U.S. patent application number 12/154419 was filed with the patent office on 2009-05-07 for compositions and methods for diagnosis and treating mood disorders.
This patent application is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Huda Akil, William E. Bunney, JR., Prabhakara V. Choudary, Simon J. Evans, Edward G. Jones, Jun Li, Juan F. Lopez, Richard Myers, Robert C. Thompson, Hiroaki Tomita, Marquis P. Vawter, Stanley Watson.
Application Number | 20090117565 12/154419 |
Document ID | / |
Family ID | 32397053 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090117565 |
Kind Code |
A1 |
Akil; Huda ; et al. |
May 7, 2009 |
Compositions and methods for diagnosis and treating mood
disorders
Abstract
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.
Inventors: |
Akil; Huda; (Ann Arbor,
MI) ; Bunney, JR.; William E.; (Laguna Beach, CA)
; Choudary; Prabhakara V.; (Davis, CA) ; Evans;
Simon J.; (Milan, MI) ; Jones; Edward G.;
(Winters, CA) ; Li; Jun; (Palo Alto, CA) ;
Lopez; Juan F.; (Ann Arbor, MI) ; Thompson; Robert
C.; (Ann Arbor, MI) ; Myers; Richard;
(Stanford, CA) ; Tomita; Hiroaki; (Irvine, CA)
; Vawter; Marquis P.; (Niguel, CA) ; Watson;
Stanley; (Ann Arbor, MI) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
Palo Alto
CA
|
Family ID: |
32397053 |
Appl. No.: |
12/154419 |
Filed: |
May 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10701263 |
Nov 3, 2003 |
7410759 |
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12154419 |
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60431454 |
Dec 6, 2002 |
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60423247 |
Nov 1, 2002 |
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Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/158 20130101; C12Q 2600/136 20130101; A61P 25/00
20180101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 38/02 20060101 A61K038/02 |
Claims
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
method.
2.-29. (canceled)
30. The method of claim 1, wherein the subject is deceased.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] 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
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] 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
[0004] 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).
[0005] 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).
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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
[0016] Table 1: Table 1 lists genes differentially expressed in
mood disorder subjects.
[0017] Table 2: Table 2 lists 72 genes differentially expressed in
mood disorder subjects.
[0018] Table 3: Table 3 lists 16 genes differentially expressed in
specific brain regions and mood disorder.
[0019] 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.
[0020] Table 5: Table 5 lists Real time PCR results on sample genes
that are differentially expressed in mood disorder subjects.
[0021] Table 6: Table 6 lists anti-depressant treatment results for
genes that are differentially expressed in mood disorder
subjects.
[0022] Table 7: Tables 7A-D lists the gene ontology of selected
genes differentially expressed in mood disorder subjects.
[0023] Table 8: Table 8 lists sample of genes that are
differentially expressed in mood disorder subjects and are
potential druggable targets.
[0024] FIG. 1 shows selected biochemical pathways for genes
differentially expressed in mood disorder subjects.
[0025] FIG. 2 summarizes functions for signal transduction
transcripts differentially expressed in MDD subjects.
[0026] FIG. 3 shows bFGF pathway transcripts differentially
expressed in MDD subjects.
[0027] FIG. 4 shows values for differential expression of bFGF
transcripts in MDD subjects.
[0028] FIG. 5 shows selected biochemical pathways that are
dysregulated in mood disorders.
[0029] FIG. 6 shows selected biochemical pathways that are
dysregulated in BP subjects.
[0030] FIG. 7 shows three genes overexpressed in mood disorder
subjects that are located in the same chromosomal region.
[0031] FIG. 8 shows three genes overexpressed in mood disorder
subjects that are located on 15q11-13 in the Prader-Willi
region.
[0032] FIG. 9 shows certain genes regulated in human postmortem
tissue and by antidepressants in rats.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] FIG. 13 shows an example of a growth factor system (e.g.,
FGF) that is altered in mood disorders.
[0037] FIG. 14 shows RealTime PCR results which confirm that
selected FGF-related genes first identified using microarray
analysis are differentially expressed in mood disorders.
[0038] FIG. 15 shows selected genes in biochemical pathways
involving GABA and glutamate that are differentially expressed in
mood disorder subjects.
DEFINITIONS
[0039] 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.
[0040] "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.
[0041] "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.
[0042] 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).
[0043] "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.
[0044] "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.
[0045] 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.
[0046] 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.
[0047] "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.
[0048] 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.
[0049] 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.
