more Such markers immobilized on a Gene Chip, an oligonucle favorable or less favorable prognosis as described in the otide, a polypeptide, a peptide, an antibody, a label, marker, tables herein, and performance of comparative genetic reporter, a pharmaceutically acceptable carrier, a physiologi analysis on the computer, thereby identifying those patients cally acceptable carrier, instructions for use, a container, a having increased risk ADHD. vessel for administration, an assay Substrate, or any combi 0066 ADHD-related CNV or SNP-containing nucleic nation thereof. acids, including but not limited to those listed below may be used for a variety of purposes in accordance with the present IV. METHODS OF USING ADHD-ASSOCIATED invention. ADHD-associated CNV or SNP-containing CNVS AND/OR SNPS FOR THE DNA, RNA, or fragments thereof may be used as probes to DEVELOPMENT OF THERAPEUTICAGENTS detect the presence of and/or expression of ADHD specific markers. Methods in which ADHD specific marker nucleic 0073. Since the CNVs and SNPs identified herein have acids may be utilized as probes for Such assays include, but been associated with the etiology of ADHD, methods for are not limited to: (1) in situ hybridization; (2) Southern identifying agents that modulate the activity of the genes and hybridization (3) northern hybridization; and (4) assorted their encoded products containing such CNVs and/or SNPs amplification reactions such as polymerase chain reactions should result in the generation of efficacious therapeutic (PCR). agents for the treatment of this disorder. 0067 Further, assays for detecting ADHD-associated 0074. Several regions of the human genome provide CNVs or SNPs may be conducted on any type of biological Suitable targets for the rational design of therapeutic agents. sample, including but not limited to body fluids (including Small nucleic acid molecules or peptide molecules corre blood, urine, serum, gastric lavage, cerebral spinal fluid), sponding to these regions may be used to advantage in the any type of cell (such as brain cells, white blood cells, design of therapeutic agents that effectively modulate the mononuclear cells, fetal cells in maternal circulation) or activity of the encoded proteins. body tissue. 0075 Molecular modeling should facilitate the identifi 0068 Clearly, ADHD-associated CNV or SNP-contain cation of specific organic molecules with capacity to bind to ing nucleic acids, vectors expressing the same, ADHD CNV the active site of the proteins encoded by the CNV or or SNP-containing marker proteins and anti-ADHD specific SNP-containing nucleic acids based on conformation or key marker antibodies of the invention can be used to detect amino acid residues required for function. A combinatorial ADHD associated CNVs or SNPs in body tissue, cells, or chemistry approach will be used to identify molecules with fluid, and alter ADHD CNV or SNP-containing marker greatest activity and then iterations of these molecules will protein expression for purposes of assessing the genetic and be developed for further cycles of screening. Several of the protein interactions involved in the development of ADHD. molecules available in this screening assay, while not lim 0069. In most embodiments for screening for ADHD iting the method, include metabotropic glutamate receptor associated CNVs or SNPs, the ADHD-associated CNV or (mGluR) positive allosteric modulators (PAM), negative SNP-containing nucleic acid in the sample will initially be allosteric modulators (NAM), and tachykinin-3/neuroki amplified, e.g. using PCR, to increase the amount of the nin-3 receptor (TACR-3/NK3R) antagonists. A specific list templates as compared to other sequences present in the includes ADX63365, ADX50938, ADX71149, ADX48621, sample. This allows the target sequences to be detected with AMN082, 1-(hetero)aryl-3-amino-pyrrolidine derivatives a high degree of sensitivity if they are present in the sample. (e.g. those provided in U.S. Patent Application Publication This initial step may be avoided by using highly sensitive No. 2008/0300266), LY341495, GSK1144814, and array techniques that are important in the art. SB223412 (Table 1). US 2017/000929.6 A1 Jan. 12, 2017

TABLE 1. Molecules and therapeutic agents available for a combinatorial chemistry approach. Product Name Company Name Indication Mechanism of Action ADX6336S Addex Schizophrenia Glutamate Receptor, Metabotropic 5 Pharmaceuticals (GRM5) Positive Allosteric Modulator ADX6336S Merck & Co Inc Schizophrenia Glutamate Receptor, Metabotropic 5 (GRM5) Positive Allosteric Modulator ADXSO938 Addex Schizophrenia Glutamate Receptor, Metabotropic 5 Pharmaceuticals (GRM5) Positive Allosteric Modulator ADXSO938 Addex Alzheimer Disease Glutamate Receptor, Metabotropic 5 Pharmaceuticals (GRM5) Positive Allosteric Modulator ADX71149 Addex Schizophrenia Glutamate Receptor, Metabotropic 2 Pharmaceuticals (GRM2) Positive Allosteric Modulator AMNO82 Schizophrenia, Glutamate Receptor, Metabotropic 7 Depression, Alzheimer (GRM7) Positive Allosteric Modulator Disease 1-(hetero)aryl-3- Eli Lilly & Co Migraine Glutamate Receptor, Metabotropic 3 amino-pyrrollidine (GRM3) Antagonist derivatives LY341495 Eli Lilly & Co Central Nervous System Glutamate Receptor, Metabotropic 2 Disorders (GRM2) Antagonist; Glutamate Receptor, Metabotropic 3 (GRM3) Antagonist ADX48621 Addex Parkinson's Disease Glutamate Receptor, Metabotropic 5 Pharmaceuticals (GRM5) Negative Allosteric Modulator GSK1144814 Glaxo Smith Kline Schizophrenia Antagonist for Neurokinin-3 receptors SB223412 (Talnetant) Glaxo Smith Kline Schizophrenia Antagonist for Neurokinin-3 receptors

0076. The polypeptides or fragments employed in drug DNA molecules may be introduced singly into such host screening assays may either be free in solution, affixed to a cells or in combination to assess the phenotype of cells Solid Support or within a cell. One method of drug screening conferred by such expression. Methods for introducing utilizes eukaryotic or prokaryotic host cells which are stably DNA molecules are also well known to those of ordinary transformed with recombinant polynucleotides expressing skill in the art. Such methods are set forth in Ausubel et al. the polypeptide or fragment, preferably in competitive bind eds. Current Protocols in Molecular Biology, John Wiley & ing assays. Such cells, either in viable or fixed form, can be Sons, NY, N.Y. 1995, the disclosure of which is incorporated used for standard binding assays. One may determine, for by reference herein. example, formation of complexes between the polypeptide 0079 A wide variety of expression vectors are available or fragment and the agent being tested, or examine the that can be modified to express the novel DNA sequences of degree to which the formation of a complex between the this invention. The specific vectors exemplified herein are polypeptide or fragment and a known Substrate is interfered merely illustrative, and are not intended to limit the scope of with by the agent being tested. the invention. Expression methods are described by Sam 0077. Another technique for drug screening provides brook et al. Molecular Cloning: A Laboratory Manual or high throughput Screening for compounds having Suitable Current Protocols in Molecular Biology 16.3-17.44 (1989). binding affinity for the encoded polypeptides and is Expression methods in Saccharomyces are also described in described in detail in Geysen, PCT published application Current Protocols in Molecular Biology (1989). WO 84/03564, published on Sep. 13, 1984. Briefly stated, 0080 Suitable vectors for use in practicing the invention large numbers of different, Small peptide test compounds, include prokaryotic vectors such as the pNH vectors (Strata Such as those described above, are synthesized on a solid gene Inc., 11099 N. Torrey Pines Rd., La Jolla, Calif. Substrate, such as plastic pins or Some other Surface. The 92037), pET vectors (Novogen Inc., 565 Science Dr. Madi peptide test compounds are reacted with the target polypep son, Wis. 53711) and the pGEX vectors (Pharmacia LKB tide and washed. Bound polypeptide is then detected by Biotechnology Inc., Piscataway, N.J. 08854). Examples of methods well known in the art. eukaryotic vectors useful in practicing the present invention 0078 A further technique for drug screening involves the include the vectors pRc/CMV, pRc/RSV, and pREP (Invit use of host eukaryotic cell lines or cells (such as described rogen, 11588 Sorrento Valley Rd., San Diego, Calif. 92121); above) which have a nonfunctional or altered ADHD asso pcDNA3.1/V5&His (Invitrogen); baculovirus vectors such ciated gene. These host cell lines or cells are defective at the as pVL 1392, pVL 1393, or p AC360 (Invitrogen); and yeast polypeptide level. The host cell lines or cells are grown in vectors such as YRP17, YIPS, and YEP24 (New England the presence of drug compound. Altered glutaminergic func Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 tion of the host cells is measured to determine if the Stratagene Inc.); Picchia vectors such as pHIL-D1 (Phillips compound is capable of regulating this function in the Petroleum Co., Bartlesville, Okla. 74004); retroviral vectors defective cells. Host cells contemplated for use in the such as PLNCX and pLPCX (Clontech); and adenoviral and present invention include but are not limited to bacterial adeno-associated viral vectors. cells, fungal cells, insect cells, mammalian cells, and plant I0081 Promoters for use in expression vectors of this cells. However, mammalian cells, particularly neuronal cells invention include promoters that are operable in prokaryotic are preferred. The ADHD-associated CNV or SNP encoding or eukaryotic cells. Promoters that are operable in prokary US 2017/000929.6 A1 Jan. 12, 2017

otic cells include lactose (lac) control elements, bacterio acid residues of the peptide is analyzed in this manner to phage lambda (pL) control elements, arabinose control ele determine the important regions of the peptide. ments, tryptophan (trp) control elements, bacteriophage T7 I0086. It is also possible to isolate a target-specific anti control elements, and hybrids thereof. Promoters that are body, selected by a functional assay, and then to solve its operable in eukaryotic cells include Epstein Barr virus crystal structure. In principle, this approach yields a phar promoters, adenovirus promoters, SV40 promoters, Rous macore upon which Subsequent drug design can be based. Sarcoma Virus promoters, cytomegalovirus (CMV) promot I0087. One can bypass protein crystallography altogether ers, baculovirus promoters such as AcMNPV polyhedrin by generating anti-idiotypic antibodies (anti-ids) to a func promoter, Picchia promoters such as the alcohol oxidase tional, pharmacologically active antibody. As a mirror image promoter, and Saccharomyces promoters such as the gal4 of a mirror image, the binding site of the anti-ids would be inducible promoter and the PGK constitutive promoter, as expected to be an analog of the original molecule. The well as neuronal-specific platelet-derived growth factor pro anti-id could then be used to identify and isolate peptides moter (PDGF), the Thy-1 promoter, the hamster and mouse from banks of chemically or biologically produced banks of Prion promoter (MoPrP), and the Glial fibrillar acidic pro peptides. Selected peptides would then act as the pharma tein (GFAP) for the expression of transgenes in glial cells. COC. 0082 In addition, a vector of this invention may contain 0088. Thus, one may design drugs which have, e.g., any one of a number of various markers facilitating the improved polypeptide activity or stability or which act as selection of a transformed host cell. Such markers include inhibitors, agonists, antagonists, etc. of polypeptide activity. genes associated with temperature sensitivity, drug resis By virtue of the availability of CNV or SNP-containing tance, or enzymes associated with phenotypic characteristics nucleic acid sequences described herein, Sufficient amounts of the host organisms. of the encoded polypeptide may be made available to 0083) Host cells expressing the ADHD-associated CNVs perform Such analytical studies as X-ray crystallography. In and/or SNPs of the present invention or functional fragments addition, the knowledge of the protein sequence provided thereof provide a system in which to screen potential com herein will guide those employing computer modeling tech pounds or agents for the ability to modulate the development niques in place of, or in addition to X-ray crystallography. of ADHD. Thus, in one embodiment, the nucleic acid I0089. In another embodiment, the availability of ADHD molecules of the invention may be used to create recombi associated CNV or SNP-containing nucleic acids enables the nant cell lines for use in assays to identify agents which production of strains of laboratory mice carrying the ADHD modulate aspects of cellular metabolism associated with associated SNPs or CNVs of the invention. Transgenic mice ADHD and aberrant glutaminergic function. Also provided expressing the ADHD-associated CNV or SNP of the inven herein are methods to screen for compounds capable of tion provide a model system in which to examine the role of modulating the function of proteins encoded by CNV and the protein encoded by the CNV or SNP-containing nucleic SNP-containing nucleic acids. acid in the development and progression towards ADHD. Methods of introducing transgenes in laboratory mice are 0084 Another approach entails the use of phage display known to those of skill in the art. Three common methods libraries engineered to express fragment of the polypeptides include: 1. integration of retroviral vectors encoding the encoded by the CNV or SNP-containing nucleic acids on the foreign gene of interest into an early embryo. 2. injection of phage surface. Such libraries are then contacted with a DNA into the pronucleus of a newly fertilized egg; and 3. the combinatorial chemical library under conditions wherein incorporation of genetically manipulated embryonic stem binding affinity between the expressed peptide and the cells into an early embryo. Production of the transgenic mice components of the chemical library may be detected. U.S. described above will facilitate the molecular elucidation of Pat. Nos. 6,057,098 and 5,965,456 provide methods and the role that a target protein plays in various cellular apparatus for performing Such assays. metabolic processes, including: aberrant glutaminergic func 0085. The goal of rational drug design is to produce tion, altered neuroactive ligand receptor signaling and aber structural analogs of biologically active polypeptides of rant neurotransmission, or altered neuronal morphology and interest or of Small molecules with which they interact (e.g., neurite outgrowth. Such mice provide an in Vivo Screening agonists, antagonists, inhibitors) in order to fashion drugs tool to study putative therapeutic drugs in a whole animal which are, for example, more active or stable forms of the model and are encompassed by the present invention. polypeptide, or which, e.g., enhance or interfere with the 0090. The term “animal' is used herein to include all function of a polypeptide in vivo. See, e.g., Hodgson, (1991) vertebrate animals, except humans. It also includes an Bio/Technology 9:19-21. In one approach, discussed above, individual animal in all stages of development, including the three-dimensional structure of a protein of interest or, for embryonic and fetal stages. A “transgenic animal' is any example, of the protein-Substrate complex, is solved by animal containing one or more cells bearing genetic infor X-ray crystallography, by nuclear magnetic resonance, by mation altered or received, directly or indirectly, by delib computer modeling or most typically, by a combination of erate genetic manipulation at the Subcellular level. Such as approaches. Less often, useful information regarding the by targeted recombination or microinjection or infection structure of a polypeptide may begained by modeling based with recombinant virus. The term “transgenic animal' is not on the structure of homologous proteins. An example of meant to encompass classical cross-breeding or in vitro rational drug design is the development of HIV protease fertilization, but rather is meant to encompass animals in inhibitors (Erickson et al., (1990) Science 249:527-533). In which one or more cells are altered by or receive a recom addition, peptides may be analyzed by an alanine Scan binant DNA molecule. This molecule may be specifically (Wells, (1991) Meth. Enzym. 202:390-411). In this tech targeted to a defined genetic locus, be randomly integrated nique, an amino acid residue is replaced by Ala, and its effect within a chromosome, or it may be extrachromosomally on the peptide's activity is determined. Each of the amino replicating DNA. The term 'germ cell line transgenic ani US 2017/000929.6 A1 Jan. 12, 2017 mal” refers to a transgenic animal in which the genetic approaches developed for selecting homologous recombi alteration or genetic information was introduced into a germ nants is the positive-negative selection (PNS) method devel line cell, thereby conferring the ability to transfer the genetic oped for genes for which no direct selection of the alteration information to offspring. If Such offspring, in fact, possess exists. The PNS method is more efficient for targeting genes Some or all of that alteration or genetic information, then which are not expressed at high levels because the marker they, too, are transgenic animals. gene has its own promoter. Non-homologous recombinants 0091. The alteration of genetic information may be for are selected against by using the Herpes Simplex virus eign to the species of animal to which the recipient belongs, thymidine kinase (HSV-TK) gene and selecting against its or foreign only to the particular individual recipient, or may nonhomologous insertion with effective herpes drugs such be genetic information already possessed by the recipient. In as gancyclovir (GANC) or (1-(2-deoxy-2-fluoro-B-D arab the last case, the altered or introduced gene may be inofluranosyl)-5-iodou-racil, (FIAU). By this counter selec expressed differently than the native gene. Such altered or tion, the number of homologous recombinants in the Sur foreign genetic information would encompass the introduc viving transformants can be increased. Utilizing ADHD tion of ADHD-associated CNV or SNP-containing nucleo associated CNV or SNP-containing nucleic acid as a tide sequences. targeted insertional cassette provides means to detect a 0092. The DNA used for altering a target gene may be Successful insertion as visualized, for example, by acquisi obtained by a wide variety of techniques that include, but are tion of immunoreactivity to an antibody immunologically not limited to, isolation from genomic sources, preparation specific for the polypeptide encoded by ADHD-associated of cDNAs from isolated mRNA templates, direct synthesis, CNV or SNP nucleic acid and, therefore, facilitates screen or a combination thereof. ing/selection of ES cells with the desired genotype. 0093. A preferred type of target cell for transgene intro duction is the embryonal stem cell (ES). ES cells may be 0097. As used herein, a knock-in animal is one in which obtained from pre-implantation embryos cultured in vitro the endogenous murine gene, for example, has been replaced (Evans et al., (1981) Nature 292:154-156: Bradley et al., with human ADHD-associated CNV or informative frag (1984) Nature 309:255-258; Gossler et al., (1986) Proc. ment thereof or SNP-containing gene of the invention. Such Natl. Acad. Sci. 83:9065-9069). Transgenes can be effi knock-in animals provide an ideal model system for study ciently introduced into the ES cells by standard techniques ing the development of ADHD. such as DNA transfection or by retrovirus-mediated trans 0098. As used herein, the expression of a ADHD-asso duction. The resultant transformed ES cells can thereafter be ciated CNV or SNP-containing nucleic acid, partial infor combined with blastocysts from a non-human animal. The mative CNV fragment thereof, or an ADHD-associated introduced ES cells thereafter colonize the embryo and fusion protein in which the CNV or SNP is encoded can be contribute to the germ line of the resulting chimeric animal. targeted in a “tissue specific manner” or “cell type specific 0094. One approach to the problem of determining the manner” using a vector in which nucleic acid sequences contributions of individual genes and their expression prod encoding all or a portion of an ADHD-associated CNV or ucts is to use isolated ADHD-associated CNV or SNP genes SNP are operably linked to regulatory sequences (e.g., as insertional cassettes to selectively inactivate a wild-type promoters and/or enhancers) that direct expression of the gene in totipotent ES cells (such as those described above) encoded protein in a particular tissue or cell type. Such and then generate transgenic mice. The use of gene-targeted regulatory elements may be used to advantage for both in ES cells in the generation of gene-targeted transgenic mice vitro and in Vivo applications. Promoters for directing tissue was described, and is reviewed elsewhere (Frohman et al., specific proteins are well known in the art and described (1989) Cell 56:145-147: Bradley et al., (1992) Bio/Technol herein. ogy 10:534-539). 0095 Techniques are available to inactivate or alter any 0099. The nucleic acid sequence encoding the ADHD genetic region to a mutation desired by using targeted associated CNV or SNP of the invention may be operably homologous recombination to insert specific changes into linked to a variety of different promoter sequences for chromosomal alleles. However, in comparison with homolo expression in transgenic animals. Such promoters include, gous extrachromosomal recombination, which occurs at a but are not limited to a prion gene promoter Such as hamster frequency approaching 100%, homologous plasmid-chro and mouse Prion promoter (MoPrP), described in U.S. Pat. mosome recombination was originally reported to only be No. 5,877,399 and in Borchelt et al., Genet. Anal. 13(6) detected at frequencies between 10° and 10. Nonhomolo (1996) pages 159-163; a rat neuronal specific enolase pro gous plasmid-chromosome interactions are more frequent moter, described in U.S. Pat. Nos. 5,612,486, and 5,387,742: occurring at levels 10-fold to 10 fold greater than compa a platelet-derived growth factor B gene promoter, described rable homologous insertion. in U.S. Pat. No. 5,811,633; a brain specific dystrophin 0096. To overcome this low proportion of targeted promoter, described in U.S. Pat. No. 5,849,999; a Thy-1 recombination in murine ES cells, various strategies have promoter; a PGK promoter; a CMV promoter; a neuronal been developed to detect or select rare homologous recom specific platelet-derived growth factor B gene promoter, a binants. One approach for detecting homologous alteration NEGR1 promoter, a GRM5 promoter, a promotor of any events uses the polymerase chain reaction (PCR) to screen gene listed in the tables below, and Glial fibrillar acidic pools of transformant cells for homologous insertion, fol protein (GFAP) promoter for the expression of transgenes in lowed by screening of individual clones. Alternatively, a glial cells. positive genetic selection approach has been developed in 0100 Methods of use for the transgenic mice of the which a marker gene is constructed which will only be active invention are also provided herein. Transgenic mice into if homologous insertion occurs, allowing these recombi which a nucleic acid containing the ADHD-associated CNV nants to be selected directly. One of the most powerful or SNP or its encoded protein have been introduced are US 2017/000929.6 A1 Jan. 12, 2017

