US20130203814A1 - Association of Rare Recurrent Genetic Variations to Attention-Deficit, Hyperactivity Disorder (ADHD) and Methods of Use Thereof for the Diagnosis and Treatment of the Same - Google Patents

Association of Rare Recurrent Genetic Variations to Attention-Deficit, Hyperactivity Disorder (ADHD) and Methods of Use Thereof for the Diagnosis and Treatment of the Same Download PDF

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US20130203814A1
US20130203814A1 US13/776,662 US201313776662A US2013203814A1 US 20130203814 A1 US20130203814 A1 US 20130203814A1 US 201313776662 A US201313776662 A US 201313776662A US 2013203814 A1 US2013203814 A1 US 2013203814A1
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adhd
snp
cnv
nucleic acid
cnvs
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Joseph Glessner
Josephine Elia
Hakon Hakonarson
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Childrens Hospital of Philadelphia CHOP
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Priority to US15/063,482 priority patent/US10844434B2/en
Assigned to THE CHILDREN'S HOSPITAL OF PHILADELPHIA reassignment THE CHILDREN'S HOSPITAL OF PHILADELPHIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ELIA, JOSEPHINE, GLESSNER, JOSEPH, HAKONARSON, HAKON
Priority to US17/065,506 priority patent/US20210254160A1/en
Priority to US18/179,825 priority patent/US20230407399A1/en
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • This invention relates to the fields of genetics and the diagnosis of attention deficit hyperactivity disorder (ADHD). More specifically, the invention provides compositions and methods useful for the diagnosis and treatment of ADHD.
  • ADHD attention deficit hyperactivity disorder
  • ADHD Attention Deficit Hyperactivity Disorder
  • GWA genome wide association
  • CNV loci The applicants reported previously on copy number variation (CNV) loci observed in the first 335 ADHD cases we recruited (Elia, et al. 2009). While none of the CNV loci detected in that study met criteria for significance, it is noteworthy that one family was observed to have a GRM5 deletion impacting all three affected children, inherited from their affected father. A GRM7 deletion in one family with ADHD was additionally detected (Elia, et al. 2009). CNVs of metabotropic glutamate receptors (mGluR) in addition to the discovery of the NK3 gene in ADHD have suggested new therapeutic approaches to the treatment of ADHD.
  • mGluR metabotropic glutamate receptors
  • An exemplary method entails detecting the presence of at least one CNV in a target polynucleotide wherein if said CNV(s) is/are present, said patient has an increased risk for developing ADHD.
  • a method for detecting a propensity for developing attention deficit hyperactivity disorder (ADHD) in a patient in need thereof is provided.
  • An exemplary method entails detecting the presence of at least 1, 2, 3, 4, 5, 6, 10, 20, 30 or all of the SNP containing nucleic acid in a target polynucleotide, said SNP being informative of a the presence of an ADHD associated copy number variation (CNV), wherein if said SNP is present, said patient has an increased risk for developing ADHD, wherein said SNP containing nucleic acid is provided in Table 13.
  • CNV ADHD associated copy number variation
  • a method for identifying agents which alter neuronal signaling and/or morphology comprises providing cells expressing at least one nucleic acid comprising the ADHD associated CNVs of the invention, (step a); providing cells which express the cognate wild type sequences which lack the CNV (step b); contacting the cells from each sample with a test agent and analyzing whether said agent alters neuronal signaling and/or morphology of cells of step a) relative to those of step b), thereby identifying agents which alter neuronal signaling and morphology.
  • the test agent modulates metabotropic glutamate receptor (mGluR) gene activity.
  • test agent is selected from a group consisting of an mGluR positive allosteric modulators (PAM) (e.g., mGluR5 PAM, mGluR7 PAM), an mGluR negative allosteric modulator (NAM) (e.g., mGluR2/3 NAM), and a tachykinin-3/neurokinin-3 receptor (TAC3/NK3R) antagonist.
  • PAM mGluR positive allosteric modulators
  • NAM mGluR negative allosteric modulator
  • TAC3/NK3R tachykinin-3/neurokinin-3 receptor
  • test agent is selected from the group consisting of ADX63365, ADX50938, ADX71149, ADX48621, AMN082, 1-(hetero)aryl-3-amino-pyrrolidine derivatives (e.g. those provided in U.S. Patent Application Publication No.
  • the invention also provides at least one isolated ADHD related SNP-containing nucleic acid selected from the group listed in Table 13.
  • a multiplex SNP panel containing all of the informative SNPs from Table 13 is provided.
  • Such SNP containing nucleic acids which indicate the presence of ADHD associated CNV(s) may optionally be contained in a suitable expression vector for expression in neuronal cells. Alternatively, they may be immobilized on a solid support. In yet another alternative, the panel may be provided in silico.
  • This method provides a test and treat paradigm, whereby a patient's genetic profile is used to personalize treatment with therapeutics targeted towards specific neurophysiological defects found in individuals exhibiting ADHD.
  • Such a test and treat model may benefit up to 50% of patients with ADHD with greater efficacy and fewer side effects than non-personalized treatment.
  • any of the patients exhibiting an alteration in glutaminergic signaling can be tested for the presence of such a genetic alteration and then treated with the appropriate pharmaceutical such as the agents listed above.
  • FIG. 1 A graphical distribution of CNV calls per individual cases (top panel) compared to controls (bottom panel).
  • FIG. 2 A graphical display of the Normalized SNP Level Perlegen 600K Data.
  • the X axis shows base pair position in Megabases on chromosome 11.
  • Raw SNP Level Data Showing GRM5 Deletion in five samples from IMAGE Perlegen 600K Data Normalized by Adapted PennCNV-Affy Protocol.
  • Genotype data termed B-allele frequency (BAF) and intensity data termed Log R Ratio (LRR) plotted.
  • BAF B-allele frequency
  • LRR Log R Ratio
  • FIG. 3 Graphs of the full SNP-Level data: A) Normalized Perlegen 600K data, B) Normalized Illumina 1M PUWMa data, and C) Normalized Affymetrix 5.0 IMAGE II data.
