Patent application title: Compositions and methods for diagnosis and treating mood disorders
Huda Akil (Ann Arbor, MI, US)
Huda Akil (Ann Arbor, MI, US)
William E. Bunney, Jr. (Laguna Beach, CA, US)
Prabhakara V. Choudary (Davis, CA, US)
Simon J. Evans (Milan, MI, US)
Edward G. Jones (Winters, CA, US)
Jun Li (Palo Alto, CA, US)
Jun Li (Palo Alto, CA, US)
Juan F. Lopez (Ann Arbor, MI, US)
Robert C. Thompson (Ann Arbor, MI, US)
Richard Myers (Stanford, CA, US)
Hiroaki Tomita (Irvine, CA, US)
Marquis P. Vawter (Niguel, CA, US)
Stanley Watson (Ann Arbor, MI, US)
The Board of Trustees of the Leland Stanford Junior University
IPC8 Class: AC12Q168FI
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2009-05-07
Patent application number: 20090117565
Patent application title: Compositions and methods for diagnosis and treating mood disorders
Simon J. Evans
Prabhakara V. Choudary
Robert C. Thompson
William E. Bunney, JR.
Edward G. Jones
Juan F. Lopez
Marquis P. Vawter
TOWNSEND AND TOWNSEND AND CREW, LLP
The Board of Trustees of the Leland Stanford Junior University
Origin: SAN FRANCISCO, CA US
IPC8 Class: AC12Q168FI
The present invention provides methods for diagnosing mental disorders
such as mood disorders, including bipolar disorder I and II and major
depression; The invention also provides methods of identifying modulators
of such mental disorders as well as methods of using these modulators to
treat patients suffering from such mental disorders.
1. A method for determining whether a subject is predisposed for major
depression disorder, the method comprising the steps of:(i) isolating a
subject's brain tissue, wherein the brain tissue is dorsolateral
prefrontal cortex tissue;(ii) contacting the subject's isolated brain
tissue with a nucleic acid reagent that selectively associates with a
polynucleotide with 95% identity to SEQ ID NO. 1;(iii) detecting the
level of reagent that selectively associates with the said
polynucleotide; and(iv) comparing the detected level of selectively
associated reagent with a control, whereby if the detected level is
significantly less than the control, an increased likelihood that the
subject has or is predisposed for major depression disorder is
determined; and whereby, if the detected level is not significantly less
than the control, an increase in said likelihood is not determined by the
30. The method of claim 1, wherein the subject is deceased.
CROSS-REFERENCES TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Ser. No. 60/423,247, filed Nov. 1, 2002 and U.S. Ser. No. 60/431,454, filed Dec. 6, 2002, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
BACKGROUND OF THE INVENTION
Clinical depression, including both bipolar disorders and major depression disorders, is a major public health problem, affecting an estimated 9.5% of the adult population of the United States each year. While it has been hypothesized that mental disorders, including mood disorders such as major depression and bipolar disorder as well as psychotic disorders such as schizophrenia, have complex genetic roots, little progress has been made in identifying gene sequences and gene products that play a role in causing these disorders, as is true for many diseases with a complex genetic origin (see, e.g. Burmeister, Biol. Psychiatry 45:522-532 (1999)). Relying on the discovery that certain genes expressed in particular brain pathways and regions are likely involved in the development of mental disorders, the present invention provides methods for diagnosis and treatment of mental disorders, as well as methods for identifying compounds effective in treating mental disorders.
BRIEF SUMMARY OF THE INVENTION
In order to further understand the neurobiology of mood disorders such as bipolar disorders (BP) and major depression disorders (MDD), the inventors of the present application have used DNA microarrays to study expression profiles of human post-mortem brains from patients diagnosed with BP or MDD. The work has focused on three brain regions: the anterior cingulated cortex (AnCg), the dorsolateral prefrontal cortex (DLPFC), and the cerebellum (CB).
The present invention demonstrates, for the first time, differential expression of the 72 nucleic acids listed in Table 2, the 16 nucleic acids listed in Table 3, or the 967 nucleic acids listed in Table 4, in the brains of patients suffering from mood disorders, such as bipolar disorder and major depression disorder, in comparison with normal control subjects. In addition, the present invention identifies biochemical pathways involved in mood disorders, where the proteins encoded by the nucleic acids listed in Table 2, 3, or 4 are components of the biochemical pathways (e.g., the bFGF signal transduction pathway, the GPCR and cAMP/PI/Rho pathways, the proteasome pathway, the oxidative phosphorylation pathway, Myelination, Cytochrome P450, or the GABA and glutamate pathways; see also FIGS. 1-5, 10-13, and 15).
Finally, genes that are differentially expressed in MDD or BP and by gender are useful in diagnosing mood disorders, as the prevalence of certain mood disorders shows a gender bias. Differential expression by brain region similarly is a useful diagnostic and therapeutic tool, as certain mood disorders primarily affect certain brain regions.
This invention thus provides methods for determining whether a subject has or is predisposed for a mental disorder such as bipolar disorder or major depression disorder. The invention also provides methods of providing a prognosis and for monitoring disease progression and treatment. Furthermore, the present invention provides nucleic acid and protein targets for assays for drugs for the treatment of mental disorders such as bipolar disorder and major depression disorder.
In some embodiments, the methods comprise the steps of: (i) obtaining a biological sample from a subject; (ii) contacting the sample with a reagent that selectively associates with a polynucleotide or polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence listed in Table 2, 3 or 4; and (iii) detecting the level of reagent that selectively associates with the sample, thereby determining whether the subject has or is predisposed for a mental disorder.
In some embodiments, the reagent is an antibody. In some embodiments, the reagent is a nucleic acid. In some embodiments, the reagent associates with a polynucleotide. In some embodiments, the reagent associates with a polypeptide. In some embodiments, the polynucleotide comprises a nucleotide sequence of a gene listed in Table 2, 3, or 4. In some embodiment, the polypeptide comprises an amino acid sequence of a gene listed in Table 2, 3, or 4. In some embodiments, the level of reagent that associates with the sample is different (i.e., higher or lower) from a level associated with humans without a mental disorder. In some embodiments, the biological sample is obtained from amniotic fluid. In some embodiments, the mental disorder is a mood disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder and major depression disorder.
The invention also provides methods of identifying a compound for treatment of a mental disorder. In some embodiments, the methods comprises the steps of: (i) contacting the compound with a polypeptide, which is encoded by a polynucleotide that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of Table 2, 3, or 4; and (ii) determining the functional effect of the compound upon the polypeptide, thereby identifying a compound for treatment of a mental disorder.
In some embodiments, the contacting step is performed in vitro. In some embodiment, the polypeptide comprises an amino acid sequence of a gene listed in Table 2, 3, or 4. In some embodiments, the polypeptide is expressed in a cell or biological sample, and the cell or biological sample is contacted with the compound. In some embodiments, the mental disorder is a mood disorder or psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder I and II and major depression. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the methods further comprise administering the compound to an animal, e.g., an animal subjected to stress as a model for depression and determining the effect on the animal, e.g., an invertebrate, a vertebrate, or a mammal. In some embodiments, the determining step comprises testing the animal's mental function.
In some embodiments, the methods comprise the steps of (i) contacting the compound to a cell, the cell comprising a polynucleotide that hybridizes under stringent conditions to a nucleotide sequence of Table 2, 3, or 4; and (ii) selecting a compound that modulates expression of the polynucleotide, thereby identifying a compound for treatment of a mental disorder. In some embodiments, the polynucleotide comprises a nucleotide sequence listed in Table 2, 3, or 4. In some embodiment, the expression of the polynucleotide is enhanced. In some embodiments, the expression of the polynucleotide is decreased. In some embodiments, the methods further comprise administering the compound to an animal and determining the effect on the animal. In some embodiments, the determining step comprises testing the animal's mental function. In some embodiments, the mental disorder is a mood disorder or psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder I and II and major depression. In some embodiments, the psychotic disorder is schizophrenia.
The invention also provides methods of treating a mental disorder in a subject. In some embodiments, the methods comprise the step of administering to the subject a therapeutically effective amount of a compound identified using the methods described above. In some embodiments, the mental disorder is a mood disorder or psychotic disorder. In some embodiments, the mood disorder is selected from the group consisting of bipolar disorder I and II and major depression. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the compound is a small organic molecule, an antibody, an antisense molecule, aptamer, or a peptide.
The invention also provides methods of treating mental disorders in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide, which is encoded by a polynucleotide that hybridizes under stringent conditions to a nucleic acid of Table 2, 3, or 4. In some embodiments, the polypeptide comprises an amino acid sequence encoded by a gene listed in Table 2, 3, or 4. In some embodiments, the mental disorder is a mood disorder or psychotic disorder. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the mood disorder is a bipolar disorder or major depression.
The invention also provides methods of treating mental disorders in a subject, comprising the step of administering to the subject a therapeutically effective amount of a polynucleotide, which hybridizes under stringent conditions to a nucleic acid of Table 2, 3, or 4. In some embodiments, the mental disorder is a mood disorder or psychotic disorder. In some embodiments, the psychotic disorder is schizophrenia. In some embodiments, the mood disorder is a bipolar disorder or major depression.
BRIEF DESCRIPTION OF THE DRAWINGS
Table 1: Table 1 lists genes differentially expressed in mood disorder subjects.
Table 2: Table 2 lists 72 genes differentially expressed in mood disorder subjects.
Table 3: Table 3 lists 16 genes differentially expressed in specific brain regions and mood disorder.
Table 4: Table 4 lists 967 genes differentially expressed in mood disorder subjects as determined by microarray analysis. Flag 1 indicates that the differential expression of the gene was confirmed by Real time PCR. Flag 2 indicates that differential expression of the gene was confirmed by anti-depressant studies. Flag 3 indicates that the gene belongs to an enriched gene ontology. Up and down indicates the direction of the changes compared to controls.
Table 5: Table 5 lists Real time PCR results on sample genes that are differentially expressed in mood disorder subjects.
Table 6: Table 6 lists anti-depressant treatment results for genes that are differentially expressed in mood disorder subjects.
Table 7: Tables 7A-D lists the gene ontology of selected genes differentially expressed in mood disorder subjects.
Table 8: Table 8 lists sample of genes that are differentially expressed in mood disorder subjects and are potential druggable targets.
FIG. 1 shows selected biochemical pathways for genes differentially expressed in mood disorder subjects.
FIG. 2 summarizes functions for signal transduction transcripts differentially expressed in MDD subjects.
FIG. 3 shows bFGF pathway transcripts differentially expressed in MDD subjects.
FIG. 4 shows values for differential expression of bFGF transcripts in MDD subjects.
FIG. 5 shows selected biochemical pathways that are dysregulated in mood disorders.
FIG. 6 shows selected biochemical pathways that are dysregulated in BP subjects.
FIG. 7 shows three genes overexpressed in mood disorder subjects that are located in the same chromosomal region.
FIG. 8 shows three genes overexpressed in mood disorder subjects that are located on 15q11-13 in the Prader-Willi region.
FIG. 9 shows certain genes regulated in human postmortem tissue and by antidepressants in rats.
FIG. 10 shows selected biochemical pathways (i.e., the GPCR and cAMP/PI/Rho pathways) for genes differentially expressed in mood disorder subjects. Two G protein coupled receptors, GPR37 and GPRC5B, are increased in both AnCg and DLPFC of BP patients, and decreased in MD. As downstream signaling pathways of GPCR, genes involved in cAMP pathway signaling are increased n BP patients, and decreased in MD. Genes involved in phosphatidylinositol pathways are deregulated specifically in MD.
FIG. 11 shows a selected biochemical pathway (i.e., the proteasome pathway) for genes differentially expressed in mood disorder subjects. The proteasome is an assembly of 28 alpha and beta subunits that functions to degrade proteins. The proteasome is involved in regulation of protein turnover and in particular oxidized proteins. There is an over representation of proteasome genes found in cortical regions of BP, but not in the cerebellum, suggesting that some functional compensation in the proteasome is occurring in BP patients.
FIG. 12 shows a selected biochemical pathway (i.e., the oxidative phosphorylation pathway) for genes differentially expressed in mood disorder subjects. The oxidative phosphorylation classification is involved in bioenergetics, metabolism, and as a byproduct can produce reactive oxygen species. This pathway is overly expressed in both bipolar and major depression, with differences between cortical regions and cerebellum.
FIG. 13 shows an example of a growth factor system (e.g., FGF) that is altered in mood disorders.
FIG. 14 shows RealTime PCR results which confirm that selected FGF-related genes first identified using microarray analysis are differentially expressed in mood disorders.
FIG. 15 shows selected genes in biochemical pathways involving GABA and glutamate that are differentially expressed in mood disorder subjects.
A "mental disorder" or "mental illness" or "mental disease" or "psychiatric or neuropsychiatric disease or illness or disorder" refers to mood disorders (e.g., major depression, mania, and bipolar disorders), psychotic disorders (e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, and shared psychotic disorder), personality disorders, anxiety disorders (e.g., obsessive-compulsive disorder) as well as other mental disorders such as substance-related disorders, childhood disorders, dementia, autistic disorder, adjustment disorder, delirium, multi-infarct dementia, and Tourette's disorder as described in Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV). Typically, such disorders have a complex genetic and/or a biochemical component.
"A psychotic disorder" refers to a condition that affects the mind, resulting in at least some loss of contact with reality. Symptoms of a psychotic disorder include, e.g., hallucinations, changed behavior that is not based on reality, delusions and the like. See, e.g., DSM IV. Schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, substance-induced psychotic disorder, and shared psychotic disorder are examples of psychotic disorders.
"Schizophrenia" refers to a psychotic disorder involving a withdrawal from reality by an individual. Symptoms comprise for at least a part of a month two or more of the following symptoms: delusions (only one symptom is required if a delusion is bizarre, such as being abducted in a space ship from the sun); hallucinations (only one symptom is required if hallucinations are of at least two voices talking to one another or of a voice that keeps up a running commentary on the patient's thoughts or actions); disorganized speech (e.g., frequent derailment or incoherence); grossly disorganized or catatonic behavior; or negative symptoms, i.e., affective flattening, alogia, or avolition. Schizophrenia encompasses disorders such as, e.g., schizoaffective disorders. Diagnosis of schizophrenia is described in, e.g., DSM IV. Types of schizophrenia include, e.g., paranoid, disorganized, catatonic, undifferentiated, and residual.
A "mood disorder" refers to disruption of feeling tone or emotional state experienced by an individual for an extensive period of time. Mood disorders include major depression disorder (i.e., unipolar disorder), mania, dysphoria, bipolar disorder, dysthymia, cyclothymia and many others. See, e.g., Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV).
"Major depression disorder," "major depressive disorder," or "unipolar disorder" refers to a mood disorder involving any of the following symptoms: persistent sad, anxious, or "empty" mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being "slowed down"; difficulty concentrating, remembering, or making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss or overeating and weight gain; thoughts of death or suicide or suicide attempts; restlessness or irritability; or persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain. Various subtypes of depression are described in, e.g., DSM IV.
