EP3149211A1 - Modifications génétiques associées à l'autisme età un phénotype autistique, et procédés de diagnostic et de traitement de l'autisme - Google Patents

Modifications génétiques associées à l'autisme età un phénotype autistique, et procédés de diagnostic et de traitement de l'autisme

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Publication number
EP3149211A1
EP3149211A1 EP15829843.0A EP15829843A EP3149211A1 EP 3149211 A1 EP3149211 A1 EP 3149211A1 EP 15829843 A EP15829843 A EP 15829843A EP 3149211 A1 EP3149211 A1 EP 3149211A1
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EP
European Patent Office
Prior art keywords
autism
cnv
asd
gene
genes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP15829843.0A
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German (de)
English (en)
Inventor
Hakon Hakonarson
Tara Wenger
Charlly Kao
Dexter HADLEY
Zhi-liang WU
Joseph Glessner
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Childrens Hospital of Philadelphia CHOP
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Childrens Hospital of Philadelphia CHOP
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Priority claimed from US14/292,480 external-priority patent/US20160244831A9/en
Application filed by Childrens Hospital of Philadelphia CHOP filed Critical Childrens Hospital of Philadelphia CHOP
Publication of EP3149211A1 publication Critical patent/EP3149211A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • This invention relates to the fields of genetics and the diagnosis and treatment of autism and autism spectrum disorders.
  • MIM Autism
  • Autism is a severe and relatively common neuropsychiatric disorder characterized by abnormalities in social behavior and communication skills, with tendencies towards patterns of abnormal repetitive movements and other behavior disturbances.
  • Current prevalence estimates are -0.2% of the population for autism and 0.9 % of the population for ASDs (MMWR Surveill Summ. 2009).
  • autism poses a major public health concern of unknown cause that extends into adulthood and places an immense economic burden on society.
  • the most prominent features of autism are social and communication deficits.
  • the former are manifested in reduced sociability (reduced tendency to seek or pay attention to social interactions), a lack of awareness of social rules, difficulties in social imitation and symbolic play, impairments in giving and seeking comfort and forming social relationships with other individuals, failure to use nonverbal communication such as eye contact, deficits in perception of others' mental and emotional states, lack of reciprocity, and failure to share experience with others.
  • Communication deficits are manifested as a delay in or lack of language, impaired ability to initiate or sustain a conversation with others, and stereotyped or repetitive use of language.
  • Psychiatry 11 18-28; Veenstra-VanderWeele and Cook, (2004) Mol. Psychiatry 9: 819-32).
  • Searches for chromosomal abnormalities in autism have revealed terminal and interstitial deletions, balanced and unbalanced translocations, and inversions on a large number of chromosomes, with abnormalities on chromosomes 15, 7, and X being most frequently reported.
  • the importance of the regions indicated by cytogenetic studies was evaluated by several whole genome screens in the multiplex autistic families (International Molecular Genetic Study of Autism Consortium, 1998).
  • autism focus on changes in neuronal connectivity as the potential underlying cause of these disorders. Imaging studies reveal changes in local and global connectivity (Just et al., (2004) Brain 127: 1811-1821 ; Herbert et al., (2005) Ann Neurol 55(4): 530-40) and developmental studies of activity- dependent cortical development suggest that autism might result from an imbalance of inhibitory and excitatory synaptic connections during development (Rubenstein and Merzenich, (2004; Genes Brain Behav 2(5): 255-67). The fundamental unit of neuronal connectivity is the synapse; thus, if autism is a disorder of neuronal connectivity, then it can likely be understood in neuronal terms as a disorder of synaptic connections.
  • neuronal connectivity anomalies revealed, for example, by direct white matter tractography, or by observable delays in characteristic electrical activity, can be directly linked to behavioral and clinical manifestations of ASD, allowing these neuron-level phenotypes to be interpreted as neural correlates of behavior.
  • kits are provided for performance of a method for detecting a propensity for developing autism or autistic spectrum disorder.
  • An exemplary kit comprises means for obtaining a sample from a patient and testing the sample for the presence or absence of at least one deletion containing CNV in a target polynucleotide, wherein if the CNV is present, the patient has an increased risk for developing autism and/or autistic spectrum disorder.
  • the deletion containing CNV is selected from the group of CNVs provided in Table II.