useful, for example, to develop screening methods to Screen TABLE 2-continued therapeutic agents to identify those capable of modulating the development of ADHD. Perlegen Data Reformatted File Samples to match Affymetrix Power Tools output format. V. PHARMACEUTICAL AND PEPTIDE THERAPIES probeset id 10009 10010 10O21 10O22 0101. The elucidation of the role played by the ADHD B) Genotype Calls Confidence Scores (All set to 1). associated CNVs and SNPs described herein in neuroactive ligand receptor signaling facilitates the development of SNP rs1OOOOO23 1 1 1 1 pharmaceutical compositions useful for treatment and diag SNP rs10000030 1 1 1 1 SNP rs1OOOOO37 1 1 1 1 nosis of ADHD. These compositions may comprise, in SNP rs1OOOOO68 1 1 1 1 addition to one of the above Substances, a pharmaceutically C) Intensity Summary (-A = log10(X), -B = log10(Y) (X and Y acceptable excipient, carrier, buffer, stabilizer or other mate value from dbGaP Single Sample Final Report files). rials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the SNP rs10000023-A 2.85 2.78 2.07 2.89 efficacy of the active ingredient. The precise nature of the SNP rs1OOOOO23-B 2.86 2.84 2.98 2.96 carrier or other material may depend on the route of admin SNP rs10000030-A 2.9 2.99 2.95 3.02 istration, e.g. oral, intravenous, cutaneous or subcutaneous, SNP rs10000030-B 2.91 2.4 2.38 3.05 nasal, intramuscular, intraperitoneal routes. 0102. Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, Small molecule or other pharmaceu tically useful compound according to the present invention CNV Calls and Review of Significant Loci that is to be given to an individual, administration is pref erably in a “prophylactically effective amount’ or a “thera 0105. No additional “CNV burden' was observed in peutically effective amount' (as the case may be, although cases vs. controls, rather the distribution of calls made was prophylaxis may be considered therapy), this being Sufi highly comparable (FIG. 1). We established CNV call reli cient to show benefit to the individual. ability in Illumina and Perlegen data by observing Mende 0103. The materials and methods set forth below are lian patterns of inheritance. Trios were first verified by provided to facilitate the practice of the following examples. genotype inheritance and analyzed to establish the quality of CNV calls from both Illumina and Perlegen platforms based Illumina Infinium Assay for CNV Discovery on observed inheritance. Based on all CNV calls called in 0104. We performed high-throughput, genome-wide SNP trios from the Illumina CHOP data, 8,647 CNVs observed in genotyping, using the Infinium II Human Hap550 BeadChip offspring were inherited from a parent while 437 CNVs were technology (Illumina San Diego Calif.), at the Center for putatively de novo which is a de novo rate of 4.811%. Based Applied Genomics at CHOP. The genotype data content on all CNV calls called in trios from the Perlegen IMAGE together with the intensity data provided by the genotyping data, 1,862 CNVs observed in offspring were inherited from array provides high confidence for CNV calls. Importantly, a parent while 505 CNVs were putatively de novo which is the simultaneous analysis of intensity data and genotype a de novo rate of 21.335%. 51 IMAGE cases, 22 deletion data in the same experimental setting establishes a highly loci, and 5 duplication loci had multiple de novo events due accurate definition for normal diploid states and any devia to low data quality and were excluded as outliers; once tion thereof. To call CNVs, we used the PennCNV algo excluded, 785 CNVs were inherited and 63 were denovo rithm, which combines multiple sources of information, which lowered the observed denovo rate to an acceptable including Log R Ratio (LRR) and B Allele Frequency (BAF) at each SNP marker, along with SNP spacing and population level of 7.42.9%. Based on CNVs observed in parents from frequency of the Ballele to generate CNV calls. The Illumina CHOP data, 9,305 CNVs were passed to the child replication case and control cohorts utilized genome-wide while 7,432 CNVs were not inherited resulting in a 55.595% SNP genotyping using the Perlegen 600K, Illumina 1M, and inheritance rate. Based on all CNVs observed in parents Affymetrix 5.0 arrays. Raw X and Y values were normalized from Perlegen IMAGE data, 2,114 CNVs were passed to the with log(10) and clustered to establish BAF and LRR with child while 3,789 CNVs were not inherited resulting in a PennCNV-Afly protocol (Table 2). Rare recurrent CNVs 35.812% inheritance rate. We excluded 65 parent samples were the focus of our study. that were outliers with 20 or greater CNVs not inherited to offspring and filtering these samples out resulted in 1.204 TABLE 2 CNVs were passed to the child while 1.221 were not inherited resulting in a 49.650% inheritance rate which Perlegen Data Reformatted File Samples to match Affymetrix Power Tools established confidence in this CNV call set. Output format. 01.06. It is intractable to review all PennCNV calls and probeset id 10009 10010 10O21 10O22 wasteful to exclude CNVs smaller than a size threshold. A) Genotype Calls File (O = AA, 1 = AB, 2 = BB, -1 = NoCall). Instead, we statistically score the loci based on all CNVs detected and review these nominally associated CNVR loci SNP rs1OOOOO23 1 SNP rs1OOOOO30 1 for appropriate overlap, signal quality, and Mendelian SNP rs10000037 O inheritance. As shown in Table 3, all reported loci show at SNP rs1OOOOO68 2 : least one case with the CNV inherited from a parent, in cases where both parents were available. US 2017/000929.6 A1 Jan. 12, 2017

TABLE 3 Novel CNVRs Over-represented in ADHD Patients CHOP CHOP Replication Replication Cases Controls Cases Controls Combined OR Exon CNVR n = 1013 n = 4105 n = 2493 n = 9222 Inh P-value CI (95%) Type Gene Distance A) Loci Significantly Associated with ADHD chr11: 88269449-88351661 4i.(3*) O 6 1 4:1:3 1.36 x 10° 38.12 De GRMS 5,858 62.5% (5-298) chirf: 12652S124- 3 O 5 O 0:1:0 3.52 x 10 infinity De GRM8 O 1265.362O2 100% chr3: 7183953-7197236 4t (1*) O 2 O 0:2:0 8.14 x 10 infinity De GRM7 20,598 100% chr6: 146657O76- 5 2 3 O 2:0:0 1.05 x 10' 15.24 Dup, GRM1 O 146694047 100% (3-72) B) ADHD Loci with Nominal Significance chr1: 72317292-72328395 4: O 1 O O:3:0 3.91 x 10' infinity Dup NEGR1 10,621 100% chirf: 153495598- 5(1*) O 3 2 1:2:0 4.08 x 10' 15.24 Dub DPP6 68,453 153564827 100% (3-72) chrS: 65027976-6SO46S2O 4 O 2 1 2:0:2 4.68 x 10' 22.85 De SGTB, O SO% (3-190) NLN chr1: 560S3497-56064495 2 O 4 2 1:0:3 1.54 x 10 11.42 De USP248 80.234 25% (2-57) chr19:3842772O-384.44834 S 2 2 3 2:2:1 495 x 10 5.33 De SLC7A10* 19,172 80% (2-17) chr3: 1844168-1859889 4t O 3 6 1:3:0 8.81 x 10 4.44 De CNTN48 255,661 100% (1-13) chr2: 81419297-81.446082 2 O 2 3 1:0:1, 3.83 x 102 5.07 Dub CTNNA28 152,417 SO% (1-23) chr4: 11377,2340- 2 O 2 3 0:0:0 3.83 x 102 5.07 Dub LARP7 O 11378.8584 NA (1-23) *Cases presented in (13); 3 Cases are present in the same family; 2 Cases are present in the same family; The “Inh” column lists the inheritance pattern of each CNV from parents to cases in the format:

0107. In total, there are 3,506 cases and 13,327 controls, analysis was further performed using the AAC method. representing greater than a three-fold abundance of control Reference genes, chosen from COBL, GUSB, and SNCA, samples to robustly define CNVs to be absent or at a lower were included based on the minimal coefficient of variation frequency than case samples. Although the number of CNVs and then data was normalized by setting a normal control to detected per sample was as high as 70, there are actually a value of 1. inferred normal diploid (CN=2) calls which make every sample equivalent. These CNVs are very rare and thus the 0109. The CNV calling on Perlegen platform used a highly similar algorithm to those used on the Illumina number of observed CNV calls will vary between samples. arrays, but the signal pre-processing steps differ. Unlike the Illumina platform, where normalized signal intensities (Log CNV Validation by Quantitative PCR (QPCR) R Ratio and B Allele Frequency) can be exported directly 0108 Universal Probe Library (UPL: Roche, Indianapo from the BeadStudio software, these signal intensity mea lis, Ind.) probes were selected using the ProbeFinder v2.41 sures in the Perlegen 600K platform need to be calculated software (Roche, Indianapolis, Ind.). Quantitative PCR was from the collection of genotyped samples based on raw X performed on an ABI 7500 RealTime PCR Instrument or on and Y values. To perform data normalization and signal an ABI PrismTM 7900HT Sequence Detection System (Ap extraction from raw final report files generated in genotyp plied Biosystems, Foster City, Calif.). Each sample was ing experiments, we first reformatted data from dbGaP into analyzed in quadruplicate either in 25 Jul reaction mixture the format produced by Affymetrix Power Tools: birdseed. (250 nM probe, 900 nM each primer, Fast Start Taq Man calls.txt, birdseed.confidences.txt, and quant-norm.pm-only. Probe Master from Roche, and 10 ng genomic DNA) or in med-polish.expr.summary.txt (see Table 2). The X and Y 10 ul reaction mixture (100 nM probe, 200 nM each primer, values provided in the sample based report files from dbGaP lx Platinum Quantitative PCR SuperMix-Uracil-DNA-Gly were reduced to a more finite range by taking the logarithm cosylase (UDG) with ROX from Invitrogen, and 25 ng base 10. For each SNP marker, we then relied on the genomic DNA). The values were evaluated using Sequence allele-specific signal intensity for the AA, AB and BB Detection Software v2.2.1 (Applied Biosystems, CA). Data genotypes on all genotyped samples to construct three US 2017/000929.6 A1 Jan. 12, 2017

canonical genotype clusters in polar coordinates theta and R. TABLE 4 similar to the Illumina clustering generation approach. The “-conf 2 option was included in running generate afly Sample exclusion based on quality control measures. geno cluster.pl since 1 was coded as the best score. Once the canonical genotype clusters were constructed, we then trans Exclusion Criteria CHOP Control formed the signal intensity values for each SNP to Log R Call Rate <98% 170 271 SD LRR -0.35 73 124 Ratio (LRR) and B Allele Frequency (BAF) values using Ethnicity non-Caucasian 71 48 normalize affy geno cluster.pl. For more technical details, Wawe Factor -0.5 - X > 0.6 251 1040 see www.openbioinformatics.org/penncmV/pennchV tuto Count CNVS >70 197 237 rial afly gw8.html. Monozygotic Twin 31 38 0110. To optimize the Hidden Markov Model (HMM), we used the baseline reference file hh550.hmm and ran Samples excluded based on Quality Control (QC) measures “-train' in PennCNV in three successive batches of thirty. on our HumanHap550 GWAS data based on statistical The first training used the samples with the lowest standard distributions to exclude poor quality DNA samples and false deviation of LRR while the other two runs, using the file positive CNVs. created as a new reference, included more random repre sentative samples. We also created definition files providing Statistical Analysis of CNVs inter-SNP distance and population b-allele frequency to 0113 CNV frequency between cases and controls was further inform CNV calling specifically adapted to the evaluated at each SNP using Fisher's exact test. We only observed Perlegen data. This allowed for CNV calls to be considered loci that were nominally significant between made in 1887 (642 cases and 1,245 parents) out of 2,789 cases and controls (p<0.05) where cases in the CHOP Perlegen 600K samples available. Although the global stan discovery cohort had the same variation, replicated in dard deviation of LRR was below 0.2 for the majority (84%) IMAGE, PUWMa, or IMAGE II or were not observed in any of samples, the intensity data was notably noisier in regions of the control subjects, and validated with an independent of called CNV and often showed a subpopulation of SNPs method. We report statistical local minimums to narrow the unable to differentiate a deletion signal, perhaps due to PCR association in reference to a region of nominal significance saturation during the lab processing. Nevertheless, the dele including SNPs residing within 1 Mb of each other (FIG. 4). tion and duplication features were still detected with con Resulting nominally significant CNVRs were excluded if firmation of homozygote and AAB/ABB genotypes respec they met any of the following criteria: i) residing on telo tively shown for the same SNPs (see FIGS. 2 and 3). mere or centromere proximal cytobands; ii) arising in a 0111 Lastly, Perlegen CNV calls were screened for over “peninsula' of common CNV arising from variation in lap with the 11 loci associated based on the CHOP Illumina boundary truncation of CNV calling (FIG. 7); iii) genomic data. The SNP level data underlying each CNV call was regions with extremes in GC content which produces reviewed to ensure clean signal quality. To ensure that each hybridization bias; or iv) samples contributing to multiple detected CNV was a true DNA feature and not in any way CNVRs. We statistically adjusted for relatedness of cases an artifact of the Perlegen 600K array used or our bioinfor with permutation (1000x). Three lines of evidence establish matics manipulations of the data, we validated each CNV statistical significance: independent replication p<0.05, per with qPCR at an independent lab (see FIG. 4). mutation of observations, and no loci observed with control enriched significance. We used DAVID (Database for Anno CNV Quality Control tation, Visualization, and Integrated Discovery) to assess 0112 We calculated Quality Control (QC) measures on the significance of functional annotation clustering of inde our Human Hap550 GWAS data based on statistical distri pendently associated CNV results into InterPro categories. butions to exclude poor quality DNA samples and false positive CNVs. The first threshold is the percentage of Permutation to Adjust Significance for Relatedness attempted SNPs which were successfully genotyped. Only 0114. For initial Fisher's exact test, related individuals samples with call rate >98% were included. The genome are not controlled for since our primary objective is to detect wide intensity signal must have as little noise as possible. CNVs in multiple samples regardless of relatedness. CNVRs Only samples with the standard deviation (SD) of normal passing this initial screen are scored for statistical signifi ized intensity (LRR)<0.35 were included. All samples must cance based on a permuted P-value which permutes case and have Caucasian ethnicity based on principle components control labels randomly of all samples with the condition analysis (FIG. 5) and all other samples were excluded. that related individuals must have the same label. Each Furthermore, case and control matching was insured by unrelated individual is assigned a case or control label and calculating a genomic inflation factor (GIF=1.024) between their related sibling is assigned the same label. Based on the groups. Wave artifacts roughly correlating with GC content number of samples with the CNVR being calculated in resulting from hybridization bias of low full length DNA randomly assigned “cases” and “controls” a Fisher's exact quantity are known to interfere with accurate inference of test P-value is assigned. The number of hypothetical sce copy number variations". Only samples where the wave narios with significance equal or greater (lower P-value) factor of LRR to wave model ranged between -0.5

TDT Analysis of 397 ADHD Cases and Parents from CHOP genotyped on the Illumina HH550 chip.