"Bipolar disorder" is a mood disorder characterized by alternating periods of extreme moods. A person with bipolar disorder experiences cycling of moods that usually swing from being overly elated or irritable (mania) to sad and hopeless (depression) and then back again, with periods of normal mood in between. Diagnosis of bipolar disorder is described in, e.g., DSM IV. Bipolar disorders include bipolar disorder I (mania with or without major depression) and bipolar disorder II (hypomania with major depression), see, e.g., DSM IV.
An "agonist" refers to an agent that binds to a polypeptide or polynucleotide of the invention, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide or polynucleotide of the invention.
An "antagonist" refers to an agent that inhibits expression of a polypeptide or polynucleotide of the invention or binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of a polypeptide or polynucleotide of the invention.
"Inhibitors," "activators," and "modulators" of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term "modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity. Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%. Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
The term "test compound" or "drug candidate" or "modulator" or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, oligonucleotide, etc. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
A "small organic molecule" refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 Daltons and less than about 2500 Daltons, preferably less than about 2000 Daltons, preferably between about 100 to about 1000 Daltons, more preferably between about 200 to about 500 Daltons.
"Determining the functional effect" refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a polynucleotide or polypeptide of the invention (such as a polynucleotide of Table 2, 3, or 4 or a polypeptide encoded by a gene of Table 2, 3, or 4), e.g., measuring physical and chemical or phenotypic effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g. binding to antibodies; measuring changes in ligand binding affinity; measurement of calcium influx; measurement of the accumulation of an enzymatic product of a polypeptide of the invention or depletion of an substrate; measurement of changes in protein levels of a polypeptide of the invention; measurement of RNA stability; G-protein binding; GPCR phosphorylation or dephosphorylation; signal transduction, e.g., receptor-ligand interactions, second messenger concentrations (e.g., cAMP, IP3, or intracellular Ca2+); identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, calorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
Samples or assays comprising a nucleic acid or protein disclosed herein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
"Biological sample" includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood, spinal fluid, sputum, tissue, lysed cells, brain biopsy, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
"Antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH--CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).
The terms "peptidomimetic" and "mimetic" refer to a synthetic chemical compound that has substantially the same structural and functional characteristics of the polynucleotides, polypeptides, antagonists or agonists of the invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics" (Fauchere, Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as a CCX CKR, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of, e.g., --CH2NH--, --CH2S--, --CH2--CH2--, --CH═CH-- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CH2SO--. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. For example, a mimetic composition is within the scope of the invention if it is capable of carrying out the binding or enzymatic activities of a polypeptide or polynucleotide of the invention or inhibiting or increasing the enzymatic activity or expression of a polypeptide or polynucleotide of the invention.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
The term "isolated," when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs (haplotypes), and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an cl carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
"Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat.'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. Nucleic acids that hybridize to the genes listed in Tables 1-8 are encompassed by the invention.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (1990).
The phrase "a nucleic acid sequence encoding" refers to a nucleic acid that contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans-acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to conform with codon preference in a specific host cell.
The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
The phrase "specifically (or selectively) binds to an antibody" or "specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background.
One who is "predisposed for a mental disorder" as used herein means a person who has an inclination or a higher likelihood of developing a mental disorder when compared to an average person in the general population.
DETAILED DESCRIPTION OF THE INVENTION
To understand the complex genetic basis of mental disorders, the present invention provides studies that have been conducted to investigate the expression patterns of genes that are differentially expressed specifically in central nervous system of subjects with mood disorders. The large spectrum of symptoms associated with mental disorders is likely a reflection of the complex genetic basis and complex gene expression patterns in patients with mental disorders. Different combinations of the genes disclosed herein can be responsible for one or more mental disorders. Furthermore, brain pathways or circuits as well as subcellular pathways are important for understanding the development and diagnosis of mental disorders. The selected brain regions described herein (AnCng, DLPFC, and CB) are implicated in the clinical symptoms of mental disorders such as mood disorders. Brain imaging studies focusing on particular brain regions, cytoarchitectural changes in brain regions, expression of key neurotransmitters or related molecules in brain regions, and subcellular pathways in brain regions all contribute to the development of mental disorders, and thus are an important consideration in the diagnosis and therapeutic uses described herein.
The present invention demonstrates the altered expression (either higher or lower expression) of the genes of Tables 1-8 at the mRNA level in selected brain regions of patients diagnosed with mood disorders (e.g., bipolar disorder and major depression disorder) in comparison with normal individuals. This invention thus provides methods for diagnosis of mental disorders such as mood disorders (e.g., bipolar disorder, major depression, and the like), psychotic disorders (e.g., schizophrenia, and the like), and other mental disorders by detecting the level of a transcript or translation product of the genes listed in Tables 1-8 as well as their corresponding biochemical pathways. The chromosomal location of such genes can be used to discover other genes in the region that are linked to development of a particular disorder.
The invention further provides methods of identifying a compound useful for the treatment of such disorders by selecting compounds that modulates the functional effect of the translation products or the expression of the transcripts described herein. The invention also provides for methods of treating patients with such mental disorders, e.g., by administering the compounds of the invention or by gene therapy.
The genes and the polypeptides that they encode, which are associated with mood disorders such as bipolar disease and major depression, are useful for facilitating the design and development of various molecular diagnostic tools such as GeneChip® containing probe sets specific for all or selected mental disorders, including but not limited to mood disorders, and as an ante- and/or post-natal diagnostic tool for screening newborns in concert with genetic counseling. Other diagnostic applications include evaluation of disease susceptibility, prognosis, and monitoring of disease or treatment process, as well as providing individualized medicine via predictive drug profiling systems, e.g., by correlating specific genomic motifs with the clinical response of a patient to individual drugs. In addition, the present invention is useful for multiplex SNP or haplotype profiling, including but not limited to the identification of pharmacogenetic targets at the gene, mRNA, protein, and pathway level.
The genes and the polypeptides that they encode, described herein, as also useful as drug targets for the development of therapeutic drugs for the treatment or prevention of mental disorders, including but not limited to mood disorders. Mental disorders have a high co-morbidity with other neurological disorders, such as Parkinson's disease or Alzheimer's. Therefore, the present invention can be used for diagnosis and treatment of patients with multiple disease states that include a mental disorder such as a mood disorder.
For example, antidepressants belong to different classes, e.g., desipramine, bupropion, and fluoxetine are in general equally effect for the treatment of clinical depression, but act by different mechanisms. The similar effectiveness of the drugs for treatment of mood disorders suggests that they act through a yet as unidentified common pathway. We disclose herein that different classes of antidepressants (specific serotonin reuptake inhibitors, like fluoxetine and tricyclic antidepressants, like desipramine) regulate a common gene, and/or a common group of genes as well as a unique set of genes when the human and animal results herein are compared.
II. General Recombinant Nucleic Acid Methods for Use with the Invention
In numerous embodiments of the present invention, polynucleotides of the invention will be isolated and cloned using recombinant methods. Such polynucleotides include, e.g., those listed in Tables 1-8, which can be used for, e.g., protein expression or during the generation of variants, derivatives, expression cassettes, to monitor gene expression, for the isolation or detection of sequences of the invention in different species, for diagnostic purposes in a patient, e.g., to detect mutations or to detect expression levels of nucleic acids or polypeptides of the invention. In some embodiments, the sequences of the invention are operably linked to a heterologous promoter. In one embodiment, the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, a primate, etc.
A. General Recombinant Nucleic Acids Methods
This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression. A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).
B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding Desired Proteins
In general, the nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode cDNA or genomic DNA. The particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences of the genes listed in Tables 1-8, which provide a reference for PCR primers and defines suitable regions for isolating specific probes. Alternatively, where the sequence is cloned into an expression library, the expressed recombinant protein can be detected immunologically with antisera or purified antibodies made against a polypeptide comprising an amino acid sequence encoded by a gene listed in Table 1-8.
Methods for making and screening genomic and cDNA libraries are well known to those of skill in the art (see, e.g., Gubler and Hoffman Gene 25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977); and Sambrook, supra). Brain cells are an example of suitable cells to isolate RNA and cDNA sequences of the invention.
Briefly, to make the cDNA library, one should choose a source that is rich in mRNA. The mRNA can then be made into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. For a genomic library, the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template. Suitable primers can be designed from specific sequences of the invention. This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a polypeptide of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Pat. Nos. 4,683,195 and 4,683,202). Genes amplified by a PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
Appropriate primers and probes for identifying polynucleotides of the invention from mammalian tissues can be derived from the sequences provided herein. For a general overview of PCR, see, Innis et al. PCR Protocols. A Guide to Methods and Applications, Academic Press, San Diego (1990).
Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned.
A gene encoding a polypeptide of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression. These intermediate vectors are typically prokaryote vectors or shuttle vectors. The proteins can be expressed in either prokaryotes, using standard methods well known to those of skill in the art, or eukaryotes as described infra.
III. Purification of Proteins of the Invention
Either naturally occurring or recombinant polypeptides of the invention can be purified for use in functional assays. Naturally occurring polypeptides, e.g., polypeptides encoded by genes listed in Tables 1-8, can be purified, for example, from mouse or human tissue such as brain or any other source of an ortholog. Recombinant polypeptides can be purified from any suitable expression system.
The polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
A number of procedures can be employed when recombinant polypeptides are purified. For example, proteins having established molecular adhesion properties can be reversible fused to polypeptides of the invention. With the appropriate ligand, the polypeptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally the polypeptide can be purified using immunoaffinity columns.
A. Purification of Proteins from Recombinant Bacteria
When recombinant proteins are expressed by the transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the proteins may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook et al., both supra, and will be apparent to those of skill in the art.
The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.
Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques.
Alternatively, it is possible to purify proteins from bacteria periplasm. Where the protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al., supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
B. Standard Protein Separation Techniques For Purifying Proteins
1. Solubility Fractionation
Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
2. Size Differential Filtration
Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.
3. Column Chromatography
The proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art.
It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
IV. Detection of Gene Expression
Those of skill in the art will recognize that detection of expression of polynucleotides of the invention has many uses. For example, as discussed herein, detection of the level of polypeptides or polynucleotides of the invention in a patient is useful for diagnosing mental disorders including mood disorders or psychotic disorders or a predisposition for a mood disorder or psychotic disorder. Moreover, detection of gene expression is useful to identify modulators of expression of the polypeptides or polynucleotides of the invention.
A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention.
The selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587 (1969).
Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20).
The probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind. Thus, the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3H, 125I, 35S, 14C, or 32P-labeled probes or the like.
Other labels include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).
In general, a detector which monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
Most typically, the amount of RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent. Typically, the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation which does not comprise the modulator, or as compared to a baseline established for a particular reaction type. Means of detecting and quantifying labels are well known to those of skill in the art.
In preferred embodiments, the target nucleic acid or the probe is immobilized on a solid support. Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.
A variety of automated solid-phase assay techniques are also appropriate. For instance, very large scale immobilized polymer arrays (VLSIPS®), available from Affymetrix, Inc. (Santa Clara, Calif.) can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Nature Medicine 2(7): 753-759.
Detection can be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes). One preferred example uses an antibody that recognizes DNA-RNA heteroduplexes in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Coutlee et al. (1989) Analytical Biochemistry 181:153-162; Bogulavski (1986) et al. J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) Proc. Nat'l Acad. Sci. USA 65:993-1000; Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin. Microbial. 41:199-209; and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, including homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).
In addition to available antibodies, one of skill in the art can easily make antibodies specific for nucleic acid duplexes using existing techniques, or modify those antibodies that are commercially or publicly available. In addition to the art referenced above, general methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art (see, e.g., Paul (3rd ed.) Fundamental Immunology Raven Press, Ltd., NY (1993); Coligan Current Protocols in Immunology Wiley/Greene, NY (1991); Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY (1988); Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y., (1986); and Kohler and Milstein Nature 256: 495-497 (1975)). Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors (see, Huse et al. Science 246:1275-1281 (1989); and Ward et al. Nature 341:544-546 (1989)). Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 μM, preferably at least about 0.01 μM or better, and most typically and preferably, 0.001 μM or better.
The nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target. For example, the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.
The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system, in particular RT-PCR or real time PCR, and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present. Alternatively, the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells or tissue, preferentially human cells or tissue from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.
V. Immunological Detection of the Polypeptides of the Invention
In addition to the detection of polynucleotide expression using nucleic acid hybridization technology, one can also use immunoassays to detect polypeptides of the invention. Immunoassays can be used to qualitatively or quantitatively analyze polypeptides. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies. A Laboratory Manual (1988).
A. Antibodies to Target Polypeptides or Other Immunogens
Methods for producing polyclonal and monoclonal antibodies that react specifically with a protein of interest or other immunogen are known to those of skill in the art (see, e.g., Coligan, supra; and Harlow and Lane, supra; Stites et al., supra and references cited therein; Goding, supra; and Kohler and Milstein Nature, 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors (see, Huse et al., supra; and Ward et al., supra). For example, in order to produce antisera for use in an immunoassay, the protein of interest or an antigenic fragment thereof, is isolated as described herein. For example, a recombinant protein is produced in a transformed cell line. An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen.
Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross-reactivity against unrelated proteins or even other homologous proteins from other organisms, using a competitive binding immunoassay. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 mM, more usually at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better.
A number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein, such as one comprising an amino acid sequence encoded by a gene listed in Table 1-8 may also be used either in pure or impure form. Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein. Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.
Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the polypeptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow and Lane, supra).
Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include, e.g., transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., supra.
Once target protein specific antibodies are available, the protein can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician. For a review of immunological and immunoassay procedures in general see, Stites, supra. Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, supra; and Harlow and Lane, supra.
Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum that was raised to the protein (e.g., one has an amino acid sequence encoded by a gene listed in Table 1-8) or a fragment thereof. This antiserum is selected to have low cross-reactivity against different proteins and any such cross-reactivity is removed by immunoabsorption prior to use in the immunoassay.
B. Immunological Binding Assays
In a preferred embodiment, a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993); Stites, supra. Immunological binding assays (or immunoassays) typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case a polypeptide of the present invention or antigenic subsequences thereof). The capture agent is a moiety that specifically binds to the analyte. In a preferred embodiment, the capture agent is an antibody that specifically binds, for example, a polypeptide of the invention. The antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
In a preferred embodiment, the labeling agent is a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally, Kronval, et al. J. Immunol., 111:1401-1406 (1973); and Akerstrom, et al. J. Immunol., 135:2589-2542 (1985)).
Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10° C. to 40° C.