  • FIG. 4 A graphical display of the IMAGE Perlegen 600K independent Validation data. Fluorescent probe-based qPCR assays using Roche Universal probe were designed to validate every candidate CNV with a completely independent test (11 of the 14 IMAGE samples with replicating CNV calls for the loci reported were available for validation and all validated in comparison with control pairs; the other 3 loci were visually validated). Error bars denote the standard deviation of quadruplicate runs. Del, deletion; Dup,duplication.
  • GEF genomic inflation factor
  • FIG. 6 An example of the SNP-based statistics applied and the resulting highest significance region Called. Examples from chr 3 are shown: A) 1,327,963-2,376,095 and B) 1,847,000-1,862,261. Complex CNV overlap is simplified by producing SNP-based statistics. As seen in plots for cases deleted and controls deleted, each SNP has a specific number of CNVs. The cases and controls are compared with a Fisher's exact test and the negative log p value is shown in the third plot. Regions of significance ranging within a power of ten are reported and the region of highest significance (local minimum p-value) within 1 MB is reported. The IMAGE cases deleted plot shows only one case sample #11939 since the remaining red regions 3′ are parents.
  • FIG. 7 CHOP Illumina Human Hap550 Independent Validation using qPCR. Fluorescent probe-based qPCR assays using Roche Universal probe were designed to validate every candidate CNV with a completely independent test (representative series shown for each locus in case and control pairs). Error bars denote the standard deviation of quadruplicate runs. Del, deletion; Dup,duplication.
  • FIG. 8 Examples of CNV observance based on B-allele frequency (BAF) and Log R Ratio (LRR).
  • FIG. 9 An illustration of the deletion directly impacting GRM5, exclusive to ADHD cases and replicated in IMAGE and PUWMa.
  • SNP coverage of the Illumina 550k, Perlegen 600k, Illumina 1M, and Affymetrix 5.0 arrays are shown as vertical blue lines.
  • FIG. 10 A display of GRM receptor gene interaction networks impacted in ADHD.
  • GRM receptor genes are shown as large diamond-shaped nodes while other interacting genes within 2 degrees if interaction are shown as smaller circular nodes.
  • Nodes are colored to represent enrichment of CNVs: dark red are deletions enriched in cases, light red are deletions enriched in controls, dark green are duplications enriched in cases, light green are duplications enriched in controls, and grey are diploid and devoid of CNVs.
  • Blue thick dashed lines highlight edges connected to at least one GRM gene while grey thin dotted lines represent all other gene interactions. Highly connected modules enriched for significant functional annotations are highlighted by blue shaded ellipses.
  • FIG. 11 A schematic overview showing the interaction of GRM receptors impacted in ADHD with modules of genes enriched for functional significance.
  • GRM receptor genes are shown as diamonds colored either green or red to represent duplications and deletions respectively enriched in cases. Boxes highlight functional modules defined by the network of interacting genes that are significantly enriched for GO annotations. Functional modules describe significant functional annotations and are labeled with the cluster name and the number of component genes in parenthesis. Functional annotations that may be particularly pertinent to ADHD underlying pathophysiology are bolded.
  • Edges of the network connect GRM receptor genes to functional modules: solid lines indicate membership of the GRM interacting gene in the functional module, and dotted lines indicate a first-degree relationship between GRM receptor genes and at least one component gene of a functional module.
  • FIG. 12 A CNV peninsula false positive association example. An example from chr 2 is shown (location 51,777,616-51,784,033). All significant CNVRs are reviewed for CNV peninsulas indicating uncertainty in boundary truncation.
  • ADHD Attention-Deficit, Hyperactivity Disorder
  • GRMs serve as critical hubs that coordinate highly connected modules of interacting genes, many of which harbor CNVs and are enriched for synaptic and neuronal biological functions.
  • a or “an” entity refers to one or more of that entity; for example, “a cDNA” refers to one or more cDNA or at least one cDNA.
  • a cDNA refers to one or more cDNA or at least one cDNA.
  • the terms “a” or “an,” “one or more” and “at least one” can be used interchangeably herein.
  • the terms “comprising,” “including,” and “having” can be used interchangeably.
  • a compound “selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds.
  • an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu.
  • isolated and “biologically pure” do not necessarily reflect the extent to which the compound has been purified.
  • An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.
  • genetic alteration refers to a change from the wild-type or reference sequence of one or more nucleic acid molecules. Genetic alterations include without limitation, base pair substitutions, additions and deletions of at least one nucleotide from a nucleic acid molecule of known sequence.
  • SNP single nucleotide polymorphism
  • CNV copy number variation
  • SNP single nucleotide polymorphisms
  • a CNV represents a copy number change involving a DNA fragment that is kilobases (kb) or larger (Feuk et al. 2006a).
  • CNVs described herein do not include those variants that arise from the insertion/deletion of transposable elements (e.g., ⁇ 6-kb KpnI repeats) to minimize the complexity of future CNV analyses.
  • CNV therefore encompasses previously introduced terms such as large-scale copy number variants (LCVs; Iafrate et al. 2004), copy number polymorphisms (CNPs; Sebat et al. 2004), and intermediate-sized variants (ISVs; Tuzun et al. 2005), but not retroposon insertions.
  • LCVs large-scale copy number variants
  • CNPs copy number polymorphisms
  • ISVs intermediate-sized variants
  • ADHD-associated SNP or “ADHD-associated specific marker” or ADHD-associated informational sequence molecule” is a SNP or marker sequence which is associated with an increased or decreased risk of developing ADHD not found normal patients who do not have this disease.
  • markers may include but are not limited to nucleic acids, proteins encoded thereby, or other small molecules.
  • ADHD-associated SNP containing nucleic acid is encompassed by the above description.
  • solid matrix refers to any format, such as beads, microparticles, a microarray, the surface of a microtitration well or a test tube, a dipstick or a filter.
  • the material of the matrix may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran or agarose.
  • phrases “consisting essentially of when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO:.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.