CHR SNP BP A1 A2 T U OR CHISQ C

18 ris8095193 S883.4095 2 167 92 1.815 21.72 3.16E-06 17 rS435798O 13498.634 2 99 174 O.S69 20.6 S6SE-06 18 ris8091710 72897492 2 29 73 0.3973 18.98 1.32E-OS 14 rS899.116 97.49518.5 2 101 172 O.S872 1847 1.73E-OS 13 rs9595945 48099556 2 245 160 1531. 17.84 2.4OE-OS 4 rS1018,199 47927 632 2 3S 80 0.437S 17.61 2.71E-OS 1 rs3795324 157456184 2 1 91 157 O.S796 17.56 2.78E-OS 3 rs644418.6 188156541 2 81 36 2.25 7.31 3.18E-OS 9 rs11144627 7565.4927 2 1 46 14 3.286 17.07 3.61E-OS 8 rs1462O11 108104653 2 199 125 1592 16.9 3.94E-OS X rS599.1935 1OO480O88 2 22 S9 O.3729 16.9 3.94E-OS 7 rs101.3572 7835O227 2 63 118 O.S339 16.71 4.3SE-OS 1 rs952619 2O316347 2 108 177 0.6102 16.71 4.37E-OS 4 rs7689O18 851164.79 2 41 87 O.4713 16.53 4.79E-05 8 rs19438.25 691.28567 2 97 162 O.S988 16.31 S.37E-OS 4 rS4696821 8473961 2 210 135 1.SS6 16.3 S.39E-OS 8 rs1943823 69131624 2 157 237 0.6624 16.24 SS7E-OS 4 rs11724347 47923023 2 26 64 O.4062 16.04 6.19E-05 1 rs753O899 76950752 2 89 151 O.S894 16.02 6.28E-OS 8 rS4890560 41.457783 2 93 156 0.5962. 15.94 6.54E-OS 6 rs2677099 45527900 2 220 144 1528. 15.87 6.79E-05 2 rs11067228 113556980 2 231 153 1.51 5.84 6.88E-05 6 rs2790102 45S4O192 2 222 146 1S21 15.7 7.44E-OS 1 rS4926757 4896.1624 2 192 122 1574 15.61 7.8OE-OS 1 rs17147479 84OSSSO4 2 137 79 1.734 15.57 7.93E-05 7 rs991.3261 1202636S 2 89 150 O.S933 15.57 7.96E-OS 9 rs7041883 1353S2660 2 17 49 O.3469 1552 8.19E-OS 2 rs7309946 103478.293 2 19 188 O.633 15.51 8.22E-OS 7 rs10226468 429.07176 2 44 219 O.6575 15.5 8.27E-OS 5 rS4384.18 2902436 2 78 36 2.167 15:47 8.37E-OS 8 rs12682232 108078371 2 99 128 1.SSS 15.42 8.63E-OS X rSS956634 12309.2612 2 S9 110 O.S364 15.39 8.74E-OS 7 rs7786719 428SO3S6 1 2 133 20S 0.6488 15.34 8.99E-OS 6 rs910586 45518290 1 2 221 146 1.514 15.33 9.04E-OS 6 rs9395O10 44453984 1 2 152 91 1.67 5.31 9.11E-OS 14 rS1184.4273 97489409 1 2 100 163 O.613S 15.09 102E-04 2 rs11904235 362883SO 1 2 64 27 2.37 5.04 105E-04 11 rS4875.18 131283728. 1 2 1SO 225 0.6667 15 1.O8E-04 6 rs692O606 33105652 2 1 164 242 O.6777 14.99 1.08E-04 14 rs2O14525 97.491.178 1 2 109 174 0.6264. 14.93 1.12E-04 11 rs7948111 23403649 1 2 6S 117 O.SSS6 14.86 1.16E-04 16 rs12598067 60940O38 2 1 6S 117 O.SSS6 14.86 1.16E-04

US 2017/000929.6 A1 Jan. 12, 2017

TABLE 7-continued TDT Analysis of 623 ADHD Cases and Parents from IMAGE genotyped on the Perlegen platform. CHR SNP BP A1 A2 T U OR CHISQ 104.169047 1 2 27S 191 1.44 1514 9.97E-OS 61678.306 2 1 112 61 1836 1S.O3 1.06E-04

CHR: Chromosome number, SNP: SNP identifier, A1: Minor allele code, A2: Major allele code, T: Transmitted minor allele count, U: Untransmitte allele count, OR: TDT odds ratio, CHISQ: TDT chi-square statistic, P: TDT asymptotic p-value

Study Criteria for Inclusion in IMAGE tained via random digit dialing. Participants were screened for psychosis and bipolar disorder. Control participants were 0116 Proband diagnosis: combined subtype ADHD. not screened for ADHD. A blood sample was collected via Children aged 6-17 years (inclusive). a US national phlebotomy service. Control participants gave One or more sibling(s) in the same age range. written consent for their biological materials to be used for Both parents available to provide DNA sample or one parent medical research at the discretion of NIMH. Controls were available plus two or more siblings. genotyped using the Affymetrix 6.0 array, at the Broad IQ above 70. Free of single-gene disorders known to be associated with Institute National Center for Genotyping and Analysis. ADHD (e.g. fragile-X, phenylketonuria, hypercalcaemia, Genotype calls were made with the BIRDSEED program, a thyroid hormone resistance). module of the BIRDSUITE package. Free of neurological disease and damage (e.g. hemiplegia and other cerebral palsies, epilepsy, hydrocephalus, post Network Analysis encephalitic syndromes, psychosis, sensorimotor handi caps). 0119 We used Cytoscape Software' to identified 228 Living at home with at least one biological parent and one genes within 2 degrees of relation to 8 GRM genes based on full sibling. the merged human interactome. We clustered this network of Not meeting criteria for autism or Asperger's syndrome. genes into 17 distinct modular clusters based solely on network topology using the ClusterViz plug in for the Study Criteria for Inclusion in IMAGE II software using the FAG-EC algorithm with default param eters. Component genes of each of the 17 modules were 0117 Proband diagnosis: ADHD according to DSM-IV submitted to DAVID to assess the significance of func TR tional enrichment using Homo sapiens GO annotations. Semi-structured diagnostic interview: KSADS-PL or Kinder-DIPS 0.120. The following examples are provided to illustrate Child Behavior Checklist, Conners parent and teacher certain embodiments of the invention. They are not intended Scales or German Teachers Report on ADHD symptoms to limit the invention in any way. according to DSM-IV Children aged 6-18 years (index patients older than 8 years). Example I IQ above 70; birth weight >2000 g; no major medical events during pregnancy; no drug abuse in mother during preg Metabotropic Glutamate Receptor Gene Alterations nancy Associated with ADHD Free of single-gene disorders known to be associated with ADHD (e.g. fragile-X, phenylketonuria, hypercalcaemia, 0121 Several rare recurrent CNVs have been identified thyroid hormone resistance). that are overrepresented in multiple independent ADHD Free of neurological disease and damage (e.g. hemiplegia cohorts that impact genes involved in glutamatergic neu and other cerebral palsies, epilepsy, hydrocephalus, post rotransmission, an important mediator for the developing encephalitic syndromes, motor neuron disorder etc.). brain and normal brain function. These results implicate Not meeting criteria for autism or Asperger's syndrome, variations involving glutamatergic gene networks of the Schizophrenia, bipolar disorder, primary major depressive brain as contributors to the genetic susceptibility of ADHD. episode, and anxiety disorder, Tourette’s Syndrome. Controls for IMAGE II Study Participants 0118. The control subjects used were drawn from 0.122 The discovery cohort included a total of 1,013 Affymetrix 6.0 genotyped subjects from the NIMH genetics ADHD cases of Northern European descent genotyped at repository. They had been collected through a US Nationally Children's Hospital of Philadelphia (CHOP). This consisted representative survey panel (of approximately 60,000 adult of 664 cases without parents and 349 cases from complete individuals at any one time, with constant turnover) ascer trios recruited at CHOP (See Tables 8 and 9). US 2017/000929.6 A1 Jan. 12, 2017 18

TABLE 8 TABLE 9-continued Clinical Demographics of Study Participants. K-SADS ADHD Severity of of CHOP Study Participants in Inattentive, Impulsive, and Hyperactive Domains. ADHD Diagnostic Criteria Score 1 Score 2 Score 3 Score 4 Subjects ADHD ADHD Cohort N Age range Ancestry ascertainment Talks Excess 37 77 255 131 Difficulty Playing Quietly 98 120 233 49 CHOP ADHD trios 349 6-18 European K-SADS-IVR CHOPADHD cases 664 6-18 European Clinical ADHD *Concentration 1 record missing diagnosis & Blurts 3 records missing. Scores 1 and 2 means that symptoms are within the normal range while scores 3 and 4 are treatment with excessive, ADHD meds: K-SADS-IVR I0123 To address replication, we accessed the IMAGE on majority cohorts which are a part of the Genetic Association Infor NIMHADHD trios 128 6-12 European DICA; Conners mation Network (GAIN). There were 624 IMAGE samples Scales that met quality control criteria for the study. Access to these UTAH cases 90 19-60 European WRAADDS, genotypes and intensity data for IMAGE was provided WURS, PRS, strict DSM-IV through the database of Genotypes and Phenotypes (dbGaP). criteria, The PUWMa consortium from University of California at including age of Los Angeles, Massachusetts General Hospital, and Wash onset before 7 ington University St. Louis contributed 864 ADHD cases IMAGE ADHD trios 642 6-17 European PACS, Conners, and 1.258 parents. The IMAGE II consortium contributed SDQ, WISC 787 ADHD cases and 898 unrelated controls. Furthermore, IMAGE II ADHD 787 5-14 European K-SADS 128 cases recruited at the NIMH and 90 cases recruited at trios German The University of Utah also served for replication. The DNA version, Kinder samples from CHOP. NIMH, and Utah cohorts were geno DIPS, Conners parent and typed using the Illumina Infinium Human Hap550K Bead teacher scales, Chip at CHOP. The IMAGE cohort was genotyped using the WISC, K-ABC Perlegen 600K platform. The PUWMa cohort was geno PUWMatrios 864 6-18 European K-SADS typed on the Illumina 1M BeadChip. The IMAGE II cohort was genotyped on the Affymetrix 5.0 array. To manage PACS: Parental Account of Child Symptoms; differences in CNV detection between arrays we used con Conners: Behavioral rating scales; trols genotyped on platforms matching the case platforms, SDQ: Strength and Difficulties Questionnaire; including: 4,105 Illumina 550k from CHOP, 3,297 Perlegen WISC: Wechsler Intelligence Scale for Children (WISC-IV); 600k from GAIN psoriasis and depression projects, 3,469 KSADS-IVR: Schedule for Affective Disorders and Schizophrenia for School-Age Chil dren-IVR; Illumina 1M from PUWMa parents and SAGE, and 2.456 DICA: Diagnostic Interview for Children and Adolescents; Affymetrix 5.0 and 6.0 controls from the NIMH genetics Kinder-DIPS: Diagnostic Interview for Psychiatric Disorders in Children, repository and AGRE parents. K-ABC: Kaufman-ABC intelligence scale. CNV Size and Number in Cases and Controls WRAADDS = Wender-Reimherr Adult Attention Deficit Disorder Scale; WURS = Wender Utah Rating Scale; (0.124. To search for novel CNVs we analyzed the 1,013 PRS = Parent Rating Scale. CHOP cases as a discovery cohort in comparison with 4,105 control children, all of whom were of European ancestry. Data from the IMAGE, PUWMa, IMAGE II, NIMH, and TABLE 9 Utah cohorts were used for replication, together with an K-SADS ADHD Severity of of CHOP Study Participants in Inattentive, independent control cohort of 9.222 genotyped on the same Impulsive, and Hyperactive Domains. platforms. Thus, the control CNV frequency is robustly characterized in multiple large independent cohorts, based Diagnostic Criteria Score 1 Score 2 Score 3 Score 4 on the Illumina, Perlegen, and Affymetrix platforms. We Often Careless 7 40 372 81 note that of the 2,713 (934 cases) IMAGE samples available Loses Things 18 126 277 79 in dbGaP, 1,886 (624 cases) met strictly established data Difficulty Finishing 16 90 311 83 quality thresholds for CNVs. Listening 10 22 320 148 Concentration* 2 25 337 135 (0.125. The PennCNV software was used to produce CNV Distracted 1 10 307 182 calls for cases and controls as previously described'. The Organizing 19 79 304 98 CNV frequency of the subjects who met quality standards, Avoiding 19 55 278 148 which included removing Substantial outliers in the count Forgetful 19 75 290 116 CNV call quality metric that deviated exponentially from the interrupts 28 73 305 94 Acts Before Thinking 28 112 283 77 distribution of the majority of the cohort, resulted in 93% of Shifts Activities 72 134 247 47 subjects having 8-45 CNV calls (FIG. 1). We called four Blurts 135 82 232 48 different copy number states, including 3,172 homozygous Difficulty Waiting Turn 8O 172 200 48 Hyperactive 53 127 227 93 deletions (copy number, or CN=0), 27,810 hemizygous Fidgeting 15 47 301 137 deletions (CN=1), 14.806 one copy duplications (CN=3), Difficulty Staying Seated 45 8O 287 88 and 581 two copy duplications (CN=4). FIG. 8 shows an On the Go 49 89 255 107 example of raw Illumina data as viewed in the BeadStudio software and the resulting CNV call. The CNV calls spanned

US 2017/000929.6 A1 Jan. 12, 2017 20

TABLE 10-continued SNP GWAS Significance of Top Ranked ADHD Associated SNPs Reported by Lesch and Zhou. A) ADHD TDT CHOP Illumina 550k data; B) ADHD Case: Control CHOP Illumina 550k data: C) ADHD IMAGE Perlegen 600k data. 4 rS10516182 7143981 2 O.28O1 O.2954 O.9279 1.274 O.259 4 rs7697323 78O1488 1 O.3782 O.38 2 O.9927 O.O142 O.9051 S rs173754 651O2O81 1 0.4925 O491S 2 OO4 O.OO428S O.9478 S rs2S8082 66166352 1 0.4619 O4S21 2 .04 O4342 OSO99 6 rS160666 2719051 2 0.2857 O3O2S O. 9222 1.515 O.21.83 6 rS2842643 41758714 2 O.2932 O.2909 O11 O.O2797 0.8672 6 rs3799977 44.945334 2 04306 O4O76 O99 2.452 O.1174 6 rS8180608 89.064414 2 04101 0.4441 O.8703 S.26S O.O2176 6 rs13586O1 91S32294 1 O.3852 O3846 2 OO3 O.OO2O76 O.9637 6 rs6921403 1541S6O2O 2 O.1373 O.1405 O.9736 O.O9408 O.7591 7 rs2237349 28S362O3 2 O.4109 O.4082 O11 O.O3276 O.8564 7 rs20O2865 154132O3S 2 O.2075 O.217 O.94.45 O.606S O4361 8 rs6991017 SSO878O 2 O.1891 O.1873 O12 O.O2315 O.8791 8 rs2248529 14657354 1 O.36O4 O.363 2 O.9888 0.03305 0.8557 8 rS4.961315 142110882 2 O.2959 O.2995 O.983 O.O6846 O.7936 9 rs2418326 114759028 1 O.2534 O2S2 2 OO7 O.O1179 0.913S 9 rs2SO2731 1280S611.1 2 O.3626 O.3508 OS3 0.6767 O.4107 14 rS1048.3393 3153O23S 1 O.2272 O.22O3 2 O41 O3146 O.S749 1S rs2SS6S60 4260913S 2 O.419 O4215 O.9899 O.O2811 O.8668 16 rS8060494 788O8972 2 O.3215 O.3228 O.9943 O.OO8131 O.9282 17 rS4790372 27.01606 2 O.3014 O.3112 O.9S46 O.S122 O.4742 17 rs124.53316 69027654 1 O.3612 O.3662 2 O.9788 O. 1193 O.7298 19 rs997669 34996323 2 O.4023 O.3876 1.064 1.025 O3114 2O rs1555322 333 1259S 1 O.1279 O.1277 2 1.002 OOOO4034 O.984 C) CHR SNP BP A1 A2 T U OR CHISQ P