  • the kit includes reagents for performing the step of detecting the presence of said CNV and further comprises specific reagents for performing a process selected from the group consisting of detection of specific hybridization, measurement of allele size, restriction fragment length polymorphism analysis, allele-specific hybridization analysis, single base primer extension reaction, and sequencing of an amplified polynucleotide.
  • the present invention provides a method for identifying agents which alter neuronal signaling and/or morphology.
  • An exemplary method entails providing cells expressing at least one CNV listed in Table 2 and cells which express the cognate wild type sequences corresponding to the CNV containing sequence, contacting both cell types with a test agent and analyzing whether the agent alters neuronal signaling and/or morphology of cells comprising the CNV relative to those which lack the genetic alteration, thereby identifying agents which alter neuronal signaling and morphology in CNV containing cells.
  • vectors encoding such CNVs contain nucleic acids flanking the affected region of deletion of a suitable length, such that cloning and transformation of cells with the CNV containing nucleic acid is possible.
  • CNV copy number variation
  • patients are tested for the presence or absence of at least one CNV containing gene is selected from the group consisting of ATP 1 OA,
  • the CNV is determined to reside in a gene important for GAB A signaling and the agent is listed in Table 3 or Table 4. In a particularly preferred embodiment, the CNV alters GABA signaling and the agent is topiramide.
  • Figure 1 The design of the present study is shown. In this two-stage design, 2076 cases vs 4754 controls were used in the discovery cohort (Stage 1), and 1159 cases vs 2546 controls were used for a replication cohort (Stage 2). All samples used passed minimal quality control metrics, but the default quality calls of PennCNV were used to discriminate the discovery cohort (best quality) from the replication cohort (lesser quality.)
  • Figure 2 A graph showing the classes of gene pathways, which are disrupted by the ASD-related CNVRs disclosed herein. All genes with exons disrupted by replicated CNVRs were submitted to Ingenuity to ascertain significance of pathway enrichment.
  • Figure 3 A schematic of a first-degree interactome of the GABAR-A family highlighting copy number defects enriched in cases (red) vs controls (blue).
  • Figure 4 Test and treat model for targeting therapeutics to specific pathways defective in disease.
  • the generic test and treat model is shown in black where a molecular diagnostic is used to genetically define a population with defective pathways that are likely to benefit from a targeted intervention.
  • Examples of trastuzumab as a targeted intervention for HER2 specific breast cancer is shown in blue as well as an extrapolation of behavioral programs and novel therapeutics that are being developed to target ASDs due to defective GABAR-A pathways in red.
  • Figure 5 Significance of CNVRs by GWAS of ASDs in European-derived or African-derived populations.
  • GWAS of 4,634 cases vs 4,726 controls in Europeans is shown on top and GWAS of 312 cases vs 4,173 controls in Africans is shown below.
  • Figure 6 Enrichment of optimal CNVRs across mGluR network of genes.
  • Nodes of the network are labeled with their gene names, with red and green representing deletions and duplications respectively, while grey nodes lack CNV data. Dark and light colors represent enrichment in cases and controls respectively.
  • the genes defining the network are showed as diamonds, while all other genes are shown as circles. Blue lines indicate evidence of interaction.
  • FIG 7 Enrichment of optimal CNVRs across CALM1 network. The first degree directed interaction network defined by CALM1 is shown.
  • Figure 8 Diagram of study design. Children with ASD and gene changes in mGluR network (mGluR f ASD) were more likely to have Syndromic ASD compared to children with ASD without abnormalities of mGluR network genes (mGluR - ASD). PO.0001.
  • GABAR-A receptor signaling was the most significant disrupted canonical pathway in ASD where case-enriched defects in GABRA5, GABRB3, and GABRG3 genes were identified.
  • the CNVRs we have identified impact multiple novel genes and signaling pathways, including genes involved in GABAR-A signaling, that can provide important targets for therapeutic intervention.
  • GFINs defective gene family interaction networks
  • CALM1 calmodulin 1
  • CNV copy number variation
  • SNP single nucleotide polymorphisms
  • a CNV represents a copy number change involving a DNA fragment that is -1 kilobases (kb) or larger (Feuk et al. 2006a).
  • CNVs described herein do not include those variants that arise from the insertion/deletion of transposable elements (e.g., ⁇ 6-kb Kpnl 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.
  • SNP single nucleotide polymorphism
  • 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.
  • 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.
  • the phrase "consisting essentially of when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO:. For example, 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.