  • partial informative CNV is used herein to refer to a nucleic acid that hybridizes to sequences comprising a duplication on a chromosome however, the partial informative CNV may not be identical to the duplication, rather, the CNV may correspond to only a portion of the duplication, but yet is still informative for the same.
  • Target nucleic acid refers to a previously defined region of a nucleic acid present in a complex nucleic acid mixture wherein the defined wild-type region contains at least one known nucleotide variation which may or may not be associated with ADHD but is informative of the risk of ADHD.
  • the nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
  • the term “isolated nucleic acid” is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it was derived.
  • the “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote.
  • An “isolated nucleic acid molecule” may also comprise a cDNA molecule.
  • An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a recombinant nucleic acid molecule.
  • isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form.
  • enriched in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that “enriched” does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/ml).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity.
  • the claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 ⁇ 6 -fold purification of the native message.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest.
  • complementary describes two nucleotides that can form multiple favorable interactions with one another.
  • adenine is complementary to thymine as they can form two hydrogen bonds.
  • guanine and cytosine are complementary since they can form three hydrogen bonds.
  • a “complement” of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine.
  • the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • specific hybridization can refer to a sequence which hybridizes to any ADHD specific marker gene or nucleic acid, but does not hybridize to other nucleotides.
  • polynucleotide which “specifically hybridizes” may hybridize only to a neurospecific specific marker, such as an ADHD-specific marker shown in the Tables contained herein. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.
  • T m 81.5° C.+16.6Log [Na+]+0.41(% G+C) ⁇ 0.63 (% formamide) ⁇ 600/#bp in duplex
  • the T m is 57° C.
  • the T m of a DNA duplex decreases by 1-1.5° C. with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C.
  • the stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25° C. below the calculated T m of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20° C. below the T m of the hybrid.
  • a moderate stringency hybridization is defined as hybridization in 6 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42° C., and washed in 2 ⁇ SSC and 0.5% SDS at 55° C. for 15 minutes.
  • a high stringency hybridization is defined as hybridization in 6 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42° C., and washed in 1 ⁇ SSC and 0.5% SDS at 65° C. for 15 minutes.
  • a very high stringency hybridization is defined as hybridization in 6 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42° C., and washed in 0.1 ⁇ SSC and 0.5% SDS at 65° C. for 15 minutes.
  • oligonucleotide is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide. Oligonucleotides, which include probes and primers, can be any length from 3 nucleotides to the full length of the nucleic acid molecule, and explicitly include every possible number of contiguous nucleic acids from 3 through the full length of the polynucleotide. Preferably, oligonucleotides are at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length.
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • primer refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.
  • the primer may vary in length depending on the particular conditions and requirement of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • PCR Polymerase chain reaction
  • vector relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome.
  • a circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes.
  • restriction enzymes An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
  • a nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
  • transformation refers to methods of inserting a nucleic acid and/or expression construct into a cell or host organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like.
  • promoter element describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA.
  • the promoter element of the present invention precedes the 5′ end of the ADHD specific marker nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.
  • nucleic acid vector can contain nucleic acid elements other than the promoter element and the ADHD specific marker nucleic acid molecule.
  • nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for polypeptide purification.
  • a “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, plastid, phage or virus, that is capable of replication largely under its own control.
  • a replicon may be either RNA or DNA and may be single or double stranded.
  • an “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • transcriptional and translational control sequences such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • reporter As used herein, the terms “reporter,” “reporter system”, “reporter gene,” or “reporter gene product” shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods.
  • the nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product.
  • the required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.
  • the introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism.
  • the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid.
  • the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism.
  • the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
  • selectable marker gene refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.
  • recombinant organism or “transgenic organism” refer to organisms which have a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art.
  • the term “organism” relates to any living being comprised of a least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. Therefore, the phrase “a recombinant organism” encompasses a recombinant cell, as well as eukaryotic and prokaryotic organism.
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.
  • a “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
  • specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair comprises nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
  • Sample or “patient sample” or “biological sample” generally refers to a sample which may be tested for a particular molecule, preferably an ADHD specific marker molecule, such as a marker described hereinbelow. Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, cerebral spinal fluid, urine, saliva, tears, pleural fluid and the like.
  • agent and “compound” are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide/DNA complexes, and any nucleic acid based molecule which exhibits the capacity to modulate the activity of the CNV or SNP-containing nucleic acids described herein or their encoded proteins.
  • Agents and compounds may also be referred to as “test agents” or “test compounds” which are evaluated for potential biological activity by inclusion in screening assays described herein below.
  • modulate refers to increasing/promoting or decreasing/inhibiting a particular cellular, biological or signaling function associated with the normal activities of the CNV containing molecules described herein or the proteins encoded thereby.
  • modulate refers to the ability of a test compound or test agent to interfere with signaling or activity of a gene or protein of the present invention.
  • the present invention provides methods of diagnosing ADHD in a patient or methods for identifying a patient having an increased risk of developing ADHD.
  • Diagnosis includes not only the initial identification of ADHD associated with the genetic alterations described herein in a patient but confirmatory testing, or screening in patients who have previously been identified as having or likely to have ADHD.
  • the methods include the steps of providing a biological sample from the patient, measuring the amount of particular sets, or any all of the ADHD associated markers (Table 13) present in the biological sample, preferably a tissue and/or blood plasma sample, and determining if the patient has a greater likelihood of ADHD based on the amount and/or type of ADHD marker expression level determined relative to those expression levels identified in patient cohorts of known outcome.
  • a patient has a greater likelihood of having ADHD when the sample has a CNV marker expression profile associated with patients previously diagnosed with ADHD.
  • the compositions and methods of the invention are useful for the prognosis and diagnosis and management of ADHD
  • the patient sample may have been previously genotyped and thus the genetic expression profile in the sample may be available to the clinician.
  • the method may entail storing reference ADHD associated marker sequence information in a database, i.e., those CNVs statistically associated with a more favorable or less favorable prognosis as described in the tables herein, and performance of comparative genetic analysis on the computer, thereby identifying those patients having increased risk ADHD.