1. Non-Competitive Assay Formats
Immunoassays for detecting proteins of interest from tissue samples may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case the protein) is directly measured. In one preferred "sandwich" assay, for example, the capture agent (e.g., antibodies specific for a polypeptide encoded by a gene listed in Table 1-8) can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the polypeptide present in the test sample. The polypeptide thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
2. Competitive Assay Formats
In competitive assays, the amount of analyte (such as a polypeptide encoded by a gene listed in Table 1-8) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (e.g., an antibody specific for the analyte) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, the protein of interest is added to the sample and the sample is then contacted with a capture agent, in this case an antibody that specifically binds to a polypeptide of the invention. The amount of immunogen bound to the antibody is inversely proportional to the concentration of immunogen present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. For example, the amount of the polypeptide bound to the antibody may be determined either by measuring the amount of subject protein present in a protein/antibody complex or, alternatively, by measuring the amount of remaining uncomplexed protein. The amount of protein may be detected by providing a labeled protein molecule.
Immunoassays in the competitive binding format can be used for cross-reactivity determinations. For example, a protein of interest can be immobilized on a solid support. Proteins are added to the assay which compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein of interest. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered proteins, e.g., distantly related homologs.
The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein partially encoded by a sequence herein that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the target protein.
3. Other Assay Formats
In a particularly preferred embodiment, western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide of the invention in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest. For example, the antibodies specifically bind to a polypeptide of interest on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest.
Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev. 5:34-41).
The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
Non-radioactive labels are often attached by indirect means. The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound. A variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Pat. No. 4,391,904).
Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple calorimetric labels may be detected directly by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.
VI. Screening for Modulators of Polypeptides and Polynucleotides of the Invention
Modulators of polypeptides or polynucleotides of the invention, i.e. agonists or antagonists of their activity or modulators of polypeptide or polynucleotide expression, are useful for treating a number of human diseases, including mood disorders or psychotic disorders. Administration of agonists, antagonists or other agents that modulate expression of the polynucleotides or polypeptides of the invention can be used to treat patients with mood disorders or psychotic disorders.
A. Screening Methods
A number of different screening protocols can be utilized to identify agents that modulate the level of expression or activity of polypeptides and polynucleotides of the invention in cells, particularly mammalian cells, and especially human cells. In general terms, the screening methods involve screening a plurality of agents to identify an agent that modulates the polypeptide activity by binding to a polypeptide of the invention, modulating inhibitor binding to the polypeptide or activating expression of the polypeptide or polynucleotide, for example.
1. Binding Assays
Preliminary screens can be conducted by screening for agents capable of binding to a polypeptide of the invention, as at least some of the agents so identified are likely modulators of polypeptide activity. The binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet and Yamamura, (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61-89. The protein utilized in such assays can be naturally expressed, cloned or synthesized.
Binding assays are also useful, e.g., for identifying endogenous proteins that interact with a polypeptide of the invention. For example, antibodies, receptors or other molecules that bind a polypeptide of the invention can be identified in binding assays.
2. Expression Assays
Certain screening methods involve screening for a compound that up or down-regulates the expression of a polypeptide or polynucleotide of the invention. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing a polypeptide or polynucleotide of the invention and then detecting an increase or decrease in expression (either transcript, translation product, or catalytic product). Some assays are performed with peripheral cells, or other cells, that express an endogenous polypeptide or polynucleotide of the invention.
Polypeptide or polynucleotide expression can be detected in a number of different ways. As described infra, the expression level of a polynucleotide of the invention in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of a polynucleotide of the invention. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques. Alternatively, a polypeptide of the invention can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to a polypeptide of the invention.
Other cell-based assays are reporter assays conducted with cells that do not express a polypeptide or polynucleotide of the invention. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a promoter of a polynucleotide of the invention that is operably linked to a reporter gene that encodes a detectable product. A number of different reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes. Exemplary enzyme reporters include, but are not limited to, β-glucuronidase, chloramphenicol acetyl transferase (CAT); Alton and Vapnek (1979) Nature 282:864-869), luciferase, β-galactosidase, green fluorescent protein (GFP) and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101).
In these assays, cells harboring the reporter construct are contacted with a test compound. A test compound that either activates the promoter by binding to it or triggers a cascade that produces a molecule that activates the promoter causes expression of the detectable reporter. Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of a polynucleotide of the invention and a reporter operably linked thereto. Here, too, an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression, can be identified by the generation of signal associated with reporter expression.
The level of expression or activity can be compared to a baseline value. As indicated above, the baseline value can be a value for a control sample or a statistical value that is representative of expression levels for a control population (e.g., healthy individuals not having or at risk for mood disorders or psychotic disorders). Expression levels can also be determined for cells that do not express a polynucleotide of the invention as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells.
A variety of different types of cells can be utilized in the reporter assays. Cells that express an endogenous polypeptide or polynucleotide of the invention include, e.g., brain cells, including cells from the cerebellum, anterior cingulate cortex, or dorsolateral prefrontal cortex. Such brain regions are part of brain circuits or pathways that are implicated in mood disorders. Cells that do not endogenously express polynucleotides of the invention can be prokaryotic, but are preferably eukaryotic. The eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs. Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cell lines, and stem cells.
Various controls can be conducted to ensure that an observed activity is authentic including running parallel reactions with cells that lack the reporter construct or by not contacting a cell harboring the reporter construct with test compound. Compounds can also be further validated as described below.
3. Catalytic Activity
Catalytic activity of polypeptides of the invention can be determined by measuring the production of enzymatic products or by measuring the consumption of substrates. Activity refers to either the rate of catalysis or the ability to the polypeptide to bind (Km) the substrate or release the catalytic product (Kd).
Analysis of the activity of polypeptides of the invention are performed according to general biochemical analyses. Such assays include cell-based assays as well as in vitro assays involving purified or partially purified polypeptides or crude cell lysates. The assays generally involve providing a known quantity of substrate and quantifying product as a function of time.
Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Preferably such studies are conducted with suitable animal models. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if expression or activity of a polynucleotide or polypeptide of the invention is in fact upregulated. The animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats.
5. Animal Models
Animal models of mental disorders also find use in screening for modulators. In one embodiment, rat models of depression (both chronic and acute), in which the rats are subjected to stress, are used for screening. In one embodiment, invertebrate models such as Drosophila models can be used, screening for modulators of Drosophila orthologs of the human genes disclosed herein. In another embodiment, transgenic animal technology including gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, or gene overexpression, will result in the absence, decreased or increased expression of a polynucleotide or polypeptide of the invention. The same technology can also be applied to make knockout cells. When desired, tissue-specific expression or knockout of a polynucleotide or polypeptide of the invention may be necessary. Transgenic animals generated by such methods find use as animal models of mental disorders and are useful in screening for modulators of mental disorders.
Knockout cells and transgenic mice can be made by insertion of a marker gene or other heterologous gene into an endogenous gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting an endogenous polynucleotide of the invention with a mutated version of the polynucleotide, or by mutating an endogenous polynucleotide, e.g., by exposure to carcinogens.
For development of appropriate stem cells, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric targeted mice can be derived according to Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
B. Modulators of Polypeptides or Polynucleotides of the Invention
The agents tested as modulators of the polypeptides or polynucleotides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Alternatively, modulators can be genetically altered versions of a polypeptide or polynucleotide of the invention. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like. Modulators also include agents designed to reduce the level of mRNA of the invention (e.g. antisense molecules, ribozymes, DNAzymes and the like) or the level of translation from an mRNA.
In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (11993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., etc.).
C. Solid State and Soluble High Throughput Assays
In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.
The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non-covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-Gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to those of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc., Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank and Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
The invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of the polynucleotides or polypeptides of the invention. In a preferred embodiment, the methods of the invention include such a control reaction. For each of the assay formats described, "no modulator" control reactions that do not include a modulator provide a background level of binding activity.
In some assays it will be desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. First, a known activator of a polynucleotide or polypeptide of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of polynucleotide or polypeptide determined according to the methods herein. Second, a known inhibitor of a polynucleotide or polypeptide of the invention can be added, and the resulting decrease in signal for the expression or activity can be similarly detected.
D. Computer-Based Assays
Yet another assay for compounds that modulate the activity of a polypeptide or polynucleotide of the invention involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of the polypeptide or polynucleotide based on the structural information encoded by its amino acid or nucleotide sequence. The input sequence interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the molecule. Similar analyses can be performed on potential receptors or binding partners of the polypeptides or polynucleotides of the invention. The models of the protein or nucleotide structure are then examined to identify regions of the structure that have the ability to bind, e.g., a polypeptide or polynucleotide of the invention. These regions are then used to identify polypeptides that bind to a polypeptide or polynucleotide of the invention.
The three-dimensional structural model of a protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a potential receptor into the computer system. The amino acid sequences encoded by the nucleic acid sequences provided herein represent the primary sequences or subsequences of the proteins, which encode the structural information of the proteins. At least 10 residues of an amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM. The three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary, and quaternary structure of the protein of interest. The software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms," and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
The tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure. The user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
Once the structure has been generated, potential ligand binding regions are identified by the computer system. Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above. The three-dimensional structure of the potential ligand is then compared to that of a polypeptide or polynucleotide of the invention to identify binding sites of the polypeptide or polynucleotide of the invention. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of genes encoding a polypeptide or polynucleotide of the invention. Such mutations can be associated with disease states or genetic traits and can be used for diagnosis. As described above, GeneChip® and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes. Identification of the mutated a polypeptide or polynucleotide of the invention involves receiving input of a first amino acid sequence of a polypeptide of the invention (or of a first nucleic acid sequence encoding a polypeptide of the invention), e.g., any amino acid sequence having at least 60%, optionally at least 70% or 85%, identity with the amino acid sequence of interest, or conservatively modified versions thereof. The sequence is entered into the computer system as described above. The first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence. The second sequence is entered into the computer system in the manner described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the sequences are identified. Such sequences can represent allelic differences in various polynucleotides, including SNPs and/or haplotypes, of the invention, and mutations associated with disease states and genetic traits.
VII. Compositions, Kits and Integrated Systems
The invention provides compositions, kits and integrated systems for practicing the assays described herein using polypeptides or polynucleotides of the invention, antibodies specific for polypeptides or polynucleotides of the invention, etc.
The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more polynucleotides or polypeptides of the invention immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of polynucleotides or polypeptides of the invention can also be included in the assay compositions.
The invention also provides kits for carrying out the therapeutic and diagnostic assays of the invention. The kits typically include a probe that comprises an antibody that specifically binds to polypeptides or polynucleotides of the invention, and a label for detecting the presence of the probe. The kits may include several polynucleotide sequences encoding polypeptides of the invention. Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding the polypeptides of the invention, or on activity of the polypeptides of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of polypeptides of the invention, a robotic armature for mixing kit components or the like.
The invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of the polypeptides of the invention. The systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.
A number of robotic fluid transfer systems are available, or can easily be made from existing components. For example, a Zymate XP (Zymark Corporation; Hopkinton, Mass.) automated robot using a Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous STAT binding assays.
Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC, MACINTOSH®, or UNIX® based (e.g., SUN® work station) computers.
One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques. Lasar based systems can also be used.
VIII. Administration and Pharmaceutical Compositions
Modulators of the polynucleotides or polypeptides of the invention (e.g., antagonists or agonists) can be administered directly to a mammalian subject for modulation of activity of those molecules in vivo. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
Diseases that can be treated include the following, which include the corresponding reference number from Morrison, DSM-IV Made Easy, 1995: Schizophrenia, Catatonic, Subchronic, (295.21); Schizophrenia, Catatonic, Chronic (295.22); Schizophrenia, Catatonic, Subchronic with Acute Exacerbation (295.23); Schizophrenia, Catatonic, Chronic with Acute Exacerbation (295.24); Schizophrenia, Catatonic, in Remission (295.55); Schizophrenia, Catatonic, Unspecified (295.20); Schizophrenia, Disorganized, Subchronic (295.11); Schizophrenia, Disorganized, Chronic (295.12); Schizophrenia, Disorganized, Subchronic with Acute Exacerbation (295.13); Schizophrenia, Disorganized, Chronic with Acute Exacerbation (295.14); Schizophrenia, Disorganized, in Remission (295.15); Schizophrenia, Disorganized, Unspecified (295.10); Schizophrenia, Paranoid, Subchronic (295.31); Schizophrenia, Paranoid, Chronic (295.32); Schizophrenia, Paranoid, Subchronic with Acute Exacerbation (295.33); Schizophrenia, Paranoid, Chronic with Acute Exacerbation (295.34); Schizophrenia, Paranoid, in Remission (295.35); Schizophrenia, Paranoid, Unspecified (295.30); Schizophrenia, Undifferentiated, Subchronic (295.91); Schizophrenia, Undifferentiated, Chronic (295.92); Schizophrenia, Undifferentiated, Subchronic with Acute Exacerbation (295.93); Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation (295.94); Schizophrenia, Undifferentiated, in Remission (295.95); Schizophrenia, Undifferentiated, Unspecified (295.90); Schizophrenia, Residual, Subchronic (295.61); Schizophrenia, Residual, Chronic (295.62); Schizophrenia, Residual, Subchronic with Acute Exacerbation (295.63); Schizophrenia, Residual, Chronic with Acute Exacerbation (295.94); Schizophrenia, Residual, in Remission (295.65); Schizophrenia, Residual, Unspecified (295.60); Delusional (Paranoid) Disorder (297.10); Brief Reactive Psychosis (298.80); Schizophreniform Disorder (295.40); Schizoaffective Disorder (295.70); Induced Psychotic Disorder (297.30); Psychotic Disorder NOS (Atypical Psychosis) (298.90); Personality Disorders, Paranoid (301.00); Personality Disorders, Schizoid (301.20); Personality Disorders, Schizotypal (301.22); Personality Disorders, Antisocial (301.70); Personality Disorders, Borderline (301.83) and bipolar disorders, maniac, hypomaniac, dysthymic or cyclothymic disorders, substance-induced mood disorders, major depression, psychotic disorders, including paranoid psychosis, catatonic psychosis, delusional psychosis, having schizoaffective disorder, and substance-induced psychotic disorder.
In some embodiments, modulators of polynucleotides or polypeptides of the invention can be combined with other drugs useful for treating mental disorders including useful for treating mood disorders, e.g., schizophrenia, bipolar disorders, or major depression. In some preferred embodiments, pharmaceutical compositions of the invention comprise a modulator of a polypeptide of polynucleotide of the invention combined with at least one of the compounds useful for treating schizophrenia, bipolar disorder, or major depression, e.g., such as those described in U.S. Pat. Nos. 6,297,262; 6,284,760; 6,284,771; 6,232,326; 6,187,752; 6,117,890; 6,239,162 or 6,166,008.
The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985)).
The modulators (e.g., agonists or antagonists) of the expression or activity of the a polypeptide or polynucleotide of the invention, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation or in compositions useful for injection. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, can be administered or example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.
The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial response in the subject over time. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the mental disorder. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular compound or vector in a particular subject.
In determining the effective amount of the modulator to be administered a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies. In general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.