  • 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 autism.
  • 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.
  • cDNA synthetic substance
  • 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-
  • 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 autism 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 an autism-specific marker shown in the Table contained herein. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.
  • T ra 81.5 " C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • 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 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in 2X SSC and 0.5% SDS at 55°C for 15 minutes.
  • a high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ 3 ⁇ 4 ⁇ 1 denatured salmon sperm DNA at 42°C, and washed in IX SSC and 0.5% SDS at 65°C for 15 minutes.
  • a very high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ denatured salmon sperm DNA at 42°C, and washed in 0.1X 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 and may or may not comprise a detectable label. 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
  • 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.
  • Probes and primers having the appropriate sequence homology which specifically hybridized to CNV containing nucleic acids are useful in the detecting the presence of such nucleic acids in biological samples.
  • 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 autism 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 autism specific marker gene 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.
  • 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 autism specific marker molecule, such as a marker shown in the tables provided below. Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, urine, saliva, tears, pleural fluid and the like.
  • agent and “test 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 materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • 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 SNP and/or CNV containing nucleic acids described herein or their encoded proteins. Agents are evaluated for potential biological activity by inclusion in screening assays described hereinbelow.
  • Autism-related-CNV and/or SNP containing nucleic acids may be used for a variety of purposes in accordance with the present invention.
  • Autism-associated CNV/SNP containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of autism specific markers.
  • Methods in which autism specific marker nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).
  • assays for detecting autism-associated CNVs/SNPs may be conducted on any type of biological sample, including but not limited to body fluids (including blood, urine, serum, gastric lavage), any type of cell (such as brain cells, white blood cells, mononuclear cells) or body tissue.
  • body fluids including blood, urine, serum, gastric lavage
  • any type of cell such as brain cells, white blood cells, mononuclear cells
  • body tissue can include for example, southern and northern blotting, RFLP, direct sequencing and PCR amplification followed by hybridization of amplified products to a microarray comprising reference nucleic acid sequences.
  • CNV/SNP containing nucleic acids can be used to detect autism associated CNVs/SNPs in body tissue, cells, or fluid, and alter autism SNP containing marker protein expression for purposes of assessing the genetic and protein interactions involved in the development of autism.
  • the autism-associated CNV/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 becoming increasingly important in the art. Alternatively, new detection technologies can overcome this limitation and enable analysis of small samples containing as little as ⁇ 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 autism-associated CNV/SNP marker expression and accordingly, diagnose autism or an autism spectrum disorder.
  • kits which may contain a autism-associated CNV/SNP specific marker polynucleotide or one or more such markers immobilized on a Gene Chip, an oligonucleotide, a polypeptide, a peptide, an antibody, a label, said label being detectable and optionally, operably linked to an oligonucleotide, polypeptide or antibody marker, or reporter, a pharmaceutically acceptable carrier, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.
  • the kit contains reagents for identifying nucleic acids present in a biological sample with harbor nucleic acids comprising the genetic alterations described herein.
  • probes or primers are designed to flank the affected region in order to assess whether the CNV is present or absent.
  • chromosomes contain regions which provide suitable targets for the rational design of therapeutic agents which modulate their activity.
  • Small peptide molecules corresponding to these regions may be used to advantage in the design of therapeutic agents which effectively modulate the activity of the encoded proteins.
  • 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/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.
  • 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 autism 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. The rate of cellular metabolism, alterations in cellular morphology and/or receptor signaling of the host cells is measured to determine if the compound is capable of altering any of these parameters 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.
  • the autism-associated CNV/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).
  • 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. 92121); pcDNA3.1/V5&His (Invitrogen); baculovirus vectors such as pVL1392, pVL1393, or pAC360 (Invitrogen); and yeast vectors such as YRP17, YIP5, and YEP24 (New England Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 Stratagene Inc.); Picchia vectors such as pHIL-Dl (Phillips Petroleum Co., Bartlesville, Okla. 74004); retroviral vectors such as PLNCX and pLPCX (Clontech); and adenoviral and adeno-associated viral vectors.
  • pRc/CMV Invitrogen,
  • 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 (tip) control elements, bacteriophage T7 control elements, and hybrids thereof.
  • 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 AcMNP V 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 autism-associated CNVs/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 autism.
