U.S. patent application number 10/989191 was filed with the patent office on 2005-09-22 for biomarkers for diagnosing schizophrenia and bipolar disorder.
This patent application is currently assigned to GeneNews Inc.. Invention is credited to Chao, Samuel, Dempsey, Adam, Liew, Choong-Chin, Yager, Thomas.
Application Number | 20050208519 10/989191 |
Document ID | / |
Family ID | 36407680 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208519 |
Kind Code |
A1 |
Liew, Choong-Chin ; et
al. |
September 22, 2005 |
Biomarkers for diagnosing schizophrenia and bipolar disorder
Abstract
The invention relates to the identification and selection of
novel biomarkers and the identification and selection of novel
biomarker combinations which are differentially expressed in blood
and useful in diagnosing schizophrenia and/or bipolar disorder as
well as monitoring therapeutic efficacy of treatment for
schizophrenia or bipolar disorder. The measurement of expression
levels of the products of the biomarkers and combinations of
biomarkers of the invention can be used to diagnose schizophrenia
and/or bipolar disorder. Measurement of the expression level of
products of biomarkers of the invention using polynucleotides and
proteins which specifically and/or selectively hybridize to the
products of the biomarkers of the invention are also encompassed
within the scope of the invention as are compositions and kits
containing said polynucleotides and proteins. Further encompassed
by the invention is the use of the polynucleotides and proteins to
monitor the efficacy of therapeutic regimens. The invention also
provides for the identification of methods of using the products of
the biomarkers of the invention in the identification of novel
therapeutic targets of schizophrenia and/or bipolar disorder and a
method of screening the genes of said biomarkers for additional
markers of disease.
Inventors: |
Liew, Choong-Chin; (Toronto,
CA) ; Yager, Thomas; (Mississauga, CA) ;
Dempsey, Adam; (Toronto, CA) ; Chao, Samuel;
(Toronto, CA) |
Correspondence
Address: |
PALMER & DODGE, LLP
KATHLEEN M. WILLIAMS
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Assignee: |
GeneNews Inc.
|
Family ID: |
36407680 |
Appl. No.: |
10/989191 |
Filed: |
November 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10989191 |
Nov 15, 2004 |
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10812731 |
Mar 30, 2004 |
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10812731 |
Mar 30, 2004 |
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10802875 |
Mar 12, 2004 |
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10989191 |
Nov 15, 2004 |
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PCT/US04/20836 |
Jun 21, 2004 |
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Current U.S.
Class: |
435/6.16 ;
536/24.3 |
Current CPC
Class: |
C12Q 2600/158 20130101;
Y02A 90/10 20180101; Y02A 90/22 20180101; C12Q 1/6883 20130101;
Y02A 90/24 20180101; Y02A 90/26 20180101 |
Class at
Publication: |
435/006 ;
536/024.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A composition comprising a collection of two or more isolated
polynucleotides, said polynucleotides which selectively hybridize
to at least two biomarkers of the invention, wherein the biomarkers
are selected from the group consisting of the genes:
adenylosuccinate synthase (ADSS); apolipoprotein B mRNA editing
enzyme, catalytic polypeptide-like 3B (APOBEC3B); ataxia
telangiectasia mutated (includes complementation groups A, C and D)
(ATM); Charcot-Leyden crystal protein (CLC); C-terminal binding
protein 1 (CTBP1); chemokine (C--X--C motif) ligand 1 (melanoma
growth stimulating activity, alpha) (CXCL1); death associated
transcription factor 1 (DATF1); S100 calcium binding protein A9
(calgranulin B) (S100A9), and as set out in Table 1, and wherein
the composition is used to measure the level of expression of said
biomarker.
2. A composition comprising a collection of two or more isolated
polynucleotides which bind selectively to the RNA products of at
least two biomarkers, wherein the biomarkers are selected from the
group consisting of the genes: adenylosuccinate synthase (ADSS);
apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B
(APOBEC3B); ataxia telangiectasia mutated (includes complementation
groups A, C and D) (ATM); Charcot-Leyden crystal protein (CLC);
C-terminal binding protein 1 (CTBP1); chemokine (C--X--C motif)
ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1);
death associated transcription factor 1 (DATF1); S100 calcium
binding protein A9 (calgranulin B) (S100A9), as set out in Table
1.
3. The composition of claim 2 wherein said polynucleotides are
useful in quantitative RT-PCR (QRT-PCR).
4. A composition comprising a collection of two or more isolated
proteins which bind selectively to the protein products of at least
two biomarkers, wherein the biomarkers are selected from the group
consisting of the genes: adenylosuccinate synthase (ADSS);
apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B
(APOBEC3B); ataxia telangiectasia mutated (includes complementation
groups A, C and D) (ATM); Charcot-Leyden crystal protein (CLC);
C-terminal binding protein 1 (CTBP1); chemokine (C--X--C motif)
ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1);
death associated transcription factor 1 (DATF1); S100 calcium
binding protein A9 (calgranulin B) (S100A9), as set out in Table
1
5. A composition comprising a collection of two or more isolated
polynucleotides which bind selectively to the RNA products of one
or more biomarkers, wherein the biomarkers are selected from the
group consisting of the genes: adenylosuccinate synthase (ADSS);
death associated transcription factor 1 (DATF1); as listed in Table
3.
6. A composition comprising a collection of one or more isolated
proteins, which bind selectively to the protein products of one or
more biomarkers, wherein the biomarkers are selected from the group
consisting of the genes: adenylosuccinate synthase (ADSS); death
associated transcription factor 1 (DATF1); as set out in Table
3.
7. A composition comprising a collection of isolated
polynucleotides which bind selectively to the RNA products of
biomarkers, wherein the biomarkers are selected from the group of
genes: adenylosuccinate synthase (ADSS); death associated
transcription factor 1 (DATF1); as set out in Table 3.
8. A composition comprising a collection of two or more isolated
polynucleotides which bind selectively to the RNA products of at
least one biomarker, wherein the biomarkers are selected from the
group as set out in Table 4.
9. A composition comprising a collection of two or more isolated
polynucleotides which bind selectively to the RNA products of at
least one biomarker, wherein the biomarkers are selected from the
group as set out in Table 5.
10. A composition comprising a collection of one or more isolated
proteins which bind selectively to the protein products of at least
one biomarker, wherein the biomarkers are selected from the group
as set out in Table 4.
11. A composition comprising a collection of one or more isolated
proteins which bind selectively to the protein products of at least
one biomarker, wherein the biomarkers are selected from the group
as set out in Table 5.
12. The composition of any one of claims 4, 6, 10 or 11, wherein
the isolated proteins are ligands.
13. The composition of any one of claims 4, 6, 10 or 11, wherein
the ligands are antibodies.
14. The composition of claim 13, wherein the antibodies are
monoclonal antibodies.
15. The composition of any one of claims 1, 2, 3, 5, 7, 8 or 9,
wherein the isolated oligonucleotides are single or double stranded
RNA.
16. The composition of any one of claims 1, 2, 3, 5, 7, 8 or 9
wherein the isolated polynucleotides are single or double stranded
DNA.
17. A method of diagnosing or prognosing schizophrenia in an
individual, comprising the steps of: a) determining the level of
one or more RNA transcripts expressed in blood obtained from said
individual, wherein said one or more RNA transcripts corresponds to
said one or more biomarkers of Table 3, and b) comparing the level
of each of said one or more RNA transcripts in said blood according
to step a) with the level of each of said one or more RNA
transcripts in blood from one or more individuals not having
schizophrenia, wherein detecting differential expression of each of
said one or more RNA transcripts in the comparison of step b) is
indicative of schizophrenia in the individual of step a).
18. A method of diagnosing or prognosing bipolar disorder in an
individual, comprising the steps of: a) determining the level of
one or more RNA transcripts expressed in blood obtained from said
individual, wherein said one or more RNA transcripts corresponds to
said one or more biomarkers of Table 4, and b) comparing the level
of each of said one or more RNA transcripts in said blood according
to step a) with the level of each of said one or more RNA
transcripts in blood from one or more individuals not having
bipolar disorder, wherein detecting differential expression of each
of said one or more RNA transcripts in the comparison of step b) is
indicative of bipolar disorder in the individual of step a).
19. A method of diagnosing or prognosing and individual as having
either bipolar disorder or schizophrenia, comprising the steps of:
a) determining the level of one or more RNA transcripts expressed
in blood obtained from said individual, wherein said one or more
RNA transcripts corresponds to said one or more biomarkers of Table
5, and b) comparing the level of each of said one or more RNA
transcripts in said blood according to step a) with the level of
each of said one or more RNA transcripts in blood from one or more
individuals having schizophrenia, wherein detecting differential
expression of each of said one or more RNA transcripts in the
comparison of step b) is indicative of bipolar disorder in the
individual of step a).
20. A method of diagnosing or prognosing and individual as having
either bipolar disorder or schizophrenia, comprising the steps of:
a) determining the level of one or more RNA transcripts expressed
in blood obtained from said individual, wherein said one or more
RNA transcripts corresponds to said one or more biomarkers of Table
5, and b) comparing the level of each of said one or more RNA
transcripts in said blood according to step a) with the level of
each of said one or more RNA transcripts in blood from one or more
individuals having bipolar disorder, wherein detecting differential
expression of each of said one or more RNA transcripts in the
comparison of step b) is indicative of schizophrenia in the
individual of step a).
21. A method of diagnosing or prognosing schizophrenia in an
individual, comprising the steps of: a) determining the level of
two or more RNA transcripts expressed in blood obtained from said
individual, wherein said two or more RNA transcripts corresponds to
said one or more biomarkers of Table 1 and b) comparing the level
of each of said two or more RNA transcripts in said blood according
to step a) with the level of each of said two or more RNA
transcripts in blood from one or more individuals having
schizophrenia, c) comparing the level of each of said two or more
RNA transcripts in said blood according to step a) with the level
of each of said two or more RNA transcripts in blood from one or
more individuals not having schizophrenia, d) determining whether
the level of said two or more RNA transcripts of step a) classify
with the levels of said transcripts in step b) as compared with
levels of said transcripts in step c), wherein said determination
is indicative of said individual of step a) having
schizophrenia.
22. A method of diagnosing or prognosing bipolar disorder in an
individual, comprising the steps of: a) determining the level of
two or more RNA transcripts expressed in blood obtained from said
individual, wherein said two or more RNA transcripts corresponds to
said two or more biomarkers of Table 1 and b) comparing the level
of each of said two or more RNA transcripts in said blood according
to step a) with the level of each of said two or more RNA
transcripts in blood from one or more individuals having bipolar
disorder, c) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals not having bipolar disorder, d) determining whether the
level of said two or more RNA transcripts of step a) classify with
the levels of said transcripts in step b) as compared with levels
of said transcripts in step c), wherein said determination is
indicative of said individual of step a) having bipolar
disorder.
23. A method of diagnosing or prognosing an individual as having
bipolar disorder or schizophrenia, comprising the steps of: a)
determining the level of two or more RNA transcripts expressed in
blood obtained from said individual, wherein said two or more RNA
transcripts corresponds to said two or more biomarkers of Table 1
and b) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals having bipolar disorder, c) comparing the level of each
of said two or more RNA transcripts in said blood according to step
a) with the level of each of said two or more RNA transcripts in
blood from one or more individuals having schizophrenia, d)
determining whether the level of said two or more RNA transcripts
of step a) classify with the levels of said transcripts in step b)
as compared with levels of said transcripts in step c), wherein
said determination is indicative of said individual of step a)
having bipolar disorder.
24. A method of diagnosing or prognosing schizophrenia in an
individual, comprising the steps of: a) determining the level of
one or more RNA transcripts expressed in blood obtained from said
individual, wherein said one or more RNA transcripts corresponds to
said one or more biomarkers of Table 1 and b) using the results
from step (a) in combination with a classifier so as to determine a
diagnosis with respect to schizophrenia.
25. A method of diagnosing or prognosing bipolar disorder in an
individual, comprising the steps of: a) determining the level of
one or more RNA transcripts expressed in blood obtained from said
individual, wherein said one or more RNA transcripts corresponds to
said one or more biomarkers of Table 1 and b) using the results
from step (a) in combination with a classifier so as to determine a
diagnosis with respect to bipolar disorder.
26. The method of any one of claims 17-25, wherein said blood
sample consists of whole blood.
27. The method of any one of claims 17-25, wherein said blood
sample consists of a drop of blood.
28. The method of any one of claims 17-25, wherein said blood
sample consists of blood that has been lysed.
29. The method of any one of claims 17-25, further comprising the
step of isolating RNA from said blood samples.
30. The method of any one of claims 17-25, wherein the step of
determining the level of each of said one or more RNA transcripts
comprises quantitative RT-PCR (QRT-PCR), wherein said one or more
transcripts are from step a) and/or step b) of claims 17-25.
31. The method of claim 30, wherein said QRT-PCR utilizes primers
which hybridize to said one or more transcripts or the complement
thereof, wherein said one or more transcripts are from step a)
and/or step b) of claims 17-25.
32. The method of claim 31, wherein said primers are 15-25
nucleotides in length.
33. The method of any one of claims 17-25, wherein the step of
determining the level of each of said one or more RNA transcripts
comprises hybridizing a first plurality of isolated nucleic acid
molecules that correspond to said one or more transcripts, to an
array comprising a second plurality of isolated nucleic acid
molecules.
34. The method of claim 33, wherein said first plurality of
isolated nucleic acid molecules comprises RNA, DNA, cDNA, PCR
products or ESTs.
35. The method of claim 33, wherein said array comprises a
plurality of isolated nucleic acid molecules comprising RNA, DNA,
cDNA, PCR products or ESTs.
36. The method of claim 33, wherein said second plurality of
isolated nucleic acid molecules on said array comprises
polynucleotides corresponding to one or more of the biomarkers of
Table 1.
37. A kit for diagnosing or prognosing schizophrenia comprising: a)
at least two sets of biomarker specific priming means wherein each
set of biomarker specific priming means produces double stranded
DNA complementary to a unique biomarker selected from Table 1;
wherein each first priming means of said sets contains a sequence
which can selectively hybridize to RNA, cDNA or an EST
complementary to one of said biomarkers to create an extension
product and each said second priming means of said sets is capable
of selectively hybridizing to said extension product; b) an enzyme
with reverse transcriptase activity; c) an enzyme with thermostable
DNA polymerase activity, and d) a labeling means; wherein each of
said primer sets is used to detect the quantitative expression
levels of said biomarker in a test subject.
38. A kit for diagnosing or prognosing bipolar disorder comprising:
a) at least two sets of biomarker specific priming means wherein
each set of biomarker specific priming means produces double
stranded DNA complementary to a unique biomarker selected from
Table 1; wherein each first priming means of said sets contains a
sequence which can selectively hybridize to RNA, cDNA or an EST
complementary to one of said biomarkers to create an extension
product and each said second priming means of said sets is capable
of selectively hybridizing to said extension product; b) an enzyme
with reverse transcriptase activity; c) an enzyme with thermostable
DNA polymerase activity, and d) a labeling means; wherein each said
primer set is used to detect the quantitative expression levels of
a biomarker in a test subject.
39. A method of diagnosing or prognosing schizophrenia in an
individual, comprising the steps of: a) determining the level of
two or more proteins expressed in blood obtained from said
individual, wherein said two or more proteins are encoded by two or
more biomarkers of Table 1, and b) comparing the level of each of
said two or more proteins in said blood according to step a) with
the level of each of said two or more proteins in blood from one or
more individuals having schizophrenia, c) comparing the level of
each of said two or more proteins in said blood according to step
a) with the level of each of said two or more proteins in blood
from one or more individuals not having schizophrenia, d)
determining whether the level of said two or more proteins of step
a) classify with the levels of said proteins in step b) as compared
with levels of said proteins in step c), wherein said determination
is indicative of said individual of step a) having
schizophrenia.
40. A method of diagnosing or prognosing bipolar disorder in an
individual, comprising the steps of: a) determining the level of
two or more proteins expressed in blood obtained from said
individual, wherein said two or more proteins are encoded by two or
more biomarkers of Table 1, and b) comparing the level of each of
said two or more proteins in said blood according to step a) with
the level of each of said two or more proteins in blood from one or
more individuals having bipolar disorder c) comparing the level of
each of said two or more proteins in said blood according to step
a) with the level of each of said two or more proteins in blood
from one or more individuals not having bipolar disorder, d)
determining whether the level of said two or more proteins of step
a) classify with the levels of said proteins in step b) as compared
with levels of said proteins in step c), wherein said determination
is indicative of said individual of step a) having bipolar
disorder.
41. A method of diagnosing or prognosing an individual with bipolar
disorder as compared with schizophrenia, comprising the steps of:
a) determining the level of two or more proteins expressed in blood
obtained from said individual, wherein said two or more proteins
are encoded by two or more biomarkers of Table 1, and b) comparing
the level of each of said two or more proteins in said blood
according to step a) with the level of each of said two or more
proteins in blood from one or more individuals having bipolar
disorder c) comparing the level of each of said two or more
proteins in said blood according to step a) with the level of each
of said two or more proteins in blood from one or more individuals
having schizophrenia, d) determining whether the level of said two
or more proteins of step a) classify with the levels of said
proteins in step b) as compared with levels of said proteins in
step c), wherein said determination is indicative of said
individual of step a) having bipolar disorder.
42. A method of diagnosing or prognosing schizophrenia in an
individual, comprising the steps of: a) determining the level of
two or more protein products expressed in blood obtained from said
individual, wherein said two or more protein products corresponds
to two or more biomarkers of Table 1 and b) using the results from
step (a) in combination with a classifier designed to differentiate
schizophrenia from non schizophrenia so as to determine a diagnosis
with respect to schizophrenia.
43. A method of diagnosing or prognosing bipolar disorder in an
individual, comprising the steps of: a) determining the level of
two or more protein products expressed in blood obtained from said
individual, wherein said two or more protein products corresponds
to two or more biomarkers of Table 1 and b) using the results from
step (a) in combination with a classifier designed to differentiate
bipolar disorder from non bipolar disorder so as to determine a
diagnosis with respect to bipolar disorder.
44. A method of diagnosing or prognosing and individual as having
bipolar disorder or schizophrenial, comprising the steps of: a)
determining the level of two or more protein products expressed in
blood obtained from said individual, wherein said two or more
protein products corresponds to two or more biomarkers of Table 1
and b) using the results from step (a) in combination with a
classifier designed to differentiate bipolar disorder from
schizophrenia so as to determine a diagnosis with respect to
bipolar disorder or schizophrenia.
45. The method of any one of claims 39-44, wherein the step of
determining the level of each of said one or more proteins
comprises the use of two or more antibodies, wherein each of said
two or more antibodies is specific for a protein product of a
biomarker listed in Table 1.
46. The method of claim 45, wherein said one or more antibodies is
selected from the group consisting of a monoclonal antibody, fv.
scfv, dab, fd, fab, and fab'2.
47. A method of developing a classifier useful for diagnosing
schizophrenia, said method comprising: (a) measuring the level of
expression of the products of the biomarkers identified in Table 1
in a training population wherein said training population is
comprised of two subgroups, a first subgroup diagnosed as having
schizophrenia and said second subgroup diagnosed as not having
schizophrenia. (b) apply one or more mathematical models to the
levels of expression of step (a) to develop one or more classifiers
which differentiate between said first subgroup and said second
subgroup.
48. A method of developing a classifier useful for diagnosing
bipolar disorder, said method comprising: (a) measuring the level
of expression of the products of the biomarkers identified in Table
1 in a training population wherein said training population is
comprised of two subgroups, a first subgroup diagnosed as having
bipolar disorder and said second subgroup diagnosed as not having
bipolar disorder. (b) apply one or more mathematical models to the
levels of expression of step (a) to develop one or more classifiers
which differentiate between said first subgroup and said second
subgroup.
49. A method of developing a classifier useful for diagnosing
bipolar disorder or schizophrenia, said method comprising: (a)
measuring the level of expression of the products of the biomarkers
identified in Table 1 in a training population wherein said
training population is comprised of two subgroups, a first subgroup
diagnosed as having bipolar disorder and said second subgroup
diagnosed as having schizophrenia. (b) apply one or more
mathematical models to the levels of expression of step (a) to
develop one or more classifiers which differentiate between said
first subgroup and said second subgroup.
50. The method of any one of claims 47-49, further comprising the
step of evaluating one or more of said classifiers of step (b) for
the classifier's ability to properly characterize each individual
of the training population.
51. The method of any one of claims 47-49, further comprising the
step of evaluating one or more of said classifiers of step (b) for
the classifier's ability to properly characterize one or more
individuals of a population which is not the training population.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of 10/812,731,
filed Mar. 30, 2004 which is a continuation in part of 10/802,875,
filed Mar. 12, 2004, which a continuation in part of application
Ser. No. 10/601,518, filed on Jun. 20, 2003, which is a
continuation-in-part of application Ser. No. 10/268,730 filed on
Oct. 9, 2002, which is a continuation of U.S. application Ser. No.
09/477,148 filed Jan. 4, 2000, now abandoned, which claims the
benefit of U.S. Provisional Application No. 60/115,125 filed on
Jan. 6, 1999. Each of these applications is incorporated herein by
reference in their entirety, including figures and drawings.
1. FIELD OF THE INVENTION
[0002] The invention relates to the identification and selection of
novel biomarkers and the identification and selection of novel
biomarker combinations which are differentially expressed in
individuals with schizophrenia and/or bipolar disorder as well as a
means of selecting the novel biomarker combinations. Further
encompassed by the invention is the use of polynucleotides and/or
proteins which specifically hybridize to the products of the
biomarkers of the invention to diagnose schizophrenia, diagnose
bipolar disorder and differentially diagnose as between
schizophrenia and bipolar disorder. Also included in the invention
are kits of said polynucleotides and/or proteins. The invention
also encompasses screening methods to monitor the efficacy of
therapeutic regimens and identify therapeutic targets for treating
schizophrenia and/or bipolar disorder, as well as providing a means
of identifying single nucleotide point mutations related to
schizophrenia and/or bipolar disorder.
2. BACKGROUND OF THE INVENTION
[0003] Schizophrenia:
[0004] Schizophrenia is a debilitating mental disorder
characterized primarily by psychotic symptoms including
hallucinations, delusions, disorganized speech, thought and
behaviour, and difficulty expressing emotion. The lifetime
prevalence of schizophrenia is about 1% of the population
worldwide, with some countries slightly lower and others slightly
higher. In the United States, roughly 2,500,000 people are affected
by it.
[0005] Diagnosis of Schizophrenia
[0006] Currently diagnosis of schizophrenia relies solely on the
analysis of a person's symptoms. Diagnosis is made from information
obtained from physicial examination, taking a person's family
history and emotional history, as well as a medical evaluation, and
a mental status examination. Relying on symptomatic history makes
diagnosis of schizophrenia difficult, particularly since no single
symptom is definitive for diagnosis. Rather, the diagnosis
encompasses a pattern of signs and symptoms, in conjunction with
impaired occupational or social functioning. Currently diagnosis
includes looking for delusions (false beliefs strongly held in
spite of invalidating evidence); visual, auditory, tactile,
olfactory or gustatory hallucinations; disorganized speech;
disorganized thinking; grossly disorganized thinking and/or
catatonic behaviour; negative symptoms, such as emotional deficit,
avolition (inability to initiate and persist in goal-directed
activities) and alogia (poverty of speech) are also symptoms of
schizophrenia. Continuous signs of the disturbance must persist for
at least 6 months. This 6-month period must include at least 1
month of active-phase symptoms (listed above) (or less if
successfully treated) and may include periods of prodromal or
residual symptoms. During these prodromal or residual periods, the
signs of the disturbance may be manifested by only negative
symptoms or two or more active-phase symptoms in an attenuated form
(e.g., odd beliefs, unusual perceptual experiences).
[0007] Diagnosis of schizophrenia is made even harder because it is
often difficult to differentiate schizophrenia from other mental
disorders including bipolar disorder, schizoaffective disorder, and
brief psychotic disorder. In addition, diagnosis of schizophrenia
is often confused with other organic medical conditions (e.g.
encephalitis) or substance conditions (drugs of abuse, such as
amphetamines and phencyclidine, or other medications). Although
recently brain imaging techniques have been utilized as a tool
towards diagnosis, this is costly, is inconvenient to patients, and
is not considered very reliable.
[0008] Bipolar Disorder:
[0009] Bipolar disorder, (also termed manic-depressive disorder),
is a mood disorder in which people experience alternating episodes
of mania and major depression. Mania is characterized by elation,
irritability, excitability, racing thought and speech, and
hyperactivity. Major depression is characterized by sadness,
withdrawal, despair, and suicidal thoughts. Bipolar disorder
affects approximately 3% of people in the United States. The age of
onset is usually the late teens or early 20s and there is usually a
history of depression. Generally, early treatment means better
prognosis.
[0010] Diagnosis of Bipolar Disorder
[0011] Traditional medical diagnostic techniques for diagnosing
bipolar disorder include: physical exam and history and mental
status exam for presence of bipolar disorder symptoms which include
a combination of at least one major depressive episode (a depressed
mood or a loss of interest or pleasure in daily activities
consistently for at least a 2 week period which represents a change
from the person's normal mood; social, occupational, educational or
other important functioning must also be negatively impaired by the
change in mood) and one manic episode (a distinct period of
persistently elevated, expansive, or irritable mood, lasting
throughout at least 4 days, that is clearly different from the
usual nondepressed mood). Diagnosis can be difficult because the
first episode of mania may go undetected, and an episode of
depression does not necessarily predict a subsequent manic episode.
Most people are symptom free for months or even years between
episodes of depression and mania. In brief, both schizophrenia and
bipolar disorder are difficult to diagnose due to the complexity of
each condition. Moreover, it can be challenging to clinically
distinguish these two conditions because of their common clinical
characteristics. It often requires a long period of observation of
a patient before the definitive diagnosis can be made. Although
there are brain-imaging tests available, they are not specific
enough. Recently there has been some advancement in analyzing gene
expression in brain tissue to identify biomarkers of mental
disorders, but one cannot translate this into a simple non invasive
diagnostic tool. Blood-based tests for diagnosis and differential
diagnosis of schizophrenia and bipolar disorder would help speed up
the diagnostic process and ensure an early administration of the
correct therapy. This is particularly important since there are
effective therapies available to manage both schizophrenia and
bipolar disorder, and early treatment in many cases means better
prognosis and decreases chances of recurrence of future acute
episodes.
[0012] Thus there is a need for a simple non-invasive diagnostic
test for diagnosing an individual as having either schizophrenia or
bipolar disorder.
3. SUMMARY OF THE INVENTION
[0013] The invention relates to the identification and selection of
novel biomarkers and the identification and selection of novel
biomarker combinations which are differentially expressed in blood
and useful in diagnosing schizophrenia and/or bipolar disorder as
well as monitoring therapeutic efficacy of treatment for
schizophrenia and/or bipolar disorder. The measurement of
expression levels of the products of the biomarkers and
combinations of biomarkers of the invention can be used to diagnose
schizophrenia and/or bipolar disorder. Measurement of the
expression level of products of biomarkers of the invention using
polynucleotides and proteins which specifically and/or selectively
hybridize to the products of the biomarkers of the invention are
also encompassed within the scope of the invention as are
compositions and kits containing said polynucleotides and proteins.
Further encompassed by the invention is the use of the
polynucleotides and proteins to monitor the efficacy of therapeutic
regimens. The invention also provides for the identification of
methods of using the biomarkers of the invention in the
identification of novel therapeutic targets of schizophrenia and/or
bipolar disorder and a method of screening the genes of said
biomarkers for additional markers of disease.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The objects and features of the invention can be better
understood with reference to the following detailed description and
drawings.
[0015] FIG. 1 is a figure showing, in one embodiment of the
invention, p values representing the differential expression for
each of the biomarkers of the invention when comparing
subpopulations of individuals as follows: (a) schizophrenia v. non
schizophrenia (control) (b) bipolar disorder (bpd) v. non bipolar
disorder (control) and (c) schizophrenia v. bipolar disorder.
[0016] FIG. 2 is a table showing, in one embodiment of the
invention, SNPs which have been identified in the biomarkers of the
invention
[0017] FIG. 3 is a table showing, in one embodiment of the
invention, various selections of two biomarkers of the
invention.
[0018] FIG. 4 is a table showing, in one embodiment of the
invention, various selections of three biomarkers of the
invention.
[0019] FIG. 5 is an example of a number of classifiers generated
for use in differentiating as between schizophrenia and normal (non
schizophrenia) with an ROC of >0.9.
[0020] FIG. 6 is an is an example of a number of classifiers
generated for use in differentiating as between bipolar disorder
and normal (non bipolar disorder) with an ROC of >0.9.
[0021] FIG. 7 is an is an example of a number of classifiers
generated for use in differentiating as between bipolar disorder
and schizophrenia with an ROC of >0.9.
5. DETAILED DESCRIPTION OF THE INVENTION
[0022] The invention relates to the identification and selection
from blood of genes which are differentially expressed as between
individuals with schizophrenia and normal individuals; individuals
with bipolar disorder and normal individuals; and as between
individuals with schizophrenia and individuals with bipolar
disorder. As such the invention encompasses polynucleotides and
polypeptides which can be used to detect and monitor differential
gene expression of the biomarker and biomarker combinations for
both diagnosis of schizophrenia and diagnosis of bipolar disorder
as well as to allow the monitoring of potential therapeutic
treatments for both schizophrenia and bipolar disorder. The
invention further encompasses a method of identifying particularly
useful combinations of biomarkers. In addition the invention
encompasses use of the biomarkers of the invention to screen for
therapeutic targets for schizophrenia and bipolar disorder and
identifies single nucleotide polymorphisms within the genes of the
invention which can be monitored to determine additional means of
diagnosing schizophrenia or bipolar disorder in individuals.
5.1 DEFINITIONS
[0023] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology and recombinant DNA techniques, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Sambrook, Fritsch & Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition;
Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid
Hybridization (B. D. Harnes & S. J. Higgins, eds., 1984); A
Practical Guide to Molecular Cloning (B. Perbal, 1984); and a
series, Methods in Enzymology (Academic Press, Inc.); Short
Protocols In Molecular Biology, (Ausubel et al., ed., 1995). All
patents, patent applications, and publications mentioned herein,
both supra and infra, are hereby incorporated by reference in their
entireties.
[0024] The following definitions are provided for specific terms
which are used in the following written description. As used
herein, the "5' end" refers to the end of an mRNA up to the first
1000 nucleotides or 1/3 of the mRNA(where the full length of the
mRNA does not include the poly A tail), starting at the first
nucleotide of the mRNA. The "5' region" of a gene refers to a
polynucleotide (double-stranded or single-stranded) located within
or at the 5' end of a gene, and includes, but is not limited to,
the 5' untranslated region, if that is present, and the 5' protein
coding region of a gene. The 5' region is not shorter than 8
nucleotides in length and not longer than 1000 nucleotides in
length. Other possible lengths of the 5' region include but are not
limited to 10, 20, 25, 50, 100, 200, 400, and 500 nucleotides.
[0025] As used herein, the "3' end" refers to the end of an mRNA up
to the last 1000 nucleotides or 1/3 of the mRNA, where the 3'
terminal nucleotide is that terminal nucleotide of the coding or
untranslated region that adjoins the poly-A tail, if one is
present. That is, the 3' end of an mRNA does not include the poly-A
tail, if one is present. The "3' region" of a gene refers to a
polynucleotide (double-stranded or single-stranded) located within
or at the 3' end of a gene, and includes, but is not limited to,
the 3' untranslated region, if that is present, and the 3' protein
coding region of a gene. The 3' region is not shorter than 8
nucleotides in length and not longer than 1000 nucleotides in
length. Other possible lengths of the 3' region include but are not
limited to 10, 20, 25, 50, 100, 200, 400, and 500 nucleotides. As
used herein, the "internal coding region" of a gene refers to a
polynucleotide (double-stranded or single-stranded) located between
the 5' region and the 3' region of a gene as defined herein. The
"internal coding region" is not shorter than 8 nucleotides in
length and not longer than 1000 nucleotides in length. Other
possible lengths of the "internal coding region" include but are
not limited to 10, 20, 25, 50, 100, 200, 400, and 500 nucleotides.
The 5', 3' and internal regions are non-overlapping and may, but
need not be contiguous, and may, but need not, add up to the full
length of the corresponding gene.
[0026] As used herein, the "amino terminal" region of a polypeptide
refers to the polypeptide sequences encoded by polynucleotide
sequences (double-stranded or single-stranded) located within or at
the 5' end of a gene, and includes, but is not limited to, the 5'
protein coding region of a gene. As used herein, the "amino
terminal" region refers to the amino terminal end of a polypeptide
up to the first 300 amino acids or 1/3 of the polypeptide, starting
at the first amino acid of the polypeptide. The "amino terminal"
region of a polypeptide is not shorter than 3 amino acids in length
and not longer than 350 amino acids in length. Other possible
lengths of the "amino terminal" region of a polypeptide include but
are not limited to 5, 10, 20, 25, 50, 100 and 200 amino acids.
[0027] As used herein, the "carboxy terminal" region of a
polypeptide refers to the polypeptide sequences encoded by
polynucleotide sequences (double-stranded or single-stranded)
located within or at the 3' end of a gene, and includes, but is not
limited to, the 3' protein coding region of a gene. As used herein,
the "carboxy terminal" region refers to the carboxy terminal end of
a polypeptide up to 300 amino acids or 1/3 of the polypeptide from
the last amino acid of the polypeptide. The "3"end" does not
include the polyA tail, if one is present. The "carboxy terminal"
region of a polypeptide is not shorter than 3 amino acids in length
and not longer than 350 amino acids in length. Other possible
lengths of the "carboxy terminal" region of a polypeptide include,
but are not limited to, 5, 10, 20, 25, 50, 100 and 200 amino
acids.
[0028] As used herein, the "internal polypeptide region" of a
polypeptide refers to the polypeptide sequences located between the
amino terminal region and the carboxy terminal region of a
polypeptide, as defined herein. The "internal polypeptide region"
of a polypeptide is not shorter than 3 amino acids in length and
not longer than 350 amino acids in length. Other possible lengths
of the "internal polypeptide region" of a polypeptide include, but
are not limited to, 5, 10, 20, 25, 50, 100 and 200 amino acids. The
amino terminal, carboxy terminal and internal polypeptide regions
of a polypeptide are non-overlapping and may, but need not be
contiguous, and may, but need not, add up to the full length of the
corresponding polypeptide.
[0029] As used herein, the term "amplified", when applied to a
nucleic acid sequence, refers to a process whereby one or more
copies of a particular nucleic acid sequence is generated from a
template nucleic acid, preferably by the method of polymerase chain
reaction (Mullis and Faloona, 1987, Methods Enzymol., 155:335).
"Polymerase chain reaction" or "PCR" refers to an in vitro method
for amplifying a specific nucleic acid template sequence. The PCR
reaction involves a repetitive series of temperature cycles and is
typically performed in a volume of 50-100 .quadrature.1. The
reaction mix comprises dNTPs (each of the four deoxynucleotides
dATP, dCTP, dGTP, and dTTP), primers, buffers, DNA polymerase, and
nucleic acid template. The PCR reaction comprises providing a set
of polynucleotide primers wherein a first primer contains a
sequence complementary to a region in one strand of the nucleic
acid template sequence and primes the synthesis of a complementary
DNA strand, and a second primer contains a sequence complementary
to a region in a second strand of the target nucleic acid sequence
and primes the synthesis of a complementary DNA strand, and
amplifying the nucleic acid template sequence employing a nucleic
acid polymerase as a template-dependent polymerizing agent under
conditions which are permissive for PCR cycling steps of (i)
annealing of primers required for amplification to a target nucleic
acid sequence contained within the template sequence, (ii)
extending the primers wherein the nucleic acid polymerase
synthesizes a primer extension product. "A set of polynucleotide
primers" or "a set of PCR primers" can comprise two, three, four or
more primers. In one embodiment, an exo-Pfu DNA polymerase is used
to amplify a nucleic acid template in PCR reaction. Other methods
of amplification include, but are not limited to, ligase chain
reaction (LCR), polynucleotide-specific based amplification (NSBA),
or any other method known in the art.
[0030] According to the invention, an "array" contemplates a
specific set of genes immobilized to a support, or a set of
corresponding 5' ends or a set of corresponding 3' ends or a set of
corresponding internal coding regions. Of course, mixtures of a 5'
end of one gene may be used as a target or a probe in combination
with a 3' end of another gene to achieve the same result of
schizophrenia or bipoloar disorder diagnosis.