For administration, modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
IX. Gene Therapy Applications
A variety of human diseases can be treated by therapeutic approaches that involve stably introducing a gene into a human cell such that the gene is transcribed and the gene product is produced in the cell. Diseases amenable to treatment by this approach include inherited diseases, including those in which the defect is in a single or multiple genes. Gene therapy is also useful for treatment of acquired diseases and other conditions. For discussions on the application of gene therapy towards the treatment of genetic as well as acquired diseases, see, Miller, Nature 357:455-460 (1992); and Mulligan, Science 260:926-932 (1993).
In the context of the present invention, gene therapy can be used for treating a variety of disorders and/or diseases in which the polynucleotides and polypeptides of the invention has been implicated. For example, compounds, including polynucleotides, can be identified by the methods of the present invention as effective in treating a mental disorder. Introduction by gene therapy of these polynucleotides can then be used to treat, e.g., mental disorders including mood disorders and psychotic disorders.
A. Vectors for Gene Delivery
For delivery to a cell or organism, the polynucleotides of the invention can be incorporated into a vector. Examples of vectors used for such purposes include expression plasmids capable of directing the expression of the nucleic acids in the target cell. In other instances, the vector is a viral vector system wherein the nucleic acids are incorporated into a viral genome that is capable of transfecting the target cell. In a preferred embodiment, the polynucleotides can be operably linked to expression and control sequences that can direct expression of the gene in the desired target host cells. Thus, one can achieve expression of the nucleic acid under appropriate conditions in the target cell.
B. Gene Delivery Systems
Viral vector systems useful in the expression of the nucleic acids include, for example, naturally occurring or recombinant viral vector systems. Depending upon the particular application, suitable viral vectors include replication competent, replication deficient, and conditionally replicating viral vectors. For example, viral vectors can be derived from the genome of human or bovine adenoviruses, vaccinia virus, herpes virus, adeno-associated virus, minute virus of mice (MVM), HIV, sindbis virus, and retroviruses (including but not limited to Rous sarcoma virus), and MoMLV. Typically, the genes of interest are inserted into such vectors to allow packaging of the gene construct, typically with accompanying viral DNA, followed by infection of a sensitive host cell and expression of the gene of interest.
As used herein, "gene delivery system" refers to any means for the delivery of a nucleic acid of the invention to a target cell. In some embodiments of the invention, nucleic acids are conjugated to a cell receptor ligand for facilitated uptake (e.g., invagination of coated pits and internalization of the endosome) through an appropriate linking moiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180). For example, nucleic acids can be linked through a polylysine moiety to asialo-oromucocid, which is a ligand for the asialoglycoprotein receptor of hepatocytes.
Similarly, viral envelopes used for packaging gene constructs that include the nucleic acids of the invention can be modified by the addition of receptor ligands or antibodies specific for a receptor to permit receptor-mediated endocytosis into specific cells (see, e.g., WO 93/20221, WO 93/14188, and WO 94/06923). In some embodiments of the invention, the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis (Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)). In other embodiments, molecular conjugates of the instant invention can include microtubule inhibitors (WO/9406922), synthetic peptides mimicking influenza virus hemagglutinin (Plank et al., J. Biol. Chem. 269:12918-12924 (1994)), and nuclear localization signals such as SV40 T antigen (WO93/19768).
Retroviral vectors are also useful for introducing the nucleic acids of the invention into target cells or organisms. Retroviral vectors are produced by genetically manipulating retroviruses. The viral genome of retroviruses is RNA. Upon infection, this genomic RNA is reverse transcribed into a DNA copy which is integrated into the chromosomal DNA of transduced cells with a high degree of stability and efficiency. The integrated DNA copy is referred to as a provirus and is inherited by daughter cells as is any other gene. The wild type retroviral genome and the proviral DNA have three genes: the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site) (see, Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984)).
The design of retroviral vectors is well known to those of ordinary skill in the art. In brief, if the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis-acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes from which these sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome are well known in the art and are used to construct retroviral vectors. Preparation of retroviral vectors and their uses are described in many publications including, e.g., European Patent Application EPA 0 178 220; U.S. Pat. No. 4,405,712, Gilboa Biotechniques 4:504-512 (1986); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al. Biotechniques 6:608-614 (1988); Miller et al. Biotechniques 7:981-990 (1989); Miller (1992) sipra; Mulligan (1993), supra; and WO 92/07943.
The retroviral vector particles are prepared by recombinantly inserting the desired nucleotide sequence into a retrovirus vector and packaging the vector with retroviral capsid proteins by use of a packaging cell line. The resultant retroviral vector particle is incapable of replication in the host cell but is capable of integrating into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, the patient is capable of producing, for example, a polypeptide or polynucleotide of the invention and thus restore the cells to a normal phenotype.
Packaging cell lines that are used to prepare the retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins required for packaging, but which are incapable of producing infectious virions. The defective retroviral vectors that are used, on the other hand, lack these structural genes but encode the remaining proteins necessary for packaging. To prepare a packaging cell line, one can construct an infectious clone of a desired retrovirus in which the packaging site has been deleted. Cells comprising this construct will express all structural viral proteins, but the introduced DNA will be incapable of being packaged. Alternatively, packaging cell lines can be produced by transforming a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.
A number of packaging cell lines suitable for the present invention are also available in the prior art. Examples of these cell lines include Crip, GPE86, PA317 and PG13 (see Miller et al., J. Virol. 65:2220-2224 (1991)). Examples of other packaging cell lines are described in Cone and Mulligan Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984); Danos and Mulligan Proceedings of the National Academy of Sciences, USA, 85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller (1990), supra.
Packaging cell lines capable of producing retroviral vector particles with chimeric envelope proteins may be used. Alternatively, amphotropic or xenotropic envelope proteins, such as those produced by PA317 and GPX packaging cell lines may be used to package the retroviral vectors.
In some embodiments of the invention, an antisense polynucleotide is administered which hybridizes to a gene encoding a polypeptide of the invention. The antisense polypeptide can be provided as an antisense oligonucleotide (see, e.g., Murayama et al., Antisense Nucleic Acid Drug Dev. 7:109-114 (1997)). Genes encoding an antisense nucleic acid can also be provided; such genes can be introduced into cells by methods known to those of skill in the art. For example, one can introduce an antisense nucleotide sequence in a viral vector, such as, for example, in hepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173 (1997)), in adeno-associated virus (see, e.g., Xiao et al., Brain Res. 756:76-83 (1997)), or in other systems including, but not limited, to an HVJ (Sendai virus)-liposome gene delivery system (see, e.g., Kaneda et al., Ann. NY Acad. Sci. 811:299-308 (1997)), a "peptide vector" (see, e.g., Vidal et al., CR Acad. Sci. III 32:279-287 (1997)), as a gene in an episomal or plasmid vector (see, e.g., Cooper et al., Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene Ther. 8:575-584 (1997)), as a gene in a peptide-DNA aggregate (see, e.g., Niidome et al., J. Biol. Chem. 272:15307-15312 (1997)), as "naked DNA" (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466), in lipidic vector systems (see, e.g., Lee et al., Crit. Rev Ther Drug Carrier Syst. 14:173-206 (1997)), polymer coated liposomes (U.S. Pat. Nos. 5,213,804 and 5,013,556), cationic liposomes (Epand et al., U.S. Pat. Nos. 5,283,185; 5,578,475; 5,279,833; and 5,334,761), gas filled microspheres (U.S. Pat. No. 5,542,935), ligand-targeted encapsulated macromolecules (U.S. Pat. Nos. 5,108,921; 5,521,291; 5,554,386; and 5,166,320). In another embodiment, conditional expression systems, such as those typified by the tet-regulated systems and the RU-486 system, can be used (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). These systems impart small molecule control on the expression of the target gene(s) of interest.
C. Pharmaceutical Formulations
When used for pharmaceutical purposes, the vectors used for gene therapy are formulated in a suitable buffer, which can be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al. Biochemistiy 5:467 (1966).
The compositions can additionally include a stabilizer, enhancer, or other pharmaceutically acceptable carriers or vehicles. A pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the nucleic acids of the invention and any associated vector. A physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).
D. Administration of Formulations
The formulations of the invention can be delivered to any tissue or organ using any delivery method known to the ordinarily skilled artisan. In some embodiments of the invention, the nucleic acids of the invention are formulated in mucosal, topical, and/or buccal formulations, particularly mucoadhesive gel and topical gel formulations. Exemplary permeation enhancing compositions, polymer matrices, and mucoadhesive gel preparations for transdermal delivery are disclosed in U.S. Pat. No. 5,346,701.
E. Methods of Treatment
The gene therapy formulations of the invention are typically administered to a cell. The cell can be provided as part of a tissue, such as an epithelial membrane, or as an isolated cell, such as in tissue culture. The cell can be provided in vivo, ex vivo, or in vitro.
The formulations can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods. In some embodiments of the invention, the nucleic acids of the invention are introduced into cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, or biolistics. In further embodiments, the nucleic acids are taken up directly by the tissue of interest.
In some embodiments of the invention, the nucleic acids of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient. Examples of ex vivo administration of therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).
X. Diagnosis of Mood Disorders and Psychotic Disorders
The present invention also provides methods of diagnosing mood disorders (such as major depression or bipolar disorder), psychotic disorders (such as schizophrenia) Diagnosis involves determining the level of a polypeptide or polynucleotide of the invention in a patient and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polypeptide or polynucleotide of the invention in a healthy person not suffering from a mood disorder or psychotic disorder or under the effects of medication or other drugs. Variation of levels of a polypeptide or polynucleotide of the invention from the baseline range (either up or down) indicates that the patient has a mood disorder or psychotic disorder or at risk of developing at least some aspects of a mood disorder or psychotic disorder. In some embodiments, the level of a polypeptide or polynucleotide of the invention are measured by taking a blood, urine or tissue sample from a patient and measuring the amount of a polypeptide or polynucleotide of the invention in the sample using any number of detection methods, such as those discussed herein, e.g., SNPs or haplotypes associated with this genes.
In some embodiments, the level of the enzymatic product of a polypeptide or polynucleotide of the invention is measured and compared to a baseline value of a healthy person or persons. Modulated levels of the product compared to the baseline indicates that the patient has a mood disorder or psychotic disorder or is at risk of developing at least some aspects of a mood disorder or psychotic disorder. Patient samples, for example, can be blood, saliva, CSF, urine or tissue samples.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
Identification of Genes Dysregulated in Mood Disorders
A total of twenty mood disorder brains (9 bipolar and 11 major depression disorder patients) with twenty control brains were used in this study. Each brain pair (case and control) was matched on the basis of gender, age, and postmortem interval. Three brain regions, dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (AnCg) and the cerebellum (CB) were extracted for RNA and subjected to microarray analysis using Affymetrix oligonucleotide GeneChips®. Each RNA sample was subjected to two independent analyses. The results were analyzed using multiple statistical tools and algorithms with various stringencies. Real time PCR analysis was used to confirm differential gene expression for selected genes. The genes identified using this study are listed in Tables 1, 2, and 3. Furthermore, biochemical pathways associated with the differentially expressed genes were identified (see FIGS. 1-5).
The two cortical regions DLPFC and AnCg had similar gene expression profiles in controls but differed significantly in MDD and BP, demonstrating distinct gene expression profiles. BP subject showed more changes in AnCg compared to DLPFC whereas MDD show less profound changes in both cortical regions but had greater effects in the DLPFC than in the AnCg. For BP, several candidate genes were located in chromosomal region 15q11-13, which is associated with the Prader-Willi syndrome (see FIGS. 6-8).
Identification of Additional Genes Dysregulated in Mood Disorders
The RNA from three brain regions, dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (AnCg) and the cerebellum (CB) from deceased patients diagnosed with bipolar disease or major depression, and matched controls were extracted and subjected to microarray analysis using Affymetrix oligonucleotide GeneChips®. The patient's particular conditions in their terminal phase (agonal factors, e.g., seizure, coma, hypoxia, dehydration, and pyrexia) and the conditions of the brain tissue after death (postmortem factors, e.g., postmortem interval, and freezer interval) are two major influences on RNA preservation in postmortem brain tissue. Brain pH has been evaluated as an indicator for agonal status, and as an indicator of RNA preservation. The effect of agonal factors and pH were taken into account for quality control of the RNA. Two RNA samples were subjected to independent analyses. The results were analyzed using multiple statistical tools and algorithms with various stringencies. The 967 genes identified using this study are listed in Table 4. Real time PCR analysis was used to confirm differential gene expression for selected genes. Real time PCR confirmation of differential gene expression for selected genes is listed in Table 5.
Furthermore, biochemical pathways associated with the differentially expressed genes were identified. In particular, cortical areas in BP patients showed activation of several pathways, including the proteasome pathway, the oxidative phosphorylation pathway, the ATP synthesis pathway, and chaperones (i.e., heat shock proteins). In addition, signaling pathways dysregulated in BP include, e.g., G-coupled protein receptors, the phosphatidylinositol pathway, the cAMP pathway, the mitogen activated protein kinase pathway, cytoskeletal systems, and the cortical GABA and glutamate systems. In MD, dysregulated genes includes genes involved in transmission of nerve impulses, neurogenesis, and the fibroblast growth factor system (FGF). (see FIGS. 10-12). Gene ontology (i.e., genetic signatures) for BP and MD can conveniently be used in developing diagnostic and therapeutic regiments for mood disorders.
Identification of Additional Genes Dysregulated in Mood Disorders Using Rat Models of Depression and Anti-Depressant Treatment
Rats were exposed to chronic unpredictable stress treatments in parallel with chronic anti-depressants treatment (e.g., the tricyclic antidepressant desipramine and the specific serotonin reuptake inhibitor fluoxetine). Saline treated stressed rats (SS) and saline treated non-stressed rats (SN) were used as controls. In particular, saline treated stressed rats (SS) were compared to desipramine treated stressed rats (DS); saline treated stressed rats (SS) were compared to fluoxetine treated stressed rats (FS); saline treated non-stressed rats (SN) were compared to desipramine treated non-stressed rats (DN); saline treated non-stressed rats (SN) were compared to fluoxetine treated non-stressed rats (FN); and saline treated stressed rats (SS) were compared to saline treated non-stressed rats (SN). Gene expression changes in rat cortex following treatment were measured. The genes identified in this study are shown in Table 6. This data suggests that different classes of antidepressants, i.e., antidepressants with apparently different mechanisms of action may act through a common biochemical pathway.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, Genbank sequences, GO terms, patents, and patent applications cited herein are hereby incorporated by reference.