  • ADHD-related CNV or SNP-containing nucleic acids including but not limited to those listed below may be used for a variety of purposes in accordance with the present invention.
  • ADHD-associated CNV or SNP-containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of ADHD specific markers.
  • Methods in which ADHD specific marker nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).
  • assays for detecting ADHD-associated CNVs or SNPs may be conducted on any type of biological sample, including but not limited to body fluids (including blood, urine, serum, gastric lavage, cerebral spinal fluid), any type of cell (such as brain cells, white blood cells, mononuclear cells, fetal cells in maternal circulation) or body tissue.
  • body fluids including blood, urine, serum, gastric lavage, cerebral spinal fluid
  • any type of cell such as brain cells, white blood cells, mononuclear cells, fetal cells in maternal circulation
  • body tissue including but not limited to body fluids (including blood, urine, serum, gastric lavage, cerebral spinal fluid), any type of cell (such as brain cells, white blood cells, mononuclear cells, fetal cells in maternal circulation) or body tissue.
  • ADHD-associated CNV or SNP-containing nucleic acids, vectors expressing the same, ADHD CNV or SNP-containing marker proteins and anti-ADHD specific marker antibodies of the invention can be used to detect ADHD associated CNVs or SNPs in body tissue, cells, or fluid, and alter ADHD CNV or SNP-containing marker protein expression for purposes of assessing the genetic and protein interactions involved in the development of ADHD.
  • the ADHD-associated CNV or SNP-containing nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the templates as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are important in the art.
  • new detection technologies can overcome this limitation and enable analysis of small samples containing as little as 1 ⁇ g of total RNA.
  • RLS Resonance Light Scattering
  • PWG planar wave guide technology
  • Any of the aforementioned techniques may be used to detect or quantify ADHD-associated CNV or SNP marker expression and accordingly, diagnose ADHD.
  • kits and Articles of Manufacture Any of the aforementioned products can be incorporated into a kit which may contain a ADHD-associated CNV or SNP specific marker polynucleotide or one or more such markers immobilized on a Gene Chip, an oligonucleotide, a polypeptide, a peptide, an antibody, a label, marker, reporter, a pharmaceutically acceptable carrier, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.
  • Molecular modeling should facilitate the identification of specific organic molecules with capacity to bind to the active site of the proteins encoded by the CNV or SNP-containing nucleic acids based on conformation or key amino acid residues required for function.
  • a combinatorial chemistry approach will be used to identify molecules with greatest activity and then iterations of these molecules will be developed for further cycles of screening.
  • Several of the molecules available in this screening assay include metabotropic glutamate receptor (mGluR) positive allosteric modulators (PAM), negative allosteric modulators (NAM), and tachykinin-3/neurokinin-3 receptor (TACR-3/NK3R) antagonists.
  • ADX63365 includes ADX63365, ADX50938, ADX71149, ADX48621, AMN082, 1-(hetero)aryl-3-amino-pyrrolidine derivatives (e.g. those provided in U.S. Patent Application Publication No. 2008/0300266), LY341495, GSK1144814, and SB223412 (Table 1).
  • the polypeptides or fragments employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may determine, for example, formation of complexes between the polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between the polypeptide or fragment and a known substrate is interfered with by the agent being tested.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity for the encoded polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds, such as those described above, are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the target polypeptide and washed. Bound polypeptide is then detected by methods well known in the art.
  • a further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional or altered ADHD associated gene. These host cell lines or cells are defective at the polypeptide level. The host cell lines or cells are grown in the presence of drug compound. Altered glutaminergic function of the host cells is measured to determine if the compound is capable of regulating this function in the defective cells.
  • Host cells contemplated for use in the present invention include but are not limited to bacterial cells, fungal cells, insect cells, mammalian cells, and plant cells. However, mammalian cells, particularly neuronal cells are preferred.
  • the ADHD-associated CNV or SNP encoding DNA molecules may be introduced singly into such host cells or in combination to assess the phenotype of cells conferred by such expression.
  • Methods for introducing DNA molecules are also well known to those of ordinary skill in the art. Such methods are set forth in Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y. 1995, the disclosure of which is incorporated by reference herein.
  • Suitable vectors for use in practicing the invention include prokaryotic vectors such as the pNH vectors (Stratagene Inc., 11099 N. Torrey Pines Rd., La Jolla, Calif. 92037), pET vectors (Novogen Inc., 565 Science Dr., Madison, Wis. 53711) and the pGEX vectors (Pharmacia LKB Biotechnology Inc., Piscataway, N.J. 08854).
  • Examples of eukaryotic vectors useful in practicing the present invention include the vectors pRc/CMV, pRc/RSV, and pREP (Invitrogen, 11588 Sorrento Valley Rd., San Diego, Calif.
  • pcDNA3.1/V5&His Invitrogen
  • baculovirus vectors such as pVL1392, pVL1393, or pAC360 (Invitrogen)
  • yeast vectors such as YRP17, YIPS, and YEP24 (New England Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 Stratagene Inc.
  • Picchia vectors such as pHIL-D1 (Phillips Petroleum Co., Bartlesville, Okla. 74004)
  • retroviral vectors such as PLNCX and pLPCX (Clontech)
  • adenoviral and adeno-associated viral vectors adenoviral and adeno-associated viral vectors.
  • Promoters for use in expression vectors of this invention include promoters that are operable in prokaryotic or eukaryotic cells. Promoters that are operable in prokaryotic cells include lactose (lac) control elements, bacteriophage lambda (pL) control elements, arabinose control elements, tryptophan (trp) control elements, bacteriophage T7 control elements, and hybrids thereof.
  • lac lactose
  • pL bacteriophage lambda
  • trp tryptophan
  • Promoters that are operable in eukaryotic cells include Epstein Barr virus promoters, adenovirus promoters, SV40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMV) promoters, baculovirus promoters such as AcMNPV polyhedrin promoter, Picchia promoters such as the alcohol oxidase promoter, and Saccharomyces promoters such as the gal4 inducible promoter and the PGK constitutive promoter, as well as neuronal-specific platelet-derived growth factor promoter (PDGF), the Thy-1 promoter, the hamster and mouse Prion promoter (MoPrP), and the Glial fibrillar acidic protein (GFAP) for the expression of transgenes in glial cells.