[0031] As used herein, the term "analog" in the context of
proteinaceous agent (e.g., proteins, polypeptides, peptides, and
antibodies) refers to a proteinaceous agent that possesses a
similar or identical function as a second proteinaceous agent but
does not necessarily comprise a similar or identical amino acid
sequence of the second proteinaceous agent, or possess a similar or
identical structure of the second proteinaceous agent. A
proteinaceous agent that has a similar amino acid sequence refers
to a second proteinaceous agent that satisfies at least one of the
following: (a) a proteinaceous agent having an amino acid sequence
that is at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or at least 99% identical to the amino acid sequence of a
second proteinaceous agent; (b) a proteinaceous agent encoded by a
nucleotide sequence that hybridizes under stringent conditions to a
nucleotide sequence encoding a second proteinaceous agent of at
least 5 contiguous amino acid residues, at least 10 contiguous
amino acid residues, at least 15 contiguous amino acid residues, at
least 20 contiguous amino acid residues, at least 25 contiguous
amino acid residues, at least 40 contiguous amino acid residues, at
least 50 contiguous amino acid residues, at least 60 contiguous
amino residues, at least 70 contiguous amino acid residues, at
least 80 contiguous amino acid residues, at least 90 contiguous
amino acid residues, at least 100 contiguous amino acid residues,
at least 125 contiguous amino acid residues, or at least 150
contiguous amino acid residues; and (c) a proteinaceous agent
encoded by a nucleotide sequence that is at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95% or at least 99% identical
to the nucleotide sequence encoding a second proteinaceous agent. A
proteinaceous agent with similar structure to a second
proteinaceous agent refers to a proteinaceous agent that has a
similar secondary, tertiary or quaternary structure to the second
proteinaceous agent. The structure of a proteinaceous agent can be
determined by methods known to those skilled in the art, including
but not limited to, peptide sequencing, X-ray crystallography,
nuclear magnetic resonance, circular dichroism, and
crystallographic electron microscopy.
[0032] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino acid or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions.times.100%). In one embodiment,
the two sequences are the same length.
[0033] The determination of percent identity between two sequences
can also be accomplished using a mathematical algorithm. A
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.
87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.
Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated
into the NBLAST and XBLAST programs of Altschul et al., 1990, J.
Mol. Biol. 215:403. BLAST nucleotide searches can be performed with
the NBLAST nucleotide program parameters set, e.g., for score=100,
wordlength=12 to obtain nucleotide sequences homologous to a
nucleic acid molecules of the present invention. BLAST protein
searches can be performed with the XBLAST program parameters set,
e.g., to score--50, wordlength=3 to obtain amino acid sequences
homologous to a protein molecule of the present invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., 1997, Nucleic Acids
Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform
an iterated search which detects distant relationships between
molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
of XBLAST and NBLAST) can be used (see, e.g., the NCBI website).
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated
in the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be
used.
[0034] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0035] As used herein, the term "analog" in the context of a
non-proteinaceous analog refers to a second organic or inorganic
molecule which possess a similar or identical function as a first
organic or inorganic molecule and is structurally similar to the
first organic or inorganic molecule. The term "analog" includes a
molecule whose core structure is the same as, or closely resembes
that of the first molecule, but which has a chemical or physical
modification the term "analog" inclues copolymers of the first
molecule that can be linked to other atoms or molecules. A
"biologically active analog" and "anolog" are used interchangeably
herein to cover an organic or inorganic molecule that exhibits
substantially the same agonist or antagonist effect of the first
organinc or inorganic molecule.
[0036] A "nucleotide analog", as used herein, refers to a
nucleotide in which the pentose sugar and/or one or more of the
phosphate esters is replaced with its respective analog. Exemplary
phosphate ester analogs include, but are not limited to,
alkylphosphonates, methylphosphonates, phosphoramidates,
phosphotriesters, phosphorothioates, phosphorodithioates,
phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,
phosphoroanilidates, phosphoroamidates, boronophosphates, etc.,
including any associated counterions, if present. Also included
within the definition of "nucleotide analog" are nucleobase
monomers which can be polymerized into polynucleotide analogs in
which the DNA/RNA phosphate ester and/or sugar phosphate ester
backbone is replaced with a different type of linkage. Further
included within "nucleotide analogs" are nucleotides in which the
nucleobase moiety is non-conventional, i.e., differs from one of G,
A, T, U or C. Generally a non-conventional nucleobase will have the
capacity to form hydrogen bonds with at least one nucleobase moiety
present on an adjacent counter-directional polynucleotide strand or
provide a non-interacting, non-interfering base.
[0037] The term "antibody" also encompasses antigen-binding
fragments of an antibody. The term "antigen-binding fragment" of an
antibody (or simply "antibody portion," or "fragment"), as used
herein, refers to one or more fragments of a full-length antibody
that retain the ability to specifically bind to a polypeptide
encoded by one of the genes of a biomarker of the invention.
Examples of binding fragments encompassed within the term
"antigen-binding fragment" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding fragment" of an antibody. These antibody fragments
are obtained using conventional techniques known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies. The antibody is preferably
monospecific, e.g., a monoclonal antibody, or antigen-binding
fragment thereof. The term "monospecific antibody" refers to an
antibody that displays a single binding specificity and affinity
for a particular target, e.g., epitope. This term includes a
"monoclonal antibody" or "monoclonal antibody composition," which
as used herein refer to a preparation of antibodies or fragments
thereof of single molecular composition.
[0038] As used herein, the terms "attaching" and "spotting" refer
to a process of depositing a nucleic acid onto a substrate to form
a nucleic acid array such that the nucleic acid is stably bound to
the substrate via covalent bonds, hydrogen bonds or ionic
interactions.
[0039] As used herein, the term "biomarker" refers to a gene that
is differentially regulated as between individuals with (a)
schizophrenia and normal individuals (individuals without
schizophrenia) (b) bipolar disorder and normal individuals
(individuals without bipolar disorder) and (c) schizophrenia and
bipolar disorder.
[0040] As used herein, a "blood nucleic acid sample", refers to
nucleic acids derived from blood and can include nucleic acids
derived from whole blood, centrifuged lysed blood, serum free whole
blood or peripheral blood leukocytes (PBLs). By whole blood is
meant unseparated whole blood, for example, a drop of whole blood.
By centrifuged lysed blood or `lysed blood` is meant whole blood
that is mixed with lysis buffer and centrifuged as described herein
(see Example 2). By serum free blood is meant whole blood wherein
the serum or plasma is removed by centrifugation as described
herein (see Example 2). Preferably, a blood nucleic acid sample is
whole blood or centrifuged lysed blood and is total RNA, mRNA or is
a nucleic acid corresponding to mRNA, for example, cDNA derived
from mRNA isolated from said blood. A nucleic acid sample can also
include a PCR product derived from total RNA, mRNA or cDNA.
[0041] As used herein, the term "brain cells" includes those cells
found in the brain and include neurons, and glial cells, including
Schwann's Cells, Satellite Cells, Microglia cels, Oligodendroglia
cells, and Astroglia cells and all cell lines thereof.
[0042] As used herein, the term `centrifuged` refers to the
centrifugation of serum free whole blood or lysed blood at 2000 rpm
(800 g) for 5 minutes at 4.degree. C.
[0043] As used herein, the term "classifier" is used to describe
the output of a mathematical model generated on its ability to
differentiate between two or more phenotypic traits--for example
having or not having schizophrenia, having or not having bipolar
disorder and either having schizophrenia or bipolar disorder.
[0044] As used herein, the terms "compound" and "agent" are used
interchangably.
[0045] As used herein, "consisting essentially of" refers to the
maximum number of genes that are required for the use of a
biomarker to diagnose schizophrenia or bipolar disorder. In one
embodiment, a biomarker for the diagnosis of schizophrenia consists
essentially of at least 2, 3, 4, 5, 6, 7, or all of the biomarkers
of the invention. In another embodiment, a biomarker for the
diagnosis of bipolar disorder consists essentially of at least 2,
3, 4, 5, 6, 7, or all of the biomarkers of the invention. In
another embodiment, a biomarker for differentiating between
schizophrenia and bipolar disorder consists essentially of at least
2, 3, 4, 5, 6, 7 or all of the biomarkers of the invention. In
another embodiment, a biomarker for diagnosis of schizophrenia
consists essentially of any one of the biomarkers in Table 3. In
another embodiment, a biomarker for diagnosis of bipolar disorder
consists essentially of any one of the biomarkers in Table 4. In
another embodiment, a biomarker for differentiating between
schizophrenia and bipolar disorder consists essentially of any one
of the Biomarkers in Table 5.
[0046] As used herein, the term "control" or "control sample" in
the context of this invention refers to one or more tissue nucleic
acid samples and/or a blood nucleic acid samples isolated from an
individual or group of individuals who are either classified as
having schizophrenia, having bipolar disorder or not having
schizophrenia or bipolar disorder where the diagnosis for the
"control" or "control sample" has been confirmed. The term control
or control sample can also refer to the compilation of data derived
from samples of one or more individuals whose diagnosis has been
confirmed as normal (not having schizophrenia or bipolar disorder)
or one or more individuals whose diagnosis has been confirmed as
having schizophrenia or bipolar disorder.
[0047] A "coding region" refers to a DNA sequence encoding
mRNA.
[0048] As used herein, the terms "compound" and "agent" are used
interchangably. As used herein, the term "derivative" in the
context of proteinaceous agent (e.g., proteins, polypeptides,
peptides, and antibodies) refers to a proteinaceous agent that
comprises an amino acid sequence which has been altered by the
introduction of amino acid residue substitutions, deletions, and/or
additions. The term "derivative" as used herein also refers to a
proteinaceous agent which has been modified, i.e., by the covalent
attachment of any type of molecule to the proteinaceous agent. For
example, but not by way of limitation, an antibody may be modified,
e.g., by glycosylation, acetylation, pegylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other
protein, etc. A derivative of a proteinaceous agent may be produced
by chemical modifications using techniques known to those of skill
in the art, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Further, a derivative of a proteinaceous agent
may contain one or more non-classical amino acids. A derivative of
a proteinaceous agent possesses a similar or identical function as
the proteinaceous agent from which it was derived.
[0049] As used herein, the term "derivative" in the context of a
non-proteinaceous derivative refers to a second organic or
inorganic molecule that is formed based upon the structure of a
first organic or inorganic molecule. A derivative of an organic
molecule includes, but is not limited to, a molecule modified,
e.g., by the addition or deletion of a hydroxyl, methyl, ethyl,
carboxyl or amine group. An organic molecule may also be
esterified, alkylated and/or phosphorylated.
[0050] As used herein, "diagnosis" refers to a process of
determining if an individual is afflicted with a disease or
ailment. "Diagnosis of schizophrenia" or "schizophrenia diagnosis"
refers to a process of determining if an individual is afflicted
with schizophrenia and includes both traditional medical diagnostic
techniques for diagnosing schizophrenia, as well as diagnostic
methods as encompassed by the invention. In one embodiment,
diagnosis of schizophrenia using methods as encompassed by the
invention includes determining whether a person has schizophrenia
or does not have schizophrenia. In another embodiment, diagnosis of
schizophrenia includes determining whether a person has
schizophrenia or bipolar disorder. "Diagnosis of bipolar disorder"
or "bipolar disorder diagnosis" refers to a process of determining
if an individual is afflicted with bipolar disorder and includes
both traditional medical diagnostic techniques for diagnosing
bipolar disorder, as well as diagnostic methods as encompassed by
the invention. In one embodiment, diagnosis of bipolar disorder
using methods as encompassed by the invention includes determining
whether a person has bipolar disorder or does not have bipolar
disorder. In another embodiment, diagnosis includes determining
whether a person has bipolar disorder or schizophrenia. Traditional
medical diagnostic techniques for diagnosing schizophrenia include:
physical exam and history, medical evaluation, and a mental status
exam and appropriate laboratory tests which can include an MRI. The
diagnosis often encompasses a pattern of signs and symptoms, in
conjunction with impaired occupational or social functioning.
Currently diagnosis includes looking for delusions (false beliefs
strongly held in spite of invalidating evidence); visual, auditory,
tactile, olfactory or gustatory hallucinations; disorganized
speech; disorganized thinking; grossly disorganized thinking and/or
catatonic behaviour; negative symptoms, such as emotional deficit,
avolition (inability to initiate and persist in goal-directed
activities) and alogia (poverty of speech) are also symptoms of
schizophrenia. Traditional medical diagnostic techniques for
diagnosing bipolar disorder include: physical exam and history and
mental status exam for presence of bipolar disorder symptoms which
include a combination of at least one major depressive episode (a
depressed mood or a loss of interest or pleasure in daily
activities consistently for at least a 2 week period which
represents a change from the person's normal mood; social,
occupational, educational or other important functioning must also
be negatively impaired by the change in mood) and one manic episode
(a distinct period of persistently elevated, expansive, or
irritable mood, lasting throughout at least 4 days, that is clearly
different from the usual nondepressed mood). In a specific
embodiment, "diagnosis of schizophrenia" refers to a determination
as between two options: e.g. that an individual has schizophrenia
or that an individual does not have schizophrenia; or e.g. that an
individual has schizophrenia or that an individual has bipolar
disorder; or e.g. than an individual has bipolar disorder or does
not have bipolar disorder. In another embodiment, "diagnosis" can
also refer to a determination as between three options e.g. an
individual has bipolar disorder, an individual has schizophrenia or
an individual has neither. In another embodiment diagnosis can
include an option that it cannot be determined with sufficient
degree of certainty as to whether an individual can be
characterized as having schizophrenia, having bipolar disorder or
having either. As would be understood by a person skilled in the
art, in this context a "sufficient degree of certainty" depends
upon the medical requirements for both the sensitivity and
specificity of the diagnosis. More particularly the sufficient
degree of certaintly includes greater than 50% sensitivity and/or
specificity, greater than 60% sensitivity and/or specificity,
greater than 70% sensitivity and/or specificity, greater than 80%
sensitivity and/or specificity, greater than 90% sensitivity and/or
specificity and 100% sensitivity and/or specificity. Note that
diagnosis can also refer to the results of a series of individual
diagnosis so as to make an ultimate diagnosis (e.g. a first
diagnosis to determine whether an individual has schizophrenia or
does not have schizophrenia and second test to determine whether
said individual has schizophrenia or is bipolar where the results
of both tests lead to a diagnosis of schizophrenia or bipolar
disorder).
[0051] As used herein, "normal" in the context of a conventional
diagnosis refers to an individual or group of individuals who have
not shown any symptoms of either schizophrenia or bipolar disorder
and are not known to have either schizophrenia or bipolar disorder.
Preferably said normal individual(s) is not on medication affecting
schizophrenia or bipolar disorder. More preferably said normal
individual(s) is not on medication affecting mental health. If
possible said individual or group of individuals has not been
diagnosed with any other disease. It is also helpful if the normal
individuals have similar sex, and age as compared with the test
individuals. "Normal", according to the invention, also refers to a
samples isolated from normal individuals and includes blood, total
RNA or mRNA isolated from normal individuals. A sample taken from a
normal individual can include RNA isolated from a blood sample
wherein said blood sample is whole blood, lysed blood, centrifuged
lysed blood or peripheral blood leukocytes (PBLs), and wherein the
blood is from an individual who has not been diagnosed with either
schizophrenia or bipolar disorder and does not show any symptoms of
schizophrenia or bipolar disorder at the time the blood is
isolated.
[0052] As used herein, the term "differential expression" refers to
a difference in the level of expression of the RNA of one or more
biomarkers of the invention, as measured by the amount or level
mRNA, and/or one or more spliced variants of mRNA of the biomarker
in one sample as compared with the level of expression of the same
one or more biomarkers of the invention in a second sample.
"Differentially expressed" can also include a measurement of the
protein encoded by the biomarker of the invention in a sample or
population of samples as compared with the amount or level of
protein expression in a second sample or population of samples.
Differential expression can be determined as described herein and
as would be understood by a person skilled in the art. The term
"differentially expressed" or "changes in the level of expression"
refers to an increase or decrease in the measurable expression
level of a given biomarker as measured by the amount of RNA and/or
the amount of protein in a sample as compared with the measurable
expression level of a given biomarker a second sample. The term
"differentially expressed" or "changes in the level of expression"
can also refer to an increase or decrease in the measurable
expression level of a given biomarker in a population of samples as
compared with the measurable expression level of a biomarker in a
second population of samples. As used herein, "differentially
expressed" can be measured using the ratio of the level of
expression of a given biomarker(s) as compared with the mean
expression level of the given biomarker(s) of a control wherein the
ratio is not equal to 1.0. Differentially expressed can also be
measured using p-value. When using p-value, a biomarker is
identified as being differentially expressed as between a first and
second population when the p-value is less than 0.1. More
preferably the p-value is less than 0.05. Even more preferably the
p-value is less than 0.01. More preferably still the p-value is
less than 0.005. Most preferably the p-value is less than 0.001.
When determining differentially expression on the basis of the
ratio, an RNA or protein is differentially expressed if the ratio
of the level of expression in a first sample as compared with a
second sample is greater than or less than 1.0. For example, a
ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 or a ratio
less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1. 0.05. In another
embodiment of the invention a nucleic acid transcript is
differentially expressed if the ratio of the mean of the level of
expression of a first population as compared with the mean level of
expression of the second population is greater than or less than
1.0 For example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4,
10, 20 or a ratio less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1.
0.05. In another embodiment of the invention a nucleic acid
transcript is differentially expressed if the ratio of its level of
expression in a first sample as compared with the mean of the
second population is greater than or less than 1.0 and includes for
example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or
a ratio less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1. 0.05.
[0053] "Differentially increased expression" or "up regulation"
refers to genes which demonstrate at least 10% or more, for
example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more or 1.1
fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, or more increase in
gene expression (as measured by RNA expression or protein
expression), relative to a control.
[0054] "Differentially decreased expression" or "down regulation"
refers to genes which demonstrate at least 10% or more, for
example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or a less than
1.0 fold, 0.8 fold, 0.6 fold, 0.4 fold, 0.2 fold, 0.1 fold or less
decrease in gene expression (as measured by RNA expression or
protein expression), relative to a control. For example, up
regulated genes includes genes having an increased level of
expression of mRNA or protein in blood isolated from individuals
characterized as having schizophrenia as compared with expression
of mRNA or protein isolated from normal individuals. For example,
down regulated genes includes genes having a decreased level of
expression in blood isolated from individuals characterized as
having schizophrenia as compared with blood isolated from normal
individuals. As used herein, the term "differential hybridization"
refers to a difference in the quantitative level of hybridization
of a nucleic acid sample from a first individual or individuals
with a trait to a complementary nucleic acid target as compared
with the hybridization of a nucleic acid sample from a second
individual or individuals not having said trait to the same
complementary nucleic acid target. A "differential hybridization"
means that the ratio of the level of hybridization of the first
sample as compared with the second sample is not equal to 1.0. For
example, the ratio of the level of hybridization of the first
sample to the target as compared to the second sample is greater
than or less than 1.0, and includes greater than 1.5 and less than
0.7, greater than 2 and less than 0.5. A differential hybridization
also exists if the hybridization is detectable in one sample but
not another sample.
[0055] As used herein, the term "drug efficacy" refers to the
effectiveness of a drug. "Drug efficacy" is usually measured by the
clinical response of the patient who has been or is being treated
with a drug. A drug is considered to have a high degree of
efficacy, if it achieves desired clinical results, for example, the
alteration of gene expression and the gene expression pattern
reflective of schizophrenia or bipolar disorder as described
herein. The amount of drug absorbed may be used to predict a
patient's response. A general rule is that as the dose of a drug is
increased, a greater effect is seen in the patient until a maximum
desired effect is reached. If more drug is administered after the
maximum point is reached, the side effects will normally
increase.
[0056] As used herein, the term "effective amount" refers to the
amount of a compound which is sufficient to reduce or ameliorate
the progression, severity and/or duration of schizophrenia or
bipolar disorder, or schizophrenic episodes/bipolar episodes or one
or more symptoms thereof, prevent the development, recurrence or
onset of schizophrenia and/or bipolar disorder or one or more
symptoms thereof, prevent the advancement of schizophrenia and/or
bipolar disorder or one or more symptoms thereof, or enhance or
improve the prophylactic or therapeutic effect(s) of another
therapy.
[0057] As used herein, the term "fragment" in the context of a
proteinaceous agent refers to a peptide or polypeptide comprising
an amino acid sequence of at least 5 contiguous amino acid
residues, at least 10 contiguous amino acid residues, at least 15
contiguous amino acid residues, at least 20 contiguous amino acid
residues, at least 25 contiguous amino acid residues, at least 40
contiguous amino acid residues, at least 50 contiguous amino acid
residues, at least 60 contiguous amino residues, at least 70
contiguous amino acid residues, at least contiguous 80 amino acid
residues, at least contiguous 90 amino acid residues, at least
contiguous 100 amino acid residues, at least contiguous 125 amino
acid residues, at least 150 contiguous amino acid residues, at
least contiguous 175 amino acid residues, at least contiguous 200
amino acid residues, or at least contiguous 250 amino acid residues
of the amino acid sequence of another polypeptide or a protein. In
a specific embodiment, a fragment of a protein or polypeptide
retains at least one function of the protein or polypeptide. In
another embodiment, a fragment of a protein or polypeptide retains
at least two, three, four, or five functions of the protein or
polypeptide. Preferably, a fragment of an antibody retains the
ability to immunospecifically bind to an antigen.
[0058] As used herein, the term "fusion protein" refers to a
polypeptide that comprises an amino acid sequence of a first
protein or polypeptide or functional fragment, analog or derivative
thereof, and an amino acid sequence of a heterologous protein,
polypeptide, or peptide (i.e., a second protein or polypeptide or
fragment, analog or derivative thereof different than the first
protein or fragment, analog or derivative thereof). In one
embodiment, a fusion protein comprises a prophylactic or
therapeutic agent fused to a heterologous protein, polypeptide or
peptide. In accordance with this embodiment, the heterologous
protein, polypeptide or peptide may or may not be a different type
of prophylactic or therapeutic agent.
[0059] As used herein, a "gene expression pattern" or "gene
expression profile" indicates the combined pattern of the results
of the analysis of the level of expression of two or more
biomarkers of the invention including 3, 4, 5, 6, 7, 8, 9, 10, 11,
12 or all of the biomarkers of the invention. A gene expression
pattern or gene expression profile can result from the measurement
of expression of the products of the biomarkers of the invention
and can be done using any known technique. For example techniques
to measure expression of the RNA products of the biomarkers of the
invention includes, PCR based methods (including RT-PCR) and non
PCR based method as well as microarray analysis. To measure protein
products of the biomarkers of the invention, techniques include
western blotting and ELISA analysis.
[0060] As used herein, the term "hybridizing to" or "hybridization"
refers to the sequence specific non-covalent binding interactions
with a complementary nucleic acid, for exampleinteractions between
a target nucleic acid sequence and a nucleic acid member on an
array.
[0061] As used herein, the term "immunoglobulin" refers to a
protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. The recognized human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 Kd or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH-terminus. Full-length
immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are
similarly encoded by a variable region gene (about 116 amino acids)
and one of the other aforementioned constant region genes, e.g.,
gamma (encoding about 330 amino acids).
[0062] As used herein, the term "in combination" when referring to
therapeutic treatments refers to the use of more than one type of
therapy (e.g., more than one prophylactic agent and/or therapeutic
agent). The use of the term "in combination" does not restrict the
order in which therapies (e.g., prophylactic and/or therapeutic
agents) are administered to a subject. A first therapy (e.g., a
first prophylactic or therapeutic agent) can be administered prior
to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96
hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8
weeks, or 12 weeks before), concomitantly with, or subsequent to
(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96
hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8
weeks, or 12 weeks after) the administration of a second therapy
(e.g., a second prophylactic or therapeutic agent) to a
subject.
[0063] As used herein, "indicative of disease" when referring to an
expression pattern indicates an expression pattern which is
diagnostic of disease such that the expression pattern is found
significantly more often in patients with a disease than in
patients without the disease (as determined using routine
statistical methods setting confidence levels at a minimum of 95%).
Preferably, an expression pattern which is indicative of disease is
found in at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95% or more in patients who have the disease
and is found in less than 10%, less than 8%, less than 5%, less
than 2.5%, or less than 1% of patients who do not have the disease.
"Indicative of disease" also indicates an expression pattern which
is diagnostic of disease such that the expression pattern more
properly categorizes with control expression patterns of
individuals with disease as compared with control expression
patterns of individuals without disease using statistical
algorithms for class prediction as would be understood by a person
skilled in the art and see for example commercially available
programs such as those provided by Silicon Genetics (e.g.
GeneSpring.TM.).
[0064] As used herein, "isolated" or "purified" when used in
reference to a nucleic acid means that a naturally occurring
sequence has been removed from its normal cellular (e.g.,
chromosomal) environment or is synthesized in a non-natural
environment (e.g., artificially synthesized). Thus, an "isolated"
or "purified" sequence may be in a cell-free solution or placed in
a different cellular environment. The term "purified" does not
imply that the sequence is the only nucleotide present, but that it
is essentially free (about 90-95% pure) of non-nucleotide material
naturally associated with it, and thus is distinguished from
isolated chromosomes.
[0065] As used herein, the terms "isolated" and "purified" in the
context of a proteinaceous agent (e.g., a peptide, polypeptide,
protein or antibody) refer to a proteinaceous agent which is
substantially free of cellular material and in some embodiments,
substantially free of heterologous proteinaceous agents (i.e.,
contaminating proteins) from the cell or tissue source from which
it is derived, or substantially free of chemical precursors or
other chemicals when chemically synthesized. The language
"substantially free of cellular material" includes preparations of
a proteinaceous agent in which the proteinaceous agent is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. Thus, a proteinaceous agent that is
substantially free of cellular material includes preparations of a
proteinaceous agent having less than about 30%, 20%, 10%, or 5% (by
dry weight) of heterologous proteinaceous agent (e.g., protein,
polypeptide, peptide, or antibody; also referred to as a
"contaminating protein"). When the proteinaceous agent is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, 10%, or 5% of the volume of the protein preparation. When the
proteinaceous agent is produced by chemical synthesis, it is
preferably substantially free of chemical precursors or other
chemicals, i.e., it is separated from chemical precursors or other
chemicals which are involved in the synthesis of the proteinaceous
agent. Accordingly, such preparations of a proteinaceous agent have
less than about 30%, 20%, 10%, 5% (by dry weight) of chemical
precursors or compounds other than the proteinaceous agent of
interest. Preferably, proteinaceous agents disclosed herein are
isolated.
[0066] As used herein, the term "level of expression" when
referring to RNA refers to the measurable quantity of a given
nucleic acid as determined by hybridization or measurements such as
real-time RT PCR, which includes use of both SYBR.RTM. green and
TaqMan.RTM. technology and which corresponds in direct proportion
with the extent to which the gene is expressed. The level of
expression of a nucleic acid is determined by methods well known in
the art. For microarray analysis, the level of expression is
measured by hybridization analysis using labeled nucleic acids
corresponding to RNA isolated from one or more individuals
according to methods well known in the art. The label on the
nucleic acid used for hybridization can be a luminescent label, an
enzymatic label, a radioactive label, a chemical label or a
physical label. Preferably, target nucleic acids are labeled with a
fluorescent molecule. Preferred fluorescent labels include, but are
not limited to: fluorescein, amino coumarin acetic acid,
tetramethylrhodamine isothiocyanate (TRITC), Texas Red, Cyanine 3
(Cy3) and Cyanine 5 (Cy5).
[0067] As used herein, a "ligand" is a molecule that specifically
binds to a polypeptide encoded by one of the genes of a biomarker
of the invention. A ligand can be a nucleic acid (RNA or DNA),
polypeptide, peptide or chemical compound. A ligand of the
invention can be a peptide ligand, e.g., a scaffold peptide, a
linear peptide, or a cyclic peptide. In a preferred embodiment, the
polypeptide ligand is an antibody. The antibody can be a human
antibody, a chimeric antibody, a recombinant antibody, a humanized
antibody, a monoclonal antibody, or a polyclonal antibody. The
antibody can be an intact immunoglobulin, e.g., an IgA, IgG, IgE,
IgD, IgM or subtypes thereof. The antibody can be conjugated to a
functional moiety (e.g., a compound which has a biological or
chemical function (which may be a second different polypeptide, a
therapeutic drug, a cytotoxic agent, a detectable moiety, or a
support. A polypeptide ligand e.g. antibody of the invention
interacts with a polypeptide, encoded by one of the genes of a
biomarker, with high affinity and specificity. For example, the
polypeptide ligand binds to a polypeptide, encoded by one of the
genes of a biomarker, with an affinity constant of at least
10.sup.7 M.sup.-1, preferably, at least 10.sup.8 M.sup.-1, 10.sup.9
M.sup.-1, or 10.sup.10 M.sup.-1.
[0068] An "mRNA" means an RNA complementary to a gene; an mRNA
includes a protein coding region and also may include 5' end and 3'
untranslated regions (UTR).
[0069] As used herein, the term "majority" refers to a number
representing more than 50% (e.g., 51%, 60%, or 70%, or 80% or 90%
or up to 100%) of the total members of a composition. The term
"majority", when referring to an array, it means more than 50%
(e.g., 51%, 60%, or 70%, or 80% or 90% or up to 100%) of the total
nucleic acid members that are stably associated with the solid
substrate of the array.
[0070] As used herein, the terms "manage", "managing" and
"management" refer to the beneficial effects that a subject derives
from a therapy (e.g., a prophylactic or therapeutic agent) which
does not result in a cure of schizophrenia and/or bipolar disorder.
In certain embodiments, a subject is administered one or more
therapies to "manage" schizophrenia and/or bipolar disorder so as
to ameliorate symptoms of schizophrenia and/or bipolar disorder,
and/or to prevent and/or retard the progression of these diseases
and or symptoms of these diseases.
[0071] Amelioration of schizophrenia and/or bipolar disorder is
defined herein as providing physical or physiological relief to
individuals and can include relief of symptoms as well as a
decrease in episode number or episode duration. Treatment of
schizophrenia and/or bipolar disorder is defined herein to provide
medical aid to counteract the disease itself, the symptoms and or
episodes of the disease (either in number or duration) and/or the
progression of the disease. These treatments may be given as
palliative therapy to help relieve symptoms and improve the quality
of life.
[0072] As used herein, "mRNA integrity" refers to the quality of
mRNA extracts from either tissue samples or blood samples. mRNA
extracts with good integrity do not appear to be degraded when
examined by methods well known in the art, for example, by RNA
agarose gel electrophoresis (e.g., Ausubel et al., John Weley &
Sons, Inc., 1997, Current Protocols in Molecular Biology).
Preferably, the mRNA samples have good integrity (e.g., less than
10%, preferably less than 5%, and more preferably less than 1% of
the mRNA is degraded) to truly represent the gene expression levels
of the tissue or blood samples from which they are extracted.
[0073] As used herein, the terms "non-responsive" and refractory"
describe patients treated with a currently available therapy (e.g.,
prophylactic or therapeutic agent) for schizophrenia and/or bipolar
disorder, which is not clinically adequate to relieve one or more
symptoms associated therewith. Typically, such patients suffer from
severe, persistently active schizophrenia and/or bipolar disorder
and require additional therapy to ameliorate the symptoms
associated with their disease.
[0074] As used herein, "nucleic acid(s)" is interchangeable with
the term "polynucleotide(s)" and it generally refers to any
polyribonucleotide or poly-deoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA or any combination
thereof. "Nucleic acids" include, without limitation, single- and
double-stranded nucleic acids. As used herein, the term "nucleic
acid(s)" also includes DNAs or RNAs as described above that contain
one or more modified bases. Thus, DNAs or RNAs with backbones
modified for stability or for other reasons are "nucleic acids".
The term "nucleic acids" as it is used herein embraces such
chemically, enzymatically or metabolically modified forms of
nucleic acids, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including for example, simple
and complex cells. A "nucleic acid" or "nucleic acid sequence" may
also include regions of single- or double-stranded RNA or DNA or
any combinations thereof and can include expressed sequence tags
(ESTs) according to some embodiments of the invention. An EST is a
portion of the expressed sequence of a gene (i.e., the "tag" of a
sequence), made by reverse transcribing a region of mRNA so as to
make cDNA.
[0075] As defined herein, a "nucleic acid array" refers a plurality
of nucleic acids (or "nucleic acid members") attached to a support
where each of the nucleic acid members is attached to a support in
a unique pre-selected region. In one embodiment, the nucleic acid
member attached to the surface of the support is DNA. In a
preferred embodiment, the nucleic acid member attached to the
surface of the support is either cDNA or oligonucleotides. In
another preferred embodiment, the nucleic acid member attached to
the surface of the support is cDNA synthesized by polymerase chain
reaction (PCR). The term "nucleic acid", as used herein, is
interchangeable with the term "polynucleotide". In another
preferred embodiment, a "nucleic acid array" refers to a plurality
of unique nucleic acids attached to nitrocellulose or other
membranes used in Southern and/or Northern blotting techniques.
[0076] As used herein "nucleic acid sample for hybridization to an
array" is defined as a nucleic acid capable of binding to a nucleic
acid bound to an array of complementary sequence through sets of
non-covalent bonding interactions including complementary base
pairing interactions. The nucleic acid sample for hybridization to
an array can either be an isolated nucleic acid sequence
corresponding to a gene or portion thereof, total RNA or mRNA
isolated from a sample. Preferably, the nucleic acid sample for
hybridization to an array is derived from human blood (including
whole blood, lysed blood, centrifuged lysed blood, or peripheral
blood leukocytes (PBLs)). More preferably, the nucleic acid sample
is single- or double-stranded DNA, RNA, or DNA-RNA hybrids, from
human blood and preferably from RNA or mRNA extracts.
[0077] As used herein, a "nucleic acid member on an array" or a
"nucleic acid member" includes nucleic acid immobilized on an array
and capable of binding to a nucleic acid probes or samples of
complementary sequence through sets of non-covalent bonding
interactions, including complementary base pairing interactions. As
used herein, a nucleic acid member or target may include natural
(i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine,
etc.). In addition, the bases in nucleic acids may be joined by a
linkage other than a phosphodiester bond, so long as it does not
interfere with hybridization (i.e., the nucleic acid target still
specifically binds to its complementary sequence under standard
stringent or selective hybridization conditions). Thus, nucleic
acid members may be peptide nucleic acids in which the constituent
bases are joined by peptide bonds rather than phosphodiester
linkages. In one embodiment, a conventional nucleic acid array of
`target` sequences bound to the array can be representative of the
entire human genome, e.g. Affymetrix chip, and the biomarker or
isolated biomarker consisting of or comprising two or more of the 3
genes described in FIG. 1 or gene probes is applied to the
conventional array. In another embodiment, sequences bound to the
array can be the biomarker or isolated biomarker according to the
invention and total cellular RNA is applied to the array.
[0078] As used herein, the term "oligonucleotide" is defined as a
molecule comprised of two or more deoxyribonucleotides and/or
ribonucleotides, and preferably more than three. Its exact size
will depend upon many factors which, in turn, depend upon the
ultimate function and use of the oligonucleotide. The
oligonucleotides may be from about 8 to about 1,000 nucleotides
long. Although oliognucleotides of 8 to 100 nucleotides are useful
in the invention, preferred oligonucleotides range from about 8 to
about 15 bases in length, from about 8 to about 20 bases in length,
from about 8 to about 25 bases in length, from about 8 to about 30
bases in length, from about 8 to about 40 bases in length or from
about 8 to about 50 bases in length.
[0079] As used herein, "patient" or "individual" refers to a mammal
who is diagnosed with schizophrenia and/or bipolar disorder.
[0080] As used herein, the phrase "pharmaceutically acceptable
salt(s)," includes, but is not limited to, salts of acidic or basic
groups that may be present in compounds identified using the
methods of the present invention. Compounds that are basic in
nature are capable of forming a wide variety of salts with various
inorganic and organic acids. The acids that can be used to prepare
pharmaceutically acceptable acid addition salts of such basic
compounds are those that form non-toxic acid addition salts, i.e.,
salts containing pharmacologically acceptable anions, including but
not limited to sulfuric, citric, maleic, acetic, oxalic,
hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,
bisulfate, phosphate, acid phosphate, isonicotinate, acetate,
lactate, salicylate, citrate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds that
include an amino moiety may form pharmaceutically acceptable salts
with various amino acids, in addition to the acids mentioned above.
Compounds that are acidic in nature are capable of forming base
salts with various pharmacologically acceptable cations. Examples
of such salts include alkali metal or alkaline earth metal salts
and, particularly, calcium, magnesium, sodium lithium, zinc,
potassium, and iron salts.