TABLE-US-00001 TABLE 1 GenBank DLPFC-MDD Chromosome Accession# Gene Description OMIM Location NM1964 Early growth response protein 1 (EGR1) EGR1 5q31.1 NM599 human Insulin-like growth factor binding protein IGFBP5 2q33-34 5 (IGFBP5) M87771 Fibroblast growth factor receptor k-sam, Splice k-sam-III 10q26 3 (k-sam-III) Z24725 H sapiens Mitogen-inducible gene (mig-2) mig-2 14q22.1 M64347 human Novel growth factor receptor (FGFR3) FGFR3 4p16.3 M80634 human Keratinocyte growth factor receptor FGFR2 10q26 (FGFR2) (SEQ ID NO: 1) Z14228 Nuclear mitotic apparatus protein 1, Alt. Splice NUMA U4 11q13 Form 2 (NuMA Clone U4) X67951 human Proliferation-associated gene (PAGA) PAGA 1p34.1 GenBank DLPFC-MDD Accession # Gene Description AF036268 SH3-domain GRB2-like 2 OMIM - SH3 DOMAIN, GRB2-LIKE, 2; SH3GL2 AF060877 regulator of G-protein signalling 20 OMIM - REGULATOR OF G PROTEIN SIGNALING 20; RGS20 AL049538 ras association (RalGDS/AF-6) domain containing OMIM - RAL GUANINE NUCLEOTIDE DISSOCIATION STIMULATOR; protein JC265 RALGDS D14838 fibroblast growth factor 9 (glia-activating factor) OMIM - FIBROBLAST GROWTH FACTOR 9; FGF9 D26070 inositol 1,4,5-triphosphate receptor, type 1 OMIM - INOSITOL 1,4,5-TRIPHOSPHATE RECEPTOR, TYPE 1; ITPR1 J02902 protein phosphatase 2 (formerly 2A), regulatory OMIM - PROTEIN PHOSPHATASE 2, STRUCTURAL/REGULATORY subunit A (PR 65), alpha isoform SUBUNIT A, ALPHA; PPP2R1A J04513 fibroblast growth factor 2 (basic) OMIM - FIBROBLAST GROWTH FACTOR 2; FGF2 L05624 mitogen-activated protein kinase kinase 1 OMIM - MITOGEN-ACTIVATED PROTEIN KINASE KINASE 1; MAP2K1 M64788 RAP1, GTPase activating protein 1 OMIM - RAP1, GTPase-ACTIVATING PROTEIN 1; RAP1GA1 M87771 fibroblast growth factor receptor 2 (bacteria- OMIM - FIBROBLAST GROWTH FACTOR RECEPTOR 2; FGFR2 expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson- Weiss syndrome) M96995 growth factor receptor-bound protein 2 OMIM - GROWTH FACTOR RECEPTOR-BOUND PROTEIN 2; GRB2 U09759 mitogen-activated protein kinase 9 OMIM - MITOGEN-ACTIVATED PROTEIN KINASE 9; MAPK9 U24152 p21/Cdc42/Rac1-activated kinase 1 (STE OMIM - p21/CDC42/RAC1-ACTIVATED KINASE 1; PAK1 20 homolog, yeast) U49857 transcriptional activator of the c-fos promoter W28432 Cluster Incl. W28432: 47f2 Homo sapiens OMIM - NEUROTROPHIC TYROSINE KINASE, RECEPTOR, TYPE 2; NTRK2 cDNA /gb = W28432 /gi = 1308443/ug = Hs.92030 /len = 921 X07109 protein kinase C, beta 1 OMIM - PROTEIN KINASE C, BETA-1; PRKCB1 X54938 inositol 1,4,5-trisphosphate 3-kinase A OMIM - INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE A; ITPKA Z71929 fibroblast growth factor receptor 2 (bacteria- OMIM - FIBROBLAST GROWTH FACTOR RECEPTOR 2; FGFR2 expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson- Weiss syndrome) GenBank Antcg BP Accession # Description Symbol NM_004794 RAB33A, member RAS oncogene family RAB33A NM_002844 protein tyrosine phosphatase, receptor type, K PTPRK M14752 M14752 HUMABLA Human c-abl gene |Gen ABL1 Bank==M14752 NM_005252 v-fos FBJ murine osteosarcoma viral oncogene FOS homolog NM_002229 jun B proto-oncogene JUNB NM_014813 KIAA0806 gene product KIAA0806 AB007943 AB007943: Homo sapiens mRNA for KIAA0474 RAP1GA1 protein |GenBank==AB007943 NM_004067 chimerin (chimaerin) 2 CHN2 NM_003676 degenerative spermatocyte homolog, lipid DEGS desaturase (Drosophila) NM_000830 glutamate receptor, ionotropic, kainate 1 GRIK1 NM_002487 necdin homolog (mouse) NDN NM_002921 retinal G protein coupled receptor RGR NM_001390 dystrobrevin, alpha DTNA NM_006000 tubulin, alpha 1 (testis specific) TUBA1 NM_001634 S-adenosylmethionine decarboxylase 1 AMD1 NM_006931 solute carrier family 2 (facilitated glucose transporter), SLC2A3 member 3 NM_003832 phosphoserine phosphatase-like PSPHL NM_005010 neuronal cell adhesion molecule NRCAM NM_002073 guanine nucleotide binding protein (G protein), GNAZ alpha z polypeptide L24123 L24123: Homo sapiens NRF1 protein (NRF1) NFE2L1 mRNA/cds = UNKNOWN /gb = L24123 /gi = 438646 /ug = Hs.83469 /len = 4992|Gen Bank==L24123 NM_000810 gamma-aminobutyric acid (GABA) A receptor, GABRA5 alpha 5 NM_005398 protein phosphatase 1, regulatory (inhibitor) PPP1R3C subunit 3C AI526089 AI526089: DU3.2-7.H07.r Homo sapiens cDNA| COX5B GenBank==AI526089 NM_000840 glutamate receptor, metabotropic 3 GRM3 NM_012249 ras-like protein TC10 TC10 NM_004791 integrin, beta-like 1 (with EGF-like repeat ITGBL1 domains) NM_000615 neural cell adhesion molecule 1 NCAM1 NM_003916 adaptor-related protein complex 1, sigma AP1S2 2 subunit NM_001406 ephrin-B3 EFNB3 NM_001718 bone morphogenetic protein 6 BMP6 X66358 X66358 cds#2 HSSTHPKB H. sapiens mRNA CDKL1 KKIALRE for serine/threonine protein kinase|GenBank==X66358 DLPC-BP D00654 actin, gamma 2, smooth muscle, enteric ACTG2 U19599 U19599 HSU19599 Human (BAX delta) mRNA| BAX GenBank==U19599 NM_006908 ras-related C3 botulinum toxin substrate 1 RAC1 (rho family, small GTP binding protein Rac1) NM_002374 microtubule-associated protein 2 MAP2 AJ001612 phosphoserine phosphatase-like PSPHL NM_000293 phosphorylase kinase, beta PHKB NM_020217 hypothetical protein DKFZp547I014 DKFZp547I014 NM_004379 cAMP responsive element binding protein 1 CREB1 NM_032041 neurocalcin delta NCALD NM_015716 Misshapen/NIK-related kinase MINK AF059274 Homo sapiens cDNA FLJ37320 fis, clone CSPG5 BRAMY2018106 NM_006158 neurofilament, light polypeptide 68 kDa NEFL NM_002730 protein kinase, cAMP-dependent, catalytic, PRKACA alpha NM_003885 cyclin-dependent kinase 5, regulatory sub CDK5R1 unit 1 (p35) NM_003020 Secretory granule, neuroendocrine protein 1 (SGNE1)(7B2 protein) located at chromosome band 15q13
TABLE-US-00002 TABLE 2 NM1964 Early growth response protein 1 (EGR1) NM599 human insulin-like growth factor binding protein 5 (IGFBP5) M87771 Fibroblast growth factor receptor k-sam, Splice 3 (k-sam-III) Z24725 H sapiens Mitogen-inducible gene (mig-2) M64347 human Novel growth factor receptor (FGFR3) M80634 human Keratinocyte growth factor receptor (FGFR2) (SEQ ID NO: 1) Z14228 Nuclear mitotic apparatus protein 1, Alt. Splice Form 2 (NuMA Clone U4) X67951 human Proliferation-associated gene (PAGA) AF036268 SH3-domain GRB2-like 2 AF060877 regulator of G-protein signalling 20 AL049538 ras association (RaIGDS/AF-6) domain containing protein JC265 D14838 fibroblast growth factor 9 (glia-activating factor) D26070 inositol 1,4,5-triphosphate receptor, type 1 J02902 protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), alpha isoform J04513 fibroblast growth factor 2 (basic) L05624 mitogen-activated protein kinase kinase 1 M64788 RAP1, GTPase activating protein 1 M87771 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) M96995 growth factor receptor-bound protein 2 U09759 mitogen-activated protein kinase 9 U24152 p21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast) U49857 transcriptional activator of the c-fos promoter W28432 Cluster Incl. W28432: 47f2 Homo sapiens cDNA /gb = W28432 /gi = 1308443 /ug = Hs.92030 /len = 921 X07109 protein kinase C, beta 1 X54938 inositol 1,4,5-trisphosphate 3-kinase A Z71929 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) NM_004794 RAB33A, member RAS oncogene family NM_002844 protein tyrosine phosphatase, receptor type, K M14752 M14752 HUMABLA Human c-abl gene|GenBank==M14752 NM_005252 v-fos FBJ murine osteosarcoma viral oncogene homolog NM_002229 jun B proto-oncogene NM_014813 KIAA0806 gene product AB007943 AB007943: Homo sapiens mRNA for KIAA0474 protein|GenBank==AB007943 NM_004067 chimerin (chimaerin) 2 NM_003676 degenerative spermatocyte homolog, lipid desaturase (Drosophila) NM_000830 glutamate receptor, ionotropic, kainate 1 NM_002487 necdin homolog (mouse) NM_002921 retinal G protein coupled receptor NM_001390 dystrobrevin, alpha NM_006000 tubulin, alpha 1 (testis specific) NM_001634 S-adenosylmethionine decarboxylase 1 NM_006931 solute carrier family 2 (facilitated glucose transporter), member 3 NM_003832 phosphoserine phosphatase-like NM_005010 neuronal cell adhesion molecule NM_002073 guanine nucleotide binding protein (G protein), alpha z polypeptide L24123 L24123: Homo sapiens NRF1 protein (NRF1) mRNA /cds = UNKNOWN /gb = L24123 /gi = 438646 /ug = Hs.83469 /len = 4992|GenBank==L24123 NM_000810 gamma-aminobutyric acid (GABA) A receptor, alpha 5 NM_005398 protein phosphatase 1, regulatory (inhibitor) subunit 3C AI526089 AI526089: DU3.2-7.H07.r Homo sapiens cDNA|GenBank==AI526089 NM_000840 glutamate receptor, metabotropic 3 NM_012249 ras-like protein TC10 NM_004791 integrin, beta-like 1 (with EGF-like repeat domains) NM_000615 neural cell adhesion molecule 1 NM_003916 adaptor-related protein complex 1, sigma 2 subunit NM_001406 ephrin-B3 NM_001718 bone morphogenetic protein 6 X66358 X66358 cds#2 HSSTHPKB H. sapiens mRNA KKIALRE for serine/threonine protein kinase|GenBank==X66358 D00654 actin, gamma 2, smooth muscle, enteric U19599 U19599 HSU19599 Human (BAX delta) mRNA|GenBank==U19599 NM_006908 ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) NM_002374 microtubule-associated protein 2 AJ001612 phosphoserine phosphatase-like NM_000293 phosphorylase kinase, beta NM_020217 hypothetical protein DKFZp547I014 NM_004379 cAMP responsive element binding protein 1 NM_032041 neurocalcin delta NM_015716 Misshapen/NIK-related kinase AF059274 Homo sapiens cDNA FLJ37320 fis, clone BRAMY2018106 NM_006158 neurofilament, light polypeptide 68 kDa NM_002730 protein kinase, cAMP-dependent, catalytic, alpha NM_003885 cyclin-dependent kinase 5, regulatory subunit 1 (p35)
TABLE-US-00003 TABLE 3 Acc. Disorder/Region Description Numb. MD DLPFC carboxypeptidase D U65090 prostaglandin D2 synthase (21 kD, brain) AI207842 NEL-like 1 (chicken) D83017 zinc finger protein 36, C3H type-like 1 X79067 phosphoribosyl pyrophosphate synthetase 1 X15331 MD AnCng solute carrier family 1 (glial high affinity glutamate transporter), member 3 D26443 clathrin, light polypeptide (Lcb) M20470 aldolase A, fructose-bisphosphate X05236 ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) X04741 BP AnCng v-raf-1 murine leukemia viral oncogene homolog 1 X03484 cytochrome c oxidase subunit Vb AI526089 proteasome (prosome, macropain) 26S subunit, non-ATPase, 1 D44466 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation X56468 protein, theta polypeptide nuclear receptor subfamily 4, group A, member 1 L13740 chondroitin sulfate proteoglycan 3 (neurocan) AF02654 fatty acid binding protein 7, brain AJ00296 BP DLPFC carboxypeptidase D U65090 indicates data missing or illegible when filed