  • Epstein Barr virus promoters adenovirus promoters, SV40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMV) promoters,
  • a vector of this invention may contain any one of a number of various markers facilitating the selection of a transformed host cell.
  • markers include genes associated with temperature sensitivity, drug resistance, or enzymes associated with phenotypic characteristics of the host organisms.
  • Host cells expressing the ADHD-associated CNVs and/or SNPs of the present invention or functional fragments thereof provide a system in which to screen potential compounds or agents for the ability to modulate the development of ADHD.
  • the nucleic acid molecules of the invention may be used to create recombinant cell lines for use in assays to identify agents which modulate aspects of cellular metabolism associated with ADHD and aberrant glutaminergic function. Also provided herein are methods to screen for compounds capable of modulating the function of proteins encoded by CNV and SNP-containing nucleic acids.
  • Another approach entails the use of phage display libraries engineered to express fragment of the polypeptides encoded by the CNV or SNP-containing nucleic acids on the phage surface. Such libraries are then contacted with a combinatorial chemical library under conditions wherein binding affinity between the expressed peptide and the components of the chemical library may be detected.
  • U.S. Pat. Nos. 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology 9:19-21.
  • the three-dimensional structure of a protein of interest or, for example, of the protein-substrate complex is solved by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches.
  • peptides may be analyzed by an alanine scan (Wells, (1991) Meth. Enzym. 202:390-411). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • anti-idiotypic antibodies As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule.
  • the anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.
  • drugs which have, e.g., improved polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of polypeptide activity.
  • CNV or SNP-containing nucleic acid sequences described herein sufficient amounts of the encoded polypeptide may be made available to perform such analytical studies as x-ray crystallography.
  • the knowledge of the protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
  • the availability of ADHD-associated CNV or SNP-containing nucleic acids enables the production of strains of laboratory mice carrying the ADHD-associated SNPs or CNVs of the invention.
  • Transgenic mice expressing the ADHD-associated CNV or SNP of the invention provide a model system in which to examine the role of the protein encoded by the CNV or SNP-containing nucleic acid in the development and progression towards ADHD.
  • Methods of introducing transgenes in laboratory mice are known to those of skill in the art. Three common methods include: 1. integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2. injection of DNA into the pronucleus of a newly fertilized egg; and 3. the incorporation of genetically manipulated embryonic stem cells into an early embryo.
  • mice described above will facilitate the molecular elucidation of the role that a target protein plays in various cellular metabolic processes, including: aberrant glutaminergic function, altered neuroactive ligand receptor signaling and aberrant neurotransmission, or altered neuronal morphology and neurite outgrowth.
  • Such mice provide an in vivo screening tool to study putative therapeutic drugs in a whole animal model and are encompassed by the present invention.
  • animal is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a “transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.
  • transgenic animal is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule.
  • This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the term “germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.
  • the alteration of genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. Such altered or foreign genetic information would encompass the introduction of ADHD-associated CNV or SNP-containing nucleotide sequences.
  • the DNA used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.
  • ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069).
  • Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction.
  • the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
  • the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated ADHD-associated CNV or SNP genes as insertional cassettes to selectively inactivate a wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice.
  • the use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described, and is reviewed elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).
  • Non-homologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with effective herpes drugs such as gancyclovir (GANC) or (1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodou-racil, (FIAU).
  • GANC Herpes Simplex virus thymidine kinase
  • FIAU (1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodou-racil
  • Utilizing ADHD-associated CNV or SNP-containing nucleic acid as a targeted insertional cassette provides means to detect a successful insertion as visualized, for example, by acquisition of immunoreactivity to an antibody immunologically specific for the polypeptide encoded by ADHD-associated CNV or SNP nucleic acid and, therefore, facilitates screening/selection of ES cells with the desired genotype.
  • a knock-in animal is one in which the endogenous murine gene, for example, has been replaced with human ADHD-associated CNV or informative fragment thereof or SNP-containing gene of the invention. Such knock-in animals provide an ideal model system for studying the development of ADHD.
  • the expression of a ADHD-associated CNV or SNP-containing nucleic acid, partial informative CNV fragment thereof, or an ADHD-associated fusion protein in which the CNV or SNP is encoded can be targeted in a “tissue specific manner” or “cell type specific manner” using a vector in which nucleic acid sequences encoding all or a portion of an ADHD-associated CNV or SNP are operably linked to regulatory sequences (e.g., promoters and/or enhancers) that direct expression of the encoded protein in a particular tissue or cell type.
  • regulatory sequences e.g., promoters and/or enhancers
  • Such regulatory elements may be used to advantage for both in vitro and in vivo applications. Promoters for directing tissue specific proteins are well known in the art and described herein.
  • the nucleic acid sequence encoding the ADHD-associated CNV or SNP of the invention may be operably linked to a variety of different promoter sequences for expression in transgenic animals.
  • promoters include, but are not limited to a prion gene promoter such as hamster and mouse Prion promoter (MoPrP), described in U.S. Pat. No. 5,877,399 and in Borchelt et al., Genet. Anal. 13(6) (1996) pages 159-163; a rat neuronal specific enolase promoter, described in U.S. Pat. Nos. 5,612,486, and 5,387,742; a platelet-derived growth factor B gene promoter, described in U.S. Pat. No.
  • a brain specific dystrophin promoter described in U.S. Pat. No. 5,849,999
  • a Thy-1 promoter a PGK promoter
  • a CMV promoter a neuronal-specific platelet-derived growth factor B gene promoter
  • a NEGR1 promoter a GRM5 promoter, a promotor of any gene listed in the tables below
  • Glial fibrillar acidic protein (GFAP) promoter for the expression of transgenes in glial cells.