[0081] As used herein, "polynucleotide" encompasses double-stranded
DNA, single-stranded DNA and double-stranded or single-stranded RNA
of more than 8 nucleotides in length. The term "polynucleotide"
includes a polymeric form of nucleotides of any length, either
ribonucleotides or deoxyribonucleotides, that comprise purine and
pyrimidine bases, or other natural, chemically or biochemically
modified, non-natural, or derivatized nucleotide bases. The
backbone of the polynucleotide can comprise sugars and phosphate
groups, as may typically be found in RNA or DNA, or modified or
substituted sugar or phosphate groups. A polynucleotide may
comprise modified nucleotides, such as methylated nucleotides and
nucleotide analogs. The sequence of nucleotides may be interrupted
by non-nucleotide components.
[0082] As used herein, "polypeptide sequences encoded by" refers to
the amino acid sequences obtained after translation of the protein
coding region of a gene, as defined herein. The mRNA nucleotide
sequence for each of the genes of the invention is identified by
its Genbank Accession number (see Table 2) and the corresponding
polypeptide sequence is identified by a Protein Accession number
(see Table 2) The Genbank Accession numbers identified in Table 2
provides the location of the 5' UTR, protein coding region (CDS)
and 3' UTR within the mRNA nucleotide sequence of each of the genes
of the invention. When a protein or fragment of a protein is used
to immunize a host animal, numerous regions of the protein may
induce the production of antibodies which bind specifically to a
given region or three-dimensional structure on the protein; these
regions or structures are referred to as epitopes or antigenic
determinants. As used herein, "antigenic fragments" refers portions
of a polypeptide that contains one or more epitopes. Epitopes can
be linear, comprising essentially a linear sequence from the
antigen, or conformational, comprising sequences which are
genetically separated by other sequences but come together
structurally at the binding site for the polypeptide ligand.
"Antigenic fragments" may be 5000, 1000, 500, 400, 300, 200, 100,
50 or 25 or 20 or 10 or 5 amino acids in length.
[0083] As used herein, the terms "prevent", "preventing" and
"prevention" refer to the prevention of the development, recurrence
or onset of schizophrenia and/or bipolar disorder or one or more
symptoms and/or episodes thereof resulting from the administration
of one or more compounds identified in accordance the methods of
the invention or the administration of a combination of such a
compound and another therapy.
[0084] The term, "primer", as used herein refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product, which
is complementary to a nucleic acid strand, is induced, i.e., in the
presence of nucleotides and an inducing agent such as a DNA
polymerase and at a suitable temperature and pH. The primer may be
either single-stranded or double-stranded and must be sufficiently
long to prime the synthesis of the desired extension product in the
presence of the inducing agent. The exact length of the primer will
depend upon many factors, including temperature, source of primer
and the method used. For example, for diagnostic applications,
depending on the complexity of the target sequence, the
oligonucleotide primer typically contains 15-25 or more
nucleotides, although it may contain fewer nucleotides. The factors
involved in determining the appropriate length of primer are
readily known to one of ordinary skill in the art.
[0085] As used herein, the term "probe" means oligonucleotides and
analogs thereof and refers to a range of chemical species that
recognize polynucleotide target sequences through hydrogen bonding
interactions with the nucleotide bases of the target sequences. The
probe or the target sequences may be single- or double-stranded RNA
or single- or double-stranded DNA or a combination of DNA and RNA
bases. A probe is at least 8 nucleotides in length and less than
the length of a complete gene. A probe may be 10, 20, 30, 50, 75,
100, 150, 200, 250, 400, 500 and up to 2000 nucleotides in length
as long as it is less than the full length of the target gene.
Probes can include oligonucleotides modified so as to have a tag
which is detectable by fluorescence, chemiluminescence and the
like. The probe can also be modified so as to have both a
detectable tag and a quencher molecule, for example Taqman.RTM. and
Molecular Beacon.RTM. probes.
[0086] The oligonucleotides and analogs thereof may be RNA or DNA,
or analogs of RNA or DNA, commonly referred to as antisense
oligomers or antisense oligonucleotides. Such RNA or DNA analogs
comprise but are not limited to 2-'O-alkyl sugar modifications,
methylphosphonate, phosphorothiate, phosphorodithioate, formacetal,
3'-thioformacetal, sulfone, sulfamate, and nitroxide backbone
modifications, and analogs wherein the base moieties have been
modified. In addition, analogs of oligomers may be polymers in
which the sugar moiety has been modified or replaced by another
suitable moiety, resulting in polymers which include, but are not
limited to, morpholino analogs and peptide nucleic acid (PNA)
analogs (Egholm, et al. Peptide Nucleic Acids
(PNA)--Oligonucleotide Analogues with an Achiral Peptide Backbone,
(1992)).
[0087] Probes may also be mixtures of any of the oligonucleotide
analog types together or in combination with native DNA or RNA. At
the same time, the oligonucleotides and analogs thereof may be used
alone or in combination with one or more additional
oliognucleotides or analogs thereof.
[0088] As used herein, "a plurality of" or "a set of" refers to
more than two, for example, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more 10 or more etc.
[0089] As used herein, "pre-selected region", "predefined region",
or "unique position" refers to a localized area on a substrate
which is, was, or is intended to be used for the deposit of a
nucleic acid and is otherwise referred to herein in the alternative
as a "selected region" or simply a "region." The pre-selected
region may have any convenient shape, e.g., circular, rectangular,
elliptical, wedge-shaped, etc. In some embodiments, a pre-selected
region is smaller than about 1 cm.sup.2, more preferably less than
1 mm.sup.2, still more preferably less than 0.5 mm.sup.2, and in
some embodiments less than 0.1 mm.sup.2. A nucleic acid member at a
"pre-selected region", "predefined region", or "unique position" is
one whose identity (e.g., sequence) can be determined by virtue of
its position at the region or unique position.
[0090] As used herein the term "product of the biomarker" or
"products of the biomarkers of the invention" refers to the RNA
and/or the protein expressed by the gene corresponding to the
biomarker of the invention. In the case of RNA it refers to the RNA
transcripts transcribed from genes corresponding to the biomarker
of the invention. In the case of protein it refers to proteins
translated from the genes corresponding to the biomarker of the
invention. The "RNA product of a biomarker of the invention"
includes mRNA transcripts, and/or specific spliced variants of mRNA
whose measure of expression can be used as a biomarker in
accordance with the teachings disclosed herein. The "protein
product of a biomarker of the invention" includes proteins
translated from the RNA products of the biomarkers of the
invention.
[0091] As used herein, the terms "prophylactic agent" and
"prophylactic agents" refer to any compound(s) which can be used in
the prevention of schizophrenia and/or bipolar disorder. In certain
embodiments, the term "prophylactic agent" refers to a compound
identified in the screening assays described herein. In certain
other embodiments, the term "prophylactic agent" refers to an agent
other than a compound identified in the screening assays described
herein which is known to be useful for, or has been or is currently
being used to prevent or impede the onset, development and/or
progression of schizophrenia and/or bipolar disorder or one or more
symptoms and/or episodes thereof.
[0092] As used herein, the phrase "prophylactically effective
amount" refers to the amount of a therapy (e.g., a prophylactic
agent) which is sufficient to result in the prevention of the
development, recurrence or onset or progression of schizophrenia
and/or bipolar disorder or one or more symptoms and/or episodes
thereof.
[0093] As used herein, the terms "protein" and "polypeptide" are
used interchangeably to refer to a chain of amino acids linked
together by peptide bonds. In a specific embodiment, a protein is
composed of less than 200, less than 175, less than 150, less than
125, less than 100, less than 50, less than 45, less than 40, less
than 35, less than 30, less than 25, less than 20, less than 15,
less than 10, or less than 5 amino acids linked together by peptide
bonds. In another embodiment, a protein is composed of at least
200, at least 250, at least 300, at least 350, at least 400, at
least 450, at least 500 or more amino acids linked together by
peptide bonds.
[0094] A "protein coding region" refers to the portion of the mRNA
encoding a polypeptide.
[0095] As used herein the "reference population" or "test
population" refers a population of "control samples" used to
develop the classifier to differentiate between (a) schizophrenic
and normal individuals; (b) bipolar disorder individuals and normal
individuals or (c) schizophrenic individuals and bipolar disorder
individuals. The "reference population" or "test population" is
comprised of a number of control samples depending upon the
classifier to be constructed and can include the following: (a)
individuals diagnosed with schizophrenia using conventional
diagnostic techniques, (b) individuals diagnosed with bipolar
disorder using conventional techniques and (c) individuals having
neither bipolar disorder or schizophrenia. In a preferred
embodiment the "reference population" or "test population" is
comprised of an equal number of "control samples" from each
phenotypic subgroup (e.g. wherein said phenotype is a determination
of status with regards to schizophrenia or bipolar disorder). In
another embodiment, the "reference population" is also matched for
other phenotypes e.g. age, sex, drug status, etc.
[0096] As used herein, the term "selectively binds" in the context
of proteins encompassed by the invention refers to the specific
interaction of a any two of a peptide, a protein, a polypeptide an
antibody, wherein the interaction preferentially occurs as between
any two of a peptide, protein, polypeptide and antibody
preferentially as compared with any other peptide, protein,
polypeptide and antibody. For example, when the two molecules are
protein molecules, a structure on the first molecule recognizes and
binds to a structure on the second molecule, rather than to other
proteins. "Selective binding", "Selective binding", as the term is
used herein, means that a molecule binds its specific binding
partner with at least 2-fold greater affinity, and preferably at
least 10-fold, 20-fold, 50-fold, 100-fold or higher affinity than
it binds a non-specific molecule.
[0097] As used herein "selective hybridization" in the context of
this invention refers to a hybridization which occurs as between a
polynucleotide encompassed by the invention and an RNA or protein
product of the biomarker of the invention wherein the hybridization
is such that the polynucleotide binds to the RNA products of the
biomarker of the invention preferentially to the RNA products of
other genes in the genome in question. In a preferred embodiment a
polynucleotide which "selectively hybridizes" is one which
hybridizes with a selectivity of greater than 70%, greater than
80%, greater than 90% and most preferably on 100% (ie cross
hybridization with other RNA species preferably occurs at less than
30%, less than 20%, less than 10%). As would be understood to a
person skilled in the art, a polynucleotide which "selectively
hybridizes" to the RNA product of a biomarker of the invention can
be determined taking into account the length and composition.
[0098] As used herein, "specifically hybridizes", "specific
hybridization" refers to hybridization which occurs when two
nucleic acid sequences are substantially complementary (at least
about 65% complementary over a stretch of at least 14 to 25
nucleotides, preferably at least about 75% complementary, more
preferably at least about 90% complementary). See Kanehisa, M.,
1984, Nucleic acids Res., 12:203, incorporated herein by reference.
As a result, it is expected that a certain degree of mismatch is
tolerated. Such mismatch may be small, such as a mono-, di- or
tri-nucleotide. Alternatively, a region of mismatch can encompass
loops, which are defined as regions in which there exists a
mismatch in an uninterrupted series of four or more nucleotides.
Numerous factors influence the efficiency and selectivity of
hybridization of two nucleic acids, for example, the hybridization
of a nucleic acid member on an array to a target nucleic acid
sequence. These factors include nucleic acid member length,
nucleotide sequence and/or composition, hybridization temperature,
buffer composition and potential for steric hindrance in the region
to which the nucleic acid member is required to hybridize. A
positive correlation exists between the nucleic acid length and
both the efficiency and accuracy with which a nucleic acid will
anneal to a target sequence. In particular, longer sequences have a
higher melting temperature (TM) than do shorter ones, and are less
likely to be repeated within a given target sequence, thereby
minimizing non-specific hybridization. Hybridization temperature
varies inversely with nucleic acid member annealing efficiency.
Similarly the concentration of organic solvents, e.g., formamide,
in a hybridization mixture varies inversely with annealing
efficiency, while increases in salt concentration in the
hybridization mixture facilitate annealing. Under stringent
annealing conditions, longer nucleic acids, hybridize more
efficiently than do shorter ones, which are sufficient under more
permissive conditions.
[0099] As used herein, "spotting" or "attaching" refers to a
process of depositing a nucleic acid member onto a solid substrate
to form a nucleic acid array such that the nucleic acid is stably
bound to the solid substrate via covalent bonds, hydrogen bonds or
ionic interactions.
[0100] As used herein, "stably associated" refers to a nucleic acid
that is stably bound to a solid substrate to form an array via
covalent bonds, hydrogen bonds or ionic interactions such that the
nucleic acid retains its unique pre-selected position relative to
all other nucleic acids that are stably associated with an array,
or to all other pre-selected regions on the solid substrate under
conditions in which an array is typically analyzed (i.e., during
one or more steps of hybridization, washes, and/or scanning,
etc.).
[0101] As used herein, "substrate" or "support" when referring to
an array refers to a material having a rigid or semi-rigid surface.
The support may be biological, non-biological, organic, inorganic,
or a combination of any of these, existing as particles, strands,
precipitates, gels, sheets, tubing, spheres, beads, containers,
capillaries, pads, slices, films, plates, slides, chips, etc.
Often, the substrate is a silicon or glass surface,
(poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,
polycarbonate, a charged membrane, such as nylon 66 or
nitrocellulose, or combinations thereof. In a preferred embodiment,
the support is glass. Preferably, at least one surface of the
substrate will be substantially flat. Preferably, the support will
contain reactive groups, including, but not limited to, carboxyl,
amino, hydroxyl, thiol, and the like. In one embodiment, the
support is optically transparent.
[0102] As used herein, "specifically hybridizes", "specific
hybridization" refers to hybridization which occurs when two
nucleic acid sequences are substantially complementary (at least
about 65% complementary over a stretch of at least 14 to 25
nucleotides, preferably at least about 75% complementary, more
preferably at least about 90% complementary). See Kanehisa, M.,
1984, Nucleic acids Res., 12:203, incorporated herein by reference.
As a result, it is expected that a certain degree of mismatch is
tolerated. Such mismatch may be small, such as a mono-, di- or
tri-nucleotide. Alternatively, a region of mismatch can encompass
loops, which are defined as regions in which there exists a
mismatch in an uninterrupted series of four or more nucleotides.
Numerous factors influence the efficiency and selectivity of
hybridization of two nucleic acids, for example, the hybridization
of a nucleic acid member on an array to a target nucleic acid
sequence. These factors include nucleic acid member length,
nucleotide sequence and/or composition, hybridization temperature,
buffer composition and potential for steric hindrance in the region
to which the nucleic acid member is required to hybridize. A
positive correlation exists between the nucleic acid length and
both the efficiency and accuracy with which a nucleic acid will
anneal to a target sequence. In particular, longer sequences have a
higher melting temperature (T.sub.M) than do shorter ones, and are
less likely to be repeated within a given target sequence, thereby
minimizing promiscuous hybridization. Hybridization temperature
varies inversely with nucleic acid member annealing efficiency.
Similarly the concentration of organic solvents, e.g., formamide,
in a hybridization mixture varies inversely with annealing
efficiency, while increases in salt concentration in the
hybridization mixture facilitate annealing. Under stringent
annealing conditions, longer nucleic acids, hybridize more
efficiently than do shorter ones, which are sufficient under more
permissive conditions.
[0103] As herein used, the term "standard stringent conditions"
means hybridization will occur only if there is at least 95% and
preferably, at least 97% identity between the sequences, wherein
the region of identity comprises at least 10 nucleotides. In one
embodiment, the sequences hybridize under stringent conditions
following incubation of the sequences overnight at 42.degree. C.,
followed by stringent washes (0.2.times.SSC at 65.degree. C.). The
degree of stringency of washing can be varied by changing the
temperature, pH, ionic strength, divalent cation concentration,
volume and duration of the washing. For example, the stringency of
hybridization may be varied by conducting the hybridization at
varying temperatures below the melting temperatures of the probes.
The melting temperature of the probe may be calculated using the
following formulas:
[0104] For oligonucleotide probes, between 14 and 70 nucleotides in
length, the melting temperature (Tm) in degrees Celcius may be
calculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fraction
G+C)-(600/N) where N is the length of the oligonucleotide.
[0105] For example, the hybridization temperature may be decreased
in increments of 5.degree. C. from 68.degree. C. to 42.degree. C.
in a hybridization buffer having a Na+ concentration of
approximately 1M. Following hybridization, the filter may be washed
with 2.times.SSC, 0.5% SDS at the temperature of hybridization.
These conditions are considered to be "moderate stringency"
conditions above 50.degree. C. and "low stringency" conditions
below 50.degree. C. A specific example of "moderate stringency"
hybridization conditions is when the above hybridization is
conducted at 55.degree. C. A specific example of "low stringency"
hybridization conditions is when the above hybridization is
conducted at 45.degree. C.
[0106] If the hybridization is carried out in a solution containing
formamide, the melting temperature may be calculated using the
equation Tm=81.5+16.6(log [Na+])+0.41 (fraction G+C)-(0.63%
formamide)-(600/N), where N is the length of the probe.
[0107] For example, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. C. In this case, the concentration of formamide in
the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to
the probe. Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate stringency" conditions above 25%
formamide and "low stringency" conditions below 25% formamide. A
specific example of "moderate stringency" hybridization conditions
is when the above hybridization is conducted at 30% formamide. A
specific example of "low stringency" hybridization conditions is
when the above hybridization is conducted at 10% formamide.
[0108] As used herein, the term "significant match", when referring
to nucleic acid sequences, means that two nucleic acid sequences
exhibit at least 65% identity, at least 70%, at least 75%, at least
80%, at least 85%, and preferably, at least 90% identity, using
comparison methods well known in the art (i.e., Altschul, S. F. et
al., 1997, Nucl. Acids Res., 25:3389-3402; Schffer, A. A. et al.,
1999, Bioinformatics 15: 1000-1011). As used herein, "significant
match" encompasses non-contiguous or scattered identical
nucleotides so long as the sequences exhibit at least 65%, and
preferably, at least 70%, at least 75%, at least 80%, at least 85%,
and preferably, at least 90% identity, when maximally aligned using
alignment methods routine in the art.
[0109] As used herein, the term "synergistic" refers to a
combination of a compound identified using one of the methods
described herein, and another therapy (e.g., agent), which is more
effective than the additive effects of the therapies. Preferably,
such other therapy has been or is currently being to prevent,
treat, manage or ameliorate schizophrenia and/or bipolar disorder
or a symptom thereof. A synergistic effect of a combination of
therapies (e.g., prophylactic or therapeutic agents) permits the
use of lower dosages of one or more of the therapies and/or less
frequent administration of said therapies to a subject with
schizophrenia or bipolar disorder. The ability to utilize lower
dosages of a therapy (e.g., a prophylactic or therapeutic agent)
and/or to administer said therapy less frequently reduces the
toxicity associated with the administration of said agent to a
subject without reducing the efficacy of said therapies in the
prevention, treatment, management or amelioration of schizophrenia
or bipolar disorder. In addition, a synergistic effect can result
in improved efficacy of therapies (e.g., agents) in the prevention,
treatment, management or amelioration of schizophrenia or bipolar
disorder. Finally, a synergistic effect of a combination of
therapies (e.g., prophylactic or therapeutic agents) may avoid or
reduce adverse or unwanted side effects associated with the use of
either therapy alone.
[0110] As used herein, a "therapeutic agent" or "agent" refers to a
compound that increases or decreases the expression of a
polynucleotide or polypeptide sequences that are differentially
expressed in a tissue or blood sample from an individual having
schizophrenia or bipolar disorder. The invention provides for a
"therapeutic agent" that 1) prevents the onset of episodes of
schizophrenia and/or bipolar disorder; 2) reduces, delays, or
eliminates advancement of episodes or severity of schizophrenia
and/or bipolar disorder and/or 3) restores one or more expression
profiles of one or more disease-indicative nucleic acids or
polypeptides of a patient to a profile more similar to that of a
normal individual when administered to a patient.
[0111] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any compound(s) which can be used in
the treatment, management or amelioration of schizophrenia and/or
bipolar disorder or one or more symptoms and/or episodes thereof.
In a specific emobodiment, the term "therapeutic agent" refers to a
compound that increases or decreases the expression of a
polynucleotide or polypeptide sequence that is differentially
expressed in a brain cell or brain cell line. The invention
provides for a "therapeutic agent" that 1) prevents the onset
schizophrenia and/or bipolar disorder or episodes thereof; 2)
reduces, delays, or eliminates schizophrenia and/or bipolar
disorder symptoms and/or episodes 3) reduces, delays, or eliminates
schizophrenia and/or bipolar disorder progression; and/or 4)
restores one or more expression profiles of one or more
disease-indicative nucleic acids or proteins of a patient to a
profile more similar to that of a normal individual when
administered to a patient. In certain embodiments, the term
"therapeutic agent" refers to a compound identified in the
screening assays described herein. In other embodiments, the term
"therapeutic agent" refers to an agent other than a compound
identified in the screening assays described herein which is known
to be useful for, or has been or is currently being used to treat,
manage or ameliorate schizophrenia and/or bipolar disorder or one
or more symptoms and/or episodes thereof.
[0112] As used herein, the term "therapeutically effective amount"
refers to that amount of a therapy (e.g., a therapeutic agent)
sufficient to result in the amelioration of schizophrenia and/or
bipolar disorder or one or more symptoms and/or episodes thereof,
prevent advancement of schizophrenia and/or bipolar disorder and/or
episodes thereof, cause regression of schizophrenia and/or bipolar
disorder and/or episodes thereof, or to enhance or improve the
therapeutic effect(s) of another therapy (e.g., therapeutic agent).
In a specific embodiment, a therapeutically effective amount refers
to the amount of a therapy (e.g., a therapeutic agent) that
modulates gene expression of the products of the biomarkers of the
inventions. Preferably, a therapeutically effective amount of a
therapy (e.g., a therapeutic agent) modulates gene expression of
the products of the biomarkers of the invention at least 5%,
preferably at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or at least 100% relative to a control therapeutic agent such as
phosphate buffered saline ("PBS").
[0113] As used herein, the terms "treat", "treatment" and
"treating" refer to the reduction or amelioration of the
progression, severity and/or duration of episodes and/or symptoms
of therapeutic agent resulting from the administration of one or
more compounds identified in accordance the methods of the
invention, or a combination of one or more compounds identified in
accordance with the invention and another therapy.
[0114] As used herein, a "tissue nucleic acid sample", refers to
nucleic acids derived from tissue, preferably brain tissue.
Preferably, a tissue nucleic acid sample is total RNA, mRNA or is a
nucleic acid corresponding to RNA, for example, cDNA. A tissue
nucleic acid sample can also include a PCR product derived from
total RNA, mRNA or cDNA.
5.2 SUMMARY
[0115] The practice of the present invention employs in part
conventional techniques of molecular biology, microbiology and
recombinant DNA techniques, which are within the skill of the art.
Such techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Second Edition; Oligonucleotide Synthesis (M. J.
Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Harnes & S.
J. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B.
Perbal, 1984); and a series, Methods in Enzymology (Academic Press,
Inc.); Short Protocols In Molecular Biology, (Ausubel et al., ed.,
1995). All patents, patent applications, and publications mentioned
herein, both supra and infra, are hereby incorporated by reference
in their entireties.
[0116] The invention as disclosed herein identifies biomarkers and
biomarker combinations as well as a method of identifying said
combinations from blood useful in diagnosing schizophrenia and/or
bipolar disorder. In order to use these biomarkers, the invention
teaches the measurement of expression of the RNA and/or the protein
products of these biomarkers. The invention further discloses the
oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA,
synthetic RNA, or other combinations of modified nucleotides that
specifically and/or selectively hybridize to the RNA products of
the biomarkers of the invention allowing measurement of the
expression of the RNA products of the biomarkers. The invention
further discloses proteins, peptides, antibodies, ligands that
specifically or selectively bind to the protein products of the
biomarkers of the invention allowing measurement of the expression
of the protein products of the invention and kits containing these
polypeptides and/or polynucleotides.
[0117] The measuring of the expression of the RNA product of the
biomarkers and combination of biomarkers of the invention, can be
done by using those polynucleotides which are specific and/or
selective for the RNA products of the biomarkers of the invention
to quantitate the expression of the RNA product. In a specific
embodiment of the invention, the polynucleotides which are specific
and/or selective for the RNA products are probes or primers. In one
embodiment, these polynucleotides are in the form of nucleic acid
probes which can be spotted onto an array to measure RNA from the
blood of an individual to be diagnosed. In another embodiment,
commercial arrays can be used to measure the expression of the RNA
product and the invention teaches which combination of genes to
analyze. In yet another embodiment, the polynucleotides which are
specific and/or selective for the RNA products of the biomarkers of
the invention are used in the form of probes and primers in
techniques such as quantitative real-time RT PCR, using for example
SYBR.RTM.Green, or using TaqMan.RTM. or Molecular Beacon
techniques, where the polynucleotides used are used in the form of
a forward primer, a reverse primer, a TaqMan labelled probe or a
Molecular Beacon labelled probe. The invention also teaches, in one
embodiment, a method of identifying useful combinations of
biomarkers by generating classifiers said classifiers able to
differentiate as between (a) schizophrenia and non schizophrenia
(b) bipolar disorder and non bipolar disorder and (c) schizophrenia
and bipolar disorder; or (d) schizophrenia, bipolar disorder and
non schizophrenia or non bipolar disorder using one or more of the
biomarkers disclosed herein. The classifiers generated are
particularly useful, in one embodiment to be used as a means to
diagnosis. Classifiers which are able to differentiate as between
(a) schizophrenia and non schizophrenia (b) bipolar disorder and
non bipolar disorder and (c) schizophrenia and bipolar disorder;
(d) schizophrenia, bipolar disorder and normal are generated by
measuring the level of expression of the RNA products and/or the
protein products of the invention and using the data resulting from
said measurement for input into a mathematical model. Classifiers
can be evaluated to determine the best combinations of biomarkers
of the invention and appropriate weightings to be accorded to said
biomarkers, so as to best classify as between two or more of the
phenotypes schizophrenia, bipolar disorder or normal of a reference
population. Note that it is not necessary that the same method used
to generate the classifier as is used to diagnose the test
individual.
[0118] The invention further contemplates the use of proteins or
polypeptides as disclosed herein and would be known by a person
skilled in the art to measure the protein products of the
biomarkers of the invention. Techniques known to persons skilled in
the art (for example, techniques such as Western Blotting,
Immunoprecipitation, ELISAs, protein microarray analysis and the
like) can then be used to measure the level of protein products
corresponding to the biomarkers of the invention. As would be
understood to a person skilled in the art, the measure of the level
of expression of the protein products of the biomarkers of the
invention requires a protein which specifically and/or selectively
binds to one or more of the protein products corresponding to each
biomarker of the invention. Data representative of the level of
expression of the protein products of the biomarker of the
invention can then be input into the model used to identify the
combination in order to determine a diagnosis as defined by the
model. In a preferred embodiment, the same method is used to
generate the expression data used to generate the mathematical
model as is used to diagnose the test individual.
[0119] The invention further contemplates the use of a combination
of proteins or polypeptides in combination with polynucleotides so
as to measure one or more products of the biomarkers of the
invention.
[0120] The invention further contemplates a composition comprising
a collection of two or more isolated polynucleotides, said
polynucleotides which selectively hybridize to at least two
biomarkers of the invention, wherein the biomarkers are selected
from the group consisting of the genes: adenylosuccinate synthase
(ADSS); apolipoprotein B mRNA editing enzyme, catalytic
polypeptide-like 3B (APOBEC3B); ataxia telangiectasia mutated
(includes complementation groups A, C and D) (ATM); Charcot-Leyden
crystal protein (CLC); C-terminal binding protein 1 (CTBP1);
chemokine (C--X--C motif) ligand 1 (melanoma growth stimulating
activity, alpha) (CXCL1); death associated transcription factor 1
(DATF1); S100 calcium binding protein A9 (calgranulin B) (S100A9),
and as set out in Table 1, and wherein the composition is used to
measure the level of expression of said biomarker.
[0121] The invention further contemplates a composition comprising
a collection of two or more isolated polynucleotides which bind
selectively to the RNA products of at least two biomarkers, wherein
the biomarkers are selected from the group consisting of the genes:
adenylosuccinate synthase (ADSS); apolipoprotein B mRNA editing
enzyme, catalytic polypeptide-like 3B (APOBEC3B); ataxia
telangiectasia mutated (includes complementation groups A, C and D)
(ATM); Charcot-Leyden crystal protein (CLC); C-terminal binding
protein 1 (CTBP1); chemokine (C--X--C motif) ligand 1 (melanoma
growth stimulating activity, alpha) (CXCL1); death associated
transcription factor 1 (DATF1); S100 calcium binding protein A9
(calgranulin B) (S100A9), as set out in Table 1. A further aspect
of this embodiment encompasses polynucleotides are useful in
quantitative RT-PCR (QRT-PCR) of one or two or more of these
biomarkers.
[0122] The invention further contemplates a composition comprising
a collection of two or more isolated proteins which bind
selectively to the protein products of at least two biomarkers,
wherein the biomarkers are selected from the group consisting of
the genes: adenylosuccinate synthase (ADSS) apolipoprotein B mRNA
editing enzyme, catalytic polypeptide-like 3B (APOBEC3B); ataxia
telangiectasia mutated (includes complementation groups A, C and D)
(ATM); Charcot-Leyden crystal protein (CLC); C-terminal binding
protein 1 (CTBP1); chemokine (C--X--C motif) ligand 1 (melanoma
growth stimulating activity, alpha) (CXCL1); death associated
transcription factor 1 (DATF1); S100 calcium binding protein A9
(calgranulin B) (S100A9), as set out in Table 1.
[0123] The invention further contemplates a composition comprising
a collection of two or more isolated polynucleotides which bind
selectively to the RNA products of one or more biomarkers, wherein
the biomarkers are selected from the group consisting of the genes:
adenylosuccinate synthase (ADSS); death associated transcription
factor 1 (DATF1); as listed in Table 3.
[0124] The invention further contemplates a composition comprising
a collection of one or more isolated proteins, which bind
selectively to the protein products of one or more biomarkers,
wherein the biomarkers are selected from the group consisting of
the genes: adenylosuccinate synthase (ADSS); death associated
transcription factor 1 (DATF1); as set out in Table 3.
[0125] The invention further contemplates a composition comprising
a collection of isolated polynucleotides which bind selectively to
the RNA products of biomarkers, wherein the biomarkers are selected
from the group of genes: adenylosuccinate synthase (ADSS); death
associated transcription factor 1 (DATF1); as set out in Table
3.
[0126] The invention further contemplates a composition comprising
a collection of two or more isolated polynucleotides which bind
selectively to the RNA products of at least one biomarker, wherein
the biomarkers are selected from the group as set out in Table
4.
[0127] The invention further contemplates a composition comprising
a collection of two or more isolated polynucleotides which bind
selectively to the RNA products of at least one biomarker, wherein
the biomarkers are selected from the group as set out in Table
5.
[0128] The invention further contemplates a composition comprising
a collection of one or more isolated proteins which bind
selectively to the protein products of at least one biomarker,
wherein the biomarkers are selected from the group as set out in
Table 4.
[0129] The invention further contemplates a composition comprising
a collection of one or more isolated proteins which bind
selectively to the protein products of at least one biomarker,
wherein the biomarkers are selected from the group as set out in
Table 5.
[0130] The invention further contemplates embodiments of any one of
the compositions of the invention, wherein the referenced isolated
proteins of said compositions are ligands, and/or wherein the
ligands are antibodies. In one aspect, these antibodies are
monoclonal antibodies.
[0131] The invention further contemplates a composition comprising
any of the oligonucleotide compositions of the invention, wherein
the isolated oligonucleotides are single or double stranded RNA,
and/or wherein the isolated polynucleotides are single or double
stranded DNA.
[0132] The invention further contemplates a method of diagnosing or
prognosing schizophrenia in an individual, comprising the steps
of:
[0133] a) determining the level of one or more RNA transcripts
expressed in blood obtained from said individual, wherein said one
or more RNA transcripts corresponds to said one or more biomarkers
of Table 3, and
[0134] b) comparing the level of each of said one or more RNA
transcripts in said blood according to step a) with the level of
each of said one or more RNA transcripts in blood from one or more
individuals not having schizophrenia,
[0135] wherein detecting differential expression of each of said
one or more RNA transcripts in the comparison of step b) is
indicative of schizophrenia in the individual of step a).
[0136] The invention further contemplates a method of diagnosing or
prognosing bipolar disorder in an individual, comprising the steps
of:
[0137] a) determining the level of one or more RNA transcripts
expressed in blood obtained from said individual, wherein said one
or more RNA transcripts corresponds to said one or more biomarkers
of Table 4, and
[0138] b) comparing the level of each of said one or more RNA
transcripts in said blood according to step a) with the level of
each of said one or more RNA transcripts in blood from one or more
individuals not having bipolar disorder,
[0139] wherein detecting differential expression of each of said
one or more RNA transcripts in the comparison of step b) is
indicative of bipolar disorder in the individual of step a).
[0140] The invention further contemplates a method of diagnosing or
prognosing and individual as having either bipolar disorder or
schizophrenia, comprising the steps of:
[0141] a) determining the level of one or more RNA transcripts
expressed in blood obtained from said individual, wherein said one
or more RNA transcripts corresponds to said one or more biomarkers
of Table 5, and
[0142] b) comparing the level of each of said one or more RNA
transcripts in said blood according to step a) with the level of
each of said one or more RNA transcripts in blood from one or more
individuals having schizophrenia,
[0143] wherein detecting differential expression of each of said
one or more RNA transcripts in the comparison of step b) is
indicative of bipolar disorder in the individual of step a).
[0144] The invention further contemplates a method of diagnosing or
prognosing and individual as having either bipolar disorder or
schizophrenia, comprising the steps of:
[0145] a) determining the level of one or more RNA transcripts
expressed in blood obtained from said individual, wherein said one
or more RNA transcripts corresponds to said one or more biomarkers
of Table 5, and
[0146] b) comparing the level of each of said one or more RNA
transcripts in said blood according to step a) with the level of
each of said one or more RNA transcripts in blood from one or more
individuals having bipolar disorder,
[0147] wherein detecting differential expression of each of said
one or more RNA transcripts in the comparison of step b) is
indicative of schizophrenia in the individual of step a).
[0148] The invention further contemplates a method of diagnosing or
prognosing schizophrenia in an individual, comprising the steps
of:
[0149] a) determining the level of two or more RNA transcripts
expressed in blood obtained from said individual, wherein said two
or more RNA transcripts corresponds to said one or more biomarkers
of Table 1 and
[0150] b) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals having schizophrenia,
[0151] c) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals not having schizophrenia,
[0152] d) determining whether the level of said two or more RNA
transcripts of step a) classify with the levels of said transcripts
in step b) as compared with levels of said transcripts in step
c),
[0153] wherein said determination is indicative of said individual
of step a) having schizophrenia.
[0154] The invention further contemplates a method of diagnosing or
prognosing bipolar disorder in an individual, comprising the steps
of:
[0155] a) determining the level of two or more RNA transcripts
expressed in blood obtained from said individual, wherein said two
or more RNA transcripts corresponds to said two or more biomarkers
of Table 1 and
[0156] b) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals having bipolar disorder,
[0157] c) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals not having bipolar disorder,
[0158] d) determining whether the level of said two or more RNA
transcripts of step a) classify with the levels of said transcripts
in step b) as compared with levels of said transcripts in step
c),
[0159] wherein said determination is indicative of said individual
of step a) having bipolar disorder.
[0160] The invention further contemplates a method of diagnosing or
prognosing an individual as having bipolar disorder or
schizophrenia, comprising the steps of:
[0161] a) determining the level of two or more RNA transcripts
expressed in blood obtained from said individual, wherein said two
or more RNA transcripts corresponds to said two or more biomarkers
of Table 1 and
[0162] b) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals having bipolar disorder,
[0163] c) comparing the level of each of said two or more RNA
transcripts in said blood according to step a) with the level of
each of said two or more RNA transcripts in blood from one or more
individuals having schizophrenia,
[0164] d) determining whether the level of said two or more RNA
transcripts of step a) classify with the levels of said transcripts
in step b) as compared with levels of said transcripts in step
c),
[0165] wherein said determination is indicative of said individual
of step a) having bipolar disorder.
[0166] The invention further contemplates a method of diagnosing or
prognosing schizophrenia in an individual, comprising the steps
of:
[0167] a) determining the level of one or more RNA transcripts
expressed in blood obtained from said individual, wherein said one
or more RNA transcripts corresponds to said one or more biomarkers
of Table 1 and
[0168] b) using the results from step (a) in combination with a
classifier so as to determine a diagnosis with respect to
schizophrenia.