TABLE-US-00004 TABLE 4 Summary Genbank Accession No. AnCg BP AnCg MD DLPFC BP DLPFC MD CB BP CB MD flags D50310 up up 1 L08485 up 1, 2, 3 U28964 up 1 AF016917 up up 1, 3 L19182 down 1 AJ001612 up down up down up 1, 3 U66879 up 1 J04046 up 1 X63575 up up up 1, 3 S74445 up 1, 2, 3 X71490 up up up up 1 AF112471 up up 1, 2, 3 AB006626 up 1 U37143 down down 1, 3 AC004131 up down up down down 1 M29273 down up down 1, 2 X76220 up up 1, 2 M12267 up 3 AF060877 down down down 1 AB018305 down 1, 2 U58334 down down down 1 AB020629 up down up down 1, 3 U37122 down down 1, 3 AL080061 down down down down 1, 3 M34309 down down 1 M80634 down down 1 (SEO ID NO: 1) M64347 down down 1 X57206 down down down 1, 3 X77196 down down 1, 3 Z24725 down down down 1 AB018342 down down 1, 3 Y10275 down up 1, 3 AB007943 down 1 AL049538 down down down 1 M14758 down down down 3 X13839 down down 3 X63432 up up 3 X04098 up up up 3 AF006082 up down 3 D67031 down down down 3 L22214 up 3 J03473 up 3 AJ236876 up up up AF072902 up up U84011 down down K02215 up up up AI800578 down down down AL049954 down R59606 down down M80899 down down 2, 3 U00957 up up AA114830 up U81607 up 3 M90360 down down X15414 up up 3 U05861 up D17793 down down 3 K03000 down down 2 U46689 up down 3 U24267 down down down M93405 down down 3 U34252 up up 3 X05236 up up up up up M21154 up up up 3 W63793 up 3 M63175 up up AB028994 down down U29926 down X81438 up up up up 3 D14662 down down down 3 AF091077 down 3 X97074 up up up up 2 D38293 up up up 3 J02611 down down M12529 down down 3 D86981 up down 3 U41518 down down U34846 down down up 2, 3 D87468 down 2 L04510 up 2, 3 AF049884 up 3 U02570 down down AB002292 down U50523 up 3 AI525393 up 3 AF006087 up up 2, 3 AF006088 up 3 Z11501 up L08424 down down 3 M27396 up up 3 S67156 down down AL096842 down down AB018258 down J05096 down down down 2 M37457 up up 2 W28508 down up AF007876 down down J04027 up 2, 3 L20977 up up 3 W28589 up up 3 AJ010953 up 2 D14710 up up up up 3 U09813 up up 3 AF087135 up up 3 AA845575 up up 3 AF047436 up up 3 AA917672 up 3 X83218 up up 3 D16469 up up AL049929 up up 3 D89052 up up 3 AI318615 down 3 AI547262 up 3 AA056747 up 3 L09235 up up up 3 AA877795 up up 2 X76228 up up up up 3 W26326 up up up X79888 up 2 X66030 down M76125 down S82297 down down AB021288 down down V00567 down down down AF029893 up up up up 3 AF082868 down down 3 U50708 down U00115 down down AL049257 up down down AB004066 down down down AF001383 up up 2, 3 U68485 up 2, 3 AF002697 up up 3 S78771 up up up up 3 AC005306 down up AB023169 up up U72649 down down down 2 AF047472 up down AB023171 up down down X94910 up up up up 3 AF054175 up 3 AF014837 down down AF009425 down down down X95592 up 2, 3 AB007948 up up up D86062 up 3 AL080097 down down down AF006621 up up up 3 J03037 down up down down 3 U79666 down 3 M76559 up 2 S60415 up 3 AF068862 up up up up 2 U12022 up up up D45887 up up AB020640 up U02390 up up 2 U02390 up up U02390 up X85030 down down U20325 up 2 AB002376 up down AL035079 up up AF070648 down down L10822 up up AF091433 down AF026166 up up 3 X74801 up up 3 AF026292 up 3 X69398 up up M38690 up down down AF023158 down M37712 down AL031282 up down M35543 up up down W27541 down down U59325 up up AF006484 down down 3 X66364 up 2 L04658 up up up X77743 up up 3 X66358 up U22398 down down down M16965 up up up W27184 up up up U65887 down 3 U60808 down 3 AI056696 up 3 U78516 down AL080084 up up 3 AA189161 up up AB023203 down down 3 U03749 up 2 Y00064 up U07223 up up 2 X70297 up W29042 up 3 D49738 up 3 Z30644 down 3 U89916 down down M59287 down down down M59287 down down down AF039704 up X91788 up 3 M20469 up up up 3 M20470 up up up up 3 AB020709 up up 2, 3 S80562 down 2, 3 D13146 up up M19650 up 3 Z21488 up up AB020675 up AB014533 up down down M92642 down down M58526 down down U65928 up up up 3 AA149486 up up 3 M22760 up 3 M19961 up 3 AI526089 up up up 3 T57872 up up 3 AA152406 down 3 AA978033 up 3 AB007618 up 3 N50520 up up 3 U65090 up down X51405 up S74445 up 2, 3 M27691 down 2 S68271 up up 3 D10656 up up 3 U49857 down down U49857 down down down AL038340 up up AL038340 up up AF039397 up up AF053641 up 3 M27826 down U89896 down up up 3 D32039 down up X15998 down X15998 down AF026547 down down down
M33146 down 2 L22569 up up X16832 down down 3 Y07593 up up up L06797 down down 3 L47738 up up up M33318 down 3 Y11307 down down down M98529 up up up up up AB002379 down down down D15057 up 3 AL050152 up down D31767 up 3 AL050084 down down AB002367 up down down 3 AF086947 up up U50733 up up up 3 W26651 up up up U48705 down L20817 down down U59321 down up AF000982 up AF007142 down down U63825 up up 3 AF021819 up 3 AL080115 up down AL049944 up up AL049934 down down down down AL050390 up up AL050272 up up AL050159 down down L08069 up up up up L08069 up up up AI810807 up up 2, 3 AI540958 up 3 AF000430 up up up 3 D50857 down down down 3 AF007875 up 3 U97105 up M97388 up up 3 D83407 up up S65738 up 3 U26742 down 3 U46744 down up 3 U84551 down up U46746 down 3 X68277 down down down 3 L05147 up AB013382 down down 3 U31930 up 3 U46461 down 3 D86550 up 2, 3 M91670 up up M91670 up up AL050282 up up M31210 down D13168 down X70940 up up 3 AB023159 up up up up up up U03877 down down U66406 up up 3 AB011542 up L18960 down AF035280 up 3 U36764 up 3 U39067 up up up 3 U94855 up up 3 U54558 up 3 AC002544 down down D13748 up up L36055 down 3 U49436 up up 2,3 AL080199 down down C18655 down AB002303 down down X51956 up 2 L35594 down up down down 2, 3 L35594 down up down down 2, 3 D45421 down 2 AF103905 down U81984 down 3 D83492 up up U12535 down M34309 down down down 3 X81625 up up down 3 J04058 up 3 AB028990 up J02931 down AJ002962 up up 3 AA977580 up 2 W26480 down down down AF035284 down down X87241 down down AF000561 down M30448 up up up AB014596 up 3 D14697 up up 3 U60060 up 3 U60061 up 3 X59065 down down down Z70276 up up up 3 U66198 up 3 D14838 up 2 Z69641 down down down Z69641 down M87770 down down down Z71929 down down down X55741 up up up AF070557 up W27472 down down W26655 up up AF052106 up up AL049949 down down down down 3 X02761 down down V01512 down down down V01512 down down down down K00650 down down AF032885 down down M84562 down U32519 up 3 AB014560 up 3 AI565760 up 3 AJ225028 up 3 M82919 up up 3 X15376 up up 3 D86181 up down S68805 down D00723 up down 3 Y13286 up up 3 S40719 down down down 3 D87467 up M65188 down 3 X52947 down down down 3 M57609 down down 3 U33267 up up up 3 X76648 up 3 AB020645 up up 2, 3 U08997 up down U08997 down X59834 down down down 3 U43083 up up 3 D90150 up up AF017656 up up up AJ238764 up 3 AL049367 down AB020662 down down down down AF047438 down 3 M22632 up up 3 AF016004 down up down down 3 D38449 up 3 U87460 up down up down down AJ011001 down X71973 up 2, 3 W28944 up up 3 M81886 up up 3 U10301 up 3 X82068 up 3 L19058 down 3 S40369 up up X77748 up up 2, 3 D87119 down down down down 3 D87119 down down down down 3 X04412 up down up down down 3 M16594 down down J05459 up 2, 3 U90313 up up up 3 M95809 up 3 X03473 up D64142 up D64142 up L19779 up M37583 up up 3 H15872 up up AA255502 down up up M25079 down down down L48215 down down AF019214 down down down AF029890 up up up U31814 up 3 AL034374 down up down down AI391567 down 3 U51004 up 3 AB014555 down 3 X58536 down down M32578 down U23803 up 3 X12671 up M16342 up 3 D89678 up 3 U01923 up 3 W27191 down 3 X92814 up 2 X99209 up AF068754 up 3 M11717 down down down L26336 up up up up up L26336 up up up up L26336 up down up down down L26336 up down L12723 up 2 X87949 up 3 X13794 up up up Y00371 up up up up L15189 up up 2, 3 AL021937 up X15183 up 3 J04988 up up up W28616 up M22382 up 3 AI912041 up 2, 3 X57830 up 2, 3 AI434146 down 3 AF012023 up X77956 down down AL022726 down U49283 up up up AA522698 up up up 3 X17025 up 3 U66042 up up M24594 up 3 X16302 up up AB017563 down 2 L42572 up 3 U26398 up up X77567 up up up 3 U96876 up 2 X53586 down down X07979 down 2 AL021786 down AA477898 up up X54938 up up up up 3 U23850 up up 3 AB016492 up up J04111 up up X51345 down down down AF070523 up D79994 down down down L02840 down 3 U52155 down 3 U39196 up Y15065 up 3 D26067 up 3 D31887 up up 3 D14663 up 3 AL049250 down down down D87074 up down D87443 up down 3 D87445 down AB002347 up AB002361 up AB007903 up 3
AB007963 down down AB011095 down down AB014526 down down AB014544 down down down down AB018335 down down AB020637 up AB020661 down down AB023152 up AB023209 down down AB023230 down down 3 AB028972 up up up AB028977 down 3 AB029034 down down AF070621 up 3 Y08319 up 3 AB002357 up 3 AF035621 up up up 3 U59919 up 3 AJ001685 down down J04182 up up down 3 U36336 down up down down 3 Y11395 up up AL050126 down 2 M90424 down 3 X02152 up up 3 X13794 up up AI535946 up up up up D55696 up 3 AF087693 up 2 X76488 up up down 3 U41060 up 3 X61118 down U79297 up up AL039458 down down 2 AB011540 down M63959 up up up up 3 M92439 up up up up AB012293 up up up 3 W26633 up up U03100 up up down 3 U03100 up down 3 D55649 up up 3 AA420624 down 3 U01828 up up U89330 up up S76756 down 3 L05624 up up up U17743 up up up 3 U71087 up up 3 Z11695 up up up 3 X14474 up 2, 3 X66867 down AF072250 up down D84557 up down X79440 up 3 S57212 up AW006742 down down down AI674208 down down up AI674208 down down up AF038186 up W26659 up AB014579 up up M16279 down 2 D25217 down AI127424 up 3 AF001359 down X70326 down up down 3 AF041080 down down down down AI670788 up up Z48051 up down U64565 down up 3 D14812 up 2, 3 AI597616 up up 3 Y11681 up up up up up 3 AL050361 up 3 Z98946 down AI547258 down down 2 AF072928 up up down 3 M55405 down down AF001548 down down down 2 AF013570 down down 2, 3 AF001548 down down down 2 D10667 down 2, 3 D10667 down 2, 3 J02854 down down 3 AF020267 down 3 AB029029 up down U42349 up up up 3 AF052142 up up up 3 AA126505 up 3 X77548 down 2 AF044209 up up U35139 up up up 2, 3 D87953 down down AF047185 up 3 AC002400 up up up AI345944 up 3 AA203354 up 2, 3 AF047181 up up up 3 AA527880 up 3 AA760866 up 3 AF050640 up up 3 AI541336 up 3 AC005329 up up AF053070 up up up up 3 Y16241 down down 3 D63878 down down down D23662 up X05608 up up up D83017 up up up 3 W27762 down down 3 X64318 up 3 Z83840 up up up AB023192 up up up up 3 AF019415 down down AF019415 down down X17620 up 2, 3 X73066 up up up 2, 3 AL038662 up 2, 3 X58965 up 2, 3 M86707 up up up 2 AI816034 up up U97669 down down W28770 up up up 3 AF002020 up down 3 AF002020 up down down down 3 AJ132583 up up 3 U61849 up up up up up up 2, 3 AI198311 up 2, 3 L13740 down down down down down down L13740 down down down down down down AB002341 up down 2 U55258 up up 2 U55258 up up 2 X99076 up AB011150 up up up up 3 U03985 up up 2, 3 X55740 down AI018523 down down down X75958 down down up down 2, 3 W28432 down down down Y10148 down down AL050066 up up up U48250 down down down U48250 down M63623 up down AF061034 up up 3 AF061034 up 3 U63717 up up 3 U62961 up 3 AB017016 up up up M80482 up down AB023211 down down down down L13385 up 2, 3 D63391 up up 3 U24152 up up up up 2, 3 U24152 up up up 2, 3 AF068864 up up up up AF068864 up up up up AF005043 up up 3 M93650 down 3 AI521453 up up up 3 X73424 up up 3 AB020631 down down D13892 up up 3 D25547 up up up up up 3 U52969 up up 3 AA535884 down U40370 up 2 AB007946 up S41458 down AF056490 down down down 3 L42451 up 3 X98248 up up up AB002345 down down down 2 AF093670 up down 3 U41816 down 3 AL096719 up 3 V00572 up up up 2, 3 M83088 up 2, 3 X84908 up U45976 up down 3 AF010312 up down up down AL120815 up up down down down Z29090 down 3 U81802 up up 3 U49070 up up W28299 up up up AL050371 up D30037 up U03090 down U60644 up up up 3 U84573 down 3 M54927 down up down down 3 M22299 up down 3 X57398 up up up up up D11428 up down up down down 3 AF001601 down down AL050161 down down down AF017786 down down up 2 AF016371 up 3 AF001691 up down 3 Y18207 down down Y18207 down N36638 down down down 3 Z50749 up 3 J02902 up J02902 up M64929 up up M64929 up M29551 up 2 X89416 up up U44772 up 3 AB014512 down 3 X67951 up 2 L19185 up up up U25182 up up M33336 up up 3 M33336 up up 3 M33336 up X07109 up up 3 X06318 up up 3 Z15108 up up 3 U29185 up 2 AB011124 up up up X15331 up up up 3 D87258 down down down D87258 down down down J03077 up up 3 M85169 up up 3 L76517 up up down 3 D00760 up 2, 3 D00762 up up 3 D00761 up up 3 D26598 up 2, 3 D26600 up up 3 D26600 up up 3 D29011 up up 2 D29012 up 3 D38048 up up up 3 AF035309 up D44466 up up 2, 3 AL031177 up up AB009398 up 2, 3 D78151 up 3 U51007 up D50063 up 3 D38047 up up up up 3 D38047 up 3 AJ001612 down up down up down up 3 D14694 up