[0050] "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
Ca.sup.2+); identification of downstream or reporter gene
expression (CAT, luciferase, .beta.-gal, GFP and the like), e.g.,
via chemiluminescence, fluorescence, calorimetric reactions,
antibody binding, inducible markers, and ligand binding assays.
[0051] 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.
[0052] "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.
[0053] "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.
[0054] 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 (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0055] 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)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H--C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.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).
[0056] 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., --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--. 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.
[0057] 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).
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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, .gamma.-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.
[0062] 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.
[0063] "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.
[0064] 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.
[0065] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0066] 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)
[0067] (see, e.g., Creighton, Proteins (1984)).
[0068] "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.
[0069] 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.
[0070] 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.
[0071] 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)).
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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).
[0076] 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.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m 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 T.sub.m, 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.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. 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.times.SSC, and 1%
SDS, incubating at 42.degree. C., or 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. 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.
[0077] 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.degree. C., and a wash in
1.times.SSC at 45.degree. 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.
[0078] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures may
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.degree. C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. 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).
[0079] 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.
[0080] 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.
[0081] 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).
[0082] 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.
[0083] 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.
[0084] 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
I. Introduction
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.TM.
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.
[0089] 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.
[0090] 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
[0091] 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
[0092] 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)).
[0093] 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.
[0094] 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).
[0095] 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
[0096] 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.
[0097] 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.
[0098] 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).
[0099] 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.
[0100] 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).
[0101] 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.
[0102] 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
[0103] 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.
[0104] 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).
[0105] 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
[0106] 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 .mu.g/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.
[0107] 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.
[0108] 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.
[0109] 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
MgSO.sub.4 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
[0110] 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
[0111] 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
[0112] 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.
[0113] 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
[0114] 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.
[0115] 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.
[0116] 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).
[0117] 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.
[0118] 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).
[0119] 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
.sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P-labeled probes
or the like.
[0120] 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).
[0121] 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.
[0122] 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.
[0123] 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.
[0124] A variety of automated solid-phase assay techniques are also
appropriate. For instance, very large scale immobilized polymer
arrays (VLSIPS.TM.), 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.
[0125] 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.).
[0126] 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 K.sub.D of at
least about 0.1 .mu.M, preferably at least about 0.01 .mu.M or
better, and most typically and preferably, 0.001 .mu.M or
better.
[0127] 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.
[0128] 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.
[0129] 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
[0130] 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
[0131] 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.
[0132] 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 10.sup.4 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 K.sub.D of at
least about 0.1 mM, more usually at least about 1 .mu.M, preferably
at least about 0.1 .mu.M or better, and most preferably, 0.01 .mu.M
or better.
[0133] 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.
[0134] 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).
[0135] 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.
[0136] 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.
[0137] 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
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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)).
[0142] 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.degree. C. to 40.degree. C.
1. Non-Competitive Assay Formats
[0143] 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
[0144] 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.
[0145] 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.
[0146] 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
[0147] 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.
[0148] 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).
4. Labels
[0149] 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.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S,
.sup.14C, or .sup.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.
[0150] 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.
[0151] 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).
[0152] 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.
[0153] 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
[0154] 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
[0155] 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
[0156] 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.
[0157] 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
[0158] 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.
[0159] 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.
[0160] 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, .beta.-glucuronidase,
chloramphenicol acetyl transferase (CAT); Alton and Vapnek (1979)
Nature 282:864-869), luciferase, .beta.-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).
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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
[0165] 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
(K.sub.m) the substrate or release the catalytic product
(K.sub.d).
[0166] 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.
4. Validation
[0167] 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
[0168] 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.
[0169] 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.
[0170] 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
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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).
[0175] 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
[0176] 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.
[0177] 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.
[0178] 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.).
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.TM. 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
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.RTM., or UNIX.RTM. based
(e.g., SUN.RTM. work station) computers.
[0197] 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
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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, 17.sup.th ed. 1985)).
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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
[0207] 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).
[0208] 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
[0209] 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
[0210] 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.
[0211] 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.
[0212] 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).
[0213] 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)).
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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
[0220] 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).
[0221] 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
[0222] 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
[0223] 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.
[0224] 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.
[0225] 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
[0226] 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.
[0227] 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.
[0228] 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.
EXAMPLES
Example 1
Identification of Genes Dysregulated in Mood Disorders
[0229] 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.TM.. 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).
[0230] 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).
Example 2
Identification of Additional Genes Dysregulated in Mood
Disorders
[0231] 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.TM..
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.
[0232] 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.
Example 3
Identification of Additional Genes Dysregulated in Mood Disorders
Using Rat Models of Depression and Anti-Depressant Treatment
[0233] 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.
[0234] 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
Sequence CWU 1
1
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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