[0169] The invention further contemplates a method of diagnosing or
prognosing bipolar disorder in an individual, comprising the steps
of:
[0170] a) determining the level of one or more RNA transcripts
expressed in blood obtained from said individual, wherein said one
or more RNA transcripts corresponds to said one or more biomarkers
of Table 1 and
[0171] b) using the results from step (a) in combination with a
classifier so as to determine a diagnosis with respect to bipolar
disorder.
[0172] The invention further contemplates that any of the methods
of the invention comprising a blood sample, that in these methods
said blood sample consists of whole blood, and/or consists of a
drop of blood, and/or consists of blood that has been lysed.
[0173] The invention further contemplates that any of the methods
of the invention comprising a blood sample, that in these methods
there comprises a further step of of isolating RNA from said blood
samples.
[0174] The invention further contemplates the instantly disclosed
methods wherein the referenced step of determining the level of
each of said one or more RNA transcripts comprises quantitative
RT-PCR (QRT-PCR), wherein said one or more transcripts are from
step a) and/or step b) of the above instantly disclosed methods.
The invention further contemplates the instantly disclosed methods
wherein the referenced QRT-PCR utilizes primers which hybridize to
said one or more transcripts or the complement thereof, wherein
said one or more transcripts are from step a) and/or step b) of the
above disclosed methods.
[0175] The invention further contemplates that any of the methods
of the invention which comprises one or more steps of determining
the level of each of said one or more RNA transcripts, comprises
quantitative RT-PCR (QRT-PCR). In one aspect, the said one or more
transcripts are from step a) and/or step b) of the instant methods.
In a further embodiment of these methods, said QRT-PCR utilizes
primers which hybridize to said one or more transcripts or the
complement thereof, wherein said one or more transcripts are from
Tables 1-6.
[0176] The invention further contemplates that any of the methods
of the invention comprising primers, said primers are 15-25
nucleotides in length.
[0177] The invention further contemplates that any of the methods
of the invention comprising one or more steps of determining the
level of each of said one or more RNA transcripts, the step of
determining the level of each of said one or more RNA transcripts
comprises hybridizing a first plurality of isolated nucleic acid
molecules that correspond to said one or more transcripts, to an
array comprising a second plurality of isolated nucleic acid
molecules. In an aspect of these embodied methods, the first
plurality of isolated nucleic acid molecules comprises RNA, DNA,
cDNA, PCR products or ESTs. In an aspect of these embodied methods,
the array comprises a plurality of isolated nucleic acid molecules
comprising RNA, DNA, cDNA, PCR products or ESTs. In an aspect of
these embodied methods, the second plurality of isolated nucleic
acid molecules on said array comprises polynucleotides
corresponding to one or more of the biomarkers of Table 1.
[0178] The invention further contemplates a kit for diagnosing or
prognosing schizophrenia comprising:
[0179] a) at least two sets of biomarker specific priming means
wherein each set of biomarker specific priming means produces
double stranded DNA complementary to a unique biomarker selected
from Table 1; wherein each first priming means of said sets
contains a sequence which can selectively hybridize to RNA, cDNA or
an EST complementary to one of said biomarkers to create an
extension product and each said second priming means of said sets
is capable of selectively hybridizing to said extension
product;
[0180] b) an enzyme with reverse transcriptase activity;
[0181] c) an enzyme with thermostable DNA polymerase activity,
and
[0182] d) a labeling means;
[0183] wherein each of said primer sets is used to detect the
quantitative expression levels of said biomarker in a test
subject.
[0184] The invention further contemplates a kit for diagnosing or
prognosing bipolar disorder comprising:
[0185] a) at least two sets of biomarker specific priming means
wherein each set of biomarker specific priming means produces
double stranded DNA complementary to a unique biomarker selected
from Table 1; wherein each first priming means of said sets
contains a sequence which can selectively hybridize to RNA, cDNA or
an EST complementary to one of said biomarkers to create an
extension product and each said second priming means of said sets
is capable of selectively hybridizing to said extension
product;
[0186] b) an enzyme with reverse transcriptase activity;
[0187] c) an enzyme with thermostable DNA polymerase activity,
and
[0188] d) a labeling means;
[0189] wherein each said primer set is used to detect the
quantitative expression levels of a biomarker in a test
subject.
[0190] The invention further contemplates a method of diagnosing or
prognosing schizophrenia in an individual, comprising the steps
of:
[0191] a) determining the level of two or more proteins expressed
in blood obtained from said individual, wherein said two or more
proteins are encoded by two or more biomarkers of Table 1, and
[0192] b) comparing the level of each of said two or more proteins
in said blood according to step a) with the level of each of said
two or more proteins in blood from one or more individuals having
schizophrenia,
[0193] c) comparing the level of each of said two or more proteins
in said blood according to step a) with the level of each of said
two or more proteins in blood from one or more individuals not
having schizophrenia,
[0194] d) determining whether the level of said two or more
proteins of step a) classify with the levels of said proteins in
step b) as compared with levels of said proteins in step c),
[0195] wherein said determination is indicative of said individual
of step a) having schizophrenia.
[0196] The invention further contemplates a method of diagnosing or
prognosing bipolar disorder in an individual, comprising the steps
of:
[0197] a) determining the level of two or more proteins expressed
in blood obtained from said individual, wherein said two or more
proteins are encoded by two or more biomarkers of Table 1, and
[0198] b) comparing the level of each of said two or more proteins
in said blood according to step a) with the level of each of said
two or more proteins in blood from one or more individuals having
bipolar disorder
[0199] c) comparing the level of each of said two or more proteins
in said blood according to step a) with the level of each of said
two or more proteins in blood from one or more individuals not
having bipolar disorder,
[0200] d) determining whether the level of said two or more
proteins of step a) classify with the levels of said proteins in
step b) as compared with levels of said proteins in step c),
[0201] wherein said determination is indicative of said individual
of step a) having bipolar disorder.
[0202] The invention further contemplates a method of diagnosing or
prognosing an individual with bipolar disorder as compared with
schizophrenia, comprising the steps of:
[0203] a) determining the level of two or more proteins expressed
in blood obtained from said individual, wherein said two or more
proteins are encoded by two or more biomarkers of Table 1, and
[0204] b) comparing the level of each of said two or more proteins
in said blood according to step a) with the level of each of said
two or more proteins in blood from one or more individuals having
bipolar disorder
[0205] c) comparing the level of each of said two or more proteins
in said blood according to step a) with the level of each of said
two or more proteins in blood from one or more individuals having
schizophrenia,
[0206] d) determining whether the level of said two or more
proteins of step a) classify with the levels of said proteins in
step b) as compared with levels of said proteins in step c),
[0207] wherein said determination is indicative of said individual
of step a) having bipolar disorder.
[0208] The invention further contemplates a method of diagnosing or
prognosing schizophrenia in an individual, comprising the steps
of:
[0209] a) determining the level of two or more protein products
expressed in blood obtained from said individual, wherein said two
or more protein products corresponds to two or more biomarkers of
Table 1 and
[0210] b) using the results from step (a) in combination with a
classifier designed to differentiate schizophrenia from non
schizophrenia so as to determine a diagnosis with respect to
schizophrenia.
[0211] The invention further contemplates a method of diagnosing or
prognosing bipolar disorder in an individual, comprising the steps
of:
[0212] a) determining the level of two or more protein products
expressed in blood obtained from said individual, wherein said two
or more protein products corresponds to two or more biomarkers of
Table 1 and
[0213] b) using the results from step (a) in combination with a
classifier designed to differentiate bipolar disorder from non
bipolar disorder so as to determine a diagnosis with respect to
bipolar disorder.
[0214] The invention further contemplates a method of diagnosing or
prognosing and individual as having bipolar disorder or
schizophrenial, comprising the steps of:
[0215] a) determining the level of two or more protein products
expressed in blood obtained from said individual, wherein said two
or more protein products corresponds to two or more biomarkers of
Table 1 and
[0216] b) using the results from step (a) in combination with a
classifier designed to differentiate bipolar disorder from
schizophrenia so as to determine a diagnosis with respect to
bipolar disorder or schizophrenia.
[0217] The invention further contemplates that in any method of the
invention which comprises one or more steps of determining the
level of each of said one or more proteins, that the step of
determining the level of each of said one or more proteins
comprises the use of two or more antibodies, wherein each of said
two or more antibodies is specific for a protein product of a
biomarker listed in Table 1. In an aspect of these methods, it is
contemplated that the one or more antibodies is selected from the
group consisting of a monoclonal antibody, fv. scfv, dab, fd, fab,
and fab'2.
[0218] The invention further contemplates a method of developing a
classifier useful for diagnosing schizophrenia, said method
comprising:
[0219] (a) measuring the level of expression of the products of the
biomarkers identified in Table 1 in a training population wherein
said training population is comprised of two subgroups, a first
subgroup diagnosed as having schizophrenia and said second subgroup
diagnosed as not having schizophrenia.
[0220] (b) apply one or more mathematical models to the levels of
expression of step (a) to develop one or more classifiers which
differentiate between said first subgroup and said second subgroup.
In one aspect of this invention, this method further comprises the
step of evaluating one or more of said classifiers of step (b) for
the classifier's ability to properly characterize each individual
of the training population. In one aspect of this invention, this
method further comprises the step of evaluating one or more of said
classifiers of step (b) for the classifier's ability to properly
characterize one or more individuals of a population which is not
the training population. In one aspect of this invention, this
method further comprises the step of evaluating one or more of said
classifiers of step (b) for the classifier's ability to properly
characterize one or more individuals of a population which is not
the training population.
[0221] The invention further contemplates a method of developing a
classifier useful for diagnosing bipolar disorder, said method
comprising:
[0222] (a) measuring the level of expression of the products of the
biomarkers identified in Table 1 in a training population wherein
said training population is comprised of two subgroups, a first
subgroup diagnosed as having bipolar disorder and said second
subgroup diagnosed as not having bipolar disorder.
[0223] (b) apply one or more mathematical models to the levels of
expression of step (a) to develop one or more classifiers which
differentiate between said first subgroup and said second subgroup.
In one aspect of this invention, this method further comprises the
step of evaluating one or more of said classifiers of step (b) for
the classifier's ability to properly characterize each individual
of the training population. In one aspect of this invention, this
method further comprises the step of evaluating one or more of said
classifiers of step (b) for the classifier's ability to properly
characterize one or more individuals of a population which is not
the training population. In one aspect of this invention, this
method further comprises the step of evaluating one or more of said
classifiers of step (b) for the classifier's ability to properly
characterize one or more individuals of a population which is not
the training population.
[0224] The invention further contemplates a method of developing a
classifier useful for diagnosing bipolar disorder or schizophrenia,
said method comprising:
[0225] (a) measuring the level of expression of the products of the
biomarkers identified in Table 1 in a training population wherein
said training population is comprised of two subgroups, a first
subgroup diagnosed as having bipolar disorder and said second
subgroup diagnosed as having schizophrenia.
[0226] (b) apply one or more mathematical models to the levels of
expression of step (a) to develop one or more classifiers which
differentiate between said first subgroup and said second subgroup.
In one aspect of this invention, this method further comprises the
step of evaluating one or more of said classifiers of step (b) for
the classifier's ability to properly characterize each individual
of the training population. In one aspect of this invention, this
method further comprises the step of evaluating one or more of said
classifiers of step (b) for the classifier's ability to properly
characterize one or more individuals of a population which is not
the training population. In one aspect of this invention, this
method further comprises the step of evaluating one or more of said
classifiers of step (b) for the classifier's ability to properly
characterize one or more individuals of a population which is not
the training population.
[0227] The invention further contemplates the instantly disclosed
methods which further comprises the step of evaluating one or more
of said classifiers of step (b) for the classifier's ability to
properly characterize one or more individuals of a population which
is not the training population.
5.3 Samples for use in the Invention
[0228] Unless otherwise indicated herein, blood samples obtained
from any subject may be used in accordance with the methods of the
invention. Examples of subjects from which such a sample may be
obtained and utilized in accordance with the methods of the
invention include, but are not limited to, asymptomatic subjects,
subjects manifesting or exhibiting 1, 2, 3, 4 or more symptoms of
schizophrenia and/or bipolar disorder, subjects clinically
diagnosed as having schizophrenia and/or bipolar disorder, subjects
predisposed to schizophrenia and/or bipolar disorder (e.g.,
subjects with a family history of schizophrenia and/or bipolar
disorder, subjects with a genetic predisposition to schizophrenia
and/or bipolar disorder, subjects suspected of having schizophrenia
and/or bipolar disorder, subjects undergoing therapy for
schizophrenia and/or bipolar disorder, subjects with schizophrenia
and/or bipolar disorder and at least one other condition (e.g.,
subjects with 2, 3, 4, 5 or more conditions), subjects not
undergoing treatment for schizophrenia and/or bipolar disorder,
subjects determined by a medical practitioner (e.g., a physician)
to be healthy or schizophrenia or bipolar disorder-free (i.e.,
normal), subjects that have been cured of schizophrenia and/or
bipolar disorder, subjects that are managing their schizophrenia
and/or bipolar disorder, and subjects that have not been diagnosed
with schizophrenia and/or bipolar disorder.
[0229] In a further embodiment, the subject from which a sample may
be obtained is a test individual wherein it is unknown whether the
person has schizophrenia or bipolar disorder, and/or it is unknown
what degree of schizophrenia or bipolar disorder the test
individual might have, if any.
[0230] 5.3.1. Blood
[0231] In one aspect of the invention, a sample of blood is
obtained from a subject according to methods well known in the art.
A sample of blood may be obtained from a subject, for example a
subject having schizophrenia, having bipolar disorder or not having
schizophrenia or bipolar disorder. In some embodiments, a drop of
blood is collected from a simple pin prick made in the skin of a
subject. Blood may be drawn from a subject from any part of the
body (e.g., a finger, a hand, a wrist, an arm, a leg, a foot, an
ankle, a stomach, and a neck) using techniques known to one of
skill in the art, in particular methods of phlebotomy known in the
art.
[0232] The amount of blood collected will vary depending upon the
site of collection, the amount required for a method of the
invention, and the comfort of the subject. However, an advantage of
one embodiment of the present invention is that the amount of blood
required to implement the methods of the present invention can be
so small that more invasive procedures are not required to obtain
the sample. For example, in some embodiments, all that is required
is a drop of blood. This drop of blood can be obtained, for
example, from a simple pinprick. In some embodiments, any amount of
blood is collected that is sufficient to detect the expression of
one, two, three, four, five, six, seven or all of the genes in
Table 1. As such, in some embodiments, the amount of blood that is
collected is 1 .mu.l or less, 0.5 .mu.l or less, 0.1 .mu.l or less,
or 0.01 .mu.l or less. However, the present invention is not
limited to such embodiments. In some embodiments more blood is
available and in some embodiments, more blood can be used to effect
the methods of the present invention. As such, in various specific
embodiments, 0.001 ml, 0.005 ml, 0.01 ml, 0.05 ml, 0.1 ml, 0.15 ml,
0.2 ml, 0.25 ml, 0.5 ml, 0.75 ml, 1 ml, 1.5 ml, 2 ml, 3 ml, 4 ml, 5
ml, 10 ml, 15 ml or more of blood is collected from a subject. In
another embodiment, 0.001 ml to 15 ml, 0.01 ml to 10 ml, 0.1 ml to
10 ml, 0.1 ml to 5 ml, 1 to 5 ml of blood is collected from a
subject.
[0233] In some embodiments of the present invention, blood is
stored within a K3/EDTA tube. In another embodiment, one can
utilize tubes for storing blood which contain stabilizing agents
such as disclosed in U.S. Pat. No. 6,617,170 (which is incorporated
herein by reference). In another embodiment the PAXgene.TM. blood
RNA system:provided by PreAnalytiX, a Qiagen/BD company may be used
to collect blood. In yet another embodiment, the Tempus.TM. blood
RNA collection tubes, offered by Applied Biosystems may be used.
Tempus.TM. collection tubes provide a closed evacuated plastic tube
containing RNA stabilizing reagent for whole blood collection.
[0234] The blood collected is preferably utilized immediately or
within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours or is
optionally stored at temperatures such as 4.degree. C., or at
-20.degree. C. prior to use in accordance with the methods of the
invention. In some embodiments, a portion of the blood sample is
used in accordance with the invention at a first instance of time
whereas one or more remaining portions of the blood sample (or
fractions thereof) are stored for a period of time for later use.
For longer term storage, storage methods well known in the art,
such as storage at cryo temperatures (e.g. below -60.degree. C.)
can be used. In some embodiments, in addition to storage of the
blood or instead of storage of the blood, plasma, serum, isolated
nucleic acid or proteins are stored for a period of time for later
use in accordance with methods known in the art.
[0235] In one aspect, whole blood is obtained from an individual
according to the methods of phlebotomy well known in the art. Whole
blood includes blood which can be used directly, and includes blood
wherein the serum or plasma has been removed and the RNA or mRNA
from the remaining blood sample has been isolated in accordance
with methods well known in the art (e.g., using, preferably, gentle
centrifugation at 300 to 800.times.g for 5 to 10 minutes). In a
specific embodiment, whole blood (i.e., unfractionated blood)
obtained from a subject is mixed with lysing buffer (e.g., Lysis
Buffer (IL): 0.6 g EDTA; 1.0 g KHCO.sub.2, 8.2 g NH.sub.4Cl
adjusted to pH 7.4 (using NaOH)), the sample is centrifuged and the
cell pellet retained, and RNA or mRNA extracted in accordance with
methods known in the art ("lysed blood") (see for example Sambrook
et al.). The use of unfractionated whole blood is preferred since
it avoids the costly and time-consuming process to separate out the
cell types within the blood (Kimoto, 1998, Mol. Gen. Genet
258:233-239; Chelly J et al., 1989, Proc. Nat. Acad. Sci. USA
86:2617-2621; Chelly J et al., 1988, Nature 333:858-860).
[0236] In some embodiments of the present invention, whole blood
collected from a subject is fractionated (i.e., separated into
components). In specific embodiments of the present invention,
blood cells are separated from whole blood collected from a subject
using techniques known in the art. For example, blood collected
from a subject can be subjected to Ficoll-Hypaque (Pharmacia)
gradient centrifugation. Such centrifugation separates erythrocytes
(red blood cells) from various types of nucleated cells and from
plasma. In particular, Ficoll-Hypaque gradient centrifugation is
useful to isolate peripheral blood leukocytes (PBLs) which can be
used in accordance with the methods of the invention.
[0237] By way of example but not limitation, macrophages can be
obtained as follows. Mononuclear cells are isolated from peripheral
blood of a subject, by syringe removal of blood followed by
Ficoll-Hypaque gradient centrifugation. Tissue culture dishes are
pre-coated with the subject's own serum or with AB+ human serum and
incubated at 37.degree. C. for one hour. Non-adherent cells are
removed by pipetting. Cold (4.degree. C.) 1 MM EDTA in
phosphate-buffered saline is added to the adherent cells left in
the dish and the dishes are left at room temperature for fifteen
minutes. The cells are harvested, washed with RPMI buffer and
suspended in RPMI buffer. Increased numbers of macrophages can be
obtained by incubating at 37.degree. C. with macrophage-colony
stimulating factor (M-CSF). Antibodies against macrophage specific
surface markers, such as Mac-1, can be labeled by conjugation of an
affinity compound to such molecules to facilitate detection and
separation of macrophages. Affinity compounds that can be used
include but are not limited to biotin, photobiotin, fluorescein
isothiocyante (FITC), or phycoerythrin (PE), or other compounds
known in the art. Cells retaining labeled antibodies are then
separated from cells that do not bind such antibodies by techniques
known in the art such as, but not limited to, various cell sorting
methods, affinity chromatography, and panning.
[0238] Blood cells can be sorted using a using a fluorescence
activated cell sorter (FACS). Fluorescence activated cell sorting
(FACS) is a known method for separating particles, including cells,
based on the fluorescent properties of the particles. See, for
example, Kamarch, 1987, Methods Enzymol 151:150-165. Laser
excitation of fluorescent moieties in the individual particles
results in a small electrical charge allowing electromagnetic
separation of positive and negative particles from a mixture. An
antibody or ligand used to detect a blood cell antigenic
determinant present on the cell surface of particular blood cells
is labeled with a fluorochrome, such as FITC or phycoerythrin. The
cells are incubated with the fluorescently labeled antibody or
ligand for a time period sufficient to allow the labeled antibody
or ligand to bind to cells. The cells are processed through the
cell sorter, allowing separation of the cells of interest from
other cells. FACS sorted particles can be directly deposited into
individual wells of microtiter plates to facilitate separation.
[0239] Magnetic beads can be also used to separate blood cells in
some embodiments of the present invention. For example, blood cells
can be sorted using a using a magnetic activated cell sorting
(MACS) technique, a method for separating particles based on their
ability to bind magnetic beads (0.5-100 m diameter). A variety of
useful modifications can be performed on the magnetic microspheres,
including covalent addition of an antibody which specifically
recognizes a cell-solid phase surface molecule or hapten. A
magnetic field is then applied, to physically manipulate the
selected beads. In a specific embodiment, antibodies to a blood
cell surface marker are coupled to magnetic beads. The beads are
then mixed with the blood cell culture to allow binding. Cells are
then passed through a magnetic field to separate out cells having
the blood cell surface markers of interest. These cells can then be
isolated.
[0240] In some embodiments, the surface of a culture dish may be
coated with antibodies, and used to separate blood cells by a
method called panning. Separate dishes can be coated with antibody
specific to particular blood cells. Cells can be added first to a
dish coated with blood cell specific antibodies of interest. After
thorough rinsing, the cells left bound to the dish will be cells
that express the blood cell markers of interest. Examples of cell
surface antigenic determinants or markers include, but are not
limited to, CD2 for T lymphocytes and natural killer cells, CD3 for
T lymphocytes, CD11a for leukocytes, CD28 for T lymphocytes, CD19
for B lymphocytes, CD20 for B lymphocytes, CD21 for B lymphocytes,
CD22 for B lymphocytes, CD23 for B lymphocytes, CD29 for
leukocytes, CD14 for monocytes, CD41 for platelets, CD61 for
platelets, CD66 for granulocytes, CD67 for granulocytes and CD68
for monocytes and macrophages.
[0241] Whole blood can be separated into cell types such as
leukocytes, platelets, erythrocytes, etc. and such cell types can
be used in accordance with the methods of the invention. Leukocytes
can be further separated into granulocytes and agranulocytes using
standard techniques and such cells can be used in accordance with
the methods of the invention. Granulocytes can be separated into
cell types such as neutrophils, eosinophils, and basophils using
standard techniques and such cells can be used in accordance with
the methods of the invention. Agranulocytes can be separated into
lymphocytes (e.g., T lymphocytes and B lymphocytes) and monocytes
using standard techniques and such cells can be used in accordance
with the methods of the invention. T lymphocytes can be separated
from B lymphocytes and helper T cells separated from cytotoxic T
cells using standard techniques and such cells can be used in
accordance with the methods of the invention. Separated blood cells
(e.g., leukocytes) can be frozen by standard techniques prior to
use in the present methods.
[0242] A blood sample that is useful according to the invention is
in an amount that is sufficient for the detection of one or more
nucleic acid or amino acid sequences according to the invention. In
a specific embodiment, a blood sample useful according to the
invention is in an amount ranging from 1 .mu.l to 100 ml,
preferably 10 .mu.l to 50 ml, more preferably 10 .mu.l to 25 ml and
most preferably 10 .mu.l to 1 ml.
5.4 RNA Preparation
[0243] In one aspect of the invention, RNA is isolated from an
individual in order to measure the RNA products of the biomarkers
of the invention. RNA is isolated from blood samples from
individuals diagnosed with schizophrenia or diagnoised with bipolar
disorder, individuals not having schizophrenia or bipolar disorder,
or test individuals.
[0244] Preferably, RNA is isolated from blood by the following
protocol. Lysis Buffer is added to blood sample in a ratio of 3
parts Lysis Buffer to 1 part blood (Lysis Buffer (IL) 0.6 g EDTA;
1.0 g KHCO.sub.2, 8.2 g NH.sub.4Cl adjusted to pH 7.4 (using
NaOH)). Sample is mixed and placed on ice for 5-10 minutes until
transparent. Lysed sample is centrifuged at 1000 rpm for 10 minutes
at 4.degree. C., and supernatant is aspirated. Pellet is
resuspended in 5 ml Lysis Buffer, and centrifuged again at 1000 rpm
for 10 minutes at 4.degree. C. Pelleted cells are homogenized using
TRIzol.RTM. (GIBCO/BRL) in a ratio of approximately 6 ml of
TRIzol.RTM. for every 10 ml of the original blood sample and
vortexed well. Samples are left for 5 minutes at room temperature.
RNA is extracted using 1.2 ml of chloroform per 1 ml of
TRIzol.RTM.. Sample is centrifuged at 12,000.times.g for 5 minutes
at 4.degree. C. and upper layer is collected. To upper layer,
isopropanol is added in ratio of 0.5 ml per 1 ml of TRIzol.RTM..
Sample is left overnight at -20.degree. C. or for one hour at
-20.degree. C. RNA is pelleted in accordance with known methods,
RNA pellet air dried, and pellet resuspended in DEPC treated
ddH.sub.2O. RNA samples can also be stored in 75% ethanol where the
samples are stable at room temperature for transportation.
[0245] Purity and integrity of RNA can be assessed by absorbance at
260/280 nm and agarose gel electrophoresis followed by inspection
under ultraviolet light. Preferably RNA integrity is assessed using
more sensitive techniques such as the Agilent 2100 Bioanalyzer 6000
RNA Nano Chip.
5.5 Biomarkers of the Invention
[0246] In one aspect, the invention provides biomarkers and
biomarker combinations wherein the measure of the level of
expression of the product or products of said biomarkers is
indicative of the existence of schizophrenia and/or bipolar
disorder.
[0247] Table 1 provides a list of the gene names and the associated
locus link ID for the biomarkers of the invention wherein the
measure of the level of expression of the biomarkers, in
combination can be used to diagnose an individual as having
schizophrenia and/or bipolar disorder and/or differentiate between
schizophrenia and bipolar disorder.
1TABLE 1 Alternative As- Locus Gene signed Link Rta_Transcript A
Symbols HGNC_Symbol Label ID notation ADSS ADS 159 adenylosuccinate
synthase APOBEC3B APO 9582 apolipoprotein B mRNA editing enzyme,
catalytic polypeptide- like 3B ATM ATM 472 ataxia telangiectasia
mutated (includes complementation groups A, C and D) CLC CLC 1178
Charcot-Leyden crystal protein CTBP1 CTB 1487 C-terminal binding
protein 1 CXCL1 CXC 2919 chemokine (C-X-C motif) ligand 1 (melanoma
growth stimulating activity, alpha) DATF1 DAT 11083 death
associated transcription factor 1 S100A9 S10 6280 S100 calcium
binding protein A9 (calgranulin B)
[0248] The genes of Table 1 are identified on the basis of their
name, Hugo Gene name and locus link ID.
[0249] As would be understood by a person skilled in the art, the
locus link ID can be used to determine the sequence of all the RNA
transcripts and all of the proteins which correspond to the
biomarkers of the invention.
[0250] Table 2 provides, in one embodiment of the invention, is a
selection of the sequences of the RNA products corresponding to the
biomarkers of the invention as disclosed in Table 1 and whose
sequences can be used to measure the level of expression of the
biomarkers of the invention using those techniques known to a
person skilled in the art. Table 2 also provides, in one embodiment
of the invention, sequences of the proteins corresponding to the
biomarkers of the invention which can be measured to determine the
level of expression of the biomarkers of the invention.
2TABLE 2 Ref Ref Sequence Sequence HGNC.sub.-- Gene Protein Locus
Gene Alternative Accession Accession Link Rta_Transcript A Symbols
Symbol Number Number ID notation ADSS NM_001126 NP_001117 159
adenylosuccinate synthase APOBEC3B NM_004900 NP_004891 9582
apolipoprotein B mRNA editing enzyme, catalytic polypeptide- like
3B ATM NM_000051 NP_000042 472 ataxia telangiectasia mutated
(includes complementation groups A, C and D) ATM NM_138292
NP_612149 472 ataxia telangiectasia mutated (includes
complementation groups A, C and D) transcript variant 2, mRNA. ATM
NM_138293 NP_612150 472 ataxia telangiectasia mutated protein
isoform 3 CLC NM_001828 NP_001819 1178 Charcot-Leyden crystal
protein CTBP1 NM_001328 NP_001319 1487 C-terminal binding protein 1
CXCL1 NM_001511 NP_001502 2919 Chemokine (C-X-C motif) ligand 1
(melanoma growth stimulating activity, alpha) DATF1 NM_022105
NP_071388 11083 death associated transcription factor 1 isoform a
DATF1 NM_080796 NP_542986 11083 death associated transcription
factor 1, isoform a DATF1 NM_080797 NP_542987 11083 death
associated transcription factor 1 isoform b S100A9 NM_002965
NP_002956 6280 S100 calcium binding protein A9 (calgranulin B)
[0251] Table 3
[0252] Table 3 provides a list of the gene names and the associated
locus link ID for the biomarkers of the invention which have been
newly identified as a biomarker which individually differentiates
as between schizophrenia and non schizophrenia individuals. Genes
are identified on the basis of their name, Hugo Gene name and locus
link ID. Genes have been selected which demonstrate a p value of
<0.2
3TABLE 3 Alternative Locus Gene Assigned Link Rta_Transcript A
Symbols HGNC_Symbol Label ID notation ADSS ADS 159 adenylosuccinate
synthase DATF1 DAT 11083 death associated transcription factor
1
[0253] Table 4 provides a list of the gene names and the associated
locus link ID for the biomarkers of the invention which have been
newly identified as a biomarker which individually can
differentiate as between individuals with bipolar disorder or not
having bipolar disorder. Genes have been selected which demonstrate
differential expression with a p value of <0.2. Table 4
identifies the genes on the basis of their name, Hugo Gene name and
locus link ID.
4TABLE 4 Alternative As- Locus Gene signed Link Rta_Transcript A
Symbols HGNC_Symbol Label ID notation ADSS ADS 159 adenylosuccinate
synthase APOBEC3B APO 9582 apolipoprotein B mRNA editing enzyme,
catalytic polypeptide-like 3B CXCL1 CXC 2919 chemokine (C-X-C
motif) ligand 1 (melanoma growth stimulating activity, alpha)
[0254] Table 5 provides a list of the gene names and the associated
locus link ID for the biomarkers of the invention which have been
newly identified as a biomarker which individually differentiates
as between individuals with bipolar disorder or individuals having
schizophrenia. Table 5 identifies the genes on the basis of their
name, Hugo Gene name and locus link ID.
5TABLE 5 Alternative Locus Gene Assigned Link Rta_Transcript A
Symbols HGNC_Symbol Label ID notation ADSS ADS 159 adenylosuccinate
synthase DATF1 DAT 11083 death associated transcription factor
1
[0255] The invention thus encompasses the use of those methods
known to a person skilled in the art to measure the expression of
these biomarkers and combinations of biomarkers for each of the
purposes outlined above.
5.6 Combinations of Biomarkers
[0256] In one embodiment, combinations of biomarkers of the present
invention includes any combination of 2, 3, 4, 5, 6, 7, or all 8,
of the biomarkers listed in Table 1. For instance, the number of
possible combinations of a subset m of n genes in any of the Tables
above is described in Feller, Intro to Probability Theory, Third
Edition, volume 1, 1968, ed. J. Wiley, using the general
formula:
m!/(n)! (m-n)!
[0257] In one embodiment of the invention, where n is 2 and m is 8,
the number of combinations of markers selected from Table 1 is: 1 8
! 2 ! ( 8 - 2 ) ! = 8 .times. 7 .times. 6 .times. 5 .times. 4
.times. 3 .times. 2 .times. 1 ( 2 .times. 1 ) ( 6 .times. 5 .times.
4 .times. 3 .times. 2 .times. 1 ) = 40320 / 1440 = 28
[0258] unique two-gene combinations. The measurement of the gene
expression of each of these two-gene combinations can independently
be used to determine whether a patient has schizophrenia. In
another specific embodiment in which m is 8 and n is three, there
are 8!/3!(8-3)! unique three-gene combinations. Each of these
unique three-gene combinations can independently serve as a model
for determining whether a patient has schizophrenia and/or bipolar
disorder.
5.7 Identifying Species of Useful Combinations of Biomarkers by
Generating Classifiers
[0259] The invention further provides a means of selecting those
combinations of biomarkers from Table 1 particularly useful for
each of the following (a) differentiating between schizophrenia and
non schizophrenia individuals (b) differentiating between bipolar
disorder and non bipolar disorder individuals (c) differentiating
between schizophrenia individuals and individuals with bipolar
disorder. The invention further provides a method of evaluating the
combinations identified for each of the utilities described above
and using the classifiers identified to diagnose an individual as
having schizophrenia or having bipolar disorder.
[0260] In order to identify useful combinations of biomarkers and
generate classifiers, a mathematical model of the invention is used
to test the possible combinations of the biomarkers of the
invention for each combination's ability to separate as between the
two (e.g. binary models such as logistic regression) or more (e.g.
models such as neural networks) phenotypic traits of a training
population used for input into the model. The phenotypic traits of
the training population used for input into the model are
phenotypic traits for use in (a) differentiating between
schizophrenia and non schizophrenia individuals (b) differentiating
between bipolar disorder and non bipolar disorder individuals (c)
differentiating between schizophrenia individuals and individuals
with bipolar disorder or (d) differentiating between schizophrenia,
bipolar disorder and individuals having neither schizophrenia or
bipolar disorder. The phenotypic traits of the training population
used for input into the mathematical model, and the model used,
will determine the utility of the combinations generated by the
model for use as a means of diagnosing schizophrenia and/or bipolar
disorder. The result of the choice of phenotypic traits of the
training population used for entry into the mathematical model is
described more thoroughly below.
[0261] The mathematical model generated can be subsequently
evaluated by determining the ability of the model to correctly call
each individual for one of the two (or more) phenotypic traits of
the population used for input into the model. In a preferred
embodiment, the individuals of the training population used to
derive the model are different from the individuals of the training
population used to test the model. As would be understood by a
person skilled in the art, this allows one to predict the ability
of the combinations as to their ability to properly characterize an
individual whose phenotypic characterization is unknown.
[0262] The data which is input into the mathematical model can be
any data which is representative of the expression level of the
product of the biomarkers being evaluated. Mathematical models
useful in accordance with the invention include those using both
supervised or unsupervised learning. In a preferred embodiment of
the invention, the mathematical model chosen uses supervised
learning in conjunction with a "training population" to evaluate
each of the possible combination of biomarkers of the invention. In
one embodiment of the invention, the mathematical model used is
selected from the following: a regression model, a logistic
regression model, a neural network, a clustering model, principal
component analysis, nearest neighbour classifier analysis, linear
discriminant analysis, quadratic discriminant analysis, a support
vector machine, a decision tree, a genetic algorithm, classifier
optimization using bagging, classifier optimization using boosting,
classifier optimization using the Random Subspace Method, a
projection pursuit, genetic programming and weighted voting. In a
preferred embodiment, a logistic regression model is used. In
another preferred embodiment, a neural network model is used.
[0263] The results of applying a mathematical model of the
invention to the data will generate one or more classifiers using
one or more biomarkers. Classifiers generated can be used to
diagnosis an unknown or test individual. As would be understood by
a person skilled in the art, the use of the classifier depends upon
the phenotypes of the population used to generate the classifier
and the mathematical model used. In one embodiment, the diagnosis
result from equations generated by logistic regression to answer
one of the following questions: (a)-does an individual have
schizophrenia, (b) does an individual have bipolar disorder or (c)
is an individual "normal". In yet another embodiment of the
invention, the answer to any of the questions above may be an
answer of non determinable.
[0264] In one preferred embodiment of the invention, each
classifier is evaluated for its ability to properly characterize
each individual of the training population using those methods
known to a person skilled in the art. For example one can evaluate
the classifier using cross validation, Leave One out Cross
Validation (LOOCV), n-fold cross validation, jackknife analysis
using standard statistical methods as disclosed. In an even more
preferred embodiment of the invention, each classifier is evaluated
for its ability to properly characterize those individuals of the
training population which were not used to generate the
classifier.