  • 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 neuronal signaling and neuronal cell communication and structure. Also provided herein are methods to screen for compounds capable of modulating the function of proteins encoded by CNV/SNP containing nucleic acids.
  • Another approach entails the use of phage display libraries engineered to express fragment of the polypeptides encoded by the CNV/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.
  • US Patents 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 therapeutic.
  • drugs which have, e.g., improved polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of polypeptide activity.
  • CNV/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 autism-associated CNV/SNP containing nucleic acids enables the production of strains of laboratory mice carrying the autism-associated CNVs/SNPs of the invention.
  • Transgenic mice expressing the autism-associated CNV/SNP of the invention provide a model system in which to examine the role of the protein encoded by the SNP containing nucleic acid in the development and progression towards autism.
  • 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 Production of the transgenic mice described above will facilitate the molecular elucidation of the role that a target protein plays in various cellular metabolic and neuronal processes.
  • 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.
  • 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 autism- associated CNV/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 retroviius-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 autism-associated CNV/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;
  • PCR polymerase chain reaction
  • PNS positive-negative selection
  • 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 (l-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5- iodou- racil, (FIAU).
  • HSV-TK Herpes Simplex virus thymidine kinase
  • GANC gancyclovir
  • FIAU l-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5- iodou- racil
  • autism- associated 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 autism-associated SNP nucleic acid and, therefore, facilitates
  • a knock-in animal is one in which the endogenous murine gene, for example, has been replaced with human autism-associated CNV/SNP containing gene of the invention. Such knock-in animals provide an ideal model system for studying the development of autism.
  • a autism-associated CNV/SNP containing nucleic acid, fragment thereof, or an autism-associated CNV/SNP fusion protein can be targeted in a "tissue specific manner" or "cell type specific manner” using a vector
  • nucleic acid sequences encoding all or a portion of autism-associated CNV/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 autism-associated CNV/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.
  • a platelet-derived growth factor B gene promoter described in U.S. Pat. No. 5,811,633
  • 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
  • Glial fibrillar acidic protein (GFAP) promoter for the expression of transgenes in glial cells.
  • Transgenic mice into which a nucleic acid containing the autism-associated CNV/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 autism.
  • compositions useful for treatment and diagnosis of autism may comprise, in addition to one of the above substances, a
  • compositions should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration, 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.
  • PennCNV was used to define CNVs across all genotyped samples. To control for potential chip-to-chip bias from the mixed SNP content introduced by genotyping across multiple chips types, only CNV calls from the 550K joint SNPs across the 550K, 610K, 660K, and 1M Illumina chips were considered. Low quality samples were excluded on a per sample basis if:
  • CNV regions CNV regions that define unambiguous sets of cases and controls impacted by CNVs which facilitates the immediate identification of "core" CNV genomic regions.
  • Fisher's exact test was used to gauge enrichment of the first order interactome of GABAR family of genes, as well as a test of 1000 random permutations of case/control labels.
  • CNVs Copy Number Variants
  • ASD autism spectrum disorders
  • Gabrb3 gene deficient mice exhibit impaired social and exploratory behaviors, deficits in non-selective attention and hypoplasia of cerebellar vermal lobules: a potential model of autism spectrum disorder (DeLorey, Sahbaie, Hashemi, Homanics, & Clark, 2008)
  • a preferred embodiment of the invention comprises clinical application of the information described herein to a patient.
  • Diagnostic compositions can be designed to identify the genetic alterations described herein in nucleic acids from a patient to assess susceptibility for developing autism or ASD. 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 CNV as described in Example I and set forth in Table II. The information obtained from the patient sample, which can optionally be amplified prior to assessment, will be used to diagnose a patient with an increased or decreased susceptibility for developing autism or ASD. Kits for performing the diagnostic method of the invention are also provided herein. Such kits comprise a microarray comprising nucleic acids containing at least one of the CNV/ SNPs provided herein in and the necessary reagents for assessing the patient samples as described above.
  • autism/ASD involved genes and the patient results will indicate which variants are present, and will identify those that possess an altered risk for developing ASD.
  • the information provided herein allows for therapeutic intervention at earlier times in disease progression than previously possible.
  • the CNV containing genes described herein provide novel targets for the development of new therapeutic agents efficacious for the treatment of this neurological disease.