1 rs2281597 34132445 O 2 O O NA NA NA 1 rs642969 97590.139 O 2 O O NA NA NA 2 rs2S87695 20O38287 1 2 320 294 1.088 1.101 O.2941 2 rs2242O73 2O8702290 2 185 182 1.016 O.02452 O.8756 3 rs10510850 60542142 1 2 109 115 O.9478 O.1607 O.6885 3 rs17233461 2S807474 2 305 322 O.9472 O4609 (0.4972 4 rs7SS403 644.0543 2 296 278 1.06S O.S645 O4S25 4 rs385.7174 7089831 2 2O2 217 O.9309 0.537 O4637 4 rs7697323 7734.317 1 2 269 278 O.9676 O.1481 (0.7004 S rs145772O 10998762 2 247 260 O.95 O.3333 O.S637 6 rS160666 2719051 2 248 262 O.946.6 O.3843 O.S353 6 rs3799977 44.945334 2 3O2 282 1.071 O.6849 0.4079 6 rs6921403 54105.599 2 149 150 O.9933 O.OO3344 O.9539 8 rs6991017 550878O 2 193 191 1.01 O.O1042 O.91.87 9 rs2418326 1671929S 1 2 236 210 1.124 1516 O.21.83 9 rs2416606 19862757 2 264 262 1.008 O.OO7605 0.93 OS 10 rs16928.529 72652991 2 277 312 O.8878 2.08 O.1493 10 rS11594082 729692.59 1 2 126 138 O.913 O.S4SS O-46O2 10 rs10786.284 98.125495 O O O NA NA NA 10 rSS15910 OS956394 2 3OO 272 1.103 1.371 O.2417 11 rs3893.215 17721406 O 2 O O NA NA NA 11 rs1083.0468 876O4834 O 2 O O NA NA NA 12 rS4964805 O2716954 O 2 O O NA NA NA 13 rs7995.215 932O6507 1 2 279 317 O.88O1 2.423 O. 1196 14 rs2239627 22705999 O 2 O O NA NA NA 14 rS10483286 24273582 O 2 O O NA NA NA 16 rS10514604 83OO3885 O 2 O O NA NA NA 17 rs244O129 6847295 O 2 O O NA NA NA

Segment-Based Comparative Analysis of CNVs frequency in cases compared to controls. To ensure reliabil ity of our CNV detection method, we experimentally vali 0128. To identify novel genomic loci harboring CNVs dated all CNVRs using quantitative PCR (qPCR), a method potentially contributing to ADHD, we applied a segment commonly used for independent validation of CNVs (FIG. based scoring approach that scans the genome for consecu tive SNPs with more frequent copy number changes in cases 7). Thus, we have applied a separate validation technique on compared to controls as we have previously described all the CNVs reported to ensure positive confirmation. Using (Glessner, et al. 2009; Wang, et al. 2007). The genomic span this approach, we have identified and experimentally vali for these consecutive SNPs delineates common copy num dated a total of 12 CNV loci that were either observed in ber variation regions, or CNVRs. In the CHOP cohort, we ADHD cases only or overrepresented in the ADHD cases identified 10 CNVRs that were observed in multiple cases that we subsequently took forward for replication in inde but not in controls, as well as 2 CNVRs that had higher pendent study cohorts. US 2017/000929.6 A1 Jan. 12, 2017

0129 Replication analysis was performed in five inde 0.130 FIG. 9 shows the CNV deletions observed at the pendent cohorts, including ADHD subjects from IMAGE, GRM5 locus (10 cases vs 1 control), using UCSC Genome PUWMa, IMAGE II, NIMH, and Utah. Based on the 10 Browser (12) with Build 36 of the human genome. Experi case-specific CNVs from the discovery cohort, 3 were also exclusive to replication cohort cases, notably GRM7, GRM8 mental validation of IMAGE, PUWMa, IMAGE II, NIMH, and NEGR1, with resulting combined P-values of 3.52x10 and Utah CNVs, using qPCR, together with Raw BAF and and 8.14x10, for GRM8 and GRM7, respectively (Table LRR plots are shown in FIGS. 2-4. 3A). A third GRM gene, GRM5, was observed in 10 ADHD I0131 Taken together, we have uncovered four genes cases (10/3.506) and one control (1/13.327) with resulting directly impacted by CNVRs in multiple independent P=1.36x10 (Table 3A). GRM1 was observed in 8 cases and 2 controls P=1.05x10'. While odds ratios (ORs) could cohorts that belong to the metabotropic glutamate receptor not be estimated for GRM7 and GRM8, since these CNVs gene family (InterPro category “GPCR, family 3, metabo were absent in the control subject, the ORs of GRM5 and tropic glutamate receptor”; P=2.1x10). It is also notewor GRM1 amounted to 38.12 and 15.24, respectively (Table 3), thy that both GRM2 and GRM6 were found to be impacted suggesting that the contribution of these CNVs to the ADHD by deletions in single ADHD cases in the IMAGE II cohort phenotype is potentially high. Thus, these 4 GRM genes and were absent in the control subjects. We additionally were impacted by CNVs that associated with ADHD and evaluated the significance of the GRM genes, using TDT in replicated successfully in the independent ADHD cohorts the same cohort, and the best support was observed for (Table 3 and Table 11), whereas the other CNV loci were GRM7, P-8.35x10 (Table 12). Furthermore, analysis was also observed to be enriched in the ADHD cases, albeit at also performed to address family based CNV statistics based nominally significant P values (Table 3b and Table 11). on transmission disequilibrium and de novo events in the TABLE 11 Discovery, Replication, and Combined Significance of CNV Regions. Permuted Permuted Permuted Discovery Replication Combined Discovery Replication Combined CNVR P-value P-value P-value P-value P-value P-value Type Gene chr11: 882694.49-88.351661 1.53 x 10 5.29 x 10 1.36 x 10 0.025 0.001 0.002 De GRMS chr7: 126441593-126621501 7.74 x 10: 4.35 x 10' 3.52 x 10 0.013 <0.001 <0.001 De GRM8 chr3: 7183953-197236 1.53 x 10' 4.53 x 102 8.14 x 10 0.011 0.039 <0.001 De GRM chr6: 146657076-146694047 4.42 x 10 9.63 x 10 1.05 x 10 0.006 <0.001 <0.001 Dup GRM1 chr1: 72317292-72328.395 1.53 x 10 2.13 x 10' 3.91 x 10 O.O36 O.213 0.011 Dup NEGR1 chirf: 153495598-153564827 1.53 x 10 6.82 x 102 4.08 x 10 <0.001 O.OS8 <0.001 Dup DPP6 chrS: 65027976-6SO46S2O 1.53 x 10 1.17 x 10' 4.68 x 10 O.OO3 O.108 O.OO1 De SGTBjNLN chr1: 560S3497-56064495 3.91 x 102 2.12 x 102 1.54 x 10 O.O3S O.O24 <0.001 De USP24 chr19:38427720-3844.4834 4.42 x 10 2.89 x 10 4.95 x 10 O.OO2 O.262 O.OO7 De SLC7A10 chr3: 1844168-1859889 1.53 x 10 4.12 x 10 8.81 x 10 O.OO8 O416 O.O15 De CNTN4 chr2: 81419297-81446082 3.91 x 102 2.89 x 10' 3.83 x 102 O.O46 O.294 0.032 Dup CTNNA2 chira: 113772340-113788584 3.91 x 102 2.89 x 10' 3.83 x 102 O.O33 O.288 0.042 Dup LARP7 The top 4 most significant loci are shown in bold.

family-based subset of 311 CHOP families and 422 IMAGE families (Tables 12 and 14). TABLE 12 ADHD Genotype GWAS of Glutamatergic Genes. The most significant SNP genotype association in each of the eight GRM gene regions. A) ADHD TDT CHOP Illumina 550k B) ADHD Case: Control CHOP Illumina 550k C) ADHD IMAGE Perlegen 600k. A) CHR SNP BP A1 A2 T U OR CHISQ P Gene

11 rS423,7549 884O7924 2 1 31 61 O.S.082 9.783 O.OO1762 GRMS 7 rs178641.59 126444172 1 2 22 46 0.4783 8.471 O.OO3609 GRM8 6 rs3887SSS 34177040 1 2 208 161 1292 5.986 OO1442 GRM4 7 rs6943,762 86O47914 2 1 69 99 O.697 S.357 O.O2O64 GRM3 3 rs7623 OSS 74.85891 1 2 151 193 0.7824 S.128 O.O23S4 GRM7 6 rs362839 146721428 2 1 125 161 O.7764 4.531 O.O3328 GRM1 3 rS4687770 5173O105 2 1 114 94 1213 1923 O.16SS GRM2 S rs2O781.83 178357150 2 1 190 21O O.9048 1 O.3173 GRM6 B) CHR SNP BP A1 F. A F U A2 OR CHISQ P Gene

3 rs7623 OSS 74.85891 1 0.3582 O4129 2 O.7936 15.48 8.35E-OS GRM7 11 rs1354411 88O16449 2 O.O3643 O.O566 1 O.63O2 1021 O.OO1396 GRMS 7 rs2283100 126643293 2 O.2281 O.193 1 1.235 9.S27 O.OO2O24 GRM8 US 2017/000929.6 A1 Jan. 12, 2017 22

TABLE 12-continued ADHD Genotype GWAS of Glutamatergic Genes. The most significant SNP genotype association in each of the eight GRM gene regions. A) ADHD TDT CHOP Illumina 550k B) ADHD Case: Control CHOP Illumina 550k C) ADHD IMAGE Perlegen 600k. 6 rs18732SO 34130718 2 O.2134 0.2455 1 0.8338 7.062 O.OO7873 GRM4 7 rs10952890 86.1931.51 1 0.027S3 O.O3917 2 O.6945 4.782 0.02877 GRM3 S rs2O781.83 1783571SO 2 0.4593 0.4897 1 O.8852 4.605 0.03189 GRM6 6 rs198363S 14670.736S 2 0.316 O.29.17 1 1.122 3.515 0.06081 GRM1 3 rS4687592 5163O896 1 O.O3442 O.O404.1 2 O.8464 1.191 0.2752 GRM2 C) CHR SNP BP A1 A2 T U OR CHISQ P Gene

6 rS122O6652 341.73960 2 1265 21 6 1.227 4.992 O.O2S47 GRM4 11 rs16O195 87932621 2 1302 2S3 1.194 4.326 O.O3753 GRMS 7 rs115634.86 126621SO1 1 2130 162 O.802S 3.507 0.06112 GRM8 3 rs11717471 7599.469 2 1238 280 O.85 3.405 0.06498 GRM7 6 rS23.00620 146745874 2, 1160 133 1.2O3 2.488 0.1147 GRM1 7 rs1468413 86271589 1 21.90 162 1173 2.227 0.1356 GRM3 5 rs7725272 178338994 2. 1289 261 1.107 1.425 0.232S GRM6 3 rs6445959 51747387 2 1169 153 1.1OS O.795 0.3726 GRM2

0.132. In view of the above finding, we hypothesized that rological processes including synaptic transmission with genes interacting with GRM receptor genes would collec effects of behavior and cognition. tively have more cases enriched with CNVs in comparison with healthy controls. We identified 228 genes within 2 Example II degrees of relation to GRM genes based on the merged human interactome provided by the Cytoscape Software Multiplex SNP Panel for Diagnosis of ADHD (Shannon, et al. 2003). We evaluated these genes in 1,231 0134. As described above in Example I, several genetic ADHD case samples and 4,105 control samples, all of which alterations have been found to be associated with the ADHD were genotyped on the same platform at CHOP for evidence phenotype. The information herein above can be applied of enrichment in the ADHD case samples (P<0.05). We clinically to patients for diagnosing an increased suscepti detected 67 GRM receptor interacting genes that were bility for developing ADHD, and therapeutic intervention. A enriched for CNVs in cases, in comparison with 16 genes in preferred embodiment of the invention comprises clinical the controls, confirming over 3-fold enrichment in CNVs in application of the information described herein to a patient. this gene network in the ADHD cases (P-4.38x10', FIG. Diagnostic compositions, including microarrays, and meth 10). ods can be designed to identify the genetic alterations 0.133 We subsequently clustered the above second described herein in nucleic acids from a patient to assess degree GRM receptor gene interaction network to define susceptibility for developing ADHD. This can occur after a highly interconnected modules of genes based on network patient arrives in the clinic; the patient has blood drawn, and topography, and looked for enrichment of gene ontology using the diagnostic methods described herein, a clinician (GO) annotations within these modules. As shown in FIG. can detect a SNP in the genetic regions listed in Tables 13A 11, GRMs do not form a large number of interactions, but and 13B below. The typical age range for a patient to be importantly serve to coordinate functional modules of other screened is between 1 and 12 years of age. The information sets of genes. For instance, GRM1 harbors duplications obtained from the patient sample (e.g., nucleic acids), which significantly enriched in ADHD cases and serves to coordi can optionally be amplified prior to assessment, will be used nate functional modules involved in housekeeping functions to diagnose a patient with an increased or decreased Sus Such as carbohydrate metabolism, phosphorylation, apopto ceptibility for developing ADHD. Kits for performing the sis and ion binding. GRM5 and GRM7 both harbor deletions diagnostic method of the invention are also provided herein. significantly enriched in cases and cluster within a func Such kits comprise a microarray comprising at least one of tional module involved in Synaptic transmission and alter the SNPs provided herein in and the necessary reagents for native splicing. Specifically, GRM5 serves to coordinate assessing the patient samples as described above. In an alternative spicing with synaptic transmission and other alternative embodiment, a multiplex SNP panel is employed neuronal processes at the post-synaptic density, while and the patient sample is assessed for the presence or GRM7 coordinates functional modules integrating neuro absence of all the SNPs listed in the Tables below. logical processes and synaptic activity with housekeeping 0.135 Table 13A provides all the genes, physical genome functions such as cytoskeletal organization and apoptosis. ranges, and SNP ranges of the ADHD markers disclosed. GRM8 also harbors deletions significantly enriched in cases Table 13B provides a “SNPList” which is a minimal set for and is itself contained within a functional module that is a "diagnostic array Such as Veracode which is technlology involved in Synaptic transmission and neurogenesis. approved by FDA. Optimally, the flanking SNPs to these Although not significantly enriched in cases, GRM3 has CNVs are included as well as intervening SNPs as they duplications that are more frequently observed in cases and could all be used to capture the CNV. serves to coordinate ubiquitination pathways, RNA binding, 0.136. When we perform the testing, the clustering algo splicing, and processing, and neuronal migration, with neu rithm GenCall will be run on the sample set and SNPs poorly US 2017/000929.6 A1 Jan. 12, 2017 clustered or significantly deviating from Hardy Weinberg continuous physical position for homozygous genotypes equilibrium will be reviewed. Copy number variations indicating deletion or AAB and ABB genotypes indicating (CNVs) will be detected using our PennCNV hidden duplication weighted with positive correlation for control Markov model (HMM) copy number variation algorithm. minor allele frequency. Median normalization which is default is turned off since the I0137 The identity of ADHD-involved genes and the data of targeted regions instead of distributed genome-wide patient results will indicate which variants are present, and does not provide enough information for median normal will identify those that possess an altered risk for developing ization. For optimization, the HMM can be trained with ADHD. The information provided herein allows for thera good quality data Suited to the specific array observed values peutic intervention at earlier times in disease progression (call rate >98% and standard deviation of log r ratio <0.3). that previously possible. Also as described herein above, the PennCNV will detect regions of contiguous SNPs with GRM receptor family provides novel targets for the devel intensity (log R Ratio) trending below 0 indicating deletion opment of new therapeutic agents efficacious for the treat or trending above 0 indicating duplication. If no such trend ment of ADHD. In particular, it would be desirable to is observed, no CNV call will be made indicating a normal modulate expression of Such genes in those patients that are diploid state. In tandem, the genotype data is evaluated in a more prone to develop the disease. TABLE 13A Multiplex SNP Panel. Del Counts Dup Counts Gene Range (B36/hg18) StartSNP End SNP (cases:controls) (cases:controls) GRMS chr11: 88269449-88351661 rS604179 rS669724 10:1 O:O GRM8 chrf: 12652S124-1265.362O2 rs7794.734 rs2237790 8:0 O:O GRM7 chr3: 7183953-7197236 rs1516302 rs6784.317 6:O O:O GRM1 chr6: 146657O76-146694047 rs12200797 rs362949 O:O 8:2 NEGR1 chr1: 72317292-72.32839S rS120331 61 rs28.21257 O:O 5:0 DPP6 chrf: 153495598-1535648.27 rS4389846 rs12703329 O:O 8:2 SGTB NLN chrS: 65027976-6SO46S2O rS1 OO73281 rs972SO1 6:1 O:O USP24 chr1: S6OS3497-56064.495 rsfS27177 rS4333889 6:2 O:O SLC7A10 chr19:38427720-384.44834 rs74868O rs4S3O278 7:5 O:O CNTN4 Chr3: 1844168-1859889 rS10510218 rs7625240 7:6 O:O CTNNA2 chr2: 81419297-81.446082 rs4430978 rs1595071 4:3 O:O LARP7 chira: 113772340-11378.8584 rs12054518 rs769.0429 4:3 O:O ACAT1 chr11: 107497467-1075.23485 rs374.1049 rs11212S25 O:O 1:O ACCN1 chr17: 283.64218-29507938 rs28.933 rS11080254 O:O 3:1 ACTR2 chr2: 653O8405-653S1891 rs268859 rS4671124 O O:1 ADCY1 chrf:4558.0645-45729237 rS472442O rs3735666 O:O 1:1 ADRBK1 chr11: 66790668-668.10933 rs12274774 rs12274774 O O:O ALDOA chr16: 29971972-29989236 rs9928448 rs2O71390 3:8 2:6 APP chr21: 26174731-2646SOO3 rs378762O rS462281 O:O 8:2 ARL15 chrS: S3216370-53642160 rS271246 rs35947 :1 2:O ATXN7L3 chr17: 39624698-39631 OSS rs11652S16 rs11652516 :1 O:O BDKRB2 cr14: 95.74O949-95780538 rs1959053 rs2O69591 :1 O:O CA8 chr3: 61263976-61356508 rsf46O476 rs6998.745 O:O 1:O CACNA1B ch: 139892O61-14O136452 rS10867084 rS2606,358 O:O 2:2 CACYBP chr1: 17323S193-173247786 rs642S310 rs11590474 O O:O CALM1 cr14: 899.3312S-89944363 rs23OO497 rs1058903 :2 O:O CHRM3 chr1: 237616487-238116519 rS413O463 rSS36477 O:O 2:1 CIC chr19: 474806S6-47.491789 rs38267O6 rs38267O6 :1 O:O CNP chr17:37372284-3.7383280 ris80786SO rs11079028 :2 O:O CRHR1 chr17: 41217448-41268973 rS4792886 rs17763104 O O:O DISC1 chr1: 2298.291.83-23O243641 rs2O82552 rs980989 O:O 4:7 DYNLL1 chr12: 119392042-11942O681 rs606443 rS58OO16 O:O 1:O FPR1 chr19: S694O837-56946962 ris867228 rS48O1891 O:O 1:1 GAPDH cr12: 6513917-6517797 rS1060619 rS1060619 O:2 1:1 GNA1S chr19:3O87229-3114741 rs1465245 rs1637656 1:1 1:O GNAI2 chr3: SO238727-SO271790 rS11716295 rs2236944 2:4 O:O G.NAO1 chr16: S478.3648-5494.86SO rs1695.6168 rs37901.16 O:O 1:1 GNAQ cr): 7952SO10-79836O12 rS6S60613 rs1930543 1:O O:O GRIK1 chr21: 298.31124-3O234153 rs2832390 rs2255821 O:O 8:2 GRIK3 chr1: 370392OO-37272431 rSS281.37 rSS63293 1:O O:O GRM1 chrö: 146390610-146800427 rS12196.298 rs2942 O:O 7:2 GRM3 chrf: 86.111165-86332.128 rsfC)1332 rS696.7992 O:O 1:O GRMS chr11: 8788.0625-88.42O888 rs3O8884 rs/931721 4:O 3:2 GRM7 chr3: 6877926-7758217 rS6443O74 rs17047886 4:O O:O GRM8 chrf: 1258.65892-12667 OS46 rs1324OSO4 rs1324.6388 3:O 1:1 GSN cr: 1230O3581-12313494.1 rs1590345 rs306772 1:O 1:O HOMER1 chrS: 78.705541-7884S456 rS3822568 rS11948804 O:O 1:O HTR2A chr13: 463OSS13-46368.995 rs38O3189 rs6312 O:O 1:O MAPK1 chr22: 2O44394-6-2O551970 rs2298.432 rS2876981 1:O O:O MTHFD1 chr14: 63924845-63996474 rS8O11839 rs2281603 1:1 O:O MX1 chr21: 41714311-417S3008 rS4579.20 rS468811 O:O 7:2 NARG1 chira: 140442.125-140531.385 rs13147688 rs2O6(O685 1:O O:O