[0265] In one embodiment, the method used to evaluate the
classifier for its ability to properly characterize each individual
of the training population is a method which evaluates the models
sensitivity (TPF, true positive fraction) and 1-specificity (TNF,
true negative fraction). In a preferred embodiment, the method used
to test the model is Receiver Operating Characteristic ("ROC")
which provides several parameters to evaluate both the sensitivity
and specificity of the diagnostic result of the equation generated.
In a particularly preferred embodiment, the ROC area (area under
the curve) is used to evaluate the equations. A ROC area greater
than 0.5, 0.6, 0.7, 0.8, 0.9 is preferred. A perfect ROC area score
of 1.0 indicates with both 100% sensitivity and 100%
specificity.
[0266] As would be understood by a person skilled in the art, the
utility of the combinations and equations determined by a
mathematical model will depend upon the phenotypes of the
populations used to generate the data for input into the model.
Examples of specific embodiments are described more thoroughly
herein.
5.8 Populations for Input into the Mathematical Models
[0267] Populations used for input should be chosen so as to result
in statistically significant resulting biomarker combinations. In
some embodiments, the reference or training population includes
between 10 and 30 subjects. In another embodiment the reference
population contains between 30-50 subjects. In still other
embodiments, the reference population includes two or more
populations each containing between 50 and 100, 100 and 500,
between 500 and 1000, or more than 1000 subjects. The reference
population includes two or more subpopulations. In a preferred
embodiment, the phenotypic characteristics of the subpopulations
are similar but for the diagnosis with respect to schizophrenia
and/or bipolar disorder, for example the subpopulations are of a
similar age and sex. It is also preferred that the subpopulations
are of roughly equivalent numbers.
[0268] For example, for populations for input into a binary
mathematical model to identify those biomarkers which are useful in
diagnosing an individual as having schizophrenia, or not having
schizophrenia, the reference population is comprised of individuals
having schizophrenia (the first subpopulation), and individuals not
have schizophrenia (the second subpopulation). For purposes of
characterizing the subpopulations as having or not having
schizophrenia, any verified method can be used. Preferably only
those individuals whose diagnoses are certain are utilized as part
of the reference population.
[0269] In another embodiment, populations for input into a binary
mathematical model to identify those biomarkers which are useful in
diagnosing an individual as having bipolar disorder, or not having
bipolar disorder are used. The reference population is comprised of
individuals having bipolar disorder (the first subpopulation), and
individuals not have bipolar disorder (the second subpopulation).
For purposes of characterizing the subpopulations as having or not
having bipolar disorder, any verified method can be used.
Preferably only those individuals whose diagnoses are certain are
utilized as part of the reference population.
[0270] In another embodiment, populations for input into a binary
mathematical model to identify those biomarkers which are useful in
diagnosing an individual as having either bipolar disorder, or
having schizophrenia are used. The reference population is
comprised of individuals having bipolar disorder (the first
subpopulation), and individuals having schizophrenia (the second
subpopulation). For purposes of characterizing the subpopulations
having schizophrenia or bipolar disorder, any verified method can
be used. Preferably only those individuals whose diagnoses are
certain are utilized as part of the reference population.
[0271] In another embodiment, populations for input into a
non-binary mathematical model is used to identify those biomarkers
which are useful in diagnosing an individual as having either
bipolar disorder, or having schizophrenia. The reference population
is comprised of individuals having bipolar disorder (the first
subpopulation), individuals having schizophrenia (the second
subpopulation) and individuals having neither (the third
subpopulation). For purposes of characterizing the subpopulations
having schizophrenia or bipolar disorder, or neither any verified
method can be used. Preferably only those individuals whose
diagnosis are certain are utilized as part of the reference
population.
[0272] 5.9 Data for Input into the Mathematical Models to Identify
Classifiers for Diagnosis
[0273] Data for input into the mathematical models is data
representative of the level of gene expression of the products of
the biomarkers of the invention. As such the data is the measure of
the products of the biomarkers of the invention including either
mRNA and/or protein expression.
[0274] In one embodiment of the invention, the RNA products of the
biomarkers of the invention which are measured are the population
of RNA products including the mRNA, and all of the spliced variants
of the mRNA. In another embodiment of the invention the products
measured are the mRNA expressed in blood. In yet another embodiment
of the invention, the products measured one or more specific
spliced variants of the mRNA which are expressed in blood. In yet
another embodiment of the invention, the products measured are the
RNA products listed in Table 2.
[0275] Protein products of the biomarkers of the invention are also
included within the scope of the invention. To practice the
invention, measurement of the protein products of the biomarkers of
the invention can be used for purposes of diagnosis. More
particularly, measurement of those populations of protein products
of the biomarkers which are differentially expressed during
schizophrenia and/or bipolar disorder are useful for purposes of
diagnosis and are encompassed herein.
[0276] In one embodiment of the invention the protein products are
those translated from the biomarkers listed in Table 1. In another
embodiment, the protein products are those which are expressed in
blood. In yet another embodiment of the invention, the protein
products are those corresponding to the proteins listed in Table
2.
[0277] In yet another embodiment, data reflective of the level of
expression of a combination of protein products and RNA products of
the biomarkers are used. As would be understood by a person skilled
in the art, other combinations of input data can be utilized to
generate classifiers useful in accordance with the invention.
5.10 Mathematical Models
[0278] Regression Models
[0279] In some embodiments the expression data for some or all of
the biomarkers identified in the present invention are used in a
regression model, preferably a logistic regression model so as to
identify classifiers useful in diagnosing schizophrenia and/or
bipolar disorder. The regression model is used to test various
combinations of two or more of the biomarkers identified in Table 1
to generate classifiers. In the case of regression models, the
classifiers which result are in the form of equations where the
data representing the expression of each of the biomarkers in the
equation is multiplied by a weighted coefficient as generated by
the regression model. The classifiers generated can be used to
analyze expression data from a test individual and provide a
diagnosis. In general, the multiple regression equation of interest
can be written
Y=.alpha.+.beta..sub.1X.sub.1+.beta..sub.2X.sub.2+. . .
+.beta..sub.kX.sub.k+.epsilon.
[0280] where Y, the dependent variable, is present (when Y is
positive) or absent (when Y is negative) of the biological feature
(e.g., absence or presence of schizophrenia and/or bipolar
disorder) associated with the first subgroup. This model says that
the dependent variable Y depends on k explanatory variables (the
measured characteristic values for the k select genes (e.g. the
biomarkers) from subjects in the first and second subgroups in the
reference population), plus an error term that encompasses various
unspecified omitted factors. In the above-identified model, the
parameter .beta..sub.1 gauges the effect of the first explanatory
variable X.sub.1 on the dependent variable Y (e.g. a weighting
factor), holding the other explanatory variables constant.
Similarly, .beta..sub.2 gives the effect of the explanatory
variable X.sub.2 on Y, holding the remaining explanatory variables
constant.
[0281] The logistic regression model is a non-linear transformation
of the linear regression. The logistic regression model is termed
the "logit" model and can be expressed as
1n[p/(1-p)]=.alpha.+.beta..sub.1X.sub.1+.beta..sub.2X.sub.2+. . .
+.beta..sub.kX.sub.k+.epsilon. or
[p/(1-p)]=exp.sup..alpha.
exp.sup..beta..sub..sup.1.sup.X.sub..sup.1
exp.sup..beta..sub..sup.2.sup.X.sub..sup.2.times.. . .
.times.exp.sup..beta..sub..sup.k.sup.X.sub..sup.k
exp.sup..epsilon.
[0282] where,
[0283] where .alpha. and .epsilon. are constants
[0284] 1n is the natural logarithm, log.sup.exp, where exp=2.71828
. . .
[0285] p is the probability that the event Y occurs, p(Y=1),
[0286] p/(1-p) is the "odds ratio",
[0287] 1n[p/(1-p)] is the log odds ratio, or "logit", and
[0288] all other components of the model are the same as the
general regression equation described above. It will be appreciated
by those of skill in the art that the term for .alpha. and
.epsilon. can be folded into the same constant. Indeed, in
preferred embodiments, a single term is used to represent .alpha.
and .epsilon.. The "logistic" distribution is an S-shaped
distribution function. The logit distribution constrains the
estimated probabilities (p) to lie between 0 and 1.
[0289] In some embodiments of the present invention, the logistic
regression model is fit by maximum likelihood estimation (MLE). In
other words, the coefficients (e.g., .alpha., .beta..sub.1,
.beta..sub.2, . . . ) are determined by maximum likelihood. A
likelihood is a conditional probability (e.g., P(Y.vertline.X), the
probability of Y given X). The likelihood function (L) measures the
probability of observing the particular set of dependent variable
values (Y.sub.1, Y.sub.2, . . . , Y.sub.n) that occur in the sample
data set. It is written as the probability of the product of the
dependent variables:
[0290] L=Prob(Y.sub.1*Y.sub.2***Y.sub.n)
[0291] The higher the likelihood function, the higher the
probability of observing the Ys in the sample. MLE involves finding
the coefficients (.alpha., .beta..sub.1, .beta..sub.2, . . . ) that
makes the log of the likelihood function (LL<0) as large as
possible or -2 times the log of the likelihood function (-2LL) as
small as possible. In MLE, some initial estimates of the parameters
.alpha., .beta..sub.1, .beta..sub.2, . . . are made. Then the
likelihood of the data given these parameter estimates is computed.
The parameter estimates are improved the likelihood of the data is
recalculated. This process is repeated until the parameter
estimates do not change much (for example, a change of less than
0.01 or 0.001 in the probability). Examples of logistic regression
and fitting logistic logistic regression models are found in
Hastie, The Elements of Statistical Learning, Springer, New York,
2001, pp. 95-100 which is incorporated herein in its entirety.
[0292] Neural Networks
[0293] In another embodiment, the expression measured for each of
the biomarkers of the present invention can be used to train a
neural network. A neural network is a two-stage regression or
classification model. A neural network can be binary or non binary.
A neural network has a layered structure that includes a layer of
input units (and the bias) connected by a layer of weights to a
layer of output units. For regression, the layer of output units
typically includes just one output unit. However, neural networks
can handle multiple quantitative responses in a seamless fashion.
As such a neural network can be applied to allow identification of
biomarkers which differentiate as between more than two
populations. In one specific example, a neural network can be
trained using expression data from the biomarkers in Table 1 to
identify those combinations of biomarkers which are specific for
schizophrenia as compared with not having schizophrenia. As a
result, the trained neural network can be used to directly identify
combination of biomarkers useful as schizophrenia diagnostic
biomarkers. In some embodiments, the back-propagation neural
network (see, for example Abdi, 1994, "A neural network primer", J.
Biol System. 2, 247-283) containing a single hidden layer of ten
neurons (ten hidden units) found in EasyNN-Plus version 4.0 g
software package (Neural Planner Software Inc.) is used.
[0294] Neural networks are described in Duda et al., 2001, Pattern
Classification, Second Edition, John Wiley & Sons, Inc., New
York; and Hastie et al., 2001, The Elements of Statistical
Learning, Springer-Verlag, New York which is incorporated herein in
its entirety.
[0295] Other Mathematical Models
[0296] The pattern classification and statistical techniques
described above are merely examples of the types of models that can
be used to construct classifiers useful for diagnosing
schizophrenia and/or bipolar disorder, for example clustering as
described on pages 211-256 of Duda and Hart, Pattern Classification
and Scene Analysis, 1973, John Wiley & Sons, Inc., New York,
incorporated herein by reference in its entirety; Principal
component analysis, (see for Jolliffe, 1986, Principal Component
Analysis, Springer, New York, incorporated herein by reference);
nearest neighbour classifier analysis, (see for example Duda,
Pattern Classification, Second Edition, 2001, John Wiley &
Sons, Inc; and Hastie, 2001, The Elements of Statistical Learning,
Springer, New York); linear discriminant analysis, (see for example
Duda, Pattern Classification, Second Edition, 2001, John Wiley
& Sons, Inc; and Hastie, 2001, The Elements of Statistical
Learning, Springer, New York; Venables & Ripley, 1997, Modern
Applied Statistics with s-plus, Springer, New York); Support Vector
Machines (see, for example, Cristianini and Shawe-Taylor, 2000, An
Introduction to Support Vector Machines, Cambridge University
Press, Cambridge, Boser et al., 1992, "A training algorithm for
optimal margin classifiers, in Proceedings of the 5.sup.th Annual
ACM Workshop on Computational Learning Theory, ACM Press,
Pittsburgh, Pa., pp. 142-152; Vapnik, 1998, Statistical Learning
Theory, Wiley, New York, incorporated herein by reference.)
5.8 Use of the Biomarkers of the Invention for Diagnosis
[0297] As would be understood by a person skilled in the art, the
identification of one or more biomarkers can be used to allow the
diagnosis of schizophrenia and/or bipolar disorder by measuring the
expression of the products of the biomarkers (gene) in an
individual to be diagnosed (the "test individual").
[0298] In one embodiment, the results from the test individual are
compared with the a control wherein the control can be results from
one or more individuals having schizophrenia and/or one or more
individuals not having schizophrenia. In another embodiment the
results from the test individual are compared with both a control
population having bipolar disorder and a control population not
having bipolar disorder.
[0299] In another embodiment, one can input expression data of the
expression of the products of the biomarkers of the test individual
into a classifier of the invention resulting in a determination of
whether said test individual has schizophrenia or has bipolar
disorder. In a preferred embodiment, one would use the same
classifier used to generate the biomarkers as to diagnose an
individual, but this is not necessary. Data representative of the
RNA or protein products of the biomarkers of the invention is input
into a classifier of the invention so as to determine a diagnosis.
The data can be generated using any technique known to measure the
level of expression of either the RNA and protein products of the
biomarkers of the invention.
[0300] In one embodiment, use of the classifier results in a
determination of whether the test individual has schizophrenia or
does not have schizophrenia. For example, using logistic regression
as the model, Y is used as a predictor of schizophrenia, where when
Y>0 a person is diagnosed as having schizophrenia and where
Y<0, a person is diagnosed as not having schizophrenia. In yet
another embodiment, one can also include a third category of
prediction wherein diagnosis is indeterminable. For example, one
can determine the standard deviation inherent within the
methodology used to measure gene expression of the biomarkers
(.delta.). If Y<.delta. but >0 or Y>-.delta. but <0,
then the diagnosis is considered indeterminable.
[0301] 5.11 Polynucleotides Used to Measure the Products of the
Biomarkers of the Invention
[0302] Polynucleotides capable of specifically or selectively
binding to the RNA products of the biomarkers of the invention are
used to measure the level of expression of the biomarkers. For
example: oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic
DNA, synthetic RNA, or other combinations of naturally occurring or
modified nucleotides which specifically and/or selectively
hybridize to one or more of the RNA products of the biomarker of
the invention are useful in accordance with the invention.
[0303] In a preferred embodiment, the oligonucleotides, cDNA, DNA,
RNA, PCR products, synthetic DNA, synthetic RNA, or other
combinations of naturally occurring or modified nucleotides
oligonucleotides which both specifically and selectively hybridize
to one or more of the RNA products of the biomarker of the
invention are used.
[0304] 5.12 Techniques to Measure the RNA Products of the
Biomarkers of the Invention
[0305] 5.12.1 Array Hybridization
[0306] In one embodiment of the invention, the polynucleotide used
to measure the RNA products of the invention can be used as nucleic
acid members stably associated with a support to comprise an array
according to one aspect of the invention. The length of a nucleic
acid member can range from 8 to 1000 nucleotides in length and are
chosen so as to be specific for the RNA products of the biomarkers
of the invention. In one embodiment, these members are selective
for the RNA products of the biomarkers of the invention. The
nucleic acid members may be single or double stranded, and/or may
be oligonucleotides or PCR fragments amplified from cDNA.
Preferably oligonucleotides are approximately 20-30 nucleotides in
length. ESTs are preferably 100 to 600 nucleotides in length. It
will be understood to a person skilled in the art that one can
utilize portions of the expressed regions of the biomarkers of the
invention as a probe on the array. More particularly
oligonucleotides complementary to the genes of the invention and or
cDNA or ESTs derived from the genes of the invention are useful.
For oligonucleotide based arrays, the selection of oligonucleotides
corresponding to the gene of interest which are useful as probes is
well understood in the art. More particularly it is important to
choose regions which will permit hybridization to the target
nucleic acids. Factors such as the Tm of the oligonucleotide, the
percent GC content, the degree of secondary structure and the
length of nucleic acid are important factors. See for example U.S.
Pat. No. 6,551,784.
[0307] Construction of a Nucleic Acid Array
[0308] In the subject methods, an array of nucleic acid members
stably associated with the surface of a substantially support is
contacted with a sample comprising target nucleic acids under
hybridization conditions sufficient to produce a hybridization
pattern of complementary nucleic acid members/target complexes in
which one or more complementary nucleic acid members at unique
positions on the array specifically hybridize to target nucleic
acids. The identity of target nucleic acids which hybridize can be
determined with reference to location of nucleic acid members on
the array.
[0309] The nucleic acid members may be produced using established
techniques such as polymerase chain reaction (PCR) and reverse
transcription (RT). These methods are similar to those currently
known in the art (see e.g., PCR Strategies, Michael A. Innis
(Editor), et al. (1995) and PCR: Introduction to Biotechniques
Series, C. R. Newton, A. Graham (1997)). Amplified nucleic acids
are purified by methods well known in the art (e.g., column
purification or alcohol precipitation). A nucleic acid is
considered pure when it has been isolated so as to be substantially
free of primers and incomplete products produced during the
synthesis of the desired nucleic acid. Preferably, a purified
nucleic acid will also be substantially free of contaminants which
may hinder or otherwise mask the specific binding activity of the
molecule.
[0310] An array, according to one aspect of the invention,
comprises a plurality of nucleic acids attached to one surface of a
support at a density exceeding 20 different nucleic acids/cm.sup.2,
wherein each of the nucleic acids is attached to the surface of the
support in a non-identical pre-selected region (e.g. a microarray).
Each associated sample on the array comprises a nucleic acid
composition, of known identity, usually of known sequence, as
described in greater detail below. Any conceivable substrate may be
employed in the invention.
[0311] In one embodiment, the nucleic acid attached to the surface
of the support is DNA. In a preferred embodiment, the nucleic acid
attached to the surface of the support is cDNA or RNA. In another
preferred embodiment, the nucleic acid attached to the surface of
the support is cDNA synthesized by polymerase chain reaction (PCR).
Preferably, a nucleic acid member in the array, according to the
invention, is at least 10, 25 or 50 nucleotides in length. In one
embodiment, a nucleic acid member is at least 150 nucleotides in
length. Preferably, a nucleic acid member is less than 1000
nucleotides in length. More preferably, a nucleic acid member is
less than 500 nucleotides in length.
[0312] In the arrays of the invention, the nucleic acid
compositions are stably associated with the surface of a support,
where the support may be a flexible or rigid support. By "stably
associated" is meant that each nucleic acid member maintains a
unique position relative to the support under hybridization and
washing conditions. As such, the samples are non-covalently or
covalently stably associated with the support surface. Examples of
non-covalent association include non-specific adsorption, binding
based on electrostatic interactions (e.g., ion pair interactions),
hydrophobic interactions, hydrogen bonding interactions, specific
binding through a specific binding pair member covalently attached
to the support surface, and the like. Examples of covalent binding
include covalent bonds formed between the nucleic acids and a
functional group present on the surface of the rigid support (e.g.,
--OH), where the functional group may be naturally occurring or
present as a member of an introduced linking group, as described in
greater detail below.
[0313] The amount of nucleic acid present in each composition will
be sufficient to provide for adequate hybridization and detection
of target nucleic acid sequences during the assay in which the
array is employed. Generally, the amount of each nucleic acid
member stably associated with the support of the array is at least
about 0.001 ng, preferably at least about 0.02 ng and more
preferably at least about 0.05 ng, where the amount may be as high
as 1000 ng or higher, but will usually not exceed about 20 ng.
Where the nucleic acid member is "spotted" onto the support in a
spot comprising an overall circular dimension, the diameter of the
"spot" will generally range from about 10 to 5,000 .mu.m, usually
from about 20 to 2,000 .mu.m and more usually from about 100 to 200
.mu.m.
[0314] Control nucleic acid members may be present on the array
including nucleic acid members comprising oligonucleotides or
nucleic acids corresponding to genomic DNA, housekeeping genes,
vector sequences, plant nucleic acid sequence, negative and
positive control genes, and the like. Control nucleic acid members
are calibrating or control genes whose function is not to tell
whether a particular "key" gene of interest is expressed, but
rather to provide other useful information, such as background or
basal level of expression.
[0315] Other control nucleic acids are spotted on the array and
used as target expression control nucleic acids and mismatch
control nucleotides to monitor non-specific binding or
cross-hybridization to a nucleic acid in the sample other than the
target to which the probe is directed. Mismatch probes thus
indicate whether a hybridization is specific or not. For example,
if the target is present, the perfectly matched probes should be
consistently brighter than the mismatched probes. In addition, if
all control mismatches are present, the mismatch probes are used to
detect a mutation.
[0316] Spotting Method
[0317] In one aspect, the invention provides for arrays where each
nucleic acid member comprising the array is spotted onto a
support.
[0318] Preferably, spotting is carried out as follows. PCR products
(.about.40 ul) biomarkers of the invention, in the same 96-well
tubes used for amplification, are precipitated with 4 ul ({fraction
(1/10)} volume) of 3M sodium acetate (pH 5.2) and 100 ul (2.5
volumes) of ethanol and stored overnight at -20.degree. C. They are
then centrifuged at 3,300 rpm at 4.degree. C. for 1 hour. The
obtained pellets are washed with 50 ul ice-cold 70% ethanol and
centrifuged again for 30 minutes. The pellets are then air-dried
and resuspended well in 20 ul 3.times.SSC or in 50%
dimethylsulfoxide (DMSO) overnight. The samples are then spotted,
either singly or in duplicate, onto slides using a robotic GMS 417
or 427 arrayer (Affymetrix, Ca).
[0319] The boundaries of the spots on the microarray may be marked
with a diamond scriber (as the spots become invisible after
post-processing). The arrays are rehydrated by suspending the
slides over a dish of warm particle free ddH.sub.2O for
approximately one minute (the spots will swell slightly but will
not run into each other) and snap-dried on a 70-80.degree. C.
inverted heating block for 3 seconds. Nucleic acid is then UV
crosslinked to the slide (Stratagene, Stratalinker, 65 mJ--set
display to "650" which is 650.times.100 uJ) or the array is baked
at 80 C for two to four hours prior to hybridization. The arrays
are placed in a slide rack. An empty slide chamber is prepared and
filled with the following solution: 3.0 grams of succinic anhydride
(Aldrich) was dissolved in 189 ml of 1-methyl-2-pyrrolidinone
(rapid addition of reagent is crucial); immediately after the last
flake of succinic anhydride is dissolved, -21.0 ml of 0.2 M sodium
borate is mixed in and the solution is poured into the slide
chamber. The slide rack is plunged rapidly and evenly in the slide
chamber and vigorously shaken up and down for a few seconds, making
sure the slides never leave the solution, and then mixed on an
orbital shaker for 15-20 minutes. The slide rack is then gently
plunged in 95.degree. C. ddH.sub.2O for 2 minutes, followed by
plunging five times in 95% ethanol. The slides are then air dried
by allowing excess ethanol to drip onto paper towels. The arrays
are stored in the slide box at room temperature until use.
[0320] Numerous methods may be used for attachment of the nucleic
acid members of the invention to the substrate (a process referred
to as "spotting"). For example, nucleic acids are attached using
the techniques of, for example U.S. Pat. No. 5,807,522, which is
incorporated herein by reference, for teaching methods of polymer
attachment.
[0321] Alternatively, spotting may be carried out using contact
printing technology as is known in the art.
[0322] Use of a Microarray
[0323] Nucleic acid arrays according to the invention can be used
to assay nucleic acids in a sample comprising one or more target
nucleic acid sequences. The arrays of the subject invention find
use in a variety of applications diagnosis of schizophrenia,
screening for therapeutic targets and the like.
[0324] The arrays are also useful in broad scale expression
screening for drug discovery and research, such as the effect of a
particular active agent on the expression pattern of biomarkers of
the invention, where such information is used to reveal drug
efficacy and toxicity, environmental monitoring, disease research
and the like.
[0325] Arrays can be made using at least one, more preferably a
combination of these sequences, as a means of diagnosing
schizophrenia.
[0326] The choice of a standard sample would be well understood by
a person skilled in the art, and would include a sample
complementary to RNA isolated from one or more normal individuals,
wherein a normal individual is an individual not having
schizophrenia or bipolar disorder.
[0327] Preparation of Nucleic Acid Sample for Hybridization to an
Array
[0328] The samples for hybridization with the arrays according to
the invention are preferably derived from total RNA from blood. In
another embodiment, targets for the arrays are derived from mRNA
from blood.
[0329] The nucleic acid sample is capable of binding to a nucleic
acid member of complementary sequence through one or more types of
chemical bonds, usually through complementary base pairing, usually
through hydrogen bond formation.
[0330] As used herein, a "nucleic acid derived from an mRNA
transcript: or a "nucleic acid corresponding to an mRNA" refers to
a nucleic acid for which synthesis of the mRNA transcript or a
sub-sequence thereof has ultimately served as a template. Thus, a
cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA, a DNA amplified from the cDNA, an RNA transcribed from the
amplified DNA, etc., are all derived from or correspond to the mRNA
transcript and detection of such derived or corresponding products
is indicative of or proportional to the presence and/or abundance
of the original transcript in a sample. Thus, suitable nucleic acid
samples include, but are not limited to, mRNA transcripts of a gene
or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed
from the cDNA, DNA amplified from a gene or genes, RNA transcribed
from amplified DNA, and the like. The nucleic acid samples used
herein are preferably derived from blood. Nucleic acids can be
single- or double-stranded DNA, RNA, or DNA-RNA hybrids synthesized
from human blood using methods known in the art, for example,
reverse transcription or PCR.
[0331] In the simplest embodiment, such a nucleic acid sample
comprises total mRNA or a nucleic acid sample corresponding to mRNA
(e.g., cDNA) isolated from blood samples. In another embodiment,
total mRNA is isolated from a given sample using, for example, an
acid guanidinium-phenol-chloroform extraction method and polyA+mRNA
is isolated by oligo dT column chromatography or by using
(dT).sub.n magnetic beads (see, e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring
Harbor Laboratory, (1989), or Current Protocols in Molecular
Biology, F. Ausubel et al., ed. Greene Publishing and
Wiley-Interscience, New York (1987). In a preferred embodiment,
total RNA is extracted using TRIzol.RTM. reagent (GIBCO/BRL,
Invitrogen Life Technologies, Cat. No. 15596). Purity and integrity
of RNA is assessed by absorbance at 260/280 nm and agarose gel
electrophoresis followed by inspection under ultraviolet light.
[0332] In some embodiments, it is desirable to amplify the nucleic
acid sample prior to hybridization, for example, when only limited
amounts of sample can be used (e.g. drop of blood). One of skill in
the art will appreciate that whatever amplification method is used,
if a quantitative result is desired, care must be taken to use a
method that maintains or controls for the relative frequencies of
the amplified nucleic acids. Methods of "quantitative"
amplification are well known to those of skill in the art. For
example, quantitative PCR involves simultaneously co-amplifying a
known quantity of a control sequence using the same primers. This
provides an internal standard that may be used to calibrate the PCR
reaction. The high density array may then include probes specific
to the internal standard for quantification of the amplified
nucleic acid. Detailed protocols for quantitative PCR are provided
in PCR Protocols, A Guide to Methods and Applications, Innis et
al., Academic Press, Inc. N.Y., (1990).
[0333] Other suitable amplification methods include, but are not
limited to polymerase chain reaction (PCR) (Innis, et al., PCR
Protocols. A Guide to Methods and Application. Academic Press, Inc.
San Diego, (1990)), ligase chain reaction (LCR) (see Wu and
Wallace, 1989, Genomics, 4:560; Landegren, et al., 1988, Science,
241:1077 and Barringer, et al., 1990, Gene, 89:117, transcription
amplification (Kwoh, et al., 1989, Proc. Natl. Acad. Sci. USA, 86:
1173), and self-sustained sequence replication (Guatelli, et al.,
1990, Proc. Nat. Acad. Sci. USA, 87: 1874).
[0334] In a particularly preferred embodiment, the nucleic acid
sample mRNA is reverse transcribed with a reverse transcriptase and
a primer consisting of oligo dT and a sequence encoding the phage
T7 promoter to provide single-stranded DNA template. The second DNA
strand is polymerized using a DNA polymerase. After synthesis of
double-stranded cDNA, T7 RNA polymerase is added and RNA is
transcribed from the cDNA template. Successive rounds of
transcription from each single cDNA template results in amplified
RNA. Methods of in vitro transcription are well known to those of
skill in the art (see, e.g., Sambrook, supra.) and this particular
method is described in detail by Van Gelder, et al., 1990, Proc.
Natl. Acad. Sci. USA, 87: 1663-1667 who demonstrate that in vitro
amplification according to this method preserves the relative
frequencies of the various RNA transcripts. Moreover, Eberwine et
al. Proc. Natl. Acad. Sci. USA, 89: 3010-3014 provide a protocol
that uses two rounds of amplification via in vitro transcription to
achieve greater than 10.sup.6 fold amplification of the original
starting material thereby permitting expression monitoring even
where biological samples are limited.
[0335] Labeling of Nucleic Acid Sample or Nucleic Acid Probe.
[0336] Nucleic acid samples are labelled so as to allow detection
of hybridization to an array of the invention. Any analytically
detectable marker that is attached to or incorporated into a
molecule may be used in the invention. An analytically detectable
marker refers to any molecule, moiety or atom which is analytically
detected and quantified.
[0337] Detectable labels suitable for use in the present invention
include any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include biotin for staining
with labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., .sup.3H, .sup.125I, 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. Patents teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241, the entireties of
which are incorporated by reference herein.
[0338] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and detecting the reaction product produced
by the action of the enzyme on the substrate, and calorimetric
labels are detected by simply visualizing the colored label.
[0339] The labels may be incorporated by any of a number of means
well known to those of skill in the art. However, in a preferred
embodiment, the label is simultaneously incorporated during the
amplification step in the preparation of the sample nucleic acids.
Thus, for example, polymerase chain reaction (PCR) with labeled
primers or labeled nucleotides will provide a labeled amplification
product. In a preferred embodiment, transcription amplification, as
described above, using a labeled nucleotide (e.g.
fluorescein-labeled UTP and/or CTP) incorporates a label into the
transcribed nucleic acids.
[0340] Alternatively, a label may be added directly to the original
nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the
amplification product after the amplification is completed. Means
of attaching labels to nucleic acids are well known to those of
skill in the art and include, for example, nick translation or
end-labeling (e.g. with a labeled RNA) by kinasing of the nucleic
acid and subsequent attachment (ligation) of a nucleic acid linker
joining the sample nucleic acid to a label (e.g., a
fluorophore).
[0341] In a preferred embodiment, the fluorescent modifications are
by cyanine dyes e.g. Cy-3/Cy-5 dUTP, Cy-3/Cy-5 dCTP (Amersham
Pharmacia) or alexa dyes (Khan, et al., 1998, Cancer Res.
58:5009-5013).
[0342] In a preferred embodiment, the two Nucleic Acid Sample
samples used for comparison are labeled with different fluorescent
dyes which produce distinguishable detection signals, for example,
nucleic acid samples made from normal brain cells are labeled with
Cy5 and nucleic acid samples made from brain tissue cells are
labeled with Cy3. The differently labeled target samples are
hybridized to the same microarray simultaneously. In a preferred
embodiment, the labeled nucleic acid samples are purified using
methods known in the art, e.g., by ethanol purification or column
purification.
[0343] In a preferred embodiment, the nucleic acid samples will
include one or more control molecules which hybridize to control
probes on the microarray to normalize signals generated from the
microarray. Preferably, labeled normalization nucleic acid samples
are nucleic acid sequences that are perfectly complementary to
control oligonucleotides that are spotted onto the microarray as
described above. The signals obtained from the normalization
controls after hybridization provide a control for variations in
hybridization conditions, label intensity, "reading" efficiency and
other factors that may cause the signal of a perfect hybridization
to vary between arrays. In a preferred embodiment, signals (e.g.,
fluorescence intensity) read from all other probes in the array are
divided by the signal (e.g., fluorescence intensity) from the
control probes, thereby normalizing the measurements.
[0344] Preferred normalization nucleic acid samples are selected to
reflect the average length of the other nucleic acid samples
present in the sample, however, they are selected to cover a range
of lengths. The normalization control(s) also can be selected to
reflect the (average) base composition of the other probes in the
array, however, in a preferred embodiment, only one or a few
normalization probes are used and they are selected such that they
hybridize well (i.e., have no secondary structure and do not self
hybridize) and do not match any nucleic acids on the array.
[0345] Normalization probes are localized at any position in the
array or at multiple positions throughout the array to control for
spatial variation in hybridization efficiency. In a preferred
embodiment, normalization controls are located at the corners or
edges of the array as well as in the middle.
[0346] Hybridization Conditions
[0347] Nucleic acid hybridization involves providing a nucleic acid
sample under conditions where the sample and the complementary
nucleic acid member can form stable hybrid duplexes through
complementary base pairing. The nucleic acids that do not form
hybrid duplexes are then washed away leaving the hybridized nucleic
acids to be detected, typically through detection of an attached
detectable label. It is generally recognized that nucleic acids are
denatured by increasing the temperature or decreasing the salt
concentration of the buffer containing the nucleic acids. Under low
stringency conditions (e.g., low temperature and/or high salt)
hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even
where the annealed sequences are not perfectly complementary. Thus
specificity of hybridization is reduced at lower stringency.
Conversely, at higher stringency (e.g., higher temperature or lower
salt) successful hybridization requires fewer mismatches.
[0348] The invention provides for hybridization conditions
comprising the Dig hybridization mix (Boehringer); or
formamide-based hybridization solutions, for example as described
in Ausubel et al., supra and Sambrook et al. supra.
[0349] Methods of optimizing hybridization conditions are well
known to those of skill in the art (see, e.g., Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 24:
Hybridization With Nucleic acid Probes, P. Tijssen, ed. Elsevier,
N.Y., (1993)).
[0350] Following hybridization, non-hybridized labeled or unlabeled
nucleic acid is removed from the support surface, conveniently by
washing, thereby generating a pattern of hybridized target nucleic
acid on the substrate surface. A variety of wash solutions are
known to those of skill in the art and may be used. The resultant
hybridization patterns of labeled, hybridized oligonucleotides
and/or nucleic acids may be visualized or detected in a variety of
ways, with the particular manner of detection being chosen based on
the particular label of the test nucleic acid, where representative
detection means include scintillation counting, autoradiography,
fluorescence measurement, calorimetric measurement, light emission
measurement and the like.
[0351] Image Acquisition and Data Analysis
[0352] Following hybridization and any washing step(s) and/or
subsequent treatments, as described above, the resultant
hybridization pattern is detected. In detecting or visualizing the
hybridization pattern, the intensity or signal value of the label
will be not only be detected but quantified, by which is meant that
the signal from each spot of the hybridization will be measured and
compared to a unit value corresponding to the signal emitted by a
known number of end labeled target nucleic acids to obtain a count
or absolute value of the copy number of each end-labeled target
that is hybridized to a particular spot on the array in the
hybridization pattern.
[0353] Methods for analyzing the data collected from hybridization
to arrays are well known in the art. For example, where detection
of hybridization involves a fluorescent label, data analysis can
include the steps of determining fluorescent intensity as a
function of substrate position from the data collected, removing
outliers, i.e., data deviating from a predetermined statistical
distribution, and calculating the relative binding affinity of the
test nucleic acids from the remaining data. The resulting data is
displayed as an image with the intensity in each region varying
according to the binding affinity between associated
oligonucleotides and/or nucleic acids and the test nucleic
acids.
[0354] The following detection protocol is used for the
simultaneous analysis of two samples to be compared, where each
sample is labeled with a different fluorescent dye.
[0355] Each element of the microarray is scanned for the first
fluorescent color. The intensity of the fluorescence at each array
element is proportional to the expression level of that gene in the
sample.
[0356] The scanning operation is repeated for the second
fluorescent label. The ratio of the two fluorescent intensities
provides a highly accurate and quantitative measurement of the
relative gene expression level in the two samples.
[0357] In a preferred embodiment, fluorescence intensities of
immobilized nucleic acid sequences were determined from images
taken with a custom confocal microscope equipped with laser
excitation sources and interference filters appropriate for the Cy3
and Cy5 fluors. Separate scans were taken for each fluor at a
resolution of 225 .mu.m.sup.2 per pixel and 65,536 gray levels.