  • the information provided herein can also be employed in a test and treat approach. Although relatively common, the ASDs are still relatively under diagnosed, especially in rural areas where community pediatricians may not be as knowledgeable about the deluge of research that continues to define these disorders and their treatment. By and large, the mainstay of treatment for the ASDs remains behavioral therapy. There are many different types of behavioral therapies that specifically cater to the diverse phenotypic manifestations of the ASDs, and in all cases earlier intervention is correlated with better results. Therefore, early diagnosis of particular ASD subtypes is crucial to early behavioral intervention (and
  • trastuzumab a monoclonal antibody that targets HER2 expressing cancer cells
  • trastuzumab a monoclonal antibody that targets HER2 expressing cancer cells
  • Figure 4 a successful personalized therapeutic
  • trastuzumab and other personalized therapeutics for cancer patients would have to undergo relatively crude interventions such as radical mastectomy, radiation, and chemotherapy with low efficacy and severe undesirable side effects.
  • trastuzumab binds to defective HER2 proteins, it prevents the HER2 protein from causing cells in the breast to reproduce uncontrollably which increases the survival of breast cancer patients.
  • trastuzumab is only effective in HER2 expressing cancer cells so patients must undergo a genetic test to determine whether their cancer will respond to treatment. In fact, this test and treat model for targeted therapy is now the gold standard for breast cancer treatment.
  • NGS next generation sequencing
  • NLGN/NRXN pathways neuronal cell adhesion pathways, glutamatergic receptor pathways, and a host of other neurophysiological pathways and networks that have been shown to be defective in sporadic cases.
  • Phenprobamate M Phenprobamate is a centrally acting Spasm Gamma-Aminobutyric Acid skeletal muscle relaxant. It acts by Type A (GABAA) Receptor interrupting neuronal communication Agonist
  • Phenprobamate is N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl
  • Primidone M is an anticonvulsant agent Epilepsy Gamma-Aminobutyric Acid that is structurally related to Type A (GABAA) Receptor barbiturates. It is a gamma- Agonist
  • GABA aminobutyric acid type
  • Adco-Topiramate contains Epilepsy Alpha-Amino-3-Hydroxy- topiramate, a sulfamate-substituted 5-Methyl-4- monosaccharide and an Isoxazolepropionic Acid anticonvulsant agent. It blocks the (AMPA) Receptor voltage dependent sodium channels, Antagonist;Gamma- augments the activity of the Aminobutyric Acid Type neurotransmitter gamma amino A (GABAA) Receptor butyrate at some subtypes of the Agonist;Sodium Channel GABA-A receptor, antagonizes the Blocker
  • Adco-Topiramate is indicated for
  • Zaleplon is a short-acting, non- Insomnia Gamma-Aminobutyric Acid benzodiazepine sedative-hypnotic. It Type A (GABAA) Receptor acts as an agonist at type 1 Agonist
  • capsules (5mg and lOmg).
  • Zolpidem* M Zolpidem is a non-benzodiazepine Insomnia Gamma-Arninobutyric Acid hypnotic of the imidazopyridine class.
  • Type A (GABAA) Receptor It acts by increasing the activity of Agonist
  • Zolpidem tartrate* M Zolfresh contains Zolpidem tartrate, a Insomnia Gamma-Aminobutyric Acid nonbenzodiazepine sedative-hypnotic, Type A (GABAA) Receptor which belongs to the imidazopyridine Agonist
  • GABA-A gamma-amino-n- butyric acid type A
  • UBEA ubiquitin conjugation
  • GABA gamma-aminobutyric acid receptor signaling
  • CACNA voltage gated calcium channels
  • GEM metabotropic glutamate
  • Many studies have found defective genetic networks in the ASDs, [21,23,37- 40] (see [16] for review), and we complement these in this work by uncovering new networks and implicating specific defective gene families that may be enriched for novel potential therapeutic targets.
  • GPCRs G-protein- coupled receptors
  • serine/threonine and tyrosine protein kinases zinc metallopeptidases
  • serine proteases nuclear hormone receptors and phosphodiesterases
  • CAG Center for Applied Genomics
  • CNVs with the PennCNV algorithm [66] which combines multiple values, including genotyping fluorescence intensity (Log R Ratio), population frequency of SNP minor alleles (B Allele Frequency), and SNP spacing into a hidden Markov model.
  • the term 'CNV' represents individual CNV calls, whereas 'CNVR' refers to population-level variation shared across subjects.
  • CNV burden defined as the average span of CNVs, between cases and controls and estimates of significance were computed using PLINK [67].