  • Transgenic mice into which a nucleic acid containing the ADHD-associated CNV or SNP or its encoded protein have been introduced are useful, for example, to develop screening methods to screen therapeutic agents to identify those capable of modulating the development of ADHD.
  • compositions useful for treatment and diagnosis of ADHD may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • the replication case and control cohorts utilized genome-wide SNP genotyping using the Perlegen 600K, Illumina 1M, and Affymetrix 5.0 arrays.
  • Raw X and Y values were normalized with log(10) and clustered to establish BAF and LRR with PennCNV-Affy protocol (Table 2).
  • Rare recurrent CNVs were the focus of our study.
  • Each sample was analyzed in quadruplicate either in 25 ⁇ l reaction mixture (250 nM probe, 900 nM each primer, Fast Start TaqMan Probe Master from Roche, and 10 ng genomic DNA) or in 10 ⁇ l reaction mixture (100 nM probe, 200 nM each primer, 1 ⁇ Platinum Quantitative PCR SuperMix-Uracil-DNA-Glycosylase (UDG) with ROX from Invitrogen, and 25 ng genomic DNA).
  • the values were evaluated using Sequence Detection Software v2.2.1 (Applied Biosystems, CA). Data analysis was further performed using the ⁇ C T method.
  • Reference genes, chosen from COBL, GUSB, and SNCA, were included based on the minimal coefficient of variation and then data was normalized by setting a normal control to a value of 1.
  • the CNV calling on Perlegen platform used a highly similar algorithm to those used on the Illumina arrays, but the signal pre-processing steps differ. Unlike the Illumina platform, where normalized signal intensities (Log R Ratio and B Allele Frequency) can be exported directly from the BeadStudio software, these signal intensity measures in the Perlegen 600K platform need to be calculated from the collection of genotyped samples based on raw X and Y values.
  • HMM Hidden Markov Model
  • QC Quality Control
  • CNV frequency between cases and controls was evaluated at each SNP using Fisher's exact test. We only considered loci that were nominally significant between cases and controls (p ⁇ 0.05) where cases in the CHOP discovery cohort had the same variation, replicated in IMAGE, PUWMa, or IMAGE II or were not observed in any of the control subjects, and validated with an independent method. We report statistical local minimums to narrow the association in reference to a region of nominal significance including SNPs residing within 1 Mb of each other ( FIG. 4 ).
  • CNVRs were excluded if they met any of the following criteria: i) residing on telomere or centromere proximal cytobands; ii) arising in a “peninsula” of common CNV arising from variation in boundary truncation of CNV calling ( FIG. 7 ); iii) genomic regions with extremes in GC content which produces hybridization bias; or iv) samples contributing to multiple CNVRs.
  • DAVID Database for Annotation, Visualization, and Integrated Discovery
  • CNVRs passing this initial screen are scored for statistical significance based on a permuted P-value which permutes case and control labels randomly of all samples with the condition that related individuals must have the same label. Each unrelated individual is assigned a case or control label and their related sibling is assigned the same label. Based on the number of samples with the CNVR being calculated in randomly assigned “cases” and “controls” a Fisher's exact test P-value is assigned. The number of hypothetical scenarios with significance equal or greater (lower P-value) provides the permuted P-value which corrects for relatedness. The Fisher's exact test P-value and counts of cases and controls with each CNVR are provided for transparency.
  • SNPs residing around exon 4 of contactin 3 appear to replicate most consistently between Illumina and Perlegen ADHD TDT statistics.
  • P(rs4073942) 2.78 ⁇ 10 ⁇ 3
  • P(rs9869828) 8.61 ⁇ 10 ⁇ 3 in addition
  • Proband diagnosis combined subtype ADHD.
  • One or more sibling(s) in the same age range are one or more sibling(s) in the same age range.
  • control subjects used were drawn from Affymetrix 6.0 genotyped subjects from the NIMH genetics repository. They had been collected through a US Nationally representative survey panel (of approximately 60,000 adult individuals at any one time, with constant turnover) ascertained via random digit dialing. Participants were screened for psychosis and bipolar disorder. Control participants were not screened for ADHD. A blood sample was collected via a US national phlebotomy service. Control participants gave written consent for their biological materials to be used for medical research at the discretion of NIMH. Controls were genotyped using the Affymetrix 6.0 array, at the Broad Institute National Center for Genotyping and Analysis. Genotype calls were made with the BIRDSEED program, a module of the BIRDSUITE package.
  • the discovery cohort included a total of 1,013 ADHD cases of Northern European descent genotyped at Children's Hospital of Philadelphia (CHOP). This consisted of 664 cases without parents and 349 cases from complete trios recruited at CHOP (See Tables 8 and 9).
  • GAIN Genetic Association Information Network
  • the IMAGE cohort was genotyped using the Perlegen 600K platform.
  • the PUWMa cohort was genotyped on the Illumina 1M BeadChip.
  • the IMAGE II cohort was genotyped on the Affymetrix 5.0 array.
  • platforms matching the case platforms including: 4,105 Illumina 550k from CHOP, 3,297 Perlegen 600k from GAIN psoriasis and depression projects, 3,469 Illumina 1M from PUWMa parents and SAGE, and 2,456 Affymetrix 5.0 and 6.0 controls from the NIMH genetics repository and AGRE parents.
  • the PennCNV software was used to produce CNV calls for cases and controls as previously described 10 .
  • the CNV frequency of the subjects who met quality standards which included removing substantial outliers in the count CNV call quality metric that deviated exponentially from the distribution of the majority of the cohort, resulted in 93% of subjects having 8-45 CNV calls ( FIG. 1 ).
  • FIG. 8 shows an example of raw Illumina data as viewed in the BeadStudio software and the resulting CNV call.
  • the CNV calls spanned from 3 to 598 SNPs, with an average of 14 SNPs per CNV call, with the largest CNV of 2.2 Mb and an average CNV size of 62 kb.
  • Variable probe coverage allows for detection of CNVs down to a small physical size, provided at least 3 SNPs are present, and the CNVs were experimentally validated using qPCR.