US 2017/000929.6 A1 Jan. 12, 2017 27

0.138. In additional studies, we have extended our previ analysis algorithms in the context of biologically relevant ous GRM network analysis of CNVs to a more comprehen expectations like developmental pathways (HOX genexes), sive network of 335 genes which show significance in cancer (Lung cancer pathway) and neuronal signaling not ADHD. The original mGluR network was generated from implicated in ADHD (GABA). The network is highly sig 271 genes, including first and second degree interacting nificant with CNVs identified in 17% of cases overall and genes as defined by the Human Interactome. The updated 9% of controls, with OR of 2.1 and P=6.5x10'7. Table 13C analysis provides more comprehensive update of the mGluR provides the names of 64 additional genes to the 271 network definition to better capture functional interactions presented (total of 335) that were identified using the of genes with GRMs. Ingenuity software. The updated analysis provides func 0.139. To generate these additional targets, the following tional interactions of genes with GRMs based on the Inge databases were employed: nuity Knowledge Base of biological interactions and func 1. The Ingenuity Knowledge Base of biological interactions tional annotations. Each Ingenuity interaction has been and functional annotations. Each Ingenuity interaction has manually created by expert Scientists with Supporting pub been manually created by expert Scientists with Supporting lications for specific interacting molecules. publications for specific interacting molecules. 2. Published GWAS results reported in the Human Genome Results from the extended CNV analysis are as follows: Epidemiology (HuGE) Navigator, which is an integrated, searchable knowledge base of genetic associations and human genome epidemiology. Dataset it genes F cases Fontrols P OR 3. Our own literature review of PubMed for interactions with Original + Ingenuity 335 0.17 0.09 6.5 x 107 2.1 mGluR genes. (ADHD GRMs)

4. Rare variants from sequencing ADHD patients not found l 8CE it.- We COOtrol) . g 9. 8. 8. in public domain (NBA Signaling 121 O.O3 O.O7 O.99 O.3 0140. These updated networks were used to analyze 1292 (-ve control) ADHD cases compared to 7449 neurologically normal con trols. Negative controls were used to validate the CNV TABLE 13C 335 Targets are listed herein below (includes the 271 targets from Table 13A). ACAT1 CACNA1A ERBB2 GRM2 MIR1236 PCBP3 PSMD11 SET TRPV1 ACAT2 CACNA1B ERBB4 GRM3 MIR1245 PCDHA4 PSMD13 SETD4 TUBA1A ACCN1 CACYBP ESR1 GRM4 MIR1246 PCMT1 PSMD6 SF3B14 TUBA1B ACCN2 CALB2 F2R GRMS MIR1252 PDCD5 PSME1 SHANK1 TUBA8 ACP1 CALM1 F2RL2 GRM6 MIR1260 PDE1B PTK2B SHANK3 TUBB ACTB CALM2 F2RL3 GRM7 MIR1262 PDE1C PXN SHBG TUBG1 ACTN1 CALM3 F3 GRM8 MIR1272 PDE6G PYGL SIAH1 TXN ACTR2 CAMK1 FGF2 GSN MIR1275 PGM1 PYGM SIM1 TYMS ADA CAMK2B FKBP3 HBXIP MIR1276 PHKB QRICH2 SLC1A2 UBE2I ADCY1 CAMK4 FLNA HOMER1 MIR1284 PHKG2 RALA SLC2A1 UBE2M ADD CASR FOS HOMER2 MIR1291 PIAS1 RANBP1 SLC6A3 UBQLN4 ADD2 CAV1 FPR1 HOMER3 MIR1305 PIAS2 RANBP9 SLC9A3R1 UCHL1 ADORA1 CAV3 FSCN1 HSP90AB1 MIR1322 PIAS4 RAP2A SLC9A3R2 VHL ADORA2A CBX7 FURIN HTR2A MIR1323 PICK1 RCC1 SNCA VIPR1 ADRA1B, CCNB1 FYN HTT MIR1324 PIK3CA RCC2 SNRPB2 YWHAQ ADRA2A CDC42 GAPDH IFNG MIR555 PIK3R1 RGS11 SOCS6 ZAP70 ADRA2C CHGB GLP1R IMPDH2 MIRS59 PLA2G7 RGS12 SOCS 7 ADRB2 CHP GLP2R IQGAP2 MIRS91 PLCB1 RGS2 SORD ADRBK1 CHRM2 GNA1S ITGB1 MIR610 PLCB3 RGS3 SRC ADRBK2 CHRM3 GNAI1 TGB7 MIR637 PLCG2 RGS4 STAU1 ALDOA CIC GNAI2 TPR1 MIR641 PLD1 RGS9BP STRAP ANXA2 CNP GNAI3 KIAAO090 MIRA 69 POMC RHOA STX12 APTX CNR1 G.NAO1 KIAA1683 MRPL14 PPIH RIF1 SUMO1 AQP COPB2 GNAQ KLHL17 MRPS16 PPM1A ROCK2 SYK ARHGAP24 CRHR1 GNB2L1 KPNA1 MTHFD1 PPM1B RPA2 TBCA ARL15 CYCS GNBS KPNA3 MTNR1A PPM1D RPLP2 TBXA2R ARNT2 CYTH2 GOPC LAMA4 MTNR1B PPM1G RPN2 TCP1 ARRB1 CYTIP GOT1 LRP2BP MX1 PPP1CC RPS1.4 TEAD3 ARRB2 DCN GP1BA LRRC59 MYO6 PPP2R1A RRM1 TFAM ATXN7L3 DHCR7 GPR26 LTA NANS PRDX1 RUWBL2 TGFB1 BDKRB1 DLST GRASP LYAR NCK1 PRKCA RYR1 TGM2 BDKRB2 DNM3 GRB7 LYN NFKBLA PRKCG RYR2 TJP1 BDNF DRD2 GRIA1 MAGI2 NMI PRLHR S1 OOA6 TK BTBD2 DRD3 GRIK3 MAP4 NPY2R PRMT1 SACS TLR1O BTG2 DSTN GRIN1 MAPK1 NR3C1 PRPSAP1 SARS TNIK C17orf24 DYNLL1 GRIP1 MAPK3 NUDC PSAT1 SCTR TPI1 C1 orf116 ECHS1 GRK4 MARK4 OPRD1 PSEN1. SDC3 TRAF2 C7orf25 EFNB2 GRKS MC4R, OPTN PSMA1 SDCBP TRMT112 CA8 EGFR GRK6 MIR1200 PAFAH1B3 PSMC1 SELE TRPC1 ACAT1 EPHB1 GRM1 MIR1207 PCBP1 PSMD1 SERPINB9 TRPC3 US 2017/000929.6 A1 Jan. 12, 2017 28