  • CNVRs were defined based on the genomic boundaries of individual CNVs, and the significance of the difference in CNVR frequency between cases and controls was evaluated at each CNVR using Fisher's exact test.
  • GFINs Gene Family Interaction Networks
  • GFIN gene family interaction networks
  • genomic coordinates were those described by
  • Betancur (2011 Brain Res).
  • the GRM/mGluR network generated by Cytoscape from the Human Interactome database was described by Elia et al. (2012 Nat Genet) using UCSC Genome Browser definitions for gene coordinates.
  • CNV calls were analyzed for overlap to known syndromic regions and GRM network genes. All syndromic aberrations detected by clinical cytogenetic laboratory testing were confirmed on corresponding SNP arrays.
  • GWAS genome- wide association study
  • CNVs rare copy number variants
  • mGlu metabotropic glutamate receptor
  • MXD MAX Dimerization Protein
  • CALM1 Calmodulin 1
  • CNVs copy number variation
  • PARP8 poly ADP-ribose polymerase family 8
  • GFINs gene family interaction networks
  • CNVRs Copy number variable regions
  • Table 7 Top gene family interaction networks discovered.
  • GFINs gene family interaction networks
  • the table lists 5 the name and size of gene family tested, the number and frequency of network genes enriched in the second degree gene interaction network, the number and frequency of cases harboring defects across the network, the number and frequency of controls harboring defects across the network, the significance of association by Fisher's exact test, the enrichment of CNV defects in cases, and the significance of that enrichment 0 by 1000 random network permutations.
  • Table 8 Most significant individual gene interaction networks ranked by permutation testing. The table lists the name and gene family member tested, the number and frequency of network genes enriched, the number and frequency of cases harboring defects, the number and frequency of controls harboring defects, and the significance of association by Fisher's exact test, the odds ratio of the effect size, and the significance of association my random permutation of network while controlling for number of enes tested.
  • GRM1, GRM3, GRM5, GRM7, and GRM8 that we identified as unique to cases and thus enriched are the same GRMs we identified as being pathogenic in ADHD and may impact glutaminergic signaling.
  • MXD MAX dimerization protein
  • MXD family of genes encode proteins that interact with MYC/MAX network of basic helix-loop-helix leucine zipper (bHLHZ) transcription factors that regulate cell proliferation, differentiation, and apoptosis [MIM 600021] [57]; MXD genes are important candidate tumor suppressor genes as the MXD-MYC-MAX network is dysregulated in various types of cancer [58].
  • the POU5F family of genes encodes for transcription factors containing a POU homeodomain, and their role has been demonstrated in embryonic development, especially during early embryogenesis, and it is necessary for embryonic stem cell pluripotency.
  • Component genes of the SMARCC gene family are members of the SWI/SNF family of proteins, whose members display helicase and ATPase activities and which are thought to regulate transcription of certain genes by altering the chromatin structure around those genes.
  • KIAA genes have been identified in the Kazusa cDNA sequencing project [61], and are predicted from novel large human cDNAs; however, they have no known function.
  • Calmodulin contains 149 amino acids that define 4 calcium-binding domains used for Ca 2+ -mediated coordination of a large number of enzymes, ion channels and other proteins including kinases and phosphatases; its functions include roles in growth and cell cycle regulation as well as in signal transduction and the synthesis and release of neurotransmitters [M 114180] [57].
  • NCOR1 nuclear receptor co-repressor 1
  • BAG1 BCL2-associated athanogene 1
  • NCOR1 is a transcriptional co- regulatory protein that appears to assist nuclear receptors in the down regulation of DNA expression through recruitment of histone deacetylases to DNA promoter regions; it is a principal regulator in neural stem cells [51].
  • the oncogene BCL2 is a membrane protein that blocks the apoptosis pathway, and BAG1 forms a BCL2- associated athanogene and represents a link between growth factor receptors and anti- apoptotic mechanisms.
  • the BAG1 gene has been implicated in age related
  • neurodegenerative diseases including Alzheimer's disease [62,63].
  • CNV'S copy number variants
  • Autism Spectrum Disorder occurs in approximately 1/88 individuals and is characterized by impairment in social communication and repetitive interests and activities 1 . Approximately 20% of cases occur in the context of an identifiable syndrome 2 . Genetic syndromes with ASD are heterogeneous, including cytogenetically visible chromosomal alterations (e.g.