Image segmentation to identify areas of hybridization,
normalization of the intensities between the two fluor images, and
calculation of the normalized mean fluorescent values at each
target are as described (Khan, et al., 1998, Cancer Res.
58:5009-5013. Chen, et al., 1997, Biomed. Optics 2:364-374).
Normalization between the images is used to adjust for the
different efficiencies in labeling and detection with the two
different fluors. This is achieved by equilibrating to a value of
one the signal intensity ratio of a set of internal control genes
spotted on the array.
[0358] In another preferred embodiment, the array is scanned in the
Cy 3 and Cy5 channels and stored as separate 16-bit TIFF images.
The images are incorporated and analysed using software which
includes a gridding process to capture the hybridization intensity
data from each spot on the array. The fluorescence intensity and
background-subtracted hybridization intensity of each spot is
collected and a ratio of measured mean intensities of CyS to Cy3 is
calculated. A linear regression approach is used for normalization
and assumes that a scatter plot of the measured Cy5 versus Cy3
intensities should have a slope of one. The average of the ratios
is calculated and used to rescale the data and adjust the slope to
one. A ratio of expression not equal to 1 is used as an indication
of differential gene expression.
[0359] In a particularly preferred embodiment, where it is desired
to quantify the transcription level (and thereby expression) of one
or more nucleic acid sequences in a sample, the nucleic acid sample
is one in which the concentration of the mRNA transcript(s) of the
gene or genes, or the concentration of the nucleic acids derived
from the mRNA transcript(s), is proportional to the transcription
level (and therefore expression level) of that gene. Similarly, it
is preferred that the hybridization signal intensity be
proportional to the amount of hybridized nucleic acid. While it is
preferred that the proportionality be relatively strict (e.g., a
doubling in transcription rate results in a doubling in mRNA
transcript in the sample nucleic acid pool and a doubling in
hybridization signal), one of skill will appreciate that the
proportionality can be more relaxed and even non-linear and still
provide meaningful results. Thus, for example, an assay where a 5
fold difference in concentration of the sample mRNA results in a 3-
to 6-fold difference in hybridization intensity is sufficient for
most purposes. Where more precise quantification is required,
appropriate controls are run to correct for variations introduced
in sample preparation and hybridization as described herein. In
addition, serial dilutions of "standard" mRNA sampels are used to
prepare calibration curves according to methods well known to those
of skill in the art. Of course, where simple detection of the
presence or absence of a transcript is desired, no elaborate
control or calibration is required.
[0360] For example, if an nucleic acid member on an array is not
labeled after hybridization, this indicates that the gene
comprising that nucleic acid member is not expressed in either
sample. If a nucleic acid member is labeled with a single color, it
indicates that a labeled gene was expressed only in one sample. The
labeling of a nucleic acid member comprising an array with both
colors indicates that the gene was expressed in both samples. Even
genes expressed once per cell are detected (1 part in 100,000
sensitivity). A difference in expression intensity in the two
samples being compared is indicative of differential expression,
the ratio of the intensity in the two samples being not equal to
1.0, preferably less than 0.7 or greater than 1.2, more preferably
less than 0.5 or greater than 1.5.
[0361] 5.12.2 RT-PCR
[0362] In aspect of the invention, the level of the expression of
the RNA products of the biomarkers of the invention can be measured
by amplifying the RNA products of the biomarkers from a sample
using reverse transcription (RT) in combination with the polymerase
chain reaction (PCR). In accordance with one embodiment of the
invention, the RT can be quantitative as would be understood to a
person skilled in the art.
[0363] Total RNA, or mRNA from a sample is used as a template and a
primer specific to the transcribed portion of a biomarker of the
invention is used to initiate reverse transcription. Methods of
reverse transcribing RNA into cDNA are well known and described in
Sambrook et al., 1989, supra. Primer design can be accomplished
utilizing commercially available software (e.g., Primer Designer
1.0, Scientific Sofware etc.). The product of the reverse
transcription is subsequently used as a template for PCR.
[0364] PCR provides a method for rapidly amplifying a particular
nucleic acid sequence by using multiple cycles of DNA replication
catalyzed by a thermostable, DNA-dependent DNA polymerase to
amplify the target sequence of interest. PCR requires the presence
of a nucleic acid to be amplified, two single-stranded
oligonucleotide primers flanking the sequence to be amplified, a
DNA polymerase, deoxyribonucleoside triphosphates, a buffer and
salts.
[0365] The method of PCR is well known in the art. PCR, is
performed as described in Mullis and Faloona, 1987, Methods
Enzymol., 155: 335, which is incorporated herein by reference. PCR
is performed using template DNA (at least 1 fg; more usefully,
1-1000 ng) and at least 25 pmol of oligonucleotide primers. A
typical reaction mixture includes: 2 .mu.l of DNA, 25 pmol of
oligonucleotide primer, 2.5 .mu.l of 10H PCR buffer 1
(Perkin-Elmer, Foster City, Calif.), 0.4 .mu.l of 1.25 .mu.M dNTP,
0.15 .mu.l (or 2.5 units) of Taq DNA polymerase (Perkin Elmer,
Foster City, Calif.) and deionized water to a total volume of 25
.mu.l. Mineral oil is overlaid and the PCR is performed using a
programmable thermal cycler.
[0366] The length and temperature of each step of a PCR cycle, as
well as the number of cycles, are adjusted according to the
stringency requirements in effect. Annealing temperature and timing
are determined both by the efficiency with which a primer is
expected to anneal to a template and the degree of mismatch that is
to be tolerated. The ability to optimize the stringency of primer
annealing conditions is well within the knowledge of one of
moderate skill in the art. An annealing temperature of between
30.degree. C. and 72.degree. C. is used. Initial denaturation of
the template molecules normally occurs at between 92.degree. C. and
99.degree. C. for 4 minutes, followed by 20-40 cycles consisting of
denaturation (94-99.degree. C. for 15 seconds to 1 minute),
annealing (temperature determined as discussed above; 1-2 minutes),
and extension (72.degree. C. for 1 minute). The final extension
step is generally carried out for 4 minutes at 72.degree. C., and
may be followed by an indefinite (0-24 hour) step at 4.degree.
C.
[0367] QRT-PCR (Quantitative RT-PCR), which is quantitative in
nature, can also be performed to provide a quantitative measure of
gene expression levels. In QRT-PCR reverse transcription and PCR
can be performed in two steps, or reverse transcription combined
with PCR can be performed concurrently. One of these techniques,
for which there are commercially available kits such as Taqman
(Perkin Elmer, Foster City, Calif.), is performed with a
transcript-specific antisense probe. This probe is specific for the
PCR product (e.g. a nucleic acid fragment derived from a gene) and
is prepared with a quencher and fluorescent reporter probe
complexed to the 5' end of the oligonucleotide. Different
fluorescent markers are attached to different reporters, allowing
for measurement of two products in one reaction. When Taq DNA
polymerase is activated, it cleaves off the fluorescent reporters
of the probe bound to the template by virtue of its 5'-to-3'
exonuclease activity. In the absence of the quenchers, the
reporters now fluoresce. The color change in the reporters is
proportional to the amount of each specific product and is measured
by a fluorometer; therefore, the amount of each color is measured
and the PCR product is quantified. The PCR reactions can be
performed in 96 well plates, 384 well plates and the like so that
samples derived from many individuals are processed and measured
simultaneously. The Taqman system has the additional advantage of
not requiring gel electrophoresis and allows for quantification
when used with a standard curve.
[0368] A second technique useful for detecting PCR products
quantitatively without is to use an intercalating dye such as the
commercially available QuantiTect SYBR Green PCR (Qiagen, Valencia
Calif.). RT-PCR is performed using SYBR green as a fluorescent
label which is incorporated into the PCR product during the PCR
stage and produces a flourescense proportional to the amount of PCR
product.
[0369] Both Taqman and QuantiTect SYBR systems can be used
subsequent to reverse transcription of RNA. Reverse transcription
can either be performed in the same reaction mixture as the PCR
step (one-step protocol) or reverse transcription can be performed
first prior to amplification utilizing PCR (two-step protocol).
[0370] Additionally, other systems to quantitatively measure mRNA
expression products are known including Molecular Beacons.RTM.
which uses a probe having a fluorescent molecule and a quencher
molecule, the probe capable of forming a hairpin structure such
that when in the hairpin form, the fluorescence molecule is
quenched, and when hybridized the flourescense increases giving a
quantitative measurement of gene expression.
[0371] Additional techniques to quantitatively measure RNA
expression include, but are not limited to, polymerase chain
reaction, ligase chain reaction, Qbeta replicase (see, e.g.,
International Application No. PCT/US87/00880), isothermal
amplification method (see, e.g., Walker et al. (1992) PNAS
89:382-396), strand displacement amplification (SDA), repair chain
reaction, Asymmetric Quantitative PCR (see, e.g., U.S. Publication
No. U.S. 200330134307A1) and the multiplex microsphere bead assay
described in Fuja et al., 2004, Journal of Biotechnology
108:193-205.
[0372] The level of gene expression can be measured by amplifying
RNA from a sample using transcription based amplification systems
(TAS), including nucleic acid sequence amplification (NASBA) and
3SR. See, e.g., Kwoh et al (1989) PNAS USA 86:1173; International
Publication No. WO 88/10315; and U.S. Pat. No. 6,329,179. In NASBA,
the nucleic acids may be prepared for amplification using
conventional phenol/chloroform extraction, heat denaturation,
treatment with lysis buffer and minispin columns for isolation of
DNA and RNA or guanidinium chloride extraction of RNA. These
amplification techniques involve annealing a primer that has target
specific sequences. Following polymerization, DNA/RNA hybrids are
digested with RNase H while double stranded DNA molecules are heat
denatured again. In either case the single stranded DNA is made
fully double stranded by addition of second target specific primer,
followed by polymerization. The double-stranded DNA molecules are
then multiply transcribed by a polymerase such as T7 or SP6. In an
isothermal cyclic reaction, the RNA's are reverse transcribed into
double stranded DNA, and transcribed once with a polymerase such as
T7 or SP6. The resulting products, whether truncated or complete,
indicate target specific sequences.
[0373] Several techniques may be used to separate amplification
products. For example, amplification products may be separated by
agarose, agarose-acrylamide or polyacrylamide gel electrophoresis
using conventional methods. See Sambrook et al., 1989. Several
techniques for detecting PCR products quantitatively without
electrophoresis may also be used according to the invention (see
for example PCR Protocols, A Guide to Methods and Applications,
Innis et al., Academic Press, Inc. N.Y., (1990)). For example,
chromatographic techniques may be employed to effect separation.
There are many kinds of chromatography which may be used in the
present invention: adsorption, partition, ion-exchange and
molecular sieve, HPLC, and many specialized techniques for using
them including column, paper, thin-layer and gas chromatography
(Freifelder, Physical Biochemistry Applications to Biochemistry and
Molecular Biology, 2nd ed., Wm. Freeman and Co., New York, N.Y.,
1982).
[0374] Another example of a separation methodology is done by
covalently labeling the oligonucleotide primers used in a PCR
reaction with various types of small molecule ligands. In one such
separation, a different ligand is present on each oligonucleotide.
A molecule, perhaps an antibody or avidin if the ligand is biotin,
that specifically binds to one of the ligands is used to coat the
surface of a plate such as a 96 well ELISA plate. Upon application
of the PCR reactions to the surface of such a prepared plate, the
PCR products are bound with specificity to the surface. After
washing the plate to remove unbound reagents, a solution containing
a second molecule that binds to the first ligand is added. This
second molecule is linked to some kind of reporter system. The
second molecule only binds to the plate if a PCR product has been
produced whereby both oligonucleotide primers are incorporated into
the final PCR products. The amount of the PCR product is then
detected and quantified in a commercial plate reader much as ELISA
reactions are detected and quantified. An ELISA-like system such as
the one described here has been developed by the Raggio Italgene
company under the C-Track trade name.
[0375] Amplification products must be visualized in order to
confirm amplification of the nucleic acid sequences of interest.
One typical visualization method involves staining of a gel with
ethidium bromide and visualization under UV light. Alternatively,
if the amplification products are integrally labeled with radio- or
fluorometrically-labeled nucleotides, the amplification products
may then be exposed to x-ray film or visualized under the
appropriate stimulating spectra, following separation.
[0376] In one embodiment, visualization is achieved indirectly.
Following separation of amplification products, a labeled, nucleic
acid probe is brought into contact with the amplified nucleic acid
sequence of interest. The probe preferably is conjugated to a
chromophore but may be radiolabeled. In another embodiment, the
probe is conjugated to a binding partner, such as an antibody or
biotin, where the other member of the binding pair carries a
detectable moiety.
[0377] In another embodiment, detection is by Southern blotting and
hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art and
may be found in many standard books on molecular protocols. See
Sambrook et al., 1989, supra. Briefly, amplification products are
separated by gel electrophoresis. The gel is then contacted with a
membrane, such as nitrocellulose, permitting transfer of the
nucleic acid and non-covalent binding. Subsequently, the membrane
is incubated with a chromophore-conjugated probe that is capable of
hybridizing with a target amplification product. Detection is by
exposure of the membrane to x-ray film or ion-emitting detection
devices.
[0378] One example of the foregoing is described in U.S. Pat. No.
5,279,721, incorporated by reference herein, which discloses an
apparatus and method for the automated electrophoresis and transfer
of nucleic acids. The apparatus permits electrophoresis and
blotting without external manipulation of the gel and is ideally
suited to carrying out methods according to the present
invention.
[0379] 5.12.3 Nuclease Protection Assays
[0380] In another embodiment of the invention, Nuclease protection
assays (including both ribonuclease protection assays and S1
nuclease assays) can be used to detect and quantitate the RNA
products of the biomarkers of the invention. In nuclease protection
assays, an antisense probe (labeled with, e.g., radiolabeled or
nonisotopic) hybridizes in solution to an RNA sample. Following
hybridization, single-stranded, unhybridized probe and RNA are
degraded by nucleases. An acrylamide gel is used to separate the
remaining protected fragments. Typically, solution hybridization is
more efficient than membrane-based hybridization, and it can
accommodate up to 100 .mu.g of sample RNA, compared with the 20-30
.mu.g maximum of blot hybridizations.
[0381] The ribonuclease protection assay, which is the most common
type of nuclease protection assay, requires the use of RNA probes.
Oligonucleotides and other single-stranded DNA probes can only be
used in assays containing S1 nuclease. The single-stranded,
antisense probe must typically be completely homologous to target
RNA to prevent cleavage of the probe:target hybrid by nuclease.
[0382] 5.12.4 Northern Blots
[0383] A standard Northern blot assay can also be used to ascertain
an RNA transcript size, identify alternatively spliced RNA
transcripts, and the relative amounts of RNA products of the
biomarker of the invention, in accordance with conventional
Northern hybridization techniques known to those persons of
ordinary skill in the art. In Northern blots, RNA samples are first
separated by size via electrophoresis in an agarose gel under
denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Nonisotopic or
high specific activity radiolabeled probes can be used including
random-primed, nick-translated, or PCR-generated DNA probes, in
vitro transcribed RNA probes, and oligonucleotides. Additionally,
sequences with only partial homology (e.g., cDNA from a different
species or genomic DNA fragments that might contain an exon) may be
used as probes. The labeled probe, e.g., a radiolabelled cDNA,
either containing the full-length, single stranded DNA or a
fragment of that DNA sequence may be at least 20, at least 30, at
least 50, or at least 100 consecutive nucleotides in length. The
probe can be labeled by any of the many different methods known to
those skilled in this art. The labels most commonly employed for
these studies are radioactive elements, enzymes, chemicals that
fluoresce when exposed to ultraviolet light, and others. A number
of fluorescent materials are known and can be utilized as labels.
These include, but are not limited to, fluorescein, rhodamine,
auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular
detecting material is anti-rabbit antibody prepared in goats and
conjugated with fluorescein through an isothiocyanate. Proteins can
also be labeled with a radioactive element or with an enzyme. The
radioactive label can be detected by any of the currently available
counting procedures. Non-limiting examples of isotopes include
.sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36Ci, .sup.51Cr,
.sup.57Co, .sup.58Co, .sup.59Fe, .sup.90Y, .sup.125I, .sup.131I,
and .sup.186Re. Enzyme labels are likewise useful, and can be
detected by any of the presently utilized colorimetric,
spectrophotometric, fluorospectrophotometric, amperometric or
gasometric techniques. The enzyme is conjugated to the selected
particle by reaction with bridging molecules such as carbodiimides,
diisocyanates, glutaraldehyde and the like. Any enzymes known to
one of skill in the art can be utilized. Examples of such enzymes
include, but are not limited to, peroxidase, beta-D-galactosidase,
urease, glucose oxidase plus peroxidase and alkaline phosphatase.
U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to
by way of example for their disclosure of alternate labeling
material and methods.
5.13 Techniques to Measure the Protein Products of the Biomarkers
of the Invention
[0384] 5.13.1 Antibody Based Methodologies
[0385] Standard techniques can also be utilized for determining the
amount of the protein or proteins of interest present in a sample.
For example, standard techniques can be employed using, e.g.,
immunoassays such as, for example, Western blot,
immunoprecipitation followed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), immunocytochemistry,
and the like to determine the amount of the protein or proteins of
interest present in a sample. A preferred agent for detecting a
protein of interest is an antibody capable of binding to a protein
of interest, preferably an antibody with a detectable label.
[0386] For such detection methods, protein from the sample to be
analyzed can easily be isolated using techniques which are well
known to those of skill in the art. Protein isolation methods can,
for example, be such as those described in Harlow and Lane (Harlow,
E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)).
[0387] Preferred methods for the detection of the protein or
proteins of interest involve their detection via interaction with a
protein-specific antibody. For example, antibodies directed a
protein of interest can be utilized as described herein. Antibodies
can be generated utilizing standard techniques well known to those
of skill in the art. See, e.g., Section 15.13.2 of this application
and Section 5.2 of U.S. Publication No. 20040018200 for a more
detailed discussion of such antibody generation techniques, which
is incorporated herein by reference. Briefly, such antibodies can
be polyclonal, or more preferably, monoclonal. An intact antibody,
or an antibody fragment (e.g., Fab or F(ab').sub.2) can, for
example, be used. Preferably, the antibody is a human or humanized
antibody.
[0388] Table 5 is a table showing, in one embodiment of the
invention, antibodies which are used to detect the proteins of the
biomarkers of the invention.
6TABLE 6 Related Antibodies Gene Commercial Scientific Commercially
Symbol Description Reference Reference Available ADSS
adenylosuccinate synthase APOBEC3B apolipoprotein B APOBEC1 mRNA
editing (ADI enzyme, catalytic Catologue # polypeptide-like 3B
APOBEC1- 1A); (SantaCruz Biotechnology; sc11738) ATM ataxia
telangiectasia Ab2629 mutated (includes (AbCam .RTM.)
complementation groups A, C and D) CLC Charcot-Leyden
Ultrastructural crystal protein localization of the Charcot-Leyden
crystal protein (lysophospholipase) to a distinct crystalloid-free
granule population in mature human eosinophils AM Dvorak, L
Letourneau, GR Login, PF Weller and SJ Ackerman American Society of
Haemotology Vol 72 Issue 1 pg. 150. CTBP1 C-terminal binding
Ab14411 protein 1 (Abcam .RTM.) CXCL1 chemokine (C-X-C Ab14026
motif) ligand 1 (Abcam .RTM.) (melanoma growth stimulating
activity, alpha) DATF1 death associated transcription factor 1
S100A9 S100 calcium CYT402 binding protein A9 (Chemicon
(calgranulin B) International)
[0389] For example, antibodies, or fragments of antibodies,
specific for a protein of interest can be used to quantitatively or
qualitatively detect the presence of the protein. This can be
accomplished, for example, by immunofluorescence techniques.
Antibodies (or fragments thereof) can, additionally, be employed
histologically, as in immunofluorescence or immunoelectron
microscopy, for in situ detection of a protein of interest. In situ
detection can be accomplished by removing a histological specimen
(e.g., a biopsy specimen) from a patient, and applying thereto a
labeled antibody thereto that is directed to a protein. The
antibody (or fragment) is preferably applied by overlaying the
labeled antibody (or fragment) onto a biological sample. Through
the use of such a procedure, it is possible to determine not only
the presence of the protein of interest, but also its distribution,
its presence in cells (e.g., brain cells and lymphocytes) within
the sample. A wide variety of well-known histological methods (such
as staining procedures) can be utilized in order to achieve such in
situ detection.
[0390] Immunoassays for a protein of interest typically comprise
incubating a biological sample of a detectably labeled antibody
capable of identifying a protein of interest, and detecting the
bound antibody by any of a number of techniques well-known in the
art. As discussed in more detail, below, the term "labeled" can
refer to direct labeling of the antibody via, e.g., coupling (i.e.,
physically linking) a detectable substance to the antibody, and can
also refer to indirect labeling of the antibody by reactivity with
another reagent that is directly labeled. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody.
[0391] For example, the biological sample can be brought in contact
with and immobilized onto a solid phase support or carrier such as
nitrocellulose, or other support which is capable of immobilizing
cells, cell particles or soluble proteins. The support can then be
washed with suitable buffers followed by treatment with the
detectably labeled fingerprint gene-specific antibody. The solid
phase support can then be washed with the buffer a second time to
remove unbound antibody. The amount of bound label on support can
then be detected by conventional means.
[0392] By "solid phase support or carrier" in the context of
proteinaceous agents is intended any support capable of binding an
antigen or an antibody. Well-known supports or carriers include
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite. The nature of the carrier can be either
soluble to some extent or insoluble for the purposes of the present
invention. The support material can have virtually any possible
structural configuration so long as the coupled molecule is capable
of binding to an antigen or antibody. Thus, the support
configuration can be spherical, as in a bead, or cylindrical, as in
the inside surface of a test tube, or the external surface of a
rod. Alternatively, the surface can be flat such as a sheet, test
strip, etc. Preferred supports include polystyrene beads. Those
skilled in the art will know many other suitable carriers for
binding antibody or antigen, or will be able to ascertain the same
by use of routine experimentation.
[0393] One of the ways in which a specific antibody can be
detectably labeled is by linking the same to an enzyme and use in
an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked
Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7,
Microbiological Associates Quarterly Publication, Walkersville,
Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler,
J. E., 1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980,
Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et
al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The
enzyme which is bound to the antibody will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual means.
Enzymes which can be used to detectably label the antibody include,
but are not limited to, malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection can also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0394] Detection can also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect a
protein of interest through the use of a radioimmunoassay (RIA)
(see, for example, Weintraub, B., Principles of Radioimmunoassays,
Seventh Training Course on Radioligand Assay Techniques, The
Endocrine Society, March, 1986, which is incorporated by reference
herein). The radioactive isotope (e.g., .sup.125I, .sup.131I,
.sup.35S or .sup.3H) can be detected by such means as the use of a
gamma counter or a scintillation counter or by autoradiography.
[0395] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0396] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0397] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0398] Likewise, a bioluminescent compound can be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0399] 5.13.2 Protein Arrays
[0400] Polypeptides which specifically and/or selectively bind to
the protein products of the biomarkers of the invention can be
immobilized on a protein array. The protein array can be used as a
diagnostic tool, e.g., to screen individiual samples (such as
isolated cells, blood, synovial fluid, sera, biopsies, and the
like) for the presence of the polypeptides protein products of the
biomarkers of the invention. The protein array can also include
antibodies as well as other ligands, e.g., that bind to the
polypeptides encoded by the biomarkers of the invention. Methods of
producing polypeptide arrays are described, e.g., in De Wildt et
al., 2000, Nature Biotech. 18:989-994; Lueking et al., 1999, Anal.
Biochem. 270:103-111; Ge, 2000, Nuc. Acids Res. 28:e3; MacBeath and
Schreiber, 2000, Science 289:1760-1763; International Publication
Nos. WO 01/40803 and WO 99/51773A1; and U.S. Pat. No. 6,406,921.
Polypeptides for the array can be spotted at high speed, e.g.,
using commercially available robotic apparatus, e.g., from Genetic
MicroSystems and Affymetrix (Santa Clara, Calif., USA) or
BioRobotics (Cambridge, UK). The array substrate can be, for
example, nitrocellulose, plastic, glass, e.g., surface-modified
glass. The array can also include a porous matrix, e.g.,
acrylamide, agarose, or another polymer.
[0401] For example, the array can be an array of antibodies, e.g.,
as described in De Wildt, supra. Cells that produce the polypeptide
ligands can be grown on a filter in an arrayed format. Polypeptide
production is induced, and the expressed antibodies are immobilized
to the filter at the location of the cell. Information about the
extent of binding at each address of the array can be stored as a
profile, e.g., in a computer database.
[0402] In one embodiment the array is an array of protein products
of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or all or any
combination of the biomarkers of the invention. In one aspect, the
invention provides for antibodies that are bound to an array which
selectively bind to the protein products of the biomarkers of the
invention.
5.14 Protein Production
[0403] Standard recombinant nucleic acid methods can be used to
express a polypeptide or antibody of the invention (e.g., a protein
product of a biomarker of the invention). Generally, a nucleic acid
sequence encoding the polypeptide is cloned into a nucleic acid
expression vector. Of course, if the protein includes multiple
polypeptide chains, each chain must be cloned into an expression
vector, e.g., the same or different vectors, that are expressed in
the same or different cells. If the protein is sufficiently small,
i.e., the protein is a peptide of less than 50 amino acids, the
protein can be synthesized using automated organic synthetic
methods. Polypeptides comprising the 5' region, 3' region or
internal coding region of a biomarker of the invention, are
expressed from nucleic acid expression vectors containing only
those nucleotide sequences corresponding to the 5' region, 3'
region or internal coding region of a biomarker of the invention.
Methods for producing antibodies directed to protein products of a
biomarker of the invention, or polypeptides encoded by the 5'
region, 3' region or internal coding regions of a biomarker of the
invention.
[0404] The expression vector for expressing the polypeptide can
include, in addition to the segment encoding the polypeptide or
fragment thereof, regulatory sequences, including for example, a
promoter, operably linked to the nucleic acid(s) of interest. Large
numbers of suitable vectors and promoters are known to those of
skill in the art and are commercially available for generating the
recombinant constructs of the present invention. The following
vectors are provided by way of example. Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A,
pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3,
pBPV, pMSG, and pSVL (Pharmacia). One preferred class of preferred
libraries is the display library, which is described below.
[0405] Methods well known to those skilled in the art can be used
to construct vectors containing a polynucleotide of the invention
and appropriate transcriptional/translational control signals.
These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Sambrook & Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and
Ausubel et al., Current Protocols in Molecular Biology (Greene
Publishing Associates and Wiley Interscience, N.Y. (1989). Promoter
regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lac, lacZ, T3, T7,
gpt, lambda P, and trc. Eukaryotic promoters include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, mouse metallothionein-I, and various art-known tissue
specific promoters. In specific embodiments, the promoter is an
inducible promoter. In other embodiments, the promoter is a
constitutive promoter. In yet other embodiments, the promoter is a
tissue-specific promoter.
[0406] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae auxotrophic markers (such as
URA3, LEU2, HIS3, and TRP1 genes), and a promoter derived from a
highly expressed gene to direct transcription of a downstream
structural sequence. Such promoters can be derived from operons
encoding glycolytic enzymes such as 3-phosphoglycerate kinase
(PGK), a-factor, acid phosphatase, or heat shock proteins, among
others. The polynucleotide of the invention is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, a nucleic acid of the invention
can encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product. Useful
expression-vectors for bacteria are constructed by inserting a
polynucleotide of the invention together with suitable translation
initiation and termination signals, optionally in operable reading
phase with a functional promoter. The vector will comprise one or
more phenotypic selectable markers and an origin of replication to
ensure maintenance of the vector and to, if desirable, provide
amplification within the host. Suitable prokaryotic hosts for
transformation include E. coli, Bacillus subtilis, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus, although others may also be
employed as a matter of choice.
[0407] As a representative but nonlimiting example, useful
expression vectors for bacteria can comprise a selectable marker
and bacterial origin of replication derived from commercially
available plasmids comprising genetic elements of the well known
cloning vector pBR322 (ATCC 37017). Such commercial vectors
include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,
Sweden) and pGEM1 (Promega, Madison, Wis., USA).
[0408] The present invention provides host cells genetically
engineered to contain the polynucleotides of the invention. For
example, such host cells may contain nucleic acids of the invention
introduced into the host cell using known transformation,
transfection or infection methods. The present invention also
provides host cells genetically engineered to express the
polynucleotides of the invention, wherein such polynucleotides are
in operative association with a regulatory sequence heterologous to
the host cell which drives expression of the polynucleotides in the
cell.
[0409] The present invention further provides host cells containing
the vectors of the present invention, wherein the nucleic acid has
been introduced into the host cell using known transformation,
transfection or infection methods. The host cell can be a
eukaryotic host cell, such as a mammalian cell, a lower eukaryotic
host cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a bacterial cell. Introduction of the
recombinant construct into the host cell can be effected, for
example, by calcium phosphate transfection, DEAE, dextran mediated
transfection, or electroporation (Davis, L. et al., Basic Methods
in Molecular Biology (1986)). Cell-free translation systems can
also be employed to produce such proteins using RNAs derived from
the DNA constructs of the present invention.
[0410] Any host/vector system can be used to express one or more of
the proteins listed in Table 2. Appropriate cloning and expression
vectors for use with prokaryotic and eukaryotic hosts are described
by Sambrook et al., in Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of
which is incorporated herein by reference in its entirety. The most
preferred host cells are those which do not normally express the
particular polypeptide or which expresses the polypeptide at low
natural level.
[0411] In a specific embodiment, the host cells are engineered to
express an endogenous gene comprising the polynucleotides of the
invention under the control of inducible regulatory elements, in
which case the regulatory sequences of the endogenous gene may be
replaced by homologous recombination. As described herein, gene
targeting can be used to replace a gene's existing regulatory
region with a regulatory sequence isolated from a different gene or
a novel regulatory sequence synthesized by genetic engineering
methods. Such regulatory sequences may be comprised of promoters,
enhancers, scaffold-attachment regions, negative regulatory
elements, transcriptional initiation sites, regulatory protein
binding sites or combinations of said sequences. Alternatively,
sequences which affect the structure or stability of the RNA or
protein produced may be replaced, removed, added, or otherwise
modified by targeting, including polyadenylation signals. mRNA
stability elements, splice sites, leader sequences for enhancing or
modifying transport or secretion properties of the protein, or
other sequences which alter or improve the function or stability of
protein or RNA molecules.
[0412] The host of the present invention may also be a yeast or
other fungi. In yeast, a number of vectors containing constitutive
or inducible promoters may be used. For a review see, Ausubel et
al. (eds), Current Protocols in Molecular Biology, Vol. 2, Greene
Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Grant et
al., 1987, "Expression and Secretion Vectors for Yeast", Methods
Enzymol. 153:516-544; Glover, DNA Cloning, Vol. II, IRL Press,
Wash., D.C., Ch. 3 (1986); Bitter, 1987, "Heterologous Gene
Expression in Yeast", Methods Enzymol. 152:673-684; and Strathern
et al. (eds), The Molecular Biology of the Yeast Saccharomyces,
Cold Spring Harbor Press, Vols. I and II (1982).
[0413] Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins. Potentially suitable bacterial strains include
Escherichia coli, enterobacteriaceae such as Serratia marescans,
bacilli such as Bacillus subtilis, Salmonella typhimurium,
pseudomonads or any bacterial strain capable of expressing
heterologous proteins. If the protein is made in yeast or bacteria,
it may be necessary to modify the protein produced therein, for
example by phosphorylation or glycosylation of the appropriate
sites, in order to obtain the functional protein. Such covalent
attachments may be accomplished using known chemical or enzymatic
methods.
[0414] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the monkey COS cells such as COS-7 lines of monkey
kidney fibroblasts, described by Gluzman, 1981, Cell 23:175 (1981),
Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human
epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells,
normal diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK, C127, 3T3, or Jurkat cells, and other cell lines
capable of expressing a compatible vector. Mammalian expression
vectors will comprise an origin of replication, a suitable promoter
and also any necessary ribosome-binding sites, polyadenylation
site, splice donor and acceptor sites, transcriptional termination
sequences, and 5' flanking nontranscribed sequences.
[0415] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents.
Recombinant polypeptides produced in bacterial culture are usually
isolated by initial extraction from cell pellets, followed by one
or more salting-out, aqueous ion exchange or size exclusion
chromatography steps. In some embodiments, the template nucleic
acid also encodes a polypeptide tag, e.g., penta- or
hexa-histidine.
[0416] Recombinant proteins can be isolated using an techniqe
well-known in the art. Scopes (Protein Purification: Principles and
Practice, Springer-Verlag, New York (1994)), for example, provides
a number of general methods for purifying recombinant (and
non-recombinant) proteins. The methods include, e.g., ion-exchange
chromatography, size-exclusion chromatography, affinity
chromatography, selective precipitation, dialysis, and hydrophobic
interaction chromatography.
[0417] Variations, modifications, and other implementations of what
is described herein will occur to those of ordinary skill in the
art without departing from the spirit and scope of the
invention.
[0418] In order that the invention described herein may be more
fully understood, the following example is set forth. It should be
understood that this example is for illustrative purposes only and
are not to be construed as limiting this invention in any
manner.
5.15 Methods for Identifying Compounds for Use in the Prevention,
Treatment, Management or Amelioration Schizophrenia and/or Bipolar
Disorder or a Symptom Thereof
[0419] 5.15.1 Methods to Identify Compounds that Modulate the
Expression or Activity of a Biomarker
[0420] The present invention provides methods of identifying
compounds that bind to the products of the biomarkers of the
invention. The present invention also provides methods for
identifying compounds that modulate the expression and/or activity
of the products of the biomarkers of the invention. The compounds
identified via such methods are useful for the development of one
or more animal models to study schizophrenia or bipolar disorder.
Further, the compounds identified via such methods are useful as
lead compounds in the development of prophylactic and therapeutic
compositions for prevention, treatment, management and/or
amelioration of Schizophrenia and/or Bipolar Disorder or a symptom
thereof. Such methods are particularly useful in that the effort
and great expense involved in testing potential prophylactics and
therapeutics in vivo is efficiently focused on those compounds
identified via the in vitro and ex vivo methods described
herein.
[0421] The present invention provides a method for identifying a
compound to be tested for an ability to prevent, treat, manage or
ameliorate Schizophrenia and/or Bipolar Disorder or a symptom
thereof, said method comprising: (a) contacting a cell expressing a
protein product of one or more biomarkers of the invention or a
fragment thereof, or a RNA product of one or more biomarkers of the
invention or a fragment thereof with a test compound; and (b)
determining the ability of the test compound to bind to the protein
product, protein fragment, RNA product, or RNA portion so that if a
compound binds to the protein product, protein fragment, RNA
product, RNA portion, a compound to be tested for an ability to
prevent, treat, manage or ameliorate Schizophrenia and/or Bipolar
Disorder or a symptom thereof is identified. The cell, for example,
can be a prokaryotic cell, yeast cell, viral cell or a cell of
mammalian origin. Determining the ability of the test compound to
bind to the protein product, protein fragment, RNA product, or RNA
portion can be accomplished, for example, by coupling the test
compound with a radioisotope or enzymatic label such that binding
of the test compound to the protein product, protein fragment, RNA
product, or RNA portion can be determined by detecting the labeled
compound in a complex. For example, test compounds can be labeled
with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively, test
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product. In a specific embodiment, the
assay comprises contacting a cell which expresses a protein product
of one or more biomarkers of the invention or a fragment thereof,
or a RNA product of one or more biomarkers of the invention or a
fragment thereof, with a known compound which binds the protein
product, protein fragment, RNA product, or RNA portion to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
the protein product, protein fragment, RNA product, or RNA portion,
wherein determining the ability of the test compound to interact
with the protein product, protein fragment, RNA product, or RNA
portion comprises determining the ability of the test compound to
preferentially bind to the protein product, protein fragment, RNA
product, or RNA portion as compared to the known compound.