0141. In accordance with the present invention, it has ange, Conn.), Microchemistry Ltd. (Moscow, Russia), been found that 10% of patients with ADHD carry specific Otava, (Toronto, ON), PharmEx Ltd. (Moscow, Russia), types of mutations of genes that encode for metabotropic Princeton Biomolecular (Monmouth Junction, N.J.), Scien glutamate receptors (mGluRs). These mutations are sensi tific Exchange (Center Ossipee, N.H.). Specs (Delft, Neth tive and specific biomarkers for selecting and treating erlands), TimTec (Newark, Del.), Toronto Research Corp. ADHD due to defective mGluR pathways. Furthermore, the (North York ON), UkrOrgSynthesis (Kiev, Ukraine), Vitas present inventors have identified drug candidates that spe M. (Moscow, Russia), Zelinsky Institute, (Moscow, Russia), cifically activate the mGluRs, potentially restoring normal and Bicol (Shanghai, China). Combinatorial libraries are neurophysiology in ADHD patients with mutations in the available and can be prepared. Libraries of natural com GRM family of mGluR genes. See Table 1. pounds in the form of bacterial, fungal, plant and animal 0142 For example, compounds which may be adminis extracts are commercially available or can be readily pre tered in implementing the test and treat paradigm described pared by methods well known in the art. It is proposed that herein include the piracetam family of nootropic agents, as compounds isolated from natural sources, such as animals, described in F. Gualtieri et al., Curr. Pharm. Des., 8: 125-38 bacteria, fungi, plant sources, including leaves and bark, and (2002). More preferably, the treating agent is a pyrogluta marine samples may be assayed as candidates for the mide. Details regarding the preparation and formulation of presence of potentially useful pharmaceutical agents. It will pyroglutamides which may be used in the practice of this be understood that the pharmaceutical agents to be screened invention are provided in U.S. Pat. No. 5,102,882 to Kimura could also be derived or synthesized from chemical com et al. A particularly preferred agent for the treatment of positions or man-made compounds. ADHD in patients determined to have one or more of the 0146 For example, the neuronal cells can be incubated in SNPs indicative of the presence of an ADHD-associated the presence and absence of a test compound, such as copy number variation, as set forth in Table 13, is (+)-5- pyroglutamides (see, e.g., U.S. Pat. No. 5,102.882) and other oxo-D-prolinepiperidinamide monohydrate (NS-105). members of the piracetam family of nootropic agents, after which the effect of the compound on glutamate signaling is Example III assessed. Agents so identified could then be tested in whole 0143. The above-identified CNV containing mGluR animal models of ADHD to assess in vivo efficacy. genes involved in ADHD pathogenesis also provide novel 0147 Agents identified using the screening assays targets for the development of new therapeutic agents effi described herein are also encompassed by the present inven cacious for the treatment of ADHD. To that end, methods of tion. screening of candidate drug (agent or compound) that modu lates mGluR protein interactions and associated pathology Discussion can be performed based on the information provided herein. 0.148. At present, there is a notable paucity of genome Representative candidate drugs include nucleic acids, poly wide association studies in ADHD, and no study has peptides, Small molecule compounds and peptidomimetics. reported CNVs that are significantly associated with ADHD. 0144. In some cases, genetic agents can be screened by AS Such, our study represents the first large-scale, unbiased contacting the yeast cell with a nucleic acid construct coding two-stage genome-wide scanning of CNVs in ADHD. for a gene. For example, one may screen cINA libraries Although we have previously reported GRM5 deletion in a expressing a variety of genes, to identify other genes that single ADHD family with three affected children and one modulate such interactions. For example, the identified family with GRM7 deletion, along with 57 other non-GRM drugs may modulate glutamate associated neuronal signal receptor genes, most of which were single events', the ing, Subcellular protein localization and/or neuronal cell genes from the metabotropic glutamate receptor family morphology or viability. Accordingly, irrespective of the (GRM5, GRM7, GRM8 and GRM1) are for the first time exact mechanism of action, drugs identified by the screening shown to be impacted by CNVs that significantly associate methods described herein are expected to provide therapeu with ADHD and observed to replicate in multiple indepen tic benefit to patients suffering from ADHD. dent case control data sets. 0145 Suitable screening methods may employ a variety 0149 Metabotropic glutamate receptors (GRMs or of neuronal cell types obtainable from the ATCC. Candidate mGluRs) are a class of G-protein-coupled receptors that drugs can be screened from large libraries of synthetic or possess a seven transmembrane region involved in the natural compounds. One example is an FDA approved modulation of excitatory synaptic transmission in the ner library of compounds that can be used by humans. In vous system'. There are three receptor groups based on addition, compound libraries are commercially available sequence homology, putative signal transduction mecha from a number of companies including but not limited to nisms, and pharmacologic properties'. GRM5 and GRM1 Maybridge Chemical Co. (Trevillet, Cornwall, UK), Com are members of Group I expressed particularly in the basal genex (Princeton, N.J.), Microsource (New Milford, CT), ganglia and cerebellum', relevant brain areas for ADHD. Aldrich (Milwaukee, Wis.), AKos Consulting and Solutions These receptors have been shown to activate phospholipase GmbH (Basel, Switzerland), Ambinter (Paris, France), C and it has been postulated they may play a role in Asinex (Moscow, Russia), Aurora (Graz, Austria), BioFocus addiction, anxiety and behavioral disorders'". GRM7 and DPI, Switzerland, Bionet (Camelford, UK), ChemBridge, GRM8 are members of Group III which is linked to the (San Diego, Calif.), ChemDiv, (San Diego, Calif.), Chemi inhibition of the cyclic AMP cascade. GRM7 has been cal Block Lt. (Moscow, Russia), ChemStar (Moscow, Rus linked with anxiety' and is the most highly conserved of all sia), Exclusive Chemistry, Ltd (Obninsk, Russia), Enamine mGluR subtypes across different mammalian species'. (Kiev, Ukraine), Evotec (Hamburg, Germany), Indofme 0150. Evidence for glutamatergic involvement in ADHD (Hillsborough, N.J.), Interbioscreen (Moscow, Russia), is arising from diverse fields. While association studies Interchim (Montlucon, France), Life Chemicals, Inc. (Or investigating variants in glutamatergic receptors and trans US 2017/000929.6 A1 Jan. 12, 2017 29 porters have reported mixed results' a genome-wide and not observed in controls. Assessment of the mother association study investigating response to the methylpheni using an adult ADHD Self-Report Scale' indicated a likeli date in ADHD children detected nominal evidence for hood of ADHD. association of several SNPs including SNPs within GRM7 0153. There are eight CNVRs presented that directly (rs3792452). GRIN2A was reportedly associated with disrupt the respective gene in these regions (including ADHD in a genetic linkage study” and GRIN2B was GRM5, GRM7, GRM8, GRM1 NEGR1, DPP6, SGTB/ associated by TDT. Magnetic resonance spectroscopy NLN and LARP7) while the remainder are annotated with studies have shown increased glutamatergic tone in frontal the closest (Table 3A and 3B). Furthermore, GRM8, GRM1 and striatal brain regions of ADHD subjects which SGTB/NLN, and LARP7 CNVs are exonic. Further func normalizes with stimulants and atomoxetine’. The SLC6A3 tional studies to fully characterize the function of the asso KO (DAT-KO) mouse, an ADHD animal model, remains ciated genes in relation with the ADHD phenotypes will be responsive to methylphenidate in spite of the lack of a conducted. Thus, our unbiased approach to assess the entire dopamine transporter' and hyperactivity in these mice can genome in multiple independent cohorts has revealed CNVs be increased by NMDA-receptor blockers and suppressed by in novel genes that have not previously been studied for any drugs that increase glutamatergic transmission'. Increased potential biological or physiological impact on the brain in midbrain SLC6A3 and DRD4 expression were reported in ADHD and await further characterization. rats where glutamate transporter increases were found in the 0154) Given the significance of the four GRM receptor striatum suggesting that decreases in dopamine may alter genes reported in ADHD pathogenesis and the rarity of glutamate signaling. Also, glutamate receptor Subunit gene CNVs at each of the loci, we elected to evaluate GRM (GRIN2A) disruption increased DA and serotonin metabo receptor interacting genes for their frequency of CNV obser lism in the frontal cortex and striatum of mice, and increased Vations in cases and controls. This allows for inclusion of locomotor activity that was reduced by dopamine or sero marginally significant loci given the prior knowledge of tonin receptor antagonists. Moreover, dysregulated robust association of the GRM receptor gene family. CNVs expression of genes in glutametergic pathways has been are often very rare (<1%) at a given locus but their asso observed in the SHR7 and in the PCB exposed rat model ciations provide stronger direct correlation to the disease of ADHD. Increased levels of glutamate have been state than common variants as evidenced by their impressive reported in the neurometabolism of ADHD brains, suggest effect sizes (see ORs in Table 3). Based on individually ing that altered glutamate transmission may be important in significant loci alone, 3.66% of ADHD cases are strongly ADHD. Although the glutamate receptors that associated correlated with the CNVs discovered. By extending the with ADHD in our study were deleted in three instances and observations from the confident GRM family to gene net duplicated in one instance, the resulting perturbations in works of GRM receptor interacting/signaling genes provides glutamate signaling in the deleted cases could promote 9.94% of ADHD cases with genetic characterization of their ADHD through a feedback loop releasing additional gluta disease after adjusting for control frequency (i.e., net impact in cases). Major supporting hubs of this network include mate in an attempt to compensate for the disparity of sent TNIK", GNAQ', and CALM1 (FIG. 11), previously and received neurotransmission signals. associated with Schizophrenia and epilepsy. Interestingly, 0151. Apart from the GRM family of genes, we have the GRM receptor network gene, GRIK1, has also been detected association of eight other loci with ADHD, four of associated with hyperactive/impulsive symptoms of which directly impact genes (Table 3B). Among those are ADHD. genes with intriguing biology with respect to ADHD. DPP6 0155 Taken together, our analysis of CNVs and func has been previously associated with Amyotrophic Lateral tional enrichment of the GRM receptor gene interaction Sclerosis (ALS) in genome wide association studies, network Suggests that GRMs do not form a large number of and CNVs impacting DPP6 have been reported in relation interactions, but serve to coordinate functional modules of with autism'. DPP6 and CTNNA2 (although our associa other sets of genes. Encouragingly, some of these modules tion does not directly impact CTNNA2) have been impli are important in the process of synaptic transmission, neu cated by earlier ADHD SNP genotype GWAS. NLN is an rogenesis, and other neuronal processes thought to be defec interesting candidate responsible for metabolic inactivation tive in ADHD. Thus, through network analysis of CNVs of neural peptides, such as Neuropeptide Y (NPY) which has impacting ADHD, we have identified modules that are previously been implicated in ADHD. SLC7A10 has important in processes such as RNA binding, processing, been shown to play a role in the modulation of glutamatergic and alternative splicing, which have been shown to influence transmission through mobilization of D-serine at the gluta brain-specific synaptic activity'. Also, we have identified matergic synapse. LARP7 is important for snRNP integrity, functional modules involved in ubiquitination, a process that a protein complex responsible for post transcriptional splic we have previously linked to autism', which shares certain ing. NEGR1 encodes a neural cell adhesion molecule and a phenotypic features with ADHD. Furthermore, abnormal trans-neural growth-promoting factor in regenerative axon functional brain connectivity is a candidate factor in devel sprouting and neuronal growth in the mammalian brain. opmental brain disorders associated with cognitive dysfunc Interestingly, this neuronal gene was recently associated tion, including ADHD. Thus, the impact CNVRs among with obesity'. the GRM family of receptors, and in particular GRM5 and 0152. In the CHOP discovery cohort, Family 230 is GRM7, may be important to the underlying molecular impacted with both GRM5 deletion inherited from the etiology of ADHD. mother and NEGR1 duplication inherited from the father in 0156. In conclusion, using a two-stage genome-wide all three ADHD cases in the family. In spite of superior IQ association approach for high-resolution CNV detection, we levels these 3 children had severe impairment. These were have identified 12 loci demonstrating enrichment of CNVs the only CNV regions observed in all three familial cases in ADHD cases as compared to controls, and Successfully US 2017/000929.6 A1 Jan. 12, 2017 30 replicated 4 of them using independent data sets of ADHD defects of which are thought to underlie ADHD and other cases and healthy controls genotyped on three different neurodevelopmental disorders. Therefore, the enrichment of platforms matched for cases and controls. Four of the genes genes within this molecular system for CNVs associated affected belong to the metabotropic glutamate receptor fam with ADHD suggests novel susceptibility mechanisms for ily. The network of over 200 genes interacting with gluta the disease and will spur assessment of additional variations, mate receptors are collectively impacted with CNVs and including structural variations and single-base changes in capture the genetic diversity of approximately 10% of all candidate genes within these molecular networks. Our ADHD cases. Furthermore, this network of genes interacting results call for expression and other functional assays to with the metabotropic glutamate receptors defines a set of assess the biological effects of CNVs in these candidate functional modules with significant neuronal functions, genes. TABLE 1.4 ADHD CNV Family Based Transmission Disequilibrium and de novo Statistical Tests. A) Illumina CHOP Deletions Enriched for Inheritance Count de novo Parel CNVR SNPs TDTDe InDe Del Notnh Gene Distance

chr18: 74258734-74260996 3 O.OO1953 9 O O SALL3 58O267 chirf: 12009238S-120099982 3 O.OO1953 9 O O KCND2 O chira: 924.99956-92502794 8 O.OO1953 9 O O KIAA1680 O chr11: 697SSS29-69759313 12 O.OO7813 7 O O FADD 2439S chr4: 4240O885-42403451 15 O.OO7813 7 O O ATP8A1 47238 chrS: 104463O47-104518786 17 O.OO7813 7 O O NR OOOO39 O chr13: 69637654-69666685 18 O.O15625 6 O O NR 002717 25969 chr3: 195971510-195982215 5 O.O312S 5 1 O FAM43A 80455 chr19:44369918-44376749 3 O.O312S 5 1 O LOC342897 2695 chr1: 2349841-2356176 4 O.O312S 5 1 O PEX10 15971 chr21: 45777720-45782727 3 O.O312S 5 O O SLC19A1 O chr10: 67748487-67785209 30 O.O312S 5 O O CTNNA3 O B) Illumina CHOP Duplications Enriched for Inheritance Count de novo ParDup CNVR SNPs TDTDup InhDup Dup Notnh Gene Distance

chr2O: S9015708-59022667 4 O.OO7813 7 O O CDH4 238287 cr12: 728O8323-72832667 5 O.O15625 6 O O BCO61638 O chr6: 73021641-73O23171 3 O.03125 5 O O RIMS1 O chr17: 740899O3-74.106,726 9 O.03125 5 O O DNAHL.1 10904 chr1: 9243828-9310031 22 O.03125 5 O O H6PD, O SPSB1 C) Illumina CHOP Deletions Enriched for de novo Count de novo de novo Parel CNVR SNPs TDTDe InDe Del Notnh Gene Distance

chr16: 87694595-87778383 16 3.02E-OS 32 2 21 AX748415, O CDH15, LOC197322 chr18: 65358832-65367619 18 3.02E-OS 33 2 21 DOK6 O cr12: SS90228O-SS923.860 3 O.OOO367 9 3 19 NDUFA4L2, O NXPH4, SHMT2, STAC3 chr17: 711.12486-7112O734 4 O.OO1848 12 3 16 KIAA1783 O chr22: 38384374-38403731 8 O.O18158 4 4 13 CACNA1I O chr19: 15992679-15997923 2 0.025875 15 6 1S LOC126536 O D) Illumina CHOP Duplications Enriched for de novo Count de novo de novo ParDup CNVR SNPs TDTDup InhDup Dup NotInh Gene Distance

chr19: 59423491-594281.32 12 4.85E-09 74 3 38 LILRB3, O LIR-3 chr3: 145217675-145247517 4 3.OSE-OS 19 O 15 CYC1, O MAF1, SHARPIN, SIPL1A chr18: 648971.88-64906488 48 O.OOO122 9 O 13 CCDC102B 23782 US 2017/000929.6 A1 Jan. 12, 2017 31

TABLE 14-continued ADHD CNV Family Based Transmission Disequilibrium and de novo Statistical Tests. cr14: 104225150-104339273 3S O.OO293 7 1 11 ADSS, O ADSSL1, AKT1, SIVA1 ch9: 1386,06913-138647195 17 O.OOS371 10 1 1 O AF161442 15 688 chr16: 65O2S6-2O28586 41 O.O15625 8 O 6 Many O chr2O: 61642713-61668792 11 O.O312S 4 1 7 C20orf195, O PRIC285, SRMS chr16: 87399.730-8743OO19 22 O.O312S 7 1 7 APRT, O CDT1, FLJOO319, GALNS chr16: 3553OOS-3590430 2O O.O312S 8 O 5 BTBD12, O NLRC3 chr22: 17257787-17355.587 60 O.O312S 3 O 5 DGCR6, O KIAA1647, PRODH E) Perlegen IMAGE Deletions Enriched for Inheritance Count de novo Pardel CNVR SNPs TDTDe InDe Del NotInh Gene Distance cr: 180271795-180274556 5 O.OO3204 2 1 13 ZNF533 O chr14: 79919894-7992.4934 5 O.O312S 1 O 7 BCO39670 O chrif: 19828746-1984.0916 7 O.O41656 4 O 11 MGC42090 4900S F) Perlegen IMAGE Duplications Enriched for Inheritance Count de novo ParDup CNVR SNPs TDTDup InhDup Dup NotInh Gene Distance chr2: 17361563-1736902O 3 O.O15625 6 O O CR623368, O KIAA1647 chr15: 30O88094-30090949 3 O.03125 5 1 O CHRNA7 19069 chirf: 71664963-71712O86 5 O.03125 5 O O MGC87315 O G) Perlegen IMAGE Deletions Enriched for de novo Count Denovo de novo Parel CNVR SNPs TDTDe InhDel Del Notnh Gene Distance cr: 180271795-180274923 6 O.OOO854 2 1 13 ZNF533 O chr10: 854.45139-854.46804 7 O.O312S 5 1 7 GHITM 442361 H) Perlegen IMAGE Duplications Enriched for de novo Count Denovo de novo ParDup CNVR SNPs TDTDup InhDup Dup NotInh Gene Distance cr12: 31276361-3128SO14 9 6.87E-OS 15 1 17 OVOS2 26OO6 chr10: 47089854-47154881 31 6.87E-OS 11 1 17 AKOSA316 O chrif: 140018-162903 13 O.OOS371 10 1 10 AL1376SS 23.529 chr3: 24371.97-2492653 23 O.O312S 4 1 7 BC045738 O chr6: 168234697-168295618 13 O.043945 5 2 8 FLOO181 9639

TABLE 1.5 ADHD CNV Family Based Transmission Disequilibrium and de novo Statistical Tests. de novo de novo de novo Parel de novo ParDup CNVR (hg18/B36/March 2006) Type TDTDel TDTDup. TDTDel TDTDup InhDel Del NotInh InhDup Dup Notinh chrif: 126441593-126621501 Del 1 1 1 1 O chr11: 88269449-88351661 Del O.12S 1 O 1 3 chr3: 7183953-7197236 Del O.25 1 1 1 2 chr6: 146657076-146694047 Dup 1 1 1 1 O chirf: 153495598-153564827 Dup O.2OS 1 O.O16 1 4 chrS: 65027976-6SO46S2O Del 1 O.S 1 1 O chr1: 560S3497-56064495 Del 1 1 1 1 O chr1: 72317292-72328.395 Dup 1 1 1 1 O US 2017/000929.6 A1 Jan. 12, 2017 32

TABLE 15-continued ADHD CNV Family Based Transmission Disequilibrium and de novo Statistical Tests. de novo de novo de novo Parel de novo ParDup CNVR (hg18/B36/March 2006) Type TDTDel TDTDup. TDTDel TDTDup InhDel Del NotInh InhDup Dup Notinh chr19:38427720-3844.4834 Del O.183 1 O.004 1 6 O 8 O O O chr3: 1844168-1859889 Del O.063 1 O 1 4 O O O O O chr2: 81419297-81446082 Dup 1 O.S 1 1 O O O 1 O O chira: 113772340-113788584 Dup 0.375 1 O.S 1 2 O 1 O O O

TABLE 16 Sample Source Contributions to Impacting CNV Loci. Per Per CHOP CHOP NIMH. Utah IMAGE Psoriasis Depression PUWMa CNVR Cases Controls C3SS C3SS C3S(S Control Control Cases chr11: 88269449-88351661 4 O O O 5 O O 1 chrif: 126441593-126621501 3 O O O 3 O O 2 chr3: 7183953-7197236 4 O O O 2 O O O chr6: 146657076-146694047 5 2 1 1 O O O O chr1: 72317292-72328.395 4 O O O O O O 1 chirf: 153495598-153564827 5 O 1 O O O O 2 chrS: 65027976-6SO46S2O 4 O O O 1 O O O chr1: 560S3497-56064495 2 O O O 3 O O O chr19:38427720-3844.4834 5 2 O O 1 O O 1 chr3: 1844168-1859889 4 O O O O O O 2 chr2: 81419297-81446082 2 O O O 1 O 3 O chira: 113772340-113788584 2 O O O 1 O O 1

SAGE AGRE IMAGE IMAGE Illumina Afly 5.0 PUWMa II II 1M Parents CNVR Parents Cases Controls Controls Controls Type Gene chr11: 882694.49-88351661 1 O O O O De GRMS chirf: 126441593-126621501 O O O O O Del GRM8 chr3: 7183953-7197236 O O O O O De GRM7 chr6: 146657076-146694047 O 1 O O O Dup GRM1 chr1: 72317292-72328.395 O O O O O Dup NEGR1 chirf: 153495598-153564827 O O 1 O 1 Dup DPP6 chrS: 65027976-6SO4652O O 1 1 O O De SGTBjNLN chr1: S6053497-56064.495 O 1 O O 2 De USP24 chr19:38427720-3844.4834 3 O O O O Del SLC7A10 chr3: 1844168-1859889 2 1 1 4 1 De CNTN4 (inh) chr2: 81419297-81446082 O 1 O O O Dup CTNNA2 chira: 113772340-113788584 1 O O 1 1 Dup LARP7

TABLE 17

Boundaries of Individual CNVs in Table 1A and 1B. Sample Exon Validation CNVR Gene Type Sample ID Region Called in Sample Distance Run chr11: 88269449-88351661 GRMS Del 230-3 chr11: 882694.49-88351661 5,858 Y chr11: 88269449-88351661 GRMS Del 230-4 chr11: 882694.49-88351661 5,858 Y chr11: 88269449-88351661 GRMS Del 230-5 chr11: 882694.49-88351661 5,858 Y chr11: 88269449-88351661 GRMS Del 497 cr11: 838.76556-9.1038751 O Y chr11: 88269449-88351661 GRMS Del 16794 cr11: 87996654-88837360 O Y chr11: 88269449-88351661 GRMS Del 13304 cr11: 88109331-88827923 O Y chr11: 88269449-88351661 GRMS De 13270 chr11: 88115425-88.481107 O Y chr11: 88269449-88351661 GRMS Del 13761 cr11: 88.305340-883.85387 O Y chr11: 88269449-88351661 GRMS De 1758O cr11: 88.305340-883.85387 O N chr11: 88269449-88351661 GRMS Del M.OfM.CS. 604401 cr11: 88.3246.15-88342595 14,924 Y chirf: 126441.593-126621SO1 GRM8 Del 1953313026. A curf: 1265.32786-1265362O2 O Y chirf: 126441.593-126621SO1 GRM8 Del 1965.040688. A curf: 1264636O2-126478OSO 54,536 Y chirf: 126441.593-126621SO1 GRM8 Del 4011452014. A curf: 1265.32786-1265362O2 O Y chirf: 126441.593-126621SO1 GRM8 Del 14125 curf: 1256.60695-126036276 O N US 2017/000929.6 A1 Jan. 12, 2017 33