  • Trisomy 21 Trisomy 21
  • microdeletion and microduplication syndromes e.g. 22ql 1.2 deletion syndrome [22ql l .2DS]; 22ql l.2 duplication syndrome [22ql l.2DupS]
  • monogenic disorders e.g. Fragile X Syndrome [FXS], Tuberous Sclerosis [TS]
  • FXS Tuberous Sclerosis
  • prenatal exposure to thalidomide, valproic acid, misoprostol, ethanol and maternal rubella infection have been associated with an elevated risk of ASD 14-19 .
  • Tuberous Sclerosis leads to over inhibition of signaling.
  • Auerbach and colleagues (2011) demonstrated abnormal synaptic learning and atypical behavior in mouse models of FXS and TS, and reversed these effects by breeding the two strains together - mice harboring both mutations had normal mGluR signaling, and learning and behavior that was indistinguishable from control mice 20 .
  • Other studies have demonstrated normalization of learning and behavior in Fragile X mice by
  • Charts were reviewed to confirm a diagnosis of ASD and also to determine medical comorbidities for each patient. Diagnosis of ASD was confirmed in the chart, but as this was a retrospective chart review, gold-standard research instruments (e.g. Autism).
  • Structural birth defects, genetic testing and medical conditions were recorded for each patient. Cases were categorized as "Syndromic ASD” if they had ASD and presence of a medical condition orstructural birth defect (e.g. cleft palate) that occurs in less than 1% of the general population. This criteria was established to define a subset of patients whose ASD and other medical problems would be highly unlikely to occur coincidentally - With a baseline rate of ASD at 1/88 and a medical condition that occurs in ⁇ 1% of the general population, the compound likelihood of both occurring by chance would be approximately 0.001%. See Figure 8.
  • DNA from subjects with ASD were each genotyped on the Human610-Quad or HumanHap550 SNP arrays from Illumina.
  • subjects were typed either on Illumina SNP arrays (Human610-Quad vl.O or HumanHap550) or Affymetrix 6.0 SNP arrays. Clustering and SNP calling was performed using
  • HMM hidden Markov model
  • Samples with SNP arrays of poor quality were excluded from CNV calling, since typically the proportion of false positives increases considerably for these samples.
  • Those samples where the genotyping call rate > 96%, standard deviation of LRR (LRR sd) ⁇ 0.4, GC-wave factor (GCWF) is between -0.2 and 0.2 after waviness correction, and total number of CNV calls for the sample ⁇ 100 were included in analysis.
  • genomic coordinates were those described by Betancur [PMID: 21129364].
  • the GRM/mGluR network generated by Cytoscape from the Human Interactome database was described by Elia et al. [PMID: 22138692] using UCSC Genome Browser definitions for gene coordinates (UCSC genes). This network from Cytoscape was used to define mGluR+ vs. mGluR- subsets.
  • GRM/mGluR network genes were identified based on I s degree interaction network of the eight GRM genes using the program Ingenuity Pathway Analysis (Ingenuity Systems Inc./Qiagen; Redwood City, CA) as well as the genes encoding the group I mGluR signaling pathway described in Kelleher et al. [PMID: 22558107]. CNV calls were analyzed for overlap to known syndromic regions and GRM network genes. All syndromic aberrations detected by clinical cytogenetic laboratory testing were confirmed on corresponding SNP arrays.
  • CNVs mGluR network copy number variations
  • CNVs in the mGluR network were found in 74% of patients with syndromic ASD compared to 16% of patients with nonsyndromic ASD (p ⁇ 0.001). Most of the mGluR CNV's in patients with syndromic ASD (75%) were included in larger clinically significant CNV's.
  • mGluR network genes are present in the 22ql 1.2 region (RANBP1) and on chromosome 21 (APP GRIK1 MX1 PCBP3 SETD4), patients with ASD in the presence of 22ql 1.2DS, 22ql 1.2DupS or Trisomy 21 accounted for 15 (33%) of the patients with Syndromic ASD + mGluR network changes.
  • Autism Spectrum Disorder in 22qll.2 Deletion Syndrome is associated with "second hit" in mGluR pathway
  • the 22ql 1.2 DS is the most common microdeletion syndrome in humans, occurring in 1 in 4,000 individuals.