M98539 down down down AI207842 down down 3 AI207842 down down 3 U33284 up M14630 down M57399 down down 2, 3 M57399 down down down 2, 3 X54131 down L77886 up Z48541 up up 2 D64053 up M93426 down down down 2, 3 X63578 up 2 Z48054 down 3 AL031781 down AL031781 down down down AI540957 up 3 AF052113 up up 3 M28209 up up 3 AL050268 up 3 AL050268 up 3 U59877 down down down 3 AI189226 down down D14889 up up 3 D14889 up up up 3 M28212 up up 3 AJ133534 up up 3 X98001 up D25274 up U41654 up 2, 3 M35416 up M31469 up up up up up M31469 up up up up AF054183 up up up up up 3 X63465 up up X63465 up S80343 up 3 D79990 down down down U28686 down down down down down U89505 up up U23946 up M11433 down 3 X00129 up up up 3 N92548 down down AW044624 up 3 U27768 up 2, 3 U78166 up up 3 D26129 up up down down AF037204 up down 3 X13973 up up M63488 up up up D87735 up 2, 3 X55954 up up 3 AI708983 up up 3 X57958 up 3 Y00281 up up 2, 3 AL031659 up up AA977163 up up up 3 M13932 up 3 AI526078 up 3 D14530 up 3 S79522 up 3 X55715 up up 3 M84711 up up up 2, 3 Y11651 up 3 L10333 up up up 2, 3 AB020693 up up M84820 up AL049940 up up down AJ001515 down AB020658 up X91257 up up up up 3 M55580 down down AF051323 up up up D12676 up up 3 M25756 up down down 2 AF070614 up 3 L10338 down 3 AF049498 up up up up 2, 3 AB011178 down down down AB007937 down down AB015345 up up up up X97064 up 3 AJ131245 up 3 AF055006 up 3 AF054184 up up 2, 3 U73167 down down AB000220 up AB002438 down down down Z11793 down D86957 down down down 3 AI743134 down down down Z81326 up 3 D28423 up up AL031681 up down L41887 up down Y00757 up up 2 AF036268 up up up 3 AB007960 down down U33760 up up up up W26700 up up up U08989 up 3 U01824 down down 3 W28850 down 3 D26443 down down down 3 H10201 down X60036 up 3 M20681 up up 3 AF007216 down down down 2, 3 AF011390 down up 2, 3 AF015926 down down 3 D86959 up down U96094 up up up 3 D80000 up down 2, 3 X59960 up up 3 AF053136 up 2 AL049650 up up up AA733050 up up U40571 down 3 AF034546 up 3 X02317 up 3 X63753 up down 3 AJ001183 down 2 Z46629 down down down AB011088 down down J03040 down down J04765 up up AF052124 up up down AF039843 down down down Y08685 up down 3 D78130 up 3 M32313 up 2 M32886 down down 3 U88666 up 3 J00306 up 2 AI636761 up 2, 3 AB011107 down down down L78440 up up U04735 up up up up M86752 up up X99325 up up up up 2, 3 AF099989 up down 3 M31303 up 2 X85116 down down down down AL035306 up 3 U77942 down 2 D63506 down down U34804 up U40215 up up up up 3 AF039945 down 2 U93305 up up X68194 down up down down 3 D38522 up U18062 up up 3 M95787 down down AF010400 up up AL050265 up 3 AL050107 down down D50495 up 2 M80627 down down down D15050 down down down U19969 down down X52882 up 3 U49188 up up L24804 up X75861 up 3 W28869 up up 3 L06139 down down down X93512 up up S95936 up up down down 3 M55153 down 3 L12350 down L12350 down down AJ133115 down down M24748 up up X97544 up 3 X97544 up 3 L27476 down down AB028950 down AI688299 down up down down 3 R16035 up 3 U81006 up 3 M92383 up D38305 down down D13641 up 3 U09477 up 3 M12125 down 3 M12125 down down 3 U12595 up 3 X00437 down down AB011089 up 3 AF084260 up 3 AJ133769 up up X89066 up 3 AF042181 up down up AF001294 up AF035283 down down X06956 up up up up X06956 up up up 2 AF005392 up up up X01703 up up AF035316 up down down 3 X02344 up down U47634 up 3 X00734 up up S75463 up 3 D17517 up down U18934 up AI310002 up 2 AF075599 up up up up U67122 up 3 X04741 up up up 3 U27460 up up U30930 down up down 3 T79616 up 3 J04973 up up 2, 3 L32977 up AA526497 up 3 U30888 up M36200 up up AL050223 up 3 U56833 up 3 L06132 up 3 AJ002428 up up up up L08666 up 3 AF024710 down down AF022375 down down M63978 down up X51521 down Z19554 down down down 3 AF060902 up up D26068 down 2 AB011113 up down W27944 down down W26496 down down down D14661 up Y08614 up 3 J04977 up up up 3 M30938 up up U89436 up up 3 X56468 up 3 X56468 up up 3 M92843 down down down 2, 3 U07802 down down X78992 down down down AL050276 down down L11672 down AD000092 up up V00599 up down X55989 down S81916 up up up up
J00153 down down down AL049423 down down AF052148 down down AL022101 up up up AL118582 down down AI095508 up 2 W28612 down down down AL049378 down down AI700633 down up down AF070536 down down AF052119 up up up AL080113 up AL049265 down AL049390 down down AF070577 up up 2 AI827895 down down X95677 up up AL080093 up AL049969 down down down AF052141 up up up L27560 down W27522 up AL022718 up down AJ005694 down 3 AL120687 up up AL046322 up up AW043812 down H12054 up AC003007 up up J03071 down down M57417 down down down M33764 up M58028 up X74262 up U19796 up U22028 down X79568 down M55914 up M21154 up M10905 down U33429 up AB014539 down X63432 up up X56841 down Y00067 up AF007140 up X13839 down down AF023268 up AF053356 down U37122 down down AB000450 up AI126004 up AF002668 up X54304 down U57843 down X02344 up X04098 up up up U96074 up D32053 up up U59632 down X14346 down Z98046 up AL096737 down AB014598 down U17886 up AI986201 up AL080181 down AB014514 up R92331 down U24183 up D00860 up U09510 up AI635895 up U66033 down U51334 down AF020762 up U24105 up M36820 down U59912 up X63368 up AF047863 up U11861 up AL080122 up M14648 down Y14153 up X81637 down M88108 up AF042384 up AA704137 up AB011156 up AI862521 up AF047469 up AF025887 up AF091085 up AL035494 up AI540925 down D32129 down AB028972 up AF091071 up AL040137 down X15187 up U48730 down L08488 up K03460 up AF005361 up M95585 up M91670 up
TABLE-US-00005 TABLE 5 RT-PCR Confirmation Gene Bank AnCg DLPFC DLPFC Acc. No. Gene Name BP AnCg MD BP MD AB020629 ABCA8 down U37122 ADD3 down X63575 ATP2B2 down X71490 ATP6V0D1 up U66879 BAD down J04046 CALM3 up AF112471 CAMK2B up D50310 CCNI up AL080061 CLIC4 down S74445 CRABP1 up up U37143 CYP2J2 down down M34309 ERBB3 down M80634 FGFR2 down (SEQ ID NO: 1) M64347 FGFR3 down L08485 GABRA5 up AF016917 GABRD down down AC004131 GPRC5B down down AB006626 HDAC4 up L19182 IGFBP7 down down X57206 ITPKB down X77196 LAMP2 down M29273 MAG down down X76220 MAL down down Z24725 MIG2 down AB018342 MYO10 down M12267 OAT down down Y10275 PSPH down AJ001612 PSPHL down down AB007943 RAP1GA1 down AF060877 RGS20 down down AL049538 RIN2 down AB018305 SPON1 down down U58334 TP53BP2 down down U28964 YWHAZ up
TABLE-US-00006 TABLE 6 Summary of Anti-Depressant Treatment Data Genbank Accession No. Gene Name SSvDS SSvFS SNvDN SNvFN SNvSS M80899 AHNAK down down K03000 ALDH1A1 down down X97074 AP2S1 down U34846 AQP4 down down up D87468 ARC up down down down L04510 ARFD1 down AF006087 ARPC4 down J05096 ATP1A2 down M37457 ATP1A3 down down up J04027 ATP2B1 up AJ010953 ATP2C1 down AA877795 ATP6V1D down X79888 AUH down AF001383 BIN1 down U68485 BIN1 down U72649 BTG2 up X95592 C1D down M76559 CACNA2D1 up AF068862 CALB1 down down AF112471 CAMK2B down U02390 CAP2 down down up U20325 CART up X66364 CDK5 down up U03749 CHGA down U07223 CHN2 down AB020709 CNK2 down S80562 CNN3 down S74445 CRABP1 up S74445 CRABP1 up M27691 CREB1 up M33146 CSRP1 down AI810807 DNCI1 down D86550 DYRK1A down U49436 EIF5 down X51956 ENO2 up up L35594 ENPP2 down L35594 ENPP2 down D45421 ENPP2 down AA977580 FACL3 down D14838 FGF9 up up L08485 GABRA5 down AB020645 GLS down up down X71973 GPX4 up X77748 GRM3 down down J05459 GSTM3 down X92814 HRASLS3 down L12723 HSPA4 down down up L15189 HSPA9B up AI912041 HSPE1 down X57830 HTR2A down down AB017563 IGSF4 up U96876 INSIG1 down X07979 ITGB1 down AL050126 LAP1B down down AF087693 LIN7A down down AL039458 LRIG1 up M29273 MAG up X76220 MAL down down X14474 MAPT up M16279 MIC2 down down D14812 MRGX down AI547258 MT2A down down down AF013570 MYH11 down down down down D10667 MYH11 down down down down D10667 MYH11 down down down down AF001548 MYH11 down down down down AF001548 MYH11 down down down down X77548 NCOA4 up up U35139 NDN down AA203354 NDUFB3 down X73066 NME1 down X17620 NME1 down AL038662 NME1 down X58965 NME2 down M86707 NMT1 down U61849 NPTX1 down down AI198311 NPY down U55258 NRCAM down AB002341 NRCAM down U55258 NRCAM down U03985 NSF down X75958 NTRK2 down L13385 PAFAH1B1 down U24152 PAK1 down U24152 PAK1 down U40370 PDE1A up AB002345 PER2 down down down V00572 PGK1 down M83088 PGM1 up AF017786 PPAP2B down M29551 PPP3CB down down X67951 PRDX1 down U29185 PRNP up D00760 PSMA2 down D26598 PSMB3 down D29011 PSMB5 up D44466 PSMD1 up AB009398 PSMD13 down M57399 PTN down M57399 PTN down Z48541 PTPRO up M93426 PTPRZ1 down X63578 PVALB down up U41654 RAGA down U27768 RGS4 down down D87735 RPL14 down down Y00281 RPN1 down up M84711 RPS3A down down L10333 RTN1 down up M25756 SCG2 down up AF049498 SCN2B down AF054184 SEC61G down Y00757 SGNE1 down AF007216 SLC4A4 down down AF011390 SLC4A4 down down D80000 SMC1L1 down AF053136 SNCB up AJ001183 SOX10 up AB018305 SPON1 down M32313 SRD5A1 up AI636761 SST down up down J00306 SST down up down X99325 STK25 down M31303 STMN1 down U77942 STX7 down down up AF039945 SYNJ2 down D50495 TCEA2 up up X06956 TUBA1 down AI310002 UBE2D2 down J04973 UQCRC2 down D26068 WBSCR1 down M92843 ZFP36 up AI095508 down down AF070577 down
TABLE-US-00007 TABLE 7a AnCg BP Genetic Ontology Genbank Accession No. Gene Name Description 26S proteasome D00762 PSMA3 proteasome (prosome, macropain) subunit, alpha type, 3 D44466 PSMD1 proteasome (prosome, macropain) 26S subunit, non-ATPase, 1 D00761 PSMB1 proteasome (prosome, macropain) subunit, beta type, 1 D38048 PSMB7 proteasome (prosome, macropain) subunit, beta type, 7 D78151 PSMD2 proteasome (prosome, macropain) 26S subunit, non-ATPase, 2 D29012 PSMB6 proteasome (prosome, macropain) subunit, beta type, 6 D38047 PSMD8 proteasome (prosome, macropain) 26S subunit, non-ATPase, 8 D26598 PSMB3 proteasome (prosome, macropain) subunit, beta type, 3 D26600 PSMB4 proteasome (prosome, macropain) subunit, beta type, 4 synaptic transmission X82068 GRIA3 glutamate receptor, ionotrophic, AMPA 3 D11428 PMP22 peripheral myelin protein 22 AI636761 SST somatostatin AA126505 NCAM1 neural cell adhesion molecule 1 L10338 SCN1B sodium channel, voltage-gated, type I, beta polypeptide X81438 AMPH amphiphysin (Stiff-Man syndrome with breast cancer 128 kDa autoantigen) M19650 CNP 2',3'-cyclic nucleotide 3' phosphodiesterase L19058 GRIK1 glutamate receptor, ionotropic, kainate 1 AI198311 NPY neuropeptide Y U68485 BIN1 bridging integrator 1 M81886 GRIA1 glutamate receptor, ionotropic, AMPA 1 Z11695 MAPK1 mitogen-activated protein kinase 1 X77748 GRM3 glutamate receptor, metabotropic 3 AF052113 RAB14 RAB14, member RAS oncogene family U40215 SYN2 synapsin II U61849 NPTX1 neuronal pentraxin I Chaperone J04988 HSPCB heat shock 90 kDa protein 1, beta U12595 TRAP1 heat shock protein 75 L12723 HSPA4 heat shock 70 kDa protein 4 L08069 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1 AL038340 CRYAB crystallin, alpha B AL038340 CRYAB crystallin, alpha B X02344 TUBB2 tubulin, beta, 2 AF026166 CCT2 chaperonin containing TCP1, subunit 2 (beta) M63959 LRPAP1 low density lipoprotein receptor-related protein associated protein 1 L26336 HSPA2 heat shock 70 kDa protein 2 AF026292 CCT7 chaperonin containing TCP1, subunit 7 (eta) L08069 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1 AA149486 COX17 COX17 homolog, cytochrome c oxidase assembly protein (yeast) Y00371 HSPA8 heat shock 70 kDa protein 8 X74801 CCT3 chaperonin containing TCP1, subunit 3 (gamma) X56468 YWHAQ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide U41816 PFDN4 prefoldin 4 L15189 HSPA9B heat shock 70 kDa protein 9B (mortalin-2) L26336 HSPA2 heat shock 70 kDa protein 2
TABLE-US-00008 TABLE 7b AnCg MD: Genetic Ontology Genbank Accession Gene Description No. Name transporter activity T79616 UQCRB ubiquinol-cytochrome c reductase binding protein AI526089 COX5B cytochrome c oxidase subunit Vb AL049929 ATP6IP2 ATPase, H+ transporting, lysosomal interacting protein 2 AF006621 C4orf1 chromosome 4 open reading frame 1 L09235 ATP6V1A1 ATPase, H+ transporting, lysosomal 70 kDa, VI subunit A, isoform 1 M22760 COX5A cytochrome c oxidase subunit Va U01824 SLC1A2 solute carrier family 1 (glial high affinity glutamate transporter), member 2 N50520 COX7B cytochrome c oxidase subunit VIIb AF007216 SLC4A4 solute carrier family 4, sodium bicarbonate cotransporter, member 4 AF011390 SLC4A4 solute carrier family 4, sodium bicarbonate cotransporter, member 4 AA526497 UQCRH ubiquinol-cytochrome c reductase hinge protein AA845575 ATP5J ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 X52947 GJA1 gap junction protein, alpha 1, 43 kDa (connexin 43) D26443 SLC1A3 solute carrier family 1 (glial high affinity glutamate transporter), member 3 AF053070 NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51 kDa X76228 ATP6V1E1 ATPase, H+ transporting, lysosomal 31 kDa, VI subunit E isoform I X63575 ATP2B2 ATPase, Ca++ transporting, plasma membrane 2