TABLE 17-continued

Boundaries of Individual CNVs in Table 1A and 1B. Sample Exon Validation CNVR Gene Type Sample ID Region Called in Sample Distance Run chirf: 126441.593-126621SO1 GRM8 De 16794 curf: 1256.60695-126036276 O N chirf: 126441.593-126621SO1 GRM8 De 11804 curf: 125679479-125937528 O N chirf: 126441.593-126621SO1 GRM8 De 987314 curf: 126SO36O2-126S 63602 O Y chirf: 126441.593-126621SO1 GRM8 De 98.7124 chrf: 1264636O2-1266O3602 O Y chr3: 7183953-7197236 GRM7 De 2O2334O146 chr3: 70531.79-7144453 18,686 Y chr3: 7183953-7197236 GRM7 De O68-3 chr3: 71839S4-7197236 20,599 Y chr3: 7183953-7197236 GRM7 De O68-4 chr3: 71839S4-7197236 20,599 Y chr3: 7183953-7197236 GRM7 De 4079019863. A chr3: 71839S4-7197236 20,599 Y chr3: 7183953-7197236 GRM7 De 11891 chr3: 6979874-7003319 101,280 Y chr3: 7183953-7197236 GRM7 De 11923 chr3: 6980446-7001696 101,852 Y chr6: 146657O76-146694047 GRM1 Dup. 388-3 chr6: 146657077-14667SS11 O Y chr6: 146657O76-146694047 GRM1 Dup 387-3 chr6: 146657077-14667SS11 O Y chr6: 146657O76-146694047 GRM1 Dup. 386-3 chr6: 146657077-14667SS11 O Y chr6: 146657O76-146694047 GRM1 Dup 4301.337678 RO2CO1 chr6: 146657077-146675511 O Y chr6: 146657O76-146694047 GRM1 Dup 430591.0011 RO1CO2 chré: 146657077-146675511 O Y chr6: 146657O76-146694047 GRM1 Dub 1181 chr6: 146657077-146694047 O Y chr6: 146657O76-146694047 GRM1 Dup 83158 chr6: 146657077-146694047 O Y chr6: 146657O76-146694047 GRM1 Dup b3 SF O181 chrö: 146685878-1467O1196 13,883 Y chr1: 72317292-72328.395 NEGR1 Dup 230-3 cr1: 72317292-72328.395 10,621 Y chr1: 72317292-72328.395 NEGR1 Dup 230-4 cr1: 72317292-72328.395 10,621 Y chr1: 72317292-72328.395 NEGR1 Dup 230-5 cr1: 72317292-72328.395 10,621 Y chr1: 72317292-72328.395 NEGR1 Dub, TD2O7.1 cr1: 71648994-73O2SO13 O Y chr1: 72317292-72328.395 NEGR1 Dub M.Of...M.CS.63O8601 cr1: 72322424-72328.395 10,621 Y chirf: 153495598-1535648.27 DPP6 Dup 332-3 curf: 153495598-153578582 54,698 Y chirf: 153495598-1535648.27 DPP6 Dup 4079019863. A curf: 153495598-153564827 68,453 Y chirf: 153495598-1535648.27 DPP6 Dup 4193372403 B curf: 153495598-153554.210 79,070 Y chirf: 153495598-1535648.27 DPP6 Dup 4243114113 RO1CO2 chr7: 153495598-153577484 55,796 Y chr7: 153495598-153564827 DPP6 Dub 1135 chr7: 153495598-153576455 56,825 N chirf: 153495598-1535648.27 DPP6 Dub 82O1671744 curf: 153118878-153338.318 O Y chirf: 153495598-1535648.27 DPP6 Dub W.OfF.CS.140002 curf: 1535O2896-153517548 115,317 Y chirf: 153495598-1535648.27 DPP6 Dub W.Of...M.CS.234002 chr7: 153545279-153559377 73,903 Y chrS: 65027976-6SO46S2O SGTB NLN De O67-3 crS: 65027976-65046S2O O Y chrS: 65027976-6SO46S2O SGTB NLN De 17-3 crS: 65027976-65046S2O O Y chrS: 65027976-6SO46S2O SGTB NLN De 52-3 crS: 65027976-65046S2O O Y chrS: 65027976-6SO46S2O SGTB NLN De 670639198. A crS: 65027976-65046S2O O Y chrS: 65027976-6SO46S2O SGTB NLN De S962 crS: 64.483534-651O1307 O Y chrS: 65027976-6SO46S2O SGTB NLN De b11 SF 1055 crS: 65020291-6SO3OSO3 3,236 Y chr1: 560S3497-56064495 USP24 De 4147907208 B cr1: 560S3497-56064.495 80,234 Y chr1: 560S3497-56064495 USP24 De 393-3 cr1: 560S3497-56064.495 80,234 Y chr1: 560S3497-56064495 USP24 De 1411 chr1: 5604O939-561324O1 67,676 Y chr1: 560S3497-56064495 USP24 De 1804 cr1: S604O939-56263366 67,676 Y chr1: 560S3497-56064495 USP24 De 1727 cr1: 560S3497-56064840 80,234 Y chr1: 560S3497-56064495 USP24 De b2 SF OO94 cr1: S605121 S-S6057576 77,952 Y chr19:3842772O-384.44834 SLC7A10 De 20-3 chr19:3841.5546-3844.4834 6,998 Y chr19:3842772O-384.44834 SLC7A10 De 224-3 chr19:3841.5546-3844.4834 6,998 Y chr19:3842772O-384.44834 SLC7A10 De 305-3 chr19:38415545-38434210 6,997 Y chr19:3842772O-384.44834 SLC7A10 De 34-4 chr19:3841 8216-3844.4834 9,668 Y chr19:3842772O-384.44834 SLC7A10 De 68-3 chr19:38423641–3844.4834 15,093 Y chr19:3842772O-384.44834 SLC7A10 De 1931 cr19:38427721-384S5315 19,173 Y chr19:3842772O-384.44834 SLC7A10 De W.Of..F.CS.121001 chr19:38423391-38442154 14,843 Y chr3: 1844168-1859889 CNTN4 De O78-3 chr3: 1273990-1859889 O Y chr3: 1844168-1859889 CNTN4 De O78-4 chr3: 1273990-1859889 O Y chr3: 1844168-1859889 CNTN4 De 41-3 chr3: 1756625-1928413 187,137 Y chr3: 1844168-1859889 CNTN4 De 77-3 chr3: 18441 68-1936623 178,927 Y chr3: 1844168-1859889 CNTN4 De M.OfF.CS.S.3701 chr3: 1793.056-1956567 158,983 Y chr3: 1844168-1859889 CNTN4 De U.OfF.CS.852301 chr3: 1835561-1852134 263,416 Y chr3: 1844168-1859889 CNTN4 De b3 SF O253 chr3: 17971 O2-193OO71 185,479 Y chr2: 81419297-81.446082 CTNNA2 Dup 34-4 chr2: 81 035643-81654296 O Y chr2: 81419297-81.446082 CTNNA2 Dup 44-3 chr2: 81 035643-81654296 O Y chr2: 81419297-81.446082 CTNNA2 Dup 1484 chr2: 81419297-81.446082 152,417 Y chr2: 81419297-81.446082 CTNNA2 Dup b10 SF 0900 cr2: 81352586-81386.102 85,706 Y chira: 113772340-113788584 LARP7 Dup 3O3-3 chira: 113744172-113798.058 O Y chira: 113772340-113788584 LARP7 Dup, 314-3 chira: 113744172-113798.058 O Y chira: 113772340-113788584 LARP7 Dup 71.90 chira: 113772340-113788584 O Y chira: 113772340-113788584 LARP7 Dup, M.FaM.CS.63OOSO3 chira: 113769438-1138O17SS O Y

*exon distance of 0 indicates that exon is impacted by the CNV sample not available for qPCR validation (sample visually validated in Bead Studio).

US 2017/000929.6 A1 Jan. 12, 2017

PICK1 O:3 O:1 US 2017/000929.6 A1 Jan. 12, 2017 36

TABLE 19 Gene clusters based on the network of interacting genes Cluster # Genes 1 SET, HNRPA3, RRM1, SORD, PSMC1, MTHFD1, CACYBP, PCBP1, TXNL2, 40425, SARS, PCID1, GSN, PSMD6, TBCA, MRPS16, RCC2, COPB2, RANBP1, PRMT1, ANXA2, FSCN1, RCC1, ACAT1, NUDC, EIF3S3, UCHL1, FKBP3, PDCD5, ACTR2, PSAT1, LYAR, PCBP3, 4, LRRC59, ACP1, ACAT2, RUVBL2, GPR26, MAPK1, CYCS, 1082, STRAP, RAP2A, IMPDH2, ACTR2, PSMD1, SETD4, TRMT112, CMPK, MRPL14, SNRPB2, TEAD3, TMEM4, TFAM, DSTN, PRPSAP1, KIAAO090, PPIH, PSMA1, RPS14, DHCR7, PSMD13, TRAF2, K, RPN2, TYMS, NCK1, NANS, NARG1, PPP2R1A, ECHS1, GOT1, 2 GRB7, PYGL, CRHR1, PDE10, CALM1, GLP1R, PYGM, PHKG2, PTHR2, PDE1B, GLP2R, ADD2, ADCY1, SCTR, PHKB, VIPR1, ADD1, PGM1, PGM1, IQGAP2 3 HBXIPS OOA6, TXN, SLC2A1, CAMK1, RAB2, PCDHA4, QRICH2, GAPDH, BTBD2, PAFAH1B3, SERPINB9, PSMD11, PRDX1, RPA2, CAMK2B, LAMA4, ARL15, TPI1, CAMK4, TK1, FYN, PGM1, ACTB, CHP 4 SLC6A3, UBQLN4, PRLHR, PICK1, CIC, APTX, ERBB2, ATXN7L3, ACCN2, AQP1, GRLA1, ACCN1, ECHS1, SACS, BTG2, LRP2BP, PRKCA 5 RALA, CDC42, DRD3, ITGB1, ITGB7, TLR10, HSP90AB1, TJP1, FURIN, VHL, MTNR1B, PSEN SHBG, DCN, F3, GRIK3, GP1BA, RHOA, SELE, DRD2, ARHGAP24, MTNR1A, FKBP3, ARRB2, GRM8 6 NPY2R, RGS12, GNAI3, ADRA2C, GNAI2, GNAO1, CACNA1B, GNAI1, GRM6, IL8RB, PLCB3 7 ADRA2A, PDE6G, SRC, MC4R, ARRB1, SNCA, RPLP2, FPR1, BDKRB2, ADRBK1, OPRD1 8 PLCB1, TXNDC4, ITPR1, CCNB1, LYN, CA8, PLCG2

10 GNB2L1, CNP, STAU1, CHGB, PSME1, SOCS7, DLST, ALDOA, SYK, SDC3, TUBB, TGM2, HD, MARK4, MAP4, MX1, TUBA1A, SOCS6, C7orf25, PLA2G7 11 HOMER1, STX12, CENTG1, RYR2, LOC653098, HOMER3, C1orfl 16, SHANK1, RYR1 12 CNR1, GNA15, CHRM2, ADRB2 13 DYNLL1, PIK3R1, NMI, TUBA2, PXN, TUBG1, NFKBIA, TUBA1B, YWHAQ 14 HRPT2, RIF1, GRM3 15 CALM3, GRM5, MYO6, KIAA1683, GRM7, LOC642393, C17orf14, CALM2 16 CALB2, TCP1, LTA, TUBA1, ZAP70 17 ADA, ADORA1

Additional analyses were preformed using an Agilent com “source' and the “target of a biological signal i.e. “GRM pp parative genomic hybridization (CGH) array. The mGluR X”, “X pp GRM”, “X pp Y, and “Y pp X. network genes are defined by both forward and reverse 0158 An Agilent comparative genomic hybridization protein protein interactions using experimental data derived (CGH) array with 173,997 genomic probes was created to from a variety of experimental protocols including yeast 2 capture the GRM gene network. Agilent SurePrint G3 Cus hybrid assays and mass spectrometry. The merged human tom CGH Microarrays Custom 4x180K were used to assay interactome combines human interactions reported in 150 ADHD subjects. The protocol version was named “CGH 1100 Jul11 released Jul. 1, 2011. CNVs were called IntAct, DIP. BIND and HPRD, in addition to papers by Rual using Agilent Cytogenomics Software. CNVs overlapping et al. Nature 437, 1173-1178 (2005); Stelzl et al. Cell 122, ADHD GRM network genes were extracted from the CNV 957-968 (2005); Ramani et al. Genome Biol. 2005; 6(5): calls. Many of these genes have already been listed in the R40. Epub 2005 Apr. 15: Venkatesan Nat. Methods 6, 83-90 tables above. 276 GRM network genes were listed previ (2009); and Yu et al. Nat. Methods 8, 478-480 (2011). ously based on forward interactions. We now have 868 GRM 0157. The original forward entries only considers GRM network genes. The previous CNV observation counts were as the “source' of and gives other genes which are “targets” based on Illumina 550k SNP microarray data. The data i.e. “GRM pp X’ and “Xpp Y” (where pp is protein protein presented in Tables 20, 21 and 22 is based on Agilent CGH interaction). Forward and reverse includes GRM as both the data.

CNVCall (hg19) Num Probes Length CN SampleID GRMNetworkGenes

chr10: S4O16O76-54O18132 8 2056 3 S4OO43624 PRKG1 chr10: S4O15519-S4O18132 10 2613 3 634992689 PRKG1 chr12: 14094648-14095317 4 669 3 634992689 GRIN2B chr19: 3153225-3154967 3 1742 3 634992689 GNA15 chr10: S32O3699-S321.1914 28 8215 1 706896538 PRKG1 chr3: 7400107-74O1986 7 1879 1 706896538 GRM7 chr15: 67868548-678692SS 4 707 1 706896538 MAP2KS chr15.45318599-45323810 14 S211 1 777193129 SORD chr10: S4O15852-54O17933 8 2081 1 1386,063997 PRKG1