[0422] The present invention provides a method for identifying a
compound to be tested for an ability to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof, said method comprising: (a) contacting a protein product
of one or more biomarkers of the invention or a fragment thereof,
or a RNA product of one or more biomarkers of the invention or a
portion thereof with a test compound; and (b) determining the
ability of the test compound to bind to the protein product,
protein fragment, RNA product, or RNA portion so that if a compound
binds to the protein product, protein fragment, RNA product, or RNA
portion, a compound to be tested for an ability to prevent, treat,
manage or ameliorate Schizophrenia and/or Bipolar Disorder or a
symptom thereof is identified. Binding of the test compound to the
protein product or protein fragment can be determined either
directly or indirectly. In a specific embodiment, the assay
includes contacting a protein product of one or more biomarkers of
the invention or a fragment thereof, or a RNA product of one or
more biomarkers of the invention or a portion thereof with a known
compound which binds the protein product, protein fragment, RNA
product, or RNA portion to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with the protein product, protein
fragment, RNA product, or RNA portion, wherein determining the
ability of the test compound to interact with the protein product,
protein fragment, RNA product, or RNA portion comprises determining
the ability of the test compound to preferentially bind to the
protein product, protein fragment, RNA product, or RNA portion as
compared to the known compound. Techniques well known in the art
can be used to determine the binding between a test compound and a
protein product of a biomarker of the invention or a fragment
thereof, or a RNA product of a biomarker of the invention or a
portion thereof.
[0423] In some embodiments of the above assay methods of the
present invention, it may be desirable to immobilize a RNA product
of a biomarker of the invention or a portion thereof, or its target
molecule to facilitate separation of complexed from uncomplexed
forms of the RNA product or RNA portion, the target molecule or
both, as well as to accommodate automation of the assay. In more
than one embodiment of the above assay methods of the present
invention, it may be desirable to immobilize either a protein
product of a biomarker of the invention or a fragment thereof, or
its target molecule to facilitate separation of complexed from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of a test compound to
a protein product of a biomarker of the invention or a fragment
thereof can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-5-transferase (GST) fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.)
or glutathione derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or a protein product of a biomarker of
the invention or a fragment thereof, and the mixture incubated
under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components and complex formation is measured either
directly or indirectly, for example, as described above.
Alternatively, the complexes can be dissociated from the matrix,
and the level of binding of a protein product of a biomarker of the
invention or a fragment thereof can be determined using standard
techniques.
[0424] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a protein product of a biomarker of the invention or a
fragment thereof, or a target molecule can be immobilized utilizing
conjugation of biotin and streptavidin. A biotinylated protein
product of a biomarker of the invention or a target molecule can be
prepared from biotin-NHS(N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals;
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with a protein product of a
biomarker of the invention or a fragment thereof can be derivatized
to the wells of the plate, and protein trapped in the wells by
antibody conjugation. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with a protein product of a biomarker of the invention, as
well as enzyme-linked assays which rely on detecting an enzymatic
activity associated with a protein product of a biomarker of the
invention or a fragment thereof, or target molecule.
[0425] The interaction or binding of a protein product of a
biomarker of the invention or a fragment thereof to a test compound
can also be determined using such proteins or protein fragments as
"bait proteins" in a two-hybrid assay or three hybrid assay (see,
e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell
72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;
Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al.
(1993) Oncogene 8:1693-1696; and International Publication No. WO
94/10300).
[0426] The present invention provides a method for identifying a
compound to be tested for an ability to prevent, treat, manage or
ameliorate Schizophrenia and/or Bipolar Disorder or a symptom
thereof, said method comprising: (a) contacting a cell expressing a
protein or RNA product of one or more biomarkers of the invention
with a test compound; (b) determining the amount of the protein or
RNA product present in (a); and (c) comparing the amount in (a) to
that present in a corresponding control cell that has not been
contacted with the test compound, so that if the amount of the
protein or RNA product is altered relative to the amount in the
control, a compound to be tested for an ability to prevent, treat,
manage or ameliorate Schizophrenia and/or Bipolar Disorder or a
symptom thereof is identified. In a specific embodiment, the
expression level(s) is altered by 5%, 10%, 15%, 25%, 30%, 40%, 50%,
5 to 25%, 10 to 30%, at least 1 fold, at least 1.5 fold, at least 2
fold, 4 fold, 5 fold, 10 fold, 25 fold, 1 to 10 fold, or 5 to 25
fold relative to the expression level in the control as determined
by utilizing an assay described herein (e.g., a microarray or
RT-PCR) or an assay well known to one of skill in the art. In
alternate embodiments, such a method comprises determining the
amount of the protein or RNA product of at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, 1 to 3, 1 to 5, 1-7,
all or any combination of the biomarkers of the invention present
in the cell and comparing the amounts to those present in the
control.
[0427] The cells utilized in the cell-based assays described herein
can be engineered to express a biomarker of the invention utilizing
techniques known in the art. See, e.g., Section III entitled
"Recombinant Expression Vectors and Host Cells" of U.S. Pat. No.
6,245,527, which is incorporated herein by reference.
Alternatively, cells that endogenously express a biomarker of the
invention can be used. For example, brain cells may be used.
[0428] In a specific embodiment, brain cells are isolated from a
"normal" individual, or an individual with schizophrenia and/or
bipolar disorder and are incubated in the presence and absence of a
test compound for varying amounts of time (i.e., 30 min, 1 hr, 5
hr, 24 hr, 48 hr and 96 hrs). When screening for prophylactic or
therapeutic agents, a clone of the full sequence of a biomarker of
the invention or functional portion thereof is used to transfect
brain cells. The transfected brain cells are cultured for varying
amounts of time (i.e., 1, 2, 3, 5, 7, 10, or 14 days) in the
presence or absence of test compound. Following incubation, target
nucleic acid samples are prepared from the brain cells and
hybridized to a nucleic acid probe corresponding to a nucleic acid
sequence which are differentially expressed in schizophrenia and/or
bipolar disorder. The nucleic acid probe is labeled, for example,
with a radioactive label, according to methods well-known in the
art and described herein. Hybridization is carried out by northern
blot, for example as described in Ausubel et al., supra or Sambrook
et al., supra). The differential hybridization, as defined herein,
of the target to the samples on the array from normal relative to
RNA from schizophrenia and/or bipolar disorder is indicative of the
level of expression of RNA corresponding to a differentially
expressed specific nucleic acid sequence. A change in the level of
expression of the target sequence as a result of the incubation
step in the presence of the test compound, is indicative of a
compound that increases or decreases the expression of the
corresponding schizophrenia and/or bipolar disorder biomarker
specific nucleic acid sequence.
[0429] The present invention also provides a method for identifying
a compound to be tested for an ability to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof, said method comprises: (a) contacting a cell-free extract
(e.g., a brain cell extract) with a nucleic acid sequence encoding
a protein or RNA product of one or more biomarkers of the invention
and a test compound; (b) determining the amount of the protein or
RNA product present in (a); and (c) comparing the amount(s) in (a)
to that present to a corresponding control that has not been
contacted with the test compound, so that if the amount of the
protein or RNA product is altered relative to the amount in the
control, a compound to be tested for an ability to prevent, treat,
manage or ameliorate schizophrenia and/or bipolar disorder or a
symptom thereof is identified. In a specific embodiment, the
expression level(s) is altered by 5%, 10%, 15%, 25%, 30%, 40%, 50%,
5 to 25%, 10 to 30%, at least 1 fold, at least 1.5 fold, at least 2
fold, 4 fold, 5 fold, 10 fold, 25 fold, 1 to 10 fold, or 5 to 25
fold relative to the expression level in the control sample
determined by utilizing an assay described herein (e.g., a
microarray or RT-PCR) or an assay well known to one of skill in the
art. In alternate embodiments, such a method comprises determining
the amount of a protein or RNA product of at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, 1 to 3, 1 to 5,
1-7, all or any combination of the biomarkers of the invention
present in the extract and comparing the amounts to those present
in the control. In certain embodiments, the amount of RNA product
of a biomarker of the invention is determined, in other
embodiments, the amount of protein product of a biomarker of the
invention is determined, while in still other embodiments, the
amount of RNA and protein product of a biomarker of the invention
is determined. Standard methods and compositions for determining
the amount of RNA or protein product of a biomarker of the
invention can be utilized. Such methods and compositions are
described in detail above.
[0430] In specific embodiments, in a screening assay described
herein, the amount of protein or RNA product of a biomarker of the
invention is determined utilizing kits. Such kits comprise
materials and reagents required for measuring the expression of at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8 or more protein or RNA products of at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, all or any combination of the biomarkers
of the invention. In specific embodiments, the kits may further
comprise one or more additional reagents employed in the various
methods, such as: (1) reagents for purifying RNA from blood, brain
cells; (2) primers for generating test nucleic acids; (3) dNTPs
and/or rNTPs (either premixed or separate), optionally with one or
more uniquely labeled dNTPs and/or rNTPs (e.g., biotinylated or Cy3
or Cy5 tagged dNTPs); (4) post synthesis labeling reagents, such as
chemically active derivatives of fluorescent dyes; (5) enzymes,
such as reverse transcriptases, DNA polymerases, and the like; (6)
various buffer mediums, e.g., hybridization and washing buffers;
(7) labeled probe purification reagents and components, like spin
columns, etc.; and (8) protein purification reagents; (9) signal
generation and detection reagents, e.g., streptavidin-alkaline
phosphatase conjugate, chemifluorescent or chemiluminescent
substrate, and the like. In particular embodiments, the kits
comprise prelabeled quality controlled protein and or RNA
transcript (preferably, mRNA) for use as a control.
[0431] In some embodiments, the kits are RT-PCR kits. In other
embodiments, the kits are nucleic acid arrays and protein arrays.
Such kits according to the subject invention will at least comprise
an array having associated protein or nucleic acid members of the
invention and packaging means therefore. Alternatively the protein
or nucleic acid members of the invention may be prepackaged onto an
array.
[0432] In a specific embodiment, kits for measuring a RNA product
of a biomarker of the invention comprise materials and reagents
that are necessary for measuring the expression of the RNA product.
For example, a microarray or RT-PCR kit may be used and contain
only those reagents and materials necessary for measuring the
levels of RNA products of at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, all or any
combination of the biomarkers of the invention. Alternatively, in
some embodiments, the kits can comprise materials and reagents that
are not limited to those required to measure the levels of RNA
products of 1, 2, 3, 4, 5, 6, 7, 8 all or any combination of the
biomarkers of the invention. For example, a microarray kit may
contain reagents and materials necessary for measuring the levels
of RNA products 1, 2, 3, 4, 5, 6, 7, 8, all or any combination of
the biomarkers of the invention, in addition to reagents and
materials necessary for measuring the levels of the RNA products of
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, or more genes other than the
biomarkers of the invention. In a specific embodiment, a microarray
or RT-PCR kit contains reagents and materials necessary for
measuring the levels of RNA products of at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, all or any combination of the biomarkers of the invention, and
1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350,
400, 450, or more genes that are not biomarkers of the invention,
or 1-10, 1-100, 1-150, 1-200, 1-300, 1-400, 1-500, 1-1000, 25-100,
25-200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300,
100-400, 100-500, 100-1000 or 500-1000 genes that are not
biomarkers of the invention. For nucleic acid micoarray kits, the
kits generally comprise probes attached to a support surface. The
probes may be labeled with a detectable label. In a specific
embodiment, the probes are specific for the 5' region, the 3'
region, the internal coding region, an exon(s), an intron(s), an
exon junction(s), or an exon-intron junction(s), of 1, 2, 3, 4, 5,
6, 7, 8, all or any combination of the biomarkers of the invention.
The microarray kits may comprise instructions for performing the
assay and methods for interpreting and analyzing the data resulting
from the performance of the assay. The kits may also comprise
hybridization reagents and/or reagents necessary for detecting a
signal produced when a probe hybridizes to a target nucleic acid
sequence. Generally, the materials and reagents for the microarray
kits are in one or more containers. Each component of the kit is
generally in its own a suitable container.
[0433] For RT-PCR kits, the kits generally comprise pre-selected
primers specific for particular RNA products (e.g., an exon(s), an
intron(s), an exon junction(s), and an exon-intron junction(s)) of
1, 2, 3, 4, 5, 6, 7, 8, all or any combination of the biomarkers of
the invention. The RT-PCR kits may also comprise enzymes suitable
for reverse transcribing and/or amplifying nucleic acids (e.g.,
polymerases such as Taq), and deoxynucleotides and buffers needed
for the reaction mixture for reverse transcription and
amplification. The RT-PCR kits may also comprise probes specific
for 1, 2, 3, 4, 5, 6, 7, 8, all or any combination of the
biomarkers of the invention. The probes may or may not be labeled
with a detectable label (e.g., a fluorescent label). Each component
of the RT-PCR kit is generally in its own suitable container. Thus,
these kits generally comprise distinct containers suitable for each
individual reagent, enzyme, primer and probe. Further, the RT-PCR
kits may comprise instructions for performing the assay and methods
for interpreting and analyzing the data resulting from the
performance of the assay. For antibody based kits, the kit can
comprise, for example: (1) a first antibody (which may or may not
be attached to a support) which binds to protein of interest (e.g.,
a protein product of 1, 2, 3, 4, 5, 6, 7, 8, all or any combination
of the biomarkers of the invention); and, optionally, (2) a second,
different antibody which binds to either the protein, or the first
antibody and is conjugated to a detectable label (e.g., a
fluorescent label, radioactive isotope or enzyme). The
antibody-based kits may also comprise beads for conducting an
immunoprecipitation. Each component of the antibody-based kits is
generally in its own suitable container. Thus, these kits generally
comprise distinct containers suitable for each antibody. Further,
the antibody-based kits may comprise instructions for performing
the assay and methods for interpreting and analyzing the data
resulting from the performance of the assay.
[0434] Reporter gene-based assays may also be conducted to identify
a compound to be tested for an ability to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof. In a specific embodiment, the present invention provides a
method for identifying a compound to be tested for an ability to
prevent, treat, manage or ameliorate schizophrenia and/or bipolar
disorder or a symptom thereof, said method comprising: (a)
contacting a compound with a cell expressing a reporter gene
construct comprising a reporter gene operably linked to a
regulatory element of a biomarker of the invention (e.g., a
promoter/enhancer element); (b) measuring the expression of said
reporter gene; and (c) comparing the amount in (a) to that present
in a corresponding control cell that has not been contacted with
the test compound, so that if the amount of expressed reporter gene
is altered relative to the amount in the control cell, a compound
to be tested for an ability to prevent, treat, manage or ameliorate
schizophrenia and/or bipolar disorder or a symptom thereof is
identified. In accordance with this embodiment, the cell may
naturally express the biomarker or be engineered to express the
biomarker. In another embodiment, the present invention provides a
method for identifying a compound to be tested for an ability to
prevent, treat, manage or ameliorate schizophrenia and/or bipolar
disorder or a symptom thereof, said method comprising: (a)
contacting a compound with a cell-free extract and a reporter gene
construct comprising a reporter gene operably linked to a
regulatory element of a biomarker of the invention (e.g., a
promoter/enhancer element); (b) measuring the expression of said
reporter gene; and (c) comparing the amount in (a) to that present
in a corresponding control that has not been contacted with the
test compound, so that if the amount of expressed reporter gene is
altered relative to the amount in the control, a compound to be
tested for an ability to prevent, treat, manage or ameliorate
schizophrenia and/or bipolar disorder or a symptom thereof is
identified. Any reporter gene well-known to one of skill in the art
may be used in reporter gene constructs used in accordance with the
methods of the invention. Reporter genes refer to a nucleotide
sequence encoding a RNA transcript or protein that is readily
detectable either by its presence (by, e.g., RT-PCR, Northern blot,
Western Blot, ELISA, etc.) or activity. Non-limiting examples of
reporter genes are listed in Table 6, infra. Reporter genes may be
obtained and the nucleotide sequence of the elements determined by
any method well-known to one of skill in the art. The nucleotide
sequence of a reporter gene can be obtained, e.g., from the
literature or a database such as GenBank. Alternatively, a
polynucleotide encoding a reporter gene may be generated from
nucleic acid from a suitable source. If a clone containing a
nucleic acid encoding a particular reporter gene is not available,
but the sequence of the reporter gene is known, a nucleic acid
encoding the reporter gene may be chemically synthesized or
obtained from a suitable source (e.g., a cDNA library, or a cDNA
library generated from, or nucleic acid, preferably poly A+ RNA,
isolated from, any tissue or cells expressing the reporter gene) by
PCR amplification. Once the nucleotide sequence of a reporter gene
is determined, the nucleotide sequence of the reporter gene may be
manipulated using methods well-known in the art for the
manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate reporter genes having a different amino
acid sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
7TABLE 7 Reporter Genes and the Properties of the Reporter Gene
Products Reporter Gene Protein Activity & Measurement CAT
(chloramphenicol Transfers radioactive acetyl groups to
acetyltransferase) chloramphenicol or detection by thin layer
chromatography and autoradiography GAL (beta- Hydrolyzes colorless
galactosides to yield colored galactosidase) products. GUS (beta-
Hydrolyzes colorless glucuronides to yield glucuronidase) colored
products. LUC (luciferase) Oxidizes luciferin, emitting photons GFP
(green Fluorescent protein without substrate fluorescent protein)
SEAP (secreted Luminescence reaction with suitable substrates or
alkaline phosphatase) with substrates that generate chromophores
HRP (horseradish In the presence of hydrogen oxide, oxidation of
peroxidase) 3,3',5,5'-tetramethylbenzidine to form a colored
complex AP (alkaline Luminescence reaction with suitable substrates
or phosphatase) with substrates that generate chromophores
[0435] In accordance with the invention, cells that naturally or
normally express one or more, all or any combination of the
biomarkers of the invention can be used in the methods described
herein. Alternatively, cells can be engineered to express one or
more, all or any combination of the biomarkers of the invention, or
a reporter gene using techniques well-known in the art and used in
the methods described herein. Examples of such techniques include,
but are not to, calcium phosphate precipitation (see, e.g., Graham
& Van der Eb, 1978, Virol. 52:546), dextran-mediated
transfection, calcium phosphate mediated transfection, polybrene
mediated transfection, protoplast fusion, electroporation,
encapsulation of the nucleic acid in liposomes, and direct
microinjection of the nucleic acid into nuclei.
[0436] In a specific embodiment, the cells used in the methods
described herein are brain cells or cell lines, lymphocytes (T or B
lymphocytes), monocytes, neutrophils, macrophages, eosinophils,
basophils, erythrocytes or platelets. In a preferred embodiment,
the cells used in the methods described herein are brain cells. In
another preferred embodiment, the cells used in the methods
described herein are lymphocytes. In another embodiment, the cells
used in the methods described herein are immortalized cell lines
derived from a source, e.g., a tissue.
[0437] Any cell-free extract that permits the translation, and
optionally but preferably, the transcription, of a nucleic acid can
be used in accordance with the methods described herein. The
cell-free extract may be isolated from cells of any species origin.
For example, the cell-free translation extract may be isolated from
human cells, cultured mouse cells, cultured rat cells, Chinese
hamster ovary (CHO) cells, Xenopus oocytes, rabbit reticulocytes,
wheat germ, or rye embryo (see, e.g., Krieg & Melton, 1984,
Nature 308:203 and Dignam et al., 1990 Methods Enzymol.
182:194-203). Alternatively, the cell-free translation extract,
e.g., rabbit reticulocyte lysates and wheat germ extract, can be
purchased from, e.g., Promega, (Madison, Wis.). In a preferred
embodiment, the cell-free extract is an extract isolated from human
cells. In a specific embodiment, the human cells are HeLa cells,
lymphocytes, or brain cells or cell lines. In addition to the
ability to modulate the expression levels of RNA and/or protein
products a biomarker of the invention, it may be desirable, at
least in certain instances, that compounds modulate the activity of
a protein product of a biomarker of the invention. Thus, the
present invention provides methods of identifying compounds to be
tested for an ability to prevent, treat, manage or ameliorate
schizophrenia and/or bipolar disorder or a symptom thereof,
comprising methods for identifying compounds that modulate the
activity of a protein product of one or more biomarkers of the
invention. Such methods can comprise: (a) contacting a cell
expressing a protein product of one or more biomarkers of the
invention with a test compound; (b) determining the activity level
of the protein product; and (c) comparing the activity level to
that in a corresponding control cell that has not been contacted
with the test compound, so that if the level of activity in (a) is
altered relative to the level of activity in the control cell, a
compound to be tested for an ability to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof is identified. In a specific embodiment, the activity
level(s) is altered by 5%, 10%, 15%, 25%, 30%, 40%, 50%, 5 to 25%,
10 to 30%, at least 1 fold, at least 1.5 fold, at least 2 fold, 4
fold, 5 fold, 10 fold, 25 fold, 1 to 10 fold, or 5 to 25 fold
relative to the activity level in the control as determined by
utilizing an assay described herein (e.g., a microarray or RT-PCR)
or an assay well known to one of skill in the art. In alternate
embodiments, such a method comprises determining the activity level
of a protein product of at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 12, at least 15, at least 20, at least 25, 1 to 5,
1-10, 5-10, 5-25, or 10-40, all or any combination of the
biomarkers of the invention present in the cell and comparing the
activity levels to those present in the control.
[0438] The present invention provides methods of identifying
compounds to be tested for an ability to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof, comprising: (a) contacting a cell-free extract with a
nucleic acid encoding a protein product of one or more biomarkers
of the invention and a test compound; (b) determining the activity
level of the protein product; and (c) comparing the activity level
to that in a corresponding control that has not been contacted with
the test compound, so that if the level of activity in (a) is
altered relative to the level of activity in the control, a
compound to be tested for an ability to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof is identified. In a specific embodiment, the activity
level(s) is altered by 5%, 10%, 15%, 25%, 30%, 40%, 50%, 5 to 25%,
10 to 30%, at least 1 fold, at least 1.5 fold, at least 2 fold, 4
fold, 5 fold, 10 fold, 25 fold, 1 to 10 fold, or 5 to 25 fold
relative to the activity level in the control as determined by
utilizing an assay described herein (e.g., a microarray or RT-PCR)
or an assay well known to one of skill in the art. In alternate
embodiments, such a method comprises determining the activity level
of a protein product of at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, 1 to 3, 1 to 5, 1-7
all or any combination of the biomarkers of the invention present
in the sample and comparing the activity levels to those present in
the control.
[0439] Standard techniques can be utilized to determine the level
of activity of a protein product of a biomarker of the invention.
Activities of protein products of biomarkers of the invention that
can be determined using techniques well known in the art.
[0440] 5.15.2 Method to Utilize the Biological Activity of the
Compounds
[0441] Upon identification of compounds to be tested for an ability
to prevent, treat, manage or ameliorate schizophrenia and/or
bipolar disorder or a symptom thereof (for convenience referred to
herein as a "lead" compound), the compounds can be further
investigated. For example, the compounds identified via the present
methods can be further tested in vivo in accepted animal models of
schizophrenia and/or bipolar disorder. Further, the compounds
identified via the methods can be analyzed with respect to their
specificity. Techniques for such additional compound investigation
are well known to one of skill in the art.
[0442] In one embodiment, the effect of a lead compound can be
assayed by measuring the cell growth or viability of the target
cell. Such assays can be carried out with representative cells of
cell types involved in schizophrenia and/or bipolar disorder (e.g.,
brain cells; cells isolated from different portions of the brain;
stem cells and the like). Alternatively, instead of culturing cells
from a patient, a lead compound may be screened using cells of a
cell line. Many assays well-known in the art can be used to assess
the survival and/or growth of a patient cell or cell line following
exposure to a lead compound; for example, cell proliferation can be
assayed by measuring Bromodeoxyuridine (BrdU) incorporation (see,
e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al.,
1988, J. Immunol. Meth. 107:79) or (.sup.3H)-thymidine
incorporation (see, e.g., Chen, J., 1996, Oncogene 13:1395-403;
Jeoung, J., 1995, J. Biol. Chem. 270:18367-73), by direct cell
count, by detecting changes in transcription, translation or
activity of known genes such as proto-oncogenes (e.g., fos, myc) or
cell cycle markers (Rb, cdc2, cyclin A, D1, D2, D3, E, etc). The
levels of such protein and RNA (e.g., mRNA) and activity can be
determined by any method well known in the art. For example,
protein can be quantitated by known immunodiagnostic methods such
as Western blotting or immunoprecipitation using commercially
available antibodies. mRNA can be quantitated using methods that
are well known and routine in the art, for example, using northern
analysis, RNase protection, the polymerase chain reaction in
connection with the reverse transcription. Cell viability can be
assessed by using trypan-blue staining or other cell death or
viability markers known in the art. In a specific embodiment, the
level of cellular ATP is measured to determined cell viability.
Differentiation can be assessed, for example, visually based on
changes in morphology.
[0443] 5.15.3 Animal Models
[0444] Compounds can be tested in suitable animal model systems
prior to use in humans. Such animal model systems include but are
not limited to rats, mice, chicken, cows, monkeys, pigs, dogs,
rabbits, etc. Any animal system well-known in the art may be used.
In certain embodiments, compounds are tested in a mouse model.
Compounds can be administered repeatedly.
[0445] Accepted animal models can be utilized to determine the
efficacy of the compounds identified via the methods described
above for the prevention, treatment, management and/or amelioration
of schizophrenia and/or bipolar disorder or a symptom thereof.
[0446] .5.15.4 Toxicity
[0447] The toxicity and/or efficacy of a compound identified in
accordance with the invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). Cells and cell lines that can be used to
assess the cytotoxicity of a compound identified in accordance with
the invention include, but are not limited to, peripheral blood
mononuclear cells (PBMCs), Caco-2 cells, and Huh7 cells. The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD.sub.50/ED.sub.50. A
compound identified in accordance with the invention that exhibits
large therapeutic indices is preferred. While a compound identified
in accordance with the invention that exhibits toxic side effects
may be used, care should be taken to design a delivery system that
targets such agents to the site of affected tissue in order to
minimize potential damage to uninfected cells and, thereby, reduce
side effects.
[0448] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of a compound
identified in accordance with the invention for use in humans. The
dosage of such agents lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
any agent used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 (i.e., the
concentration of the compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0449] 5.15.5 Design of Congeners or Analogs
[0450] The compounds which display the desired biological activity
can be used as lead compounds for the development or design of
congeners or analogs having useful pharmacological activity. For
example, once a lead compound is identified, molecular modeling
techniques can be used to design variants of the compound that can
be more effective. Examples of molecular modeling systems are the
CHARM and QUANTA programs (Polygen Corporation, Waltham, Mass.).
CHARM performs the energy minimization and molecular dynamics
functions. QUANTA performs the construction, graphic modelling and
analysis of molecular structure. QUANTA allows interactive
construction, modification, visualization, and analysis of the
behavior of molecules with each other.
[0451] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen et al., 1988,
Acta Pharmaceutical Fennica 97:159-166; Ripka, 1998, New Scientist
54-57; McKinaly & Rossmann, 1989, Annu. Rev. Pharmacol.
Toxiciol. 29:111-122; Perry & Davies, OSAR: Quantitative
Structure-Activity Relationships in Drug Design pp. 189-193 (Alan
R. Liss, Inc. 1989); Lewis & Dean, 1989, Proc. R. Soc. Lond.
236:125-140 and 141-162; Askew et al., 1989, J. Am. Chem. Soc. 111:
1082-1090. Other computer programs that screen and graphically
depict chemicals are available from companies such as BioDesign,
Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario,
Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these
are primarily designed for application to drugs specific to
particular proteins, they can be adapted to design of drugs
specific to any identified region. The analogs and congeners can be
tested for binding to the proteins of interest (i.e., the protein
products of a biomarker of the invention) using the above-described
screens for biologic activity. Alternatively, lead compounds with
little or no biologic activity, as ascertained in the screen, can
also be used to design analogs and congeners of the compound that
have biologic activity.
[0452] 5.15.6 Compounds
[0453] Compounds that can be tested and identified methods
described herein can include, but are not limited to, compounds
obtained from any commercial source, including Aldrich (1001 West
St. Paul Ave., Milwaukee, Wis. 53233), Sigma Chemical (P.O. Box
14508, St. Louis, Mo. 63178), Fluka Chemie AG (Industriestrasse 25,
CH-9471 Buchs, Switzerland (Fluka Chemical Corp. 980 South 2nd
Street, Ronkonkoma, NY 11779)), Eastman Chemical Company, Fine
Chemicals (P.O Box 431, Kingsport, TN 37662), Boehringer Mannheim
GmbH (Sandhofer Strasse 116, D-68298 Mannheim), Takasago (4 Volvo
Drive, Rockleigh, N.J. 07647), SST Corporation (635 Brighton Road,
Clifton, N.J. 07012), Ferro (111 West Irene Road, Zachary, LA
70791), Riedel-deHaen Aktiengesellschaft (P.O. Box D-30918, Seelze,
Germany), PPG Industries Inc., Fine Chemicals (One PPG Place, 34th
Floor, Pittsburgh, Pa. 15272). Further any kind of natural products
may be screened using the methods of the invention, including
microbial, fungal, plant or animal extracts.
[0454] Compounds from large libraries of synthetic or natural
compounds can be screened. Numerous means are currently used for
random and directed synthesis of saccharide, peptide, and nucleic
acid-based compounds. Synthetic compound libraries are commercially
available from a number of companies including Maybridge Chemical
Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon
Associates (Merrimack, N.H.), and Microsource (New Milford, CT). A
rare chemical library is available from Aldrich (Milwaukee, Wis.).
Combinatorial libraries are available and are prepared.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available from
e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are
readily produceable by methods well known in the art. Additionally,
natural and synthetically produced libraries and compounds are
readily modified through conventional chemical, physical, and
biochemical means.
[0455] Furthermore, diversity libraries of test compounds,
including small molecule test compounds, may be utilized. Libraries
screened using the methods of the present invention can comprise a
variety of types of compounds. Examples of libraries that can be
screened in accordance with the methods of the invention include,
but are not limited to, peptoids; random biooligomers; diversomers
such as hydantoins, benzodiazepines and dipeptides; vinylogous
polypeptides; nonpeptidal peptidomimetics; oligocarbamates;
peptidyl phosphonates; peptide nucleic acid libraries; antibody
libraries; carbohydrate libraries; and small molecule libraries
(preferably, small organic molecule libraries). In some
embodiments, the compounds in the libraries screened are nucleic
acid or peptide molecules. In a non-limiting example, peptide
molecules can exist in a phage display library. In other
embodiments, the types of compounds include, but are not limited
to, peptide analogs including peptides comprising non-naturally
occurring amino acids, e.g., D-amino acids, phosphorous analogs of
amino acids, such as .alpha.-amino phosphoric acids and
.alpha.-amino phosphoric acids, or amino acids having non-peptide
linkages, nucleic acid analogs such as phosphorothioates and PNAs,
hormones, antigens, synthetic or naturally occurring drugs,
opiates, dopamine, serotonin, catecholamines, thrombin,
acetylcholine, prostaglandins, organic molecules, pheromones,
adenosine, sucrose, glucose, lactose and galactose. Libraries of
polypeptides or proteins can also be used in the assays of the
invention.
[0456] In a specific embodiment, the combinatorial libraries are
small organic molecule libraries including, but not limited to,
benzodiazepines, isoprenoids, thiazolidinones, metathiazanones,
pyrrolidines, morpholino compounds, and benzodiazepines. In another
embodiment, the combinatorial libraries comprise peptoids; random
bio-oligomers; benzodiazepines; diversomers such as hydantoins,
benzodiazepines and dipeptides; vinylogous polypeptides;
nonpeptidal peptidomimetics; oligocarbamates; peptidyl
phosphonates; peptide nucleic acid libraries; antibody libraries;
or carbohydrate libraries. Combinatorial libraries are themselves
commercially available For example, libraries may be commercially
obtained from, e.g., Specs and BioSpecs B.V. (Rijswijk, The
Netherlands), Chembridge Corporation (San Diego, Calif.), Contract
Service Company (Dolgoprudny, Moscow Region, Russia), Comgenex USA
Inc. (Princeton, N.J.), Maybridge Chemicals Ltd. (Cornwall PL34
OHW, United Kingdom), Asinex (Moscow, Russia), ComGenex (Princeton,
N.J.), Ru, Tripos, Inc. (St. Louis, Mo.), ChemStar, Ltd (Moscow,
Russia), 3D Pharmaceuticals (Exton, Pennsylvania), and Martek
Biosciences (Columbia, Md.).
[0457] In a preferred embodiment, the library is preselected so
that the compounds of the library are more amenable for cellular
uptake. For example, compounds are selected based on specific
parameters such as, but not limited to, size, lipophilicity,
hydrophilicity, and hydrogen bonding, which enhance the likelihood
of compounds getting into the cells. In another embodiment, the
compounds are analyzed by three-dimensional or four-dimensional
computer computation programs.
[0458] The combinatorial compound library for use in accordance
with the methods of the present invention may be synthesized. There
is a great interest in synthetic methods directed toward the
creation of large collections of small organic compounds, or
libraries, which could be screened for pharmacological, biological
or other activity. The synthetic methods applied to create vast
combinatorial libraries are performed in solution or in the solid
phase, i.e., on a support. Solid-phase synthesis makes it easier to
conduct multi-step reactions and to drive reactions to completion
with high yields because excess reagents can be easily added and
washed away after each reaction step. Solid-phase combinatorial
synthesis also tends to improve isolation, purification and
screening. However, the more traditional solution phase chemistry
supports a wider variety of organic reactions than solid-phase
chemistry. Combinatorial compound libraries of the present
invention may be synthesized using the apparatus described in U.S.
Pat. No. 6,190,619 to Kilcoin et al., which is hereby incorporated
by reference in its entirety. U.S. Pat. No. 6,190,619 discloses a
synthesis apparatus capable of holding a plurality of reaction
vessels for parallel synthesis of multiple discrete compounds or
for combinatorial libraries of compounds.
[0459] In one embodiment, the combinatorial compound library can be
synthesized in solution. The method disclosed in U.S. Pat. No.
6,194,612 to Boger et al., which is hereby incorporated by
reference in its entirety, features compounds useful as templates
for solution phase synthesis of combinatorial libraries. The
template is designed to permit reaction products to be easily
purified from unreacted reactants using liquid/liquid or
solid/liquid extractions. The compounds produced by combinatorial
synthesis using the template will preferably be small organic
molecules. Some compounds in the library may mimic the effects of
non-peptides or peptides. In contrast to solid phase synthesize of
combinatorial compound libraries, liquid phase synthesis does not
require the use of specialized protocols for monitoring the
individual steps of a multistep solid phase synthesis (Egner et
al., 1995, J. Org. Chem. 60:2652; Anderson et al., 1995, J. Org.
Chem. 60:2650; Fitch et al., 1994, J. Org. Chem. 59:7955; Look et
al., 1994, J. Org. Chem. 49:7588; Metzger et al., 1993, Angew.
Chem., Int. Ed. Engl. 32:894; Youngquist et al., 1994, Rapid
Commun. Mass Spect. 8:77; Chu et al., 1995, J. Am. Chem. Soc.
117:5419; Brummel et al., 1994, Science 264:399; and Stevanovic et
al., 1993, Bioorg. Med. Chem. Lett. 3:431).
[0460] Combinatorial compound libraries useful for the methods of
the present invention can be synthesized on supports. In one
embodiment, a split synthesis method, a protocol of separating and
mixing supports during the synthesis, is used to synthesize a
library of compounds on supports (see e.g., Lam et al., 1997, Chem.
Rev. 97:41-448; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA
90:10922-10926 and references cited therein). Each support in the
final library has substantially one type of compound attached to
its surface. Other methods for synthesizing combinatorial libraries
on supports, wherein one product is attached to each support, will
be known to those of skill in the art (see, e.g., Nefzi et al.,
1997, Chem. Rev. 97:449-472).
[0461] In some embodiments of the present invention, compounds can
be attached to supports via linkers. Linkers can be integral and
part of the support, or they may be nonintegral that are either
synthesized on the support or attached thereto after synthesis.
Linkers are useful not only for providing points of compound
attachment to the support, but also for allowing different groups
of molecules to be cleaved from the support under different
conditions, depending on the nature of the linker. For example,
linkers can be, inter alia, electrophilically cleaved,
nucleophilically cleaved, photocleavable, enzymatically cleaved,
cleaved by metals, cleaved under reductive conditions or cleaved
under oxidative conditions. In a preferred embodiment, the
compounds are cleaved from the support prior to high throughput
screening of the compounds.
[0462] If the library comprises arrays or microarrays of compounds,
wherein each compound has an address or identifier, the compound
can be deconvoluted, e.g., by cross-referencing the positive sample
to original compound list that was applied to the individual test
assays.
[0463] If the library is a peptide or nucleic acid library, the
sequence of the compound can be determined by direct sequencing of
the peptide or nucleic acid. Such methods are well known to one of
skill in the art.
[0464] A number of physico-chemical techniques can be used for the
de novo characterization of compounds. Examples of such techniques
include, but are not limited to, mass spectrometry, NMR
spectroscopy, X-ray crytallography and vibrational
spectroscopy.