TABLE-US-00009 TABLE 7c DLPFC BP Genetic Ontology Genbank Accession No. Gene Name Description hydrogen ion transporter activity AA917672 ATP5L ATP synthase, H+ transporting, mitochondrial F0 complex, subunit g AI526089 COX5B cytochrome c oxidase subunit Vb J04973 UQCRC2 ubiquinol-cytochrome c reductase core protein II AF050640 NDUFS2 NADH dehydrogenase (ubiquinone) Fe--S protein 2, 49 kDa (NADH-coenzyme Q reductase) AL049929 ATP6IP2 ATPase, H+ transporting, lysosomal interacting protein 2 AF047181 NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16 kDa AF047436 ATP5J2 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f, isoform 2 D14710 ATP5A1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1 U09813 ATP5G3 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9) isoform 3 N50520 COX7B cytochrome c oxidase subunit VIIb AF087135 ATP5H ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d D89052 ATP6V0B ATPase, H+ transporting, lysosomal 21 kDa, V0 subunit c'' X76228 ATP6V1E1 ATPase, H+ transporting, lysosomal 31 kDa, V1 subunit E isoform 1 Chaperone U56833 VBP1 von Hippel-Lindau binding protein 1 L08069 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1 AL038340 CRYAB crystallin, alpha B AL038340 CRYAB crystallin, alpha B X15183 HSPCA heat shock 90 kDa protein 1, alpha X56468 YWHAQ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide L24804 TEBP unactive progesterone receptor, 23 kD W28616 HSPCB heat shock 90 kDa protein 1, beta D49738 CKAP1 cytoskeleton-associated protein 1 AF026166 CCT2 chaperonin containing TCP1, subunit 2 (beta) M63959 LRPAP1 low density lipoprotein receptor-related protein associated protein 1 X87949 HSPA5 heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) L26336 HSPA2 heat shock 70 kDa protein 2 M22382 HSPD1 heat shock 60 kDa protein 1 (chaperonin) L08069 DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1 AF035316 TUBB tubulin, beta polypeptide AI912041 HSPE1 heat shock 10 kDa protein 1 (chaperonin 10) Y00371 HSPA8 heat shock 70 kDa protein 8 X74801 CCT3 chaperonin containing TCP1, subunit 3 (gamma) X56468 YWHAQ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide W29042 CIA30 CGI-65 protein L15189 HSPA9B heat shock 70 kDa protein 9B (mortalin-2) OXPHOS X71490 ATP6V0D1 ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d isoform 1 D14710 ATP5A1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle U09813 ATP5G3 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9) isoform 3 AF087135 ATP5H ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d AA845575 ATP5J ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 AF047436 ATP5J2 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f, isoform 2 AA917672 ATP5L ATP synthase, H+ transporting, mitochondrial F0 complex, subunit g X83218 ATP5O ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit D89052 ATP6V0B ATPase, H+ transporting, lysosomal 21 kDa, V0 subunit c'' AA056747 ATP6V1A1 ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A X76228 ATP6V1E1 ATPase, H+ transporting, lysosomal 31 kDa, V1 subunit E isoform 1 AI526089 COX5B cytochrome c oxidase subunit Vb N50520 COX7B cytochrome c oxidase subunit VIIb AC002400 NDUFAB1 NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8 kDa AA203354 NDUFB3 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12 kDa AF047181 NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16 kDa AF050640 NDUFS2 NADH dehydrogenase (ubiquinone) Fe--S protein 2, 49 kDa (NADH-coenzyme Q reductase) J04973 UQCRC2 ubiquinol-cytochrome c reductase core protein II L32977 UQCRFS1 ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 U17886 SDHB succinate dehydrogenase complex, subunit B, iron sulfur (Ip)
TABLE-US-00010 TABLE 7d DLPFC MD Genetic Ontology Genbank Accession Gene No. Name Description transmission of nerve impulse D11428 PMP22 peripheral myelin protein 22 AF049498 SCN2B sodium channel, voltage-gated, type II, beta polypeptide M82919 GABRB3 gamma-aminobutyric acid (GABA) A receptor, beta 3 X59834 GLUL glutamate-ammonia ligase (glutamine synthase) X81438 AMPH amphiphysin (Stiff-Man syndrome with breast cancer 128 kDa autoantigen) M54927 PLP1 proteolipid protein 1 (Pelizaeus-Merzbacher disease, spastic paraplegia 2, uncomplicated) Z11695 MAPK1 mitogen-activated protein kinase 1 U01824 SLC1A2 solute carrier family 1 (glial high affinity glutamate transporter), member 2 M32886 SRI sorcin U40215 SYN2 synapsin II X15376 GABRG2 gamma-aminobutyric acid (GABA) A receptor, gamma 2 D26443 SLC1A3 solute carrier family 1 (glial high affinity glutamate transporter), member 3 X68194 SYPL synaptophysin-like protein U61849 NPTX1 neuronal pentraxin I neurogenesis D11428 PMP22 peripheral myelin protein 22 U30930 UGT8 UDP glycosyltransferase 8 (UDP-galactose ceramide galactosyltransferase) W28770 NP25 neuronal protein M54927 PLP1 proteolipid protein 1 (Pelizaeus-Merzbacher disease, spastic paraplegia 2, uncomplicated) D83017 NELL1 NEL-like 1 (chicken) U34846 AQP4 aquaporin 4 Z70276 FGF12 fibroblast growth factor 12 M80899 AHNAK AHNAK nucleoprotein (desmoyokin) M57399 PTN pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) AF016004 GPM6B glycoprotein M6B X70326 MLP MARCKS-like protein AF036268 SH3GL2 SH3-domain GRB2-like 2 M93426 PTPRZ1 protein tyrosine phosphatase, receptor-type, Z polypeptide 1 U61849 NPTX1 neuronal pentraxin I M93650 PAX6 paired box gene 6 (aniridia, keratitis) phosphoric ester hydralase activity X55740 NT5E 5'-nucleotidase, ecto (CD73) AF001601 PON2 paraoxonase 2 X68277 DUSP1 dual specificity phosphatase 1 L35594 ENPP2 ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin) AB013382 DUSP6 dual specificity phosphatase 6 L05147 DUSP3 dual specificity phosphatase 3 (vaccinia virus phosphatase VH1-related) Z48541 PTPRO protein tyrosine phosphatase, receptor type, O N36638 PPP1R3C protein phosphatase 1, regulatory (inhibitor) subunit 3C AJ001612 PSPHL phosphoserine phosphatase-like AF017786 PPAP2B phosphatidic acid phosphatase type 2B U60644 PLD3 phospholipase D3 AF056490 PDE8A phosphodiesterase 8A M93426 PTPRZ1 protein tyrosine phosphatase, receptor-type, Z polypeptide 1
TABLE-US-00011 TABLE 8 Selected Potential Druggable Targets Genbank Accession Gene No. Name Target category Description AB020629 ABCA8 transporter ATP-binding cassette, sub-family A (ABC1), member 8 X63575 ATP2B2 transporter ATPase, Ca++ transporting, plasma membrane 2 X71490 ATP6V0D1 transporter ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d isoform 1 S74445 CRABP1 transporter cellular retinoic acid binding protein 1 M34309 ERBB3 tyrosine kinase v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) receptor M80634 FGFR2 tyrosine kinase fibroblast growth factor receptor 2 (bacteria-expressed kinase, (SEQ ID receptor keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon NO: 1) syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) M64347 FGFR3 tyrosine kinase fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) receptor AC004131 GPRC5B GPCR G protein-coupled receptor, family C, group 5, member B X77196 LAMP2 ligand for cell lysosomal-associated membrane protein 2 adhesion molecule U37122 ADD3 regulator of adducin 3 (gamma) kinase U66879 BAD regulator of BCL2-antagonist of cell death protease AB007943 RAP1GA1 regulator of RAP1, GTPase activating protein 1 kinase AF060877 RGS20 regulator of regulator of G-protein signalling 20 GTPase AL049538 RIN2 regulator of Ras and Rab interactor 2 GTPase U58334 TP53BP2 reuglator of tumor protein p53 binding protein, 2 protein degradation U28964 YWHAZ regulator of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide enzyme J04046 CALM3 kinase calmodulin 3 (phosphorylase kinase, delta) AF112471 CAMK2B kinase calcium/calmodulin-dependent protein kinase (CaM kinase) II beta D50310 CCNI regulator of cyclin I kinase U37143 CYP2J2 monooxygenase cytochrome P450, family 2, subfamily J, polypeptide 2 AB006626 HDAC4 enzyme histone deacetylase 4 X57206 ITPKB kinase inositol 1,4,5-trisphosphate 3-kinase B M12267 OAT enzyme ornithine aminotransferase (gyrate atrophy) Y10275 PSPH enzyme phosphoserine phosphatase AJ001612 PSPHL enzyme phosphoserine phosphatase-like AL080061 CLIC4 channel chloride intracellular channel 4 L08485 GABRAS channel gamma-aminobutyric acid (GABA) A receptor, alpha 5 AF016917 GABRD channel gamma-aminobutyric acid (GABA) A receptor, delta L19182 IGFBP7 regulator of insulin-like growth factor binding protein 7 receptor ligand M29273 MAG myelination myelin associated glycoprotein X76220 MAL myelination mal, T-cell differentiation protein Z24725 MIG2 signal mitogen inducible 2 transduction AB018342 MYO10 partner for myosin X calmodulin-like protein AB018305 SPON1 axon growth spondin 1, (f-spondin) extracellular matrix protein guidance
113106DNAHomo sapienshuman fibroblast growth factor receptor 2 (FGFR2) (keratinocyte growth factor receptor) cDNA 1cccgcgagca aagtttggtg gaggcaacgc aagcctgagt cctttcttcc tctcgttccc 60caaatccgag ggcagcccgc gggcgtcatg gcgctcctcc gcagcctggg gtacgcgtga 120agcccgggag gcttggcgcc ggcgaagacc caaggaccac tcttctgcgt ttggagttgc 180tccccgcaac cccgggctcg tcgctttctc catcccgacc cacgcggggc cggggacaac 240acaggtcgcg gaggagcgtt gccattcaag tgactgcagc agcagcgcag cgcctcggtt 300cctgagccca ccgcagctga aggcattgcg cgtagtccat gcccgtagag gaagtgtgca 360gatgggatta acgtccacat ggagatatgg aagaggaccg gggattggta ccgtaaccat 420ggtcagctgg ggtcgtttca tctgcctggt cgtggtcacc atggcaacct tgtccctggc 480ccggccctcc ttcagtttag ttgaggatac cacattagag ccagaagagc caccaaccaa 540ataccaaatc tctcaaccag aagtgtacgt ggctgcgcca ggggagtcgc tagaggtgcg 600ctgcctgttg aaagatgccg ccgtgatcag ttggactaag gatggggtgc acttggggcc 660caacaatagg acagtgctta ttggggagta cttgcagata aagggcgcca cacctagaga 720ctccggcctc tatgcttgta ctgccagtag gactgtagac agtgaaactt ggtacttcat 780ggtgaatgtc acagatgcca tctcatccgg agatgatgag gatgacaccg atggtgcgga 840agattttgtc agtgagaaca gtaacaacaa gagagcacca tactggacca acacagaaaa 900gatggaaaag cggctccatg ctgtgcctgc ggccaacact gtcaagtttc gctgcccagc 960cggggggaac ccaatgccaa ccatgcggtg gctgaaaaac gggaaggagt ttaagcagga 1020gcatcgcatt ggaggctaca aggtacgaaa ccagcactgg agcctcatta tggaaagtgt 1080ggtcccatct gacaagggaa attatacctg tgtagtggag aatgaatacg ggtccatcaa 1140tcacacgtac cacctggatg ttgtggagcg atcgcctcac cggcccatcc tccaagccgg 1200actgccggca aatgcctcca cagtggtcgg aggagacgta gagtttgtct gcaaggttta 1260cagtgatgcc cagccccaca tccagtggat caagcacgtg gaaaagaacg gcagtaaata 1320cgggcccgac gggctgccct acctcaaggt tctcaagcac tcggggataa atagttccaa 1380tgcagaagtg ctggctctgt tcaatgtgac cgaggcggat gctggggaat atatatgtaa 1440ggtctccaat tatatagggc aggccaacca gtctgcctgg ctcactgtcc tgccaaaaca 1500gcaagcgcct ggaagagaaa aggagattac agcttcccca gactacctgg agatagccat 1560ttactgcata ggggtcttct taatcgcctg tatggtggta acagtcatcc tgtgccgaat 1620gaagaacacg accaagaagc cagacttcag cagccagccg gctgtgcaca agctgaccaa 1680acgtatcccc ctgcggagac aggtaacagt ttcggctgag tccagctcct ccatgaactc 1740caacaccccg ctggtgagga taacaacacg cctctcttca acggcagaca cccccatgct 1800ggcaggggtc tccgagtatg aacttccaga ggacccaaaa tgggagtttc caagagataa 1860gctgacactg ggcaagcccc tgggagaagg ttgctttggg caagtggtca tggcggaagc 1920agtgggaatt gacaaagaca agcccaagga ggcggtcacc gtggccgtga agatgttgaa 1980agatgatgcc acagagaaag acctttctga tctggtgtca gagatggaga tgatgaagat 2040gattgggaaa cacaagaata tcataaatct tcttggagcc tgcacacagg atgggcctct 2100ctatgtcata gttgagtatg cctctaaagg caacctccga gaatacctcc gagcccggag 2160gccacccggg atggagtact cctatgacat taaccgtgtt cctgaggagc agatgacctt 2220caaggacttg gtgtcatgca cctaccagct ggccagacgg atggagtact tggcttccca 2280aaaatgtatt catcgagatt tagcagccag aaatgttttg gtaacagaaa acaatgtgat 2340gaaaatagca gactttggac tcgccagaga tatcaacaat atagactatt acaaaaagac 2400caccaatggg cggcttccag tcaagtggat ggctccagaa gccctgtttg atagagtata 2460cactcatcag agtgatgtct ggtccttcgg ggtgttaatg tgggagatct tcactttagg 2520gggctcgccc tacccaggga ttcccgtgga ggaacttttt aagctgctga aggaaggaca 2580cagaatggat aagccagcca actgcaccaa cgaactgtac atgatgatga gggactgttg 2640gcatgcagtg ccctcccaga gaccaacgtt caagcagttg gtagaagact tggatcgaat 2700tctcactctc acaaccaatg aggaatactt ggacctcagc caacctctcg aacagtattc 2760acctagttac cctgacacaa gaagttcttg ttcttcagga gatgattctg ttttttctcc 2820agaccccatg ccttacgaac catgccttcc tcagtatcca cacataaacg gcagtgttaa 2880aacatgaatg actgtgtctg cctgtcccca aacaggacag cactgggaac ctagctacac 2940tgagcaggga gaccatgcct cccagagctt gttgtctcca cttgtatata tggatcagag 3000gagtaaataa ttggaaaagt aatcagcata tgtgtaaaga tttatacagt tgaaaacttg 3060taatcttccc caggaggaga agaaggtttc tggagcagtg gactgc 3106
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