US 2017/000929.6 A1 Jan. 12, 2017 49

7900HTreal-time PCR system (Applied Biosystems). Tem (0170 4. Haberstick B C, Timberlake D, Hopfer C J, plates were cDNA synthesized from total RNA and random Lessem J. M. Ehringer M A. Hewitt J. K. Genetic and primer by using the RT cDNA synthesis kit (Applied Bio environmental contributions to retrospectively reported systems, Catil 4374867). RNA was isolated from the cells DSM-IV childhood attention deficit hyperactivity disor cultured in RPMI 1640 media containing 10% FBS. der. Psychol Med 38, 1057-1066 (2008). 0171 5. Wang K. Zhang H. Ma D, et al., Common Flow Cytometry. genetic variants on 5p14.1 associate with autism spectrum 0164. Cells were fixed with paraformaldehyde and disorders. Nature 459, 528-533 (2009). stained with specific mGluR5 monoclonal antibody (R&D, 0172 6. Franke B. Neale B M, Faraone S V. Genome Catil MAB4514) or isotype matched control antibody wide association studies in ADHD. Hum Genet. doi: (R&D, Catil MAB002) followed by phycoerythrin-conju 10.1007/s00439-009-0663-4 (2009). gated anti-mouse antibody. Stained cells were analyzed by (0173 7. Neale BM, Lasky-Su J, Anney R. etal. Genome using BD FACS Calibur flow cytometer. wide association scan of attention deficit hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet 147B, Results 1337-1344. (2008). (0174 8. Lasky-Su J, Neale B M, Franke B et al., (0165 Cells of each subject were divided into three Genome-wide association scan of quantitative traits for samples. One of them was used as background control with attention deficit hyperactivity disorder identifies novel staining, and two of them were stained by mGluR5 Ab and associations and confirms candidate gene associations. control Ab, respectively. The mean fluorescence of cells American Journal of Medical Genetics Part B: Neurop stained with mCluR5 Ab and control Ab for each subject is sychiatric Genetics 147B(8), 1345-1354 (2008). listed in Table A. Ratio of the mGluR Ab staining and (0175 9. Lesch K P Timmesfeld N, Renner T J, et al. control Ab staining was calculated and presented in Table 1 Molecular genetics of adult ADHD: converging evidence too. The mean ratio of the ADHD group was 4.6 (+0.4), from genome-wide association and extended pedigree whereas the mean of the control was 7.3 (+1.7). The differ linkage studies. J Neural Transm 115, 1573-1585 (2008). ence is statistically significant (t-test, p=0.024), representing (0176 10. Wang K, Li M, Hadley D, et al. 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0186 20. Turic D. Langley K, Mills S, et al. Follow-up model for attention-deficit hyperactivity disorder the of genetic linkage findings on chromosome 16p13: evi spontaneously hypertensive rat. Behav Brain Res 117, dence of association of N-methyl-D-aspartate glutamate 69-74 (2000). receptor 2A gene polymorphism with ADHD. Mol Psy 0201 35. Russell VA. Dopamine hypofunction possibly chiatry 9, 169-173 (2004). results from a defect in glutamate-stimulated release of 0187. 21. Mick, E. and Faraone, S. V., Genetics of dopamine in the nucleus accumbens shell of a rat model attention deficit hyperactivity disorder. Child Adolesc for attention deficit hyperactivity disorder the sponta Psychiatr ClinNAm 17, 261-284, vi-viii. (2008). neously hypertensive rat. Neurosci Biobehav Rev 27, 0188 22. Turic D. Langley K, Williams H. et al. A family 671-682 (2003). based study implicates solute carrier family 1-member 3 (0202 36. DashBanerjee T, Middleton F A, Berger D F, (SLC1A3) gene in attention-deficit/hyperactivity disor Lombardo J P Sagvolden T. Faraone S V. A comparison der. Biol Psychiatry 2005 Jun. 1: 57(11): 1461-6. of molecular alterations in environmental and genetic rat 0189 23. Elia J. Capasso M. Zaheer Z. et al. Candidate models of ADHD: a pilot study. Am J Med Genet B gene analysis in an on-going genome-wide association Neuropsychiatr Genet 147B, 1554-63 (2008). study of attention-deficit hyperactivity disorder: Sugges (0203 37. Sagvolden T. Johansen E. B. Weien G, et al. The tive association signals in ADRA1A. Psychiatr Genet. spontaneously hypertensive rat model of ADHD the PMID: 19352218 (2009). importance of selecting the appropriate reference strain. (0190. 24. Mick E. Neale B, Middleton FA, McGough J Neuropharmacology 57, 619-26 (2009). J. Faraone S V. Genome-wide association study of (0204 38. Del Bo R, Ghezzi S, Corti S, et. al., DPP6 gene response to methylphenidate in 187 children with atten variability confers increased risk of developing sporadic tion-deficit/hyperactivity disorder. Am J Med Genet B amyotrophic lateral Sclerosis in Italian patients. Journal of Neuropsychiatr Genet 147B. 1412-1418 (2008). Neurology, Neurosurgery & Psychiatry 79(9), 1085 (0191) 25. Dorval K M, Wigg K G, Crosbie J, et al., (2008). Association of the glutamate receptor Subunit gene (0205 39. Cronin S, Tomik B, Bradley D G, Slowik A, GRIN2B with attention-deficit/hyperactivity disorder. Hardiman O Screening for replication of genome-wide Genes Brain Behav. 6(5) (2007). SNP associations in sporadic ALS. European Journal of (0192. 26. Jin Z, Zang Y F. Zeng Y W. Zhang L, Wang Y Human Genetics 17, 213-218 (2009). F. Striatal neuronal loss or dysfunction and choline rise in 0206. 40. Marshall C R. 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of Schizophrenia using brain activation as a quantitative c) diagnosing the Subject as having ADHD when the phenotype. Schizophr Bull. 2009 January: 35(1): 96-108. presence of at least one ADHD-associated CNV in the Epub 2008 Nov. 20. nucleic acid sample is detected. 0215 49. Wang X, Bao X, Pal R, Agbas A, Michaelis E 33. A method of detecting at least one attention deficit K. Transcriptomic responses in mouse brain exposed to hyperactivity disorder (ADHD)-associated copy number chronic excess of the neurotransmitter glutamate. BMC variation (CNV) in a human Subject, comprising, Genomics. 2010 Jun. 7: 11:360. a) obtaining a nucleic acid sample from said Subject; 0216 50. C. de Lanerolle, Nihal: Eid, Tore; Lee, Tih b) detecting whether the sample contains at least one Shih. Genomic Expression in the Epileptogenic Hip ADHD-associated CNV by contacting the nucleic acid pocampus and Psychiatric Co-Morbidities. Current Psy sample with a probe of Sufficient length and composi chiatry Reviews, Volume 6, Number 2, May 2010, pp. tion to detect a duplication or deletion CNV in at least 135-144(10). one (ADHD)-associated copy number variation (CNV) 0217. 51. Ule, J., Stefani, G. Mele, A., Ruggiu, M., gene. Wang, X., Taneri, B., et al. (2006). An RNA map predict 34. The method of claim 1, wherein the ADHD-associated ing Nova-dependent splicing regulation. Nature, 444 CNV is in a gene selected from ACAT1, ACCN1, ACTR2, (7119), 580-6. doi:10.1038/nature05304. ADCY1, ADRB2, ADRBK1, ALDOA, ANXA2, APP, 0218 52. Ule, J., Ule, A., Spencer, J., Williams, A., Hu, APTX, AQP1, ARHGAP24, ARL1, ARRB1, ARRB2, J., Cline, M., et al. (2005). Nova regulates brain-specific ATXN7L3, BDKRB1, BDKRB2, BTBD2, BTG2, splicing to shape the synapse. Nature genetics, 37(8), C17orf44, C1orf116, C7orf25, CA8, CACNA1B, CACYBP, 844-52. doi:10.1038/ng1610. CALB2, CALM1, CALM2, CALMS, CAMK1, CAMK2B, 0219 53. Murias, M., Swanson, J. M., & Srinivasan, R. CAMK4, CCNB1, CDC42, CHGB, CHP. CHRM2, (2007). Functional connectivity of frontal cortex in CHRM3, CIC, CNP, CNR1, CNTN4, COPB2, CRHR1, healthy and ADHD children reflected in EEG coherence. CTNNA2, CYCS, DCN, DHCR7, DISC1, DLST, DPP6, Cerebral cortex (New York, N.Y.: 1991), 17(8), 1788-99. DRD2, DRD3, DSTN, DYNLL1, ECHS1, EGFR, ERBB2, doi:10.1093/cercorfbh1089. F2R, F2RL2, F2RL3, F3, FKBP3, FPR1, FSCN1, FURIN, 0220, 54. Wang, L., Zhu, C. He, Y., Zang, Y., Cao, Q., FYN, GAPDH, GLP1R, GLP2R, GNA15, GNAI1, GNAI2, Zhang, H., et al. (2009). Altered small-world brain func GNAI3, GNAO1, GNAQ. GNB2L1, GOT1, GP1 BA, tional networks in children with attention-deficit/hyper GPR26, GRB7, GRIA1, GRIK1, GRIK3, GRM1, GRM2, activity disorder Human brain mapping, 30(2), 638-49. GRM3, GRM4, GRM5, GRM6, GRM7, GRM8, GSN, doi:10.1002/hbm.20530. HBXIP, HOMER1, HOMERS, HSP90AB1, HTR2A, 0221) While certain of the preferred embodiments of the IMPDH2, IQGAP2, ITGB1, ITGB7, ITPR1, KIAA0090, present invention have been described and specifically KIAA1683, LAMA4, LARP7, LRP2BP, LRRC59, LTA, exemplified above, it is not intended that the invention be LYAR, LYN, MAP4, MAPK1, MARK4, MC4R, MRPL14, limited to such embodiments. It will be apparent to one MRPS16, MTHFD1, MTNR1A, MTNR1B, MX1, MYO6, skilled in the art that various changes and modifications can NANS, NARG1, NCK1, NEGR1, NFKBIA, NMI, NPY2R, be made therein without departing from the scope of the NUDC, OPRD1, PAFAH1B3, PCBP1, PCBP3, PCDHA4, present invention, as set forth in the following claims. PCMT1, PDCD5, PDE1B, PDE10, PDE6G, PGM1, PHKB, PHKG2, PICK1, PIK3CA, PIK3R1, PLA2G7, PLCB1, 1.-29. (canceled) PLCB3, PLCG2, PPIH, PPP2R1A, PRDX1, PRKCA, 30. A method for treating attention deficit hyperactivity PRLHR, PRMT1, PRPSAP1, PSAT1, PSEN1, PSMA1, disorder (ADHD) in a human Subject, comprising adminis PSMC1, PSMD1, PSMD11, PSMD13, PSMD6, PSME1, tering an effective amount of (+)-5-oxo-D-prolinepiperidi PXN, PYGL, PYGM, QRICH2, RALA, RANBP1, RAP2A, namide monohydrate (NS-105) to a subject having at least RCC1, RCC2, RGS12, RGS2, RHOA, RIF1, RPA2, RPLP2, one ADHD-associated CNV, thereby treating ADHD. RPN2, RPS14, RRM1, RUVBL2, RYR1, RYR2, S100A6, 31. A method for treating attention deficit hyperactivity SACS, SARS, SCTR, SDC3, SELE, SERPINB9, SET, disorder (ADHD) in a human Subject, comprising, SETD4, SF3B14, SHANK1, SHBG, SIAH1, SLC2A1, a) obtaining genetic information relating to the Subject; SLC6A3, SLC7A10, SNCA, SNRPB2, SOCS6, SOCS7, b) determining from the genetic information whether the SORD, SRC, STAU1, STRAP, STX12, SYK, TBCA, subject has at least one ADHD-associated copy number TBXA2R, TCP1, TEAD3, TFAM, TGM2, TJP1, TK1, variation (CNV); and TLR10, TNIK, TPI1, TRAF2, TRMT112, USP24, and VHL. c) administering an effective amount of (+)-5-oxo-D- 35. The method of claim 30, wherein the ADHD-associ prolinepiperidinamide monohydrate (NS-105) to the ated CNV is a duplication. subject if it is determined that the subject has at least 36. The method of claim 30, wherein the ADHD-associ one ADHD-associated copy number variation (CNV), ated CNV is a deletion. thereby treating ADHD. 37. The method of claim 30, wherein the ADHD-associ 32. A method for diagnosing attention deficit hyperactiv ated CNV is determined by detecting the presence of one or ity disorder (ADHD) in a human Subject, comprising, more single nucleotide polymorphisms (SNPs) between any a) obtaining a nucleic acid sample from said Subject; one of the following StartSNP and EndSNP ranges: b) detecting whether the sample contains at least one ADHD-associated copy number variation (CNV) by contacting the nucleic acid sample with a probe of Gene StartSNP End SNP Sufficient length and composition to detect a duplica GRMS kgp11022062 rsf123374 tion or deletion CNV in at least one ADHD-associated GRM8 rS117672O2 kgp13721602 copy number variation (CNV) gene; and

US 2017/000929.6 A1 Jan. 12, 2017 54

rs7790046, rs77941 12, rs12532924, rs4452722, rs 11212525, rs1151824, rs1151832, rs1151836, rs 11975478, rs11976255, rs10280963, rs4507681, rs11551174, rs11578320, rs11636774, rs11766192, rs6955717, rs7811481, rs6945869, rs4074568, rs 11784742, rs11881878, rs12030517, rs12084975, rs4074817, rs4726385, rs4380850, rs12703323, rs 12112953, rs12124903, rs12187625, rs12460584, rs9791911, rs4397308, rs10224365, rs10267846, rs 12550354, rs12708003, rs13050871, rs13164221, cnviO096121, cnvi0096122, cnvi0096.123, rs 13196459, rs13344984, rs13383563, rs1378954, cnviO096.124, cnvi0096.125, cnviO096126, rs10952466, rs 1521470, rs1560092, rs1564183, rs1642742, cnviO096127, rs10952467, cnviO096128, cnvi0096.129, rs 16823297, rs16830067, rs16882366, rs16882383, rs 10272007, cnviO096130, cnviO09.6131, cnvi0096132, rs 16911383, rs169382, rs17001492, rs1728.1761, rs4726389, rs12674128, rs10254647, rs12703329, rs 17288723, rs17413044, rs17804007, rs17804163, rs 12668613, rs112314, rs10073281, rs17590975, rs1783016, rs17835915, rs1787438, rs1844737, rs972501, rs252646, rs4367814, rs7527177, rs 1985858, rs2018721, rs2070746, rs2071085, rs 10888939, rs4512692, rs6588574, rs4333889, rs2082552, rs2087633, rs2113509, rs2123465, rs 10493.190, rs752503, rs748680, rs7256230, rs2130818, rs214488, rs2239206, rs2251388, rs 10500264, rs4530278, rs736289, rs9825865, rs2268203, rs2272566, rs2288888, rs2389908, rs 10510218, rs12488941, rs9860556, rs17044355, rS2432957, rs2444256, rs2444258, rs2444263, rs13322503, rs6781373, rs7625240, rs1387084, rs2444273, rs25860, rs2587888, rs26775, rs2770304, rs6547363, rs4430978, rs10208516, rs1595071, rs277340, rs28033, rs2829984, rs2829989, rs2832409, rs2862499, rs1565010, rs12054518, rs1129065, rs2835261, rs2835263, rs2838037, rs2854439, rs4834296, rs4409021, cnvi0018439, rs448.8992, rs2898449, rs2932925, rs3 18477, rs3 18489, rs363426, rs6533635, rs11722959, rs4555714, rs11946967, rs3634.52, rs363456, rs363463, rs363464, rs363472, rs2352050, rs10031435, rs7690429, rs4834302, rs363478, rs374.1049, rs3745530, rs3745532, rs 10027926, rs1014446, rs10158813, rs10226,190, rs3745844, rs3771886, rs3787625, rs3797269, rs 10247918, rs1035028, rs105.13040, rs1058903, rs380417, rs383700, rs3856557, rs3893.128, rs396969, rs 1062708, rs1065769, rs10753239, rs10760169, rs406179, rs407281, rs419793, rs431162, rs446791, rs 1078305, rs10869977, rs110402, rs11079028, rs457920, rs459498, rs466513, rs468105, rs468440, rs 11145589, rs11160322, rs11590474, rs1160752, rs468646, rs469083, rs473151 rs4767884, rs4767886, rs 11652516, rs11653,181, rs11701789, rs11716295, rs4801850, rs4801891, rs4802724, rs4802731, rs 1181.0113, rs11810325, rs12085929, rs12172554, rs4837820, rs4942587, rs505404, rs577259, rs577298, rs 12274774, rs12482750, rs1261.0125, rs12637875, rs580016, rs606443, rs647 1849, rs6505377, rs 12641989, rs12701 140, rs13116176, rs1320763, rs6509626, rs6984526, rs698.6917, rs70.1332, rs13238408, rs1361115, rs14.06674, rs1409052, rs7128029, rs7220709, rs7251605, rs7253284, rs 1436450, rs1637656, rs16844364, rs16910509, rs731779, rs7465573, rs3043226, rs3078771, rs17267677, rs173365, rs17763104, rs17786782, rs8103945, rs3106271, rs3132871, rs3133858, rs 1860790, rs1892846, rs2060685, rs2070106, rs823 161, rs323162, rs323163, rs367228, rs378691, rs2071390, rs2238798, rs2255734, rs2269497, rs880221, rs922445, rs927544, rs9314645, rs9392442, rs2279053, rs2279054, rs2292005, rs23.00497, rs9534505, rs9534507, rs9889352, rs9903823, and rs2300502, rs2302450, rs2304150, rs242939, rS997.5850. rs242942, rs2485570, rs2490365, rs2490371, 39. The method of claim 32, wherein the step of detecting rs2490372, rs2490373, rs2490385, rs2490389, the presence of the ADHD-associated CNV is performed rs2661679, rs268859, rs2835239, rs2835240, using a process selected from detection of specific hybrid rs2835244, rs2839060, rs2854701, rs2854702, ization, measurement of allele size, restriction fragment rs2876981, rs306759, rs306761, rs306784, rs3088231, length polymorphism analysis, and allele-specific hybrid rs3213718, rs346789, rs346794, rs347675, rs373617, ization analysis. rs3764622, rs3771099, rs3785877, rs3788216, 40. The method of claim 32, wherein the nucleic acid is rs3788217, rs3797251, rs3797252, rs3797255, DNA rs3803737, rs3807618, rs381083, rs3826706, 41. The method of claim 32, wherein the nucleic acid rs3827268, rs385689, rs3917410, rs3917419, sample is from blood, urine, serum, gastric lavage, cerebral rs445953, rs449114, rs4690096, rs4723 103, spinal fluid, brain cells, mononuclear cells, fetal cells in rs4789267, rs4792886, rs4802533, rs4865809, maternal circulation, or body tissue. rs4905466, rs3353, rs3361, rs3367, rs3368, rs350115, 42. The probe as described in claim 32. rs5871, rs6425310, rs6501880, rs6745303, rs684.8950, 43. A multiplex SNP panel comprising nucleic acids rs7046030, rs7048503, rs7256033, rs7258075, informative of the presence of ADHD associated CNVs, rs7517274, rs7529390, rs760436, rs762864, wherein said panel comprises nucleic acids comprising one rs7787057, rs3003379, rs301 1839, rs3013400, or more of the nucleic acid snps recited in claim 38. rs8074821, rs3078650, rs3136867, rs3141815, 44. A solid support comprising one or more of the ADHD rs952209, rs9610417, rs9897269, rs10092625, related SNP containing nucleic acid recited in claim 38. rs 10108007, rs10 128770, rs1024516, rs10412613, rs 10420138, rs10423794, rs1045288, rs1049353, 45. A kit comprising the multiplex SNP panel of claim 43 rs 10512455, rs10512456, rs1052886, rs1060619, together with instructions for use. rs10739593, rs10760165, rs10760167, rs10802802, 46. Akit comprising the Solid Support of claim 44 together rs 10846246, rs10890819, rs10902112, rs10915206, with instructions for use. rs 10925969, rs10957123, rs111223.19, rs11137372, k k k k k