5.16 Use of Identified Compounds to Prevent, Treat, Manage or
Ameliorate Schizophrenia and/or Bipolar Disorder or a Symptom
Thereof
[0465] The present invention provides methods of preventing,
treating, managing or ameliorating schizophrenia and/or bipolar
disorder or a symptom thereof, said methods comprising
administering to a subject in need thereof one or more compounds
identified in accordance with the methods of the invention. In
certain embodiments, the subject has mild, moderate, marked or
severe schizophrenia and/or bipolar disorder. In a preferred
embodiment, the subject is human. In one embodiment, the invention
provides a method of preventing, treating, managing or ameliorating
schizophrenia and/or bipolar disorder or a symptom thereof, said
method comprising administering to a subject in need thereof a dose
of a prophylactically or therapeutically effective amount of one or
more compounds identified in accordance with the methods of the
invention. In a specific embodiment, a compound identified in
accordance with the methods of the invention is not administered to
prevent, treat, or ameliorate schizophrenia and/or bipolar disorder
or a symptom thereof, if such compound has been used previously to
prevent, treat, manage or ameliorate schizophrenia and/or bipolar
disorder or a symptom thereof. In another embodiment, a compound
identified in accordance with the methods of the invention is not
administered to prevent, treat, or ameliorate schizophrenia and/or
bipolar disorder or a symptom thereof, if such compound has
suggested to be used to prevent, treat, manage or ameliorate
schizophrenia and/or bipolar disorder or a symptom thereof. In
another embodiment, a compound identified in accordance with the
methods of the invention specifically binds to and/or alters the
expression and/or activity level of a protein or RNA product of
only one biomarker of the invention. In another embodiment, a
compound identified in accordance with the methods of the invention
is not administered to prevent, treat, or ameliorate schizophrenia
and/or bipolar disorder or a symptom thereof, if such compound
binds to and/or alters the expression and/or activity of a protein
or RNA product of one, two, three, all or any combination of the
following biomarkers of Table 1. In yet another embodiment, a
compound identified in accordance with the methods of the invention
binds to and/or alters the expression and/or activity level of a
protein or RNA product of at least 2, at least 3, at least 4, at
least 5, at least 10, at least 15, at least 20, at least 25, a or
more biomarkers of the invention.
[0466] The invention also provides methods of preventing, treating,
managing or ameliorating schizophrenia and/or bipolar disorder or a
symptom thereof, said methods comprising administering to a subject
in need thereof one or more of the compounds identified utilizing
the screening methods described herein, and one or more other
therapies (e.g., prophylactic or therapeutic agents and surgery).
In a specific embodiment, such therapies are currently being used,
have been used or are known to be useful in the prevention,
treatment, management or amelioration of schizophrenia and/or
bipolar disorder or a symptom thereof (including, but not limited
to the prophylactic or therapeutic agents listed herein). The
therapies (e.g., prophylactic or therapeutic agents) of the
combination therapies of the invention can be administered
sequentially or concurrently. In a specific embodiment, the
combination therapies of the invention comprise a compound
identified in accordance with the invention and at least one other
therapy that has the same mechanism of action as said compound. In
another specific embodiment, the combination therapies of the
invention comprise a compound identified in accordance with the
methods of the invention and at least one other therapy (e.g.,
prophylactic or therapeutic agent) which has a different mechanism
of action than said compound. The combination therapies of the
present invention improve the prophylactic or therapeutic effect of
a compound of the invention by functioning together with the
compound to have an additive or synergistic effect. The combination
therapies of the present invention reduce the side effects
associated with the therapies (e.g., prophylactic or therapeutic
agents).
[0467] The prophylactic or therapeutic agents of the combination
therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, the prophylactic or
therapeutic agents of the combination therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
The prophylactic or therapeutic agents may be administered to a
subject by the same or different routes of administration.
[0468] In specific embodiment, a pharmaceutical composition
comprising one or more compounds identified in an assay described
herein is administered to a subject, preferably a human, to
prevent, treat, manage or ameliorate schizophrenia and/or bipolar
disorder or a symptom thereof. In accordance with the invention,
the pharmaceutical composition may also comprise one or more
prophylactic or therapeutic agents. Preferably, such agents are
currently being used, have been used or are known to be useful in
the prevention, treatment, management or amelioration of
schizophrenia and/or bipolar disorder or a symptom thereof.
[0469] A compound identified in accordance with the methods of the
invention may be used as a first, second, third, fourth or fifth
line of therapy for schizophrenia and/or bipolar disorder. The
invention provides methods for treating, managing or ameliorating
schizophrenia and/or bipolar disorder or a symptom thereof in a
subject refractory to conventional therapies for schizophrenia
and/or bipolar disorder, said methods comprising administering to
said subject a dose of a prophylactically or therapeutically
effective amount of a compound identified in accordance with the
methods of the invention.
[0470] The invention provides methods for treating, managing or
ameliorating schizophrenia and/or bipolar disorder or a symptom
thereof in a subject refractory to existing single agent therapies
for schizophrenia and/or bipolar disorder, said methods comprising
administering to said subject a dose of a prophylactically or
therapeutically effective amount of a compound identified in
accordance with the methods of the invention and a dose of a
prophylactically or therapeutically effective amount of one or more
other therapies (e.g., prophylactic or therapeutic agents). The
invention also provides methods for treating or managing a
schizophrenia and/or bipolar disorder by administering a compound
identified in accordance with the methods of the invention in
combination with any other therapy (e.g., surgery) to patients who
have proven refractory to other therapies but are no longer on
these therapies. The invention also provides methods for the
treatment or management of a patient having schizophrenia and/or
bipolar disorder and immunosuppressed by reason of having
previously undergone other therapies. The invention also provides
alternative methods for the treatment or management of
schizophrenia and/or bipolar disorder where hormonal therapy and/or
biological therapy/immunotherapy has proven or may prove too toxic,
i.e., results in unacceptable or unbearable side effects, for the
subject being treated or managed.
5.17 Compounds of the Invention
[0471] Representative, non-limiting examples of compounds that can
used in accordance with the methods of the invention to prevent,
treat, manage and/or ameliorate schizophrenia and/or bipolar
disorder or a symptom thereof are described in detail below.
[0472] First, such compounds can include, for example, antisense,
ribozyme, or triple helix compounds that can downregulate the
expression or activity of a protein or RNA product of a biomarker
of the invention. Such compounds are described in detail in the
subsection below.
[0473] Second, such compounds can include, for example, antibody
compositions that can modulate the expression of a protein or RNA
product of a biomarker of the invention, or the activity of a
protein product of a biomarker of the invention. In a specific
embodiment, the antibody compositions downregulate the expression a
protein or RNA product of a biomarker of the invention, or the
activity of a protein product of a biomarker of the invention. Such
compounds are described in detail in the subsection below.
[0474] Third, such compounds can include, for example, protein
products of a biomarker of the invention. The invention encompasses
the use of peptides or peptide mimetics selected to mimic a protein
product of a biomarker of the invention to prevent, treat, manage
or ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof. Further, such compounds can include, for example,
dominant-negative polypeptides that can modulate the expression a
protein or RNA product of a biomarker of the invention, or the
activity of a protein product of a biomarker of the invention.
[0475] The methods also encompasses the use derivatives, analogs
and fragments of a protein product of a biomarker of the invention
to prevent, treat, manage or ameliorate schizophrenia and/or
bipolar disorder or a symptom thereof. In particular, the invention
encompasses the use of fragments of a protein product of a
biomarker of the invention comprising one or more domains of such a
protein(s) to prevent, treat, manage or ameliorate schizophrenia
and/or bipolar disorder or a symptom thereof. In another specific
embodiment, the invention encompasses the use of a protein product
of a biomarker of the invention, or an analog, derivative or
fragment of such a protein which is expressed as a fusion, or
chimeric protein product (comprising the protein, fragment, analog,
or derivative joined via a peptide bond to a heterologous protein
sequence). In specific embodiments, an antisense oligonucleotide of
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
15, at least 20, at least 25, a, or more of biomarkers of the
invention are administered to prevent, treat, manage or ameliorate
schizophrenia and/or bipolar disorder or a symptom thereof. In
other embodiments, one or more of protein products of a biomarker
of the invention or a fragment, analog, or derivative thereof are
administered to prevent, treat, manage or ameliorate schizophrenia
and/or bipolar disorder or a symptom thereof. In other embodiment,
one or more antibodies that specifically bind to a protein product
of the invention are administered to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof. In other embodiments, one or more dominant-negative
polypeptides are administered to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof.
[0476] 5.17.1 Antisense, Ribozyme, Triple-Helix Compositions
[0477] Standard techniques can be utilized to produce antisense,
triple helix, or ribozyme molecules reactive to one or more of the
genes listed in Tables 1-4, and transcripts of the genes the genes
listed in Tables 1-4, for use as part of the methods described
herein. First, standard techniques can be utilized for the
production of antisense nucleic acid molecules, i.e., molecules
which are complementary to a sense nucleic acid encoding a
polypeptide of interest, e.g., complementary to the coding strand
of a double-stranded cDNA molecule or complementary to an mRNA
sequence. Accordingly, an antisense nucleic acid can hydrogen bond
to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire coding strand, or to only a portion
thereof, e.g., all or part of the protein coding region (or open
reading frame). An antisense nucleic acid molecule can be antisense
to all or part of a non-coding region of the coding strand of a
nucleotide sequence encoding a polypeptide of interest. The
non-coding regions ("5' and 3' untranslated regions") are the 5'
and 3' sequences that flank the coding region and are not
translated into amino acids.
[0478] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length.
An antisense nucleic acid of the invention can be constructed using
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
Antisense nucleic acid molecules administered to a subject or
generated in situ such that they hybridize with or bind to cellular
mRNA encoding the polypeptide of interest to thereby inhibit
expression, e.g., by inhibiting transcription and/or translation.
The hybridization can be by conventional nucleotide complementarity
to form a stable duplex, or, for example, in the case of an
antisense nucleic acid molecule which binds to DNA duplexes,
through specific interactions in the major groove of the double
helix. An example of a route of administration of antisense nucleic
acid molecules of the invention includes direct injection at a
tissue, e.g., a joint (e.g., a knee, hip, elbow, and knuckle),
site. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell, e.g., a T cell
or brain cell, surface, e.g., by linking the antisense nucleic acid
molecules to peptides or antibodies which bind to cell surface
receptors or antigens. The antisense nucleic acid molecules can
also be delivered to cells using vectors, e.g., gene therapy
vectors, described below. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[0479] An antisense nucleic acid molecule of interest can be an
a-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic acid
molecule forms specific double-stranded hybrids with complementary
RNA in which, contrary to the usual .alpha.-units, the strands run
parallel to each other (Gaultier et al., 1987, Nucleic Acids Res.
15:6625-6641). The antisense nucleic acid molecule can also
comprise a 2'-o-methylribonucleotide (Inoue et al., 1987, Nucleic
Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et
al., 1987, FEBS Lett. 215:327-330). Ribozymes are catalytic RNA
molecules with ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region, and can also be generated using standard
techniques. Thus, ribozymes (e.g., hammerhead ribozymes (described
in Haselhoff and Gerlach, 1988, Nature 334:585-591)) can be used to
catalytically cleave mRNA transcripts to thereby inhibit
translation of the protein encoded by the mRNA. A ribozyme having
specificity for a nucleic acid molecule encoding a polypeptide of
interest can be designed based upon the nucleotide sequence of a
cDNA disclosed herein. For example, a derivative of a Tetrahymena
L-19 IVS RNA can be constructed in which the nucleotide sequence of
the active site is complementary to the nucleotide sequence to be
cleaved in a Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.
U.S. Pat. No. 5,116,742. Alternatively, an mRNA encoding a
polypeptide of interest can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel and Szostak, 1993, Science
261:1411-1418.
[0480] Triple helical structures can also be generated using well
known techniques. For example, expression of a polypeptide of
interest can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the gene encoding the
polypeptide (e.g., the promoter and/or enhancer) to form triple
helical structures that prevent transcription of the gene in target
cells. See generally Helene, 1991, Anticancer Drug Des.
6(6):569-84; Helene, 1992, Ann. N.Y. Acad. Sci. 660:27-36; and
Maher, 1992, Bioassays 14(12):807-15.
[0481] In various embodiments, nucleic acid compositions can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve, e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids
(see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1):
5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al., 1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl.
Acad. Sci. USA 93: 14670-675. PNAs can, for example, be modified,
e.g., to enhance their stability or cellular uptake, by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras can
be generated which may combine the advantageous properties of PNA
and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H
and DNA polymerases, to interact with the DNA portion while the PNA
portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup, 1996,
supra, and Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63.
For example, a DNA chain can be synthesized on a support using
standard phosphoramidite coupling chemistry and modified nucleoside
analogs. Compounds such as
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite can be
used as a link between the PNA and the 5' end of DNA (Mag et al.,
1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled
in a stepwise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al., 1996, Nucleic Acids Res.
24(17):3357-63). Alternatively, chimeric molecules can be
synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et
al., 1975, Bioorganic Med. Chem. Lett. 5:1119-11124).
[0482] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. USA 84:648-652; International Publication No. WO 88/09810) or
the blood-brain barrier (see, e.g., International Publication No.
WO 89/10134). In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.,
1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g.,
Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide
may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent,
hybridization-triggered cleavage agent, etc.
[0483] 5.17.2 Antibody Compositions
[0484] In one embodiment, antibodies that specifically bind to one
or more protein products of one or more biomarkers of the invention
are administered to a subject, preferably a human, to prevent,
treat, manage or ameliorate schizophrenia and/or bipolar disorder
or a symptom thereof. In another embodiment, any combination of
antibodies that specifically bind to one or more protein products
of one or more biomarkers of the invention are administered to a
subject, preferably a human, to prevent, treat, manage or
ameliorate schizophrenia and/or bipolar disorder or a symptom
thereof. In a specific embodiment, one or more antibodies that
specifically bind to one or more protein products of one or more
biomarkers of the invention are administered to a subject,
preferably a human, in combination with other types of therapies
(e.g., NSAIDS) to prevent, treat, manage or ameliorate
schizophrenia and/or bipolar disorder or a symptom thereof. In
certain embodiments, antibodies known in the art that specifically
bind to one or more protein products of one or more biomarkers of
the invention are administered to a subject, preferably a human,
alone or in combination with other types of therapies (e.g.,
NSAIDS) to prevent, treat, manage or ameliorate schizophrenia
and/or bipolar disorder or a symptom thereof. In other embodiments,
antibodies known in the art that specifically bind to one or more
protein products of one or more biomarkers of the invention are not
administered to a subject, preferably a human, alone or in
combination with other types of therapies (e.g., NSAIDS) to
prevent, treat, manage or ameliorate schizophrenia and/or bipolar
disorder or a symptom thereof.
[0485] One or more antibodies that specifically bind to one or more
protein products of one or more biomarkers of the invention can be
administered to a subject, preferably a human, using various
delivery systems are known to those of skill in the art. For
example, such antibodies can be administered by encapsulation in
liposomes, microparticles or microcapsules. See, e.g., U.S. Pat.
No. 5,762,904, U.S. Pat. No. 6,004,534, and International
Publication No. WO 99/52563. In addition, such antibodies can be
administered using recombinant cells capable of expressing the
antibodies, or retroviral, other viral vectors or non-viral vectors
capable of expressing the antibodies.
[0486] Antibodies that specifically bind one or more protein
products of one or more biomarkers of the invention can be obtained
from any known source. For example, Table 5 provides a list of
commercially available antibodies specific for one or more of the
protein products of the biomarkers of the invention. Alternatively,
antibodies that specifically bind to one or more protein products
of one or more biomarkers of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0487] Antibodies include, but are not limited to, polyclonal
antibodies, monoclonal antibodies, bispecific antibodies,
multispecific antibodies, human antibodies, humanized antibodies,
camelised antibodies, chimeric antibodies, single-chain Fvs (scFv)
(see e.g., Bird et al. (1988) Science 242:423-426; and Huston et
al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883), single chain
antibodies, single domain antibodies, Fab fragments, F(ab')
fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above. The term "antibody", as used herein, refers to
immunoglobulin molecules and immunologically active fragments of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site. Immunoglobulin molecules can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2) or
subclass. Examples of immunologically active fragments of
immunoglobulin molecules include F(ab) fragments (a monovalent
fragment consisting of the VL, VH, CL and CH1 domains) and F(ab')2
fragments (a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region) which can be generated
by treating the antibody with an enzyme such as pepsin or papain.
Immunologically active fragments also include, but are not limited
to, Fd fragments (consisting of the VH and CH1 domains), Fv
fragments (consisting of the VL and VH domains of a single arm of
an antibody), dAb fragments (consisting of a VH domain; Ward et
al., (1989) Nature 341:544-546), and isolated complementarity
determining regions (CDRs). Antibodies that specifically bind to an
antigen can be produced by any method known in the art for the
synthesis of antibodies, in particular, by chemical synthesis or
preferably, by recombinant expression techniques.
[0488] Polyclonal antibodies that specifically bind to an antigen
can be produced by various procedures well-known in the art. For
example, a human antigen can be administered to various host
animals including, but not limited to, rabbits, mice, rats, etc. to
induce the production of sera containing polyclonal antibodies
specific for the human antigen. Various adjuvants may be used to
increase the immunological response, depending on the host species,
and include but are not limited to, Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and corynebacterium parvum. Such
adjuvants are also well known in the art.
[0489] The term "monospecific antibody" refers to an antibody that
displays a single binding specificity and affinity for a particular
target, e.g., epitope. This term includes monoclonal antibodies.
Monoclonal antibodies can be prepared using a wide variety of
techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. See, e.g., U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439,
4,411,993 and 4,196,265; Kennett et al (eds.), Monoclonal
Antibodies, Hybridomas: A New Dimension in Biological Analyses,
Plenum Press (1980); and Harlow and Lane (eds.), Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press (1988),
which are incorporated herein by reference. For example, monoclonal
antibodies can be produced using hybridoma techniques including
those known in the art and taught, for example, in Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies
and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said
references incorporated by reference in their entireties). Other
techniques that enable the production of antibodies through
recombinant techniques (e.g., techniques described by William D.
Huse et al., 1989, Science, 246: 1275-1281; L. Sastry et al., 1989,
Proc. Natl. Acad. Sci. USA, 86: 5728-5732; and Michelle Alting-Mees
et al., Strategies in Molecular Biology, 3: 1-9 (1990) involving a
commercial system available from Stratacyte, La Jolla, Calif.) may
also be utilized to construct monoclonal antibodies. The term
"monoclonal antibody" as used herein is not limited to antibodies
produced through hybridoma technology. The term "monoclonal
antibody" refers to an antibody that is derived from a single
clone, including any eukaryotic, prokaryotic, or phage clone, and
not the method by which it is produced.
[0490] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with a protein product of a
biomarker of the invention, and once an immune response is
detected, e.g., antibodies specific for the protein are detected in
the mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC. Hybridomas are selected and cloned by
limited dilution. Additionally, a RIMMS (repetitive immunization
multiple sites) technique can be used to immunize an animal
(Kilptrack et al., 1997, Hybridoma 16:381-9, incorporated by
reference in its entirety). The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0491] Accordingly, the present invention provides methods of
generating antibodies by culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with a protein product of a biomarker of the invention, with
myeloma cells and then screening the hybridomas resulting from the
fusion for hybridoma clones that secrete an antibody able to bind
to the protein or protein fragment.
[0492] Antibody fragments which recognize specific epitopes of a
protein product of a biomarker of the invention may be generated by
any technique known to those of skill in the art. For example, Fab
and F(ab')2 fragments of the invention may be produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes
such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2 fragments). F(ab')2 fragments contain the variable region,
the light chain constant region and the CH1 domain of the heavy
chain. Further, the antibodies of the present invention can also be
generated using various phage display methods known in the art. In
phage display methods, functional antibody domains are displayed on
the surface of phage particles which carry the polynucleotide
sequences encoding them. In particular, DNA sequences encoding VH
and VL domains are amplified from animal cDNA libraries (e.g.,
human or murine cDNA libraries of affected tissues). The DNA
encoding the VH and VL domains are recombined together with an scFv
linker by PCR and cloned into a phagemid vector. The vector is
electroporated in E. coli and the E. coli is infected with helper
phage. Phage used in these methods are typically filamentous phage
including fd and M13 and the VH and VL domains are usually
recombinantly fused to either the phage gene III or gene VIII.
Phage expressing an antigen binding domain that binds to a
particular antigen can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Examples of phage display methods that can be used
to make the antibodies of the present invention include those
disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50;
Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough
et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997,
Gene 187:9-18; Burton et al., 1994, Advances in Immunology
57:191-280; PCT Application No. PCT/GB91/O1 134; International
Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO
92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO 97/13844;
and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717,
5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637,
5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0493] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in
International Publication No. WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and
Better et al., 1988, Science 240:1041-1043 (said references
incorporated by reference in their entireties).
[0494] To generate whole antibodies, PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques known to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a VH constant region, e.g., the
human gamma 4 constant region, and the PCR amplified VL domains can
be cloned into vectors expressing a VL constant region, e.g., human
kappa or lamba constant regions. Preferably, the vectors for
expressing the VH or VL domains comprise an EF-1.alpha. promoter, a
secretion signal, a cloning site for the variable domain, constant
domains, and a selection marker such as neomycin. The VH and VL
domains may also cloned into one vector expressing the necessary
constant regions. The heavy chain conversion vectors and light
chain conversion vectors are then co-transfected into cell lines to
generate stable or transient cell lines that express full-length
antibodies, e.g., IgG, using techniques known to those of skill in
the art. For some uses, including in vivo use of antibodies in
humans and in vitro detection assays, it may be preferable to use
human or chimeric antibodies. Completely human antibodies are
particularly desirable for therapeutic treatment of human subjects.
Human antibodies can be made by a variety of methods known in the
art including phage display methods described above using antibody
libraries derived from human immunoglobulin sequences. See also
U.S. Pat. Nos. 4,444,887 and 4,716,111; and International
Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO
98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which
is incorporated herein by reference in its entirety.
[0495] Antibodies can also be produced by a transgenic animal. In
particular, human antibodies can be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the J.sub.H
region prevents endogenous antibody production. The modified
embryonic stem cells are expanded and microinjected into
blastocysts to produce chimeric mice. The chimeric mice are then be
bred to produce homozygous offspring which express human
antibodies. The transgenic mice are immunized in the normal fashion
with a selected antigen, e.g., all or a portion of a polypeptide of
the invention. Monoclonal antibodies directed against the antigen
can be obtained from the immunized, transgenic mice using
conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., International Publication
Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos.
5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806,
5,814,318, and 5,939,598, which are incorporated by reference
herein in their entirety. In addition, companies such as Abgenix,
Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0496] U.S. Pat. No. 5,849,992, for example, describes a method of
expressing an antibody in the mammary gland of a transgenic mammal.
A transgene is constructed that includes a milk-specific promoter
and nucleic acids encoding the antibody of interest and a signal
sequence for secretion. The milk produced by females of such
transgenic mammals includes, secreted-therein, the antibody of
interest. The antibody can be purified from the milk, or for some
applications, used directly.
[0497] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different immunoglobulin
molecules. Methods for producing chimeric antibodies are known in
the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al.,
1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.
Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,
4,816,397, and 6,331,415, which are incorporated herein by
reference in their entirety. A humanized antibody is an antibody or
its variant or fragment thereof which is capable of binding to a
predetermined antigen and which comprises a framework region having
substantially the amino acid sequence of a human immunoglobulin and
a CDR having substantially the amino acid sequence of a non-human
immuoglobulin. A humanized antibody comprises substantially all of
at least one, and typically two, variable domains (Fab, Fab',
F(ab').sub.2, Fabc, Fv) in which all or substantially all of the
CDR regions correspond to those of a non-human immunoglobulin
(i.e., donor antibody) and all or substantially all of the
framework regions are those of a human immunoglobulin consensus
sequence. Preferably, a humanized antibody also comprises at least
a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin. Ordinarily, the antibody will contain
both the light chain as well as at least the variable domain of a
heavy chain. The antibody also may include the CH1, hinge, CH2,
CH3, and CH4 regions of the heavy chain. The humanized antibody can
be selected from any class of immunoglobulins, including IgM, IgG,
IgD, IgA and IgE, and any isotype, including IgG.sub.1, IgG.sub.2,
IgG.sub.3 and IgG.sub.4. Usually the constant domain is a
complement fixing constant domain where it is desired that the
humanized antibody exhibit cytotoxic activity, and the class is
typically IgG.sub.1. Where such cytotoxic activity is not
desirable, the constant domain may be of the IgG.sub.2 class. The
humanized antibody may comprise sequences from more than one class
or isotype, and selecting particular constant domains to optimize
desired effector functions is within the ordinary skill in the art.
The framework and CDR regions of a humanized antibody need not
correspond precisely to the parental sequences, e.g., the donor CDR
or the consensus framework may be mutagenized by substitution,
insertion or deletion of at least one residue so that the CDR or
framework residue at that site does not correspond to either the
consensus or the import antibody. Such mutations, however, will not
be extensive. Usually, at least 75% of the humanized antibody
residues will correspond to those of the parental FR and CDR
sequences, more often 90%, and most preferably greater than 95%.
Humanized antibody can be produced using variety of techniques
known in the art, including but not limited to, CDR-grafting
(European Patent No. EP 239,400; International Publication No. WO
91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089),
veneering or resurfacing (European Patent Nos. EP 592,106 and EP
519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498;
Studnicka et al., 1994, Protein Engineering 7(6):805-814; and
Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S.
Patent No. 5,565,332), and techniques disclosed in, e.g., U.S. Pat.
No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, Tan et al.,
2002, J. Immunol. 169:1119-25, Caldas et al., 2000, Protein Eng.
13(5):353-60, Morea et al., 2000, Methods 20(3):267-79, Baca et
al., 1997, J. Biol. Chem. 272(16):10678-84, Roguska et al., 1996,
Protein Eng. 9(10):895-904, Couto et al., 1995, Cancer Res. 55 (23
Supp):5973s-5977s, Couto et al., 1995, Cancer Res. 55(8):1717-22,
Sandhu J S, 1994, Gene 150(2):409-10, and Pedersen et al., 1994, J.
Mol. Biol. 235(3):959-73. Often, framework residues in the
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and
Riechmann et al., 1988, Nature 332:323, which are incorporated
herein by reference in their entireties.)
[0498] Single domain antibodies, for example, antibodies lacking
the light chains, can be produced by methods well-known in the art.
See Riechmann et al., 1999, J. Immuno. 231:25-38; Nuttall et al.,
2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J.
Biotechnol. 74(4):277302; U.S. Pat. No. 6,005,079; and
International Publication Nos. WO 94/04678, WO 94/25591, and WO
01/44301, each of which is incorporated herein by reference in its
entirety. Further, the antibodies that specifically bind to an
antigen can, in turn, be utilized to generate anti-idiotype
antibodies that "mimic" an antigen using techniques well known to
those skilled in the art. (See, e.g., Greenspan & Bona, 1989,
FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol.
147(8):2429-2438). Such antibodies can be used, alone or in
combination with other therapies, in the prevention, treatment,
management or amelioration of schizophrenia and/or bipolar disorder
or a symptom thereof.
[0499] The invention encompasses polynucleotides comprising a
nucleotide sequence encoding an antibody or fragment thereof that
specifically binds to an antigen. The invention also encompasses
polynucleotides that hybridize under high stringency, intermediate
or lower stringency hybridization conditions to polynucleotides
that encode an antibody of the invention. The polynucleotides may
be obtained, and the nucleotide sequence of the polynucleotides
determined, by any method known in the art. The nucleotide
sequences encoding known antibodies can be determined using methods
well known in the art, i.e., nucleotide codons known to encode
particular amino acids are assembled in such a way to generate a
nucleic acid that encodes the antibody. Such a polynucleotide
encoding the antibody may be assembled from chemically synthesized
oligonucleotides (e.g., as described in Kutmeier et al., 1994,
BioTechniques 17:242), which, briefly, involves the synthesis of
overlapping oligonucleotides containing portions of the sequence
encoding the antibody, fragments, or variants thereof, annealing
and ligating of those oligonucleotides, and then amplification of
the ligated oligonucleotides by PCR.
[0500] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0501] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described
in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual,
2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and
Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY, which are both incorporated by reference
herein in their entireties), to generate antibodies having a
different amino acid sequence, for example to create amino acid
substitutions, deletions, and/or insertions.
[0502] Once a polynucleotide encoding an antibody molecule, heavy
or light chain of an antibody, or fragment thereof (preferably, but
not necessarily, containing the heavy or light chain variable
domain) of the invention has been obtained, the vector for the
production of the antibody molecule may be produced by recombinant
DNA technology using techniques well-known in the art.
[0503] In one preferred embodiment, monoclonal antibodies are
produced in mammalian cells. Preferred mammalian host cells for
expressing the clone antibodies or antigen-binding fragments
thereof include Chinese Hamster Ovary (CHO cells) (including
dhfr-CHO cells, described in Urlaub and Chasin (1980, Proc. Natl.
Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker,
e.g., as described in Kaufmnan and Sharp (1982, Mol. Biol.
159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells and
SP2 cells, COS cells, and a cell from a transgenic animal, e.g., a
transgenic mammal. For example, the cell is a mammary epithelial
cell. In addition to the nucleic acid sequence encoding the
diversified immunoglobulin domain, the recombinant expression
vectors may carry additional sequences, such as sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0504] In an exemplary system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr.sup.-
CHO cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to enhancer/promoter regulatory
elements (e.g., derived from SV40, CMV, adenovirus and the like,
such as a CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the antibody heavy and light
chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. For example, some antibodies can be isolated by
affinity chromatography with a Protein A or Protein G.
[0505] For antibodies that include an Fc domain, the antibody
production system preferably synthesizes antibodies in which the Fc
region is glycosylated. For example, the Fc domain of IgG molecules
is glycosylated at asparagine 297 in the CH2 domain. This
asparagine is the site for modification with biantennary-type
oligosaccharides. It has been demonstrated that this glycosylation
is required for effector functions mediated by Fc.gamma. receptors
and complement C1q (Burton and Woof, 1992, Adv. Immunol. 51 :1-84;
Jefferis et al., 1998, Immunol. Rev. 163:59-76). In a preferred
embodiment, the Fc domain is produced in a mammalian expression
system that appropriately glycosylates the residue corresponding to
asparagine 297. The Fc domain can also include other eukaryotic
post-translational modifications.
[0506] Once an antibody molecule has been produced by recombinant
expression, it may be purified by any method known in the art for
purification of an immunoglobulin molecule, for example, by
chromatography (e.g., ion exchange, affinity, particularly by
affinity for the specific antigen after Protein A, and sizing
column chromatography), centrifugation, differential solubility, or
by any other standard technique for the purification of proteins.
Further, the antibodies or fragments thereof may be fused to
heterologous polypeptide sequences known in the art to facilitate
purification.
[0507] 5.17.3 Gene Therapy Techniques
[0508] Gene therapy refers to therapy performed by the
administration to a subject of an expressed or expressible nucleic
acid. Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0509] In specific embodiments, one or more antisense
oligonucleotides for one or more biomarkers of the invention are
administered to prevent, treat, manage or ameliorate schizophrenia
and/or bipolar disorder or a symptom thereof, by way of gene
therapy. In other embodiments, one or more nucleic acid molecules
comprising nucleotides encoding one or more antibodies that
specifically bind to one or more protein products of one or more
biomarkers of the invention are administered to prevent, treat,
manage or ameliorate schizophrenia and/or bipolar disorder or a
symptom thereof, by way of gene therapy. In other embodiments, one
or more nucleic acid molecules comprising nucleotides encoding
protein products of one or more biomarkers of the invention or
analogs, derivatives or fragments thereof, are administered to
prevent, treat, manage or ameliorate schizophrenia and/or bipolar
disorder or a symptom thereof, by way of gene therapy. In yet other
embodiments, one or more nucleic acid molecules comprising
nucleotides encoding one or more dominant-negative polypeptides of
one or more protein products of one or more biomarker of the
invention are administered to prevent, treat, manage or ameliorate
schizophrenia and/or bipolar disorder or a symptom thereof, by way
of gene therapy. For general reviews of the methods of gene
therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505;
Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev.
Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science
260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly
known in the art of recombinant DNA technology which can be used
are described in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0510] In one aspect, a composition of the invention comprises
nucleic acid sequences encoding one or more antibodies that
specifically bind to one or more protein products of one or more
biomarkers of the invention, said nucleic acid sequences being part
of expression vectors that express one or more antibodies in a
suitable host. In particular, such nucleic acid sequences have
promoters operably linked to the antibodies, said promoter being
inducible or constitutive, and, optionally, tissue-specific.
[0511] In another aspect, a composition of the invention comprises
nucleic acid sequences encoding dominant-negative polypeptides of
one or protein products of one or more biomarkers of the invention,
said nucleic acid sequences being part of expression vectors that
express dominant-negative polypeptides in a suitable host. In
particular, such nucleic acid sequences have promoters operably
linked to the dominant-negative polypeptides, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the dominant-negative coding sequences and any other desired
sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the dominant-negative nucleic acids
(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
[0512] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy. In a
specific embodiment, the nucleic acid sequence is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that they
become intracellular, e.g., by infection using defective or
attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., International Publication
Nos. WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated
Dec. 23, 1992 (Wilson et al.); WO 92/20316 dated Nov. 26, 1992
(Findeis et al.); WO 93/14188 dated Jul. 22, 1993 (Clarke et al.),
WO 93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the
nucleic acid can be introduced intracellularly and incorporated
within host cell DNA for expression, by homologous recombination
(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
[0513] For example, a retroviral vector can be used. These
retroviral vectors have been modified to delete retroviral
sequences that are not necessary for packaging of the viral genome
and integration into host cell DNA. The nucleic acid sequences
encoding the antibodies of interest, or proteins of interest or
fragments thereof to be used in gene therapy are cloned into one or
more vectors, which facilitates delivery of the gene into a
patient. More detail about retroviral vectors can be found in
Boesen et al., 1994, Biotherapy 6:291-302, which describes the use
of a retroviral vector to deliver the mdr1 gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest.
93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and
Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and
Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
[0514] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang, et al., 1995, Gene Therapy
2:775-783. In a preferred embodiment, adenovirus vectors are
used.
[0515] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146). Another approach to gene
therapy involves transferring a gene to cells in tissue culture by
such methods as electroporation, lipofection, calcium phosphate
mediated transfection, or viral infection. Usually, the method of
transfer includes the transfer of a selectable marker to the cells.
The cells are then placed under selection to isolate those cells
that have taken up and are expressing the transferred gene. Those
cells are then delivered to a patient.
[0516] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et
al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0517] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) and/or
schizophrenia and/or bipolar disorder cells are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0518] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, schizophrenia and/or bipolar
disorder cells, fibroblasts, muscle cells, hepatocytes; blood cells
such as T lymphocytes, B lymphocytes, monocytes, macrophages,
neutrophils, eosinophils, megakaryocytes, granulocytes; various
stem or progenitor cells, in particular hematopoietic stem or
progenitor cells, e.g., as obtained from bone marrow, umbilical
cord blood, peripheral blood, fetal liver, etc.
[0519] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0520] In one embodiment in which recombinant cells are used in
gene therapy, nucleic acid sequences encoding antibodies of
interest, or proteins of interest or fragments thereof are
introduced into the cells such that they are expressible by the
cells or their progeny, and the recombinant cells are then
administered in vivo for therapeutic effect. In a specific
embodiment, stem or progenitor cells are used. Any stem and/or
progenitor cells which can be isolated and maintained in vitro can
potentially be used in accordance with this embodiment of the
present invention (see, e.g., International Publication No. WO
94/08598, dated Apr. 28, 1994; Stemple and Anderson, 1992, Cell
71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow
and Scott, 1986, Mayo Clinic Proc. 61:771).
[0521] Promoters that may be used to control the expression of
nucleic acid sequences encoding antibodies of interest, proteins of
interest or fragments thereof may be constitutive, inducible or
tissue-specific. Non-limiting examples include the SV40 early
promoter region (Bemoist and Chambon, 1981, Nature 290:304-310),
the promoter contained in the 3' long terminal repeat of Rous
sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes
thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.
Sci. USA 78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:39-42);
prokaryotic expression vectors such as the P-lactamase promoter
(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA
75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc.
Natl. Acad. Sci. USA 80:21-25); see also "Useful proteins from
recombinant bacteria" in Scientific American, 1980, 242:74-94;
plant expression vectors comprising the nopaline synthetase
promoter