  • the CNV calls spanned from 3 to 708 SNPs, with an average of 12.8 SNPs per CNV call, with the largest CNV of 2.9 Mb and an average CNV size of 53.6 kb.
  • Replication analysis was performed in five independent cohorts, including ADHD subjects from IMAGE, PUWMa, IMAGE II, NIMH, and Utah. Based on the 10 case-specific CNVs from the discovery cohort, 3 were also exclusive to replication cohort cases, notably GRM7, GRM8 and NEGRI, with resulting combined P-values of 3.52 ⁇ 10 ⁇ 6 and 8.14 ⁇ 10 ⁇ 5 , for GRM8 and GRM7, respectively (Table 3A).
  • FIG. 9 shows the CNV deletions observed at the GRM5 locus (10 cases vs 1 control), using UCSC Genome Browser (12) with Build 36 of the human genome.
  • Experimental validation of IMAGE, PUWMa, IMAGE II, NIMH, and Utah CNVs, using qPCR, together with Raw BAF and LRR plots are shown in FIGS. 2-4 .
  • ADHD Genotype GWAS of Glutamatergic Genes. The most significant SNP genotype association in each of the eight GRM gene regions.
  • GRMs do not form a large number of interactions, but importantly serve to coordinate functional modules of other sets of genes.
  • GRM1 harbors duplications significantly enriched in ADHD cases and serves to coordinate functional modules involved in housekeeping functions such as carbohydrate metabolism, phosphorylation, apoptosis and ion binding.
  • GRM5 and GRM7 both harbor deletions significantly enriched in cases and cluster within a functional module involved in synaptic transmission and alternative splicing.
  • GRM5 serves to coordinate alternative spicing with synaptic transmission and other neuronal processes at the post-synaptic density
  • GRM7 coordinates functional modules integrating neurological processes and synaptic activity with housekeeping functions such as cytoskeletal organization and apoptosis
  • GRM8 also harbors deletions significantly enriched in cases and is itself contained within a functional module that is involved in synaptic transmission and neurogenesis.
  • GRM3 has duplications that are more frequently observed in cases and serves to coordinate ubiquitination pathways, RNA binding, splicing, and processing, and neuronal migration, with neurological processes including synaptic transmission with effects of behavior and cognition.
  • Example I several genetic alterations have been found to be associated with the ADHD phenotype.
  • the information herein above can be applied clinically to patients for diagnosing an increased susceptibility for developing ADHD, and therapeutic intervention.
  • a preferred embodiment of the invention comprises clinical application of the information described herein to a patient.
  • Diagnostic compositions, including microarrays, and methods can be designed to identify the genetic alterations described herein in nucleic acids from a patient to assess susceptibility for developing ADHD. This can occur after a patient arrives in the clinic; the patient has blood drawn, and using the diagnostic methods described herein, a clinician can detect a SNP in the genetic regions listed in Tables 13A and 13B below.
  • the typical age range for a patient to be screened is between 1 and 12 years of age.
  • kits for performing the diagnostic method of the invention comprise a microarray comprising at least one of the SNPs provided herein in and the necessary reagents for assessing the patient samples as described above.
  • a multiplex SNP panel is employed and the patient sample is assessed for the presence or absence of all the SNPs listed in the Tables below.
  • Table 13A provides all the genes, physical genome ranges, and SNP ranges of the ADHD markers disclosed.
  • Table 13B provides a “SNPList” which is a minimal set for a “diagnostic array” such as veracode which is technlology approved by FDA.
  • the flanking SNPs to these CNVs are included as well as intervening SNPs as they could all be used to capture the CNV.
  • CNVs Copy number variations
  • HMM PennCNV hidden Markov model
  • genotype data is evaluated in a continuous physical position for homozygous genotypes indicating deletion or AAB and ABB genotypes indicating duplication weighted with positive correlation for control minor allele frequency.
  • the identity of ADHD-involved genes and the patient results will indicate which variants are present, and will identify those that possess an altered risk for developing ADHD.
  • the information provided herein allows for therapeutic intervention at earlier times in disease progression that previously possible.
  • the GRM receptor family provides novel targets for the development of new therapeutic agents efficacious for the treatment of ADHD. In particular, it would be desirable to modulate expression of such genes in those patients that are more prone to develop the disease.
  • mGluRs metabotropic glutamate receptors
  • compounds which may be administered in implementing the test and treat paradigm described herein include the piracetam family of nootropic agents, as described in F. Gualtieri et al., Curr. Pharm. Des., 8: 125-38 (2002).
  • the treating agent is a pyroglutamide. Details regarding the preparation and formulation of pyroglutamides which may be used in the practice of this invention are provided in U.S. Pat. No. 5,102,882 to Kimura et al.
  • a particularly preferred agent for the treatment of ADHD in patients determined to have one or more of the SNPs indicative of the presence of an ADHD-associated copy number variation, as set forth in Table 13, is (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105).
  • CNV containing mGluR genes involved in ADHD pathogenesis also provide novel targets for the development of new therapeutic agents efficacious for the treatment of ADHD.
  • candidate drug agents or compound
  • Representative candidate drugs include nucleic acids, polypeptides, small molecule compounds and peptidomimetics.
  • genetic agents can be screened by contacting the yeast cell with a nucleic acid construct coding for a gene.
  • a nucleic acid construct coding for a gene For example, one may screen cDNA libraries expressing a variety of genes, to identify other genes that modulate such interactions.
  • the identified drugs may modulate glutamate associated neuronal signaling, subcellular protein localization and/or neuronal cell morphology or viability. Accordingly, irrespective of the exact mechanism of action, drugs identified by the screening methods described herein are expected to provide therapeutic benefit to patients suffering from ADHD.
  • Suitable screening methods may employ a variety of neuronal cell types obtainable from the ATCC.
  • Candidate drugs can be screened from large libraries of synthetic or natural compounds.
  • One example is an FDA approved library of compounds that can be used by humans.
  • compound libraries are commercially available from a number of companies including but not limited to Maybridge Chemical Co.