  • the typical deletion spans approximately 3 Mb and includes approximately 45 genes, causing a variety of medical and behavioral disorders (Table 9) 25-28 .
  • ASD occurs in approximately 20%, and psychosis in 25% 5 ' 9 .
  • the 22ql 1.2DupS results in the same types of birth defects and medical comorbidities seen in 22ql 1.2 DS, but at a lower rate (among over 60 patients in our clinical cohort).
  • the prevalence of ASD is 27%, which is slightly higher than the rate in children with 22ql 1.2DS.
  • Thalidomide exposure during pregnancy causes a variety of birth defects that have all been reported in 22ql 1.2DS, including some that are extremely rare (e.g. phocomelia, radial ray defects). (Table 9). Miller and Stromland reported an elevated risk of ASD following exposure to thalidomide during early embryogenesis 16 . This study included prospective evaluation by a psychiatrist was done for adults who had been exposed to thalidomide during pregnancy and evaluation by a physician to document birth defects and associated features. All cases of ASD following thalidomide exposure had ear anomalies, suggesting exposure between days 24-28 post-fertilization. Among individuals exposed at this time, there was a 27% rate of ASD.
  • Valproic acid is widely used as an anticonvulsant, mood stabilizer, and to prevent migraine headaches. Exposure to VPA during pregnancy causes an increased rate of several birth defects, all of which have been reported in 22ql 1.2DS, and most of which have been seen in Thalidomide Embryopathy (Table 9). Table 9 compares the birth defects seen in 22ql 1.2 DS, Thalidomide Embryopathy and Fetal Valproate Syndrome. The comparison of all birth defects seen in 22ql 1.2 DS to the exposures syndromes was not made because 22ql 1.2 DS includes deletion of dozens of additional genes which we do not propose to be affected in Thalidomide Embryopathy or Fetal Valproate Syndrome.
  • VPA Deacetylase Inhibitor
  • Embryopathy are all reported in children with 22ql 1.2 Deletion Syndrome
  • Nanson, J. L. Autism in fetal alcohol syndrome a report of six cases. Alcohol. Clin. Exp. Res. 16, 558-565 (1992).
  • Embryological origin for autism developmental anomalies of the cranial nerve motor nuclei. J. Comp. Neurol. 370, 247-261 (1996).

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Abstract

La présente invention concerne des compositions et des méthodes pour le dépistage et le traitement de l'autisme et d'un trouble du spectre autistique.
EP15829843.0A 2014-05-30 2015-07-28 Modifications génétiques associées à l'autisme età un phénotype autistique, et procédés de diagnostic et de traitement de l'autisme Withdrawn EP3149211A1 (fr)

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US14/292,480 US20160244831A9 (en) 2011-07-07 2014-05-30 Genetic Alterations Associated with Autism and the Autistic Phenotype and Methods of Use Thereof for the Diagnosis and Treatment of Autism
US201414131359A 2014-07-10 2014-07-10
PCT/US2015/042354 WO2016022324A1 (fr) 2014-05-30 2015-07-28 Modifications génétiques associées à l'autisme età un phénotype autistique, et procédés de diagnostic et de traitement de l'autisme

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EP2609220B1 (fr) 2010-08-24 2018-06-27 The Children's Hospital of Philadelphia Association de variations génétiques récurrentes rares du trouble du déficit de l'attention avec hyperactivité (tdah) et procédés d'utilisation associés pour le diagnostic et le traitement du tdah
US11219617B2 (en) 2014-05-30 2022-01-11 The Children's Hospital Of Philadelphia Methods of diagnosing and treating autism
CN116919961A (zh) 2015-09-08 2023-10-24 费城儿童医院 诊断和治疗焦虑症的方法
CN109082473A (zh) * 2018-10-16 2018-12-25 兰州大学 一种公牛znf280ay基因拷贝数变异的检测方法及应用

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WO2000045812A1 (fr) * 1999-02-08 2000-08-10 Ortho-Mcneil Pharmaceutical, Inc. Dérivés d'agent anti-convulsif utilisés dans le traitement de l'autisme
US20070292962A1 (en) * 2006-04-10 2007-12-20 Duke University Methods and compositions for genetic markers for autism
CA2766246C (fr) * 2008-11-14 2020-03-10 Hakon Hakonarson Variants genetiques intervenant dans la cognition humaine et leurs procedes d'utilisation comme cibles diagnostiques et therapeutiques

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