  • the neuronal cells can be incubated in the presence and absence of a test compound, such as pyroglutamides (see, e.g., U.S. Pat. No. 5,102,882) and other members of the piracetam family of nootropic agents, after which the effect of the compound on glutamate signaling is assessed. Agents so identified could then be tested in whole animal models of ADHD to assess in vivo efficacy.
  • a test compound such as pyroglutamides (see, e.g., U.S. Pat. No. 5,102,882) and other members of the piracetam family of nootropic agents
  • Agents identified using the screening assays described herein are also encompassed by the present invention.
  • Metabotropic glutamate receptors are a class of G-protein-coupled receptors that possess a seven transmembrane region involved in the modulation of excitatory synaptic transmission in the nervous system 14 .
  • GRM5 and GRM1 are members of Group I expressed particularly in the basal ganglia and cerebellum 16 , relevant brain areas for ADHD. These receptors have been shown to activate phospholipase C and it has been postulated they may play a role in addiction, anxiety and behavioral disorders 17 .
  • GRM7 and GRM8 are members of Group III which is linked to the inhibition of the cyclic AMP cascade. GRM7 has been linked with anxiety 18 and is the most highly conserved of all mGluR subtypes across different mammalian species 19 .
  • the SLC6A3-KO (DAT-KO) mouse an ADHD animal model, remains responsive to methylphenidate in spite of the lack of a dopamine transporter 30 and hyperactivity in these mice can be increased by NMDA-receptor blockers and suppressed by drugs that increase glutamatergic transmission 31 .
  • Increased midbrain SLC6A3 and DRD4 expression were reported in rats where glutamate transporter increases were found in the striatum 32 suggesting that decreases in dopamine may alter glutamate signaling.
  • glutamate receptor subunit gene (GRIN2A) disruption increased DA and serotonin metabolism in the frontal cortex and striatum of mice, and increased locomotor activity that was reduced by dopamine or serotonin receptor antagonists 33 .
  • DPP6 has been previously associated with Amyotrophic Lateral Sclerosis (ALS) in genome wide association studies 38,39 , and CNVs impacting DPP6 have been reported in relation with autism 40 .
  • DPP6 and CTNNA2 have been implicated by earlier ADHD SNP genotype GWAS 9 .
  • NLN is an interesting candidate responsible for metabolic inactivation of neural peptides, such as Neuropeptide Y (NPY) which has previously been implicated in ADHD 45,46 .
  • NPY Neuropeptide Y
  • SLC7A10 has been shown to play a role in the modulation of glutamatergic transmission through mobilization of D-serine at the glutamatergic synapse.
  • LARP7 is important for snRNP integrity, a protein complex responsible for post transcriptional splicing.
  • NEGR1 encodes a neural cell adhesion molecule and a trans-neural growth-promoting factor in regenerative axon sprouting and neuronal growth in the mammalian brain. Interestingly, this neuronal gene was recently associated with obesity 41 .
  • GRM receptor interacting/signaling genes By extending the observations from the confident GRM family to gene networks of GRM receptor interacting/signaling genes provides 9.94% of ADHD cases with genetic characterization of their disease after adjusting for control frequency (i.e., net impact in cases).
  • Major supporting hubs of this network include TNIK 48 , GNAQ 49 , and CALM1 50 ( FIG. 11 ), previously associated with schizophrenia and epilepsy.
  • the GRM receptor network gene, GRIK1 has also been associated with hyperactive/impulsive symptoms of ADHD 8 .
  • abnormal functional brain connectivity is a candidate factor in developmental brain disorders associated with cognitive dysfunction, including ADHD 53,54 .
  • the impact CNVRs among the GRM family of receptors, and in particular GRM5 and GRM7, may be important to the underlying molecular etiology of ADHD.
  • the mGluR network genes are defined by both forward and reverse protein protein interactions using experimental data derived from a variety of experimental protocols including yeast 2 hybrid assays and mass spectrometry.
  • the merged human interactome combines human interactions reported in IntAct, DIP, BIND and HPRD, in addition to papers by Rual et al. Nature 437, 1173-1178 (2005); Stelzl et al. Cell 122, 957-968 (2005); Ramani et al. Genome Biol. 2005; 6(5):R40. Epub 2005 Apr. 15; Venkatesan Nat. Methods 6, 83-90 (2009); and Yu et al. Nat. Methods 8, 478-480 (2011).
  • GRM pp X genes which are “targets” i.e. “GRM pp X” and “X pp Y” (where pp is protein protein interaction).
  • Forward and reverse includes GRM as both the “source” and the “target” of a biological signal i.e. “GRM pp X”, “X pp GRM”, “X pp Y”, and “Y pp X”.
  • CN refers to copy number state (1 deletion; 2 normal wt; 3 duplication).
  • Count Samples Count Samples Gene Deletion Duplication
  • Table 21 shows 64 network genes which are enriched for CNVs and lists number of case subjects per CNV. These genes provide new targets for the design of therapeutics useful for the treatment and diagnosis of ADHD.
  • Reciprocal Gene Querying 2 degrees protein-protein interaction with GRM genes based on Cytoscape Human Interactome are: GRM1; GRM2; GRM3; GRM4; GRM5; GRM6; GRM7; GRM8; TUBB; ITPR1; GAPDH; ADORA1; ADRBK1; GNA15; GNAQ; RGS12; RIF1; TNIK; HRPT2; FLNA; CALM2; HOMER1; CALM3; CALM1; GNAO1; TUBA1B; TUBA1A; PICK1; TUBA1; TUBA2; PRKCA; HOMER2; PDCD8; C9orf25; SDCBP; PPM1A; PRKCZ; TUBA8; ACAT1; ACAT2; ACCN1; ACCN2; ACP1; ACTA1; ACTB; ADA; ADCY1; ADD1; ADD2; ADD3; ADRA1B; AD
  • the ratio of fluorescence value between the mGluR5 Ab staining and the control Ab staining was calculated.
  • the mean ratio of the ADHD group was 4.6 ( ⁇ 0.4), whereas the mean of the control was 7.3 ( ⁇ 1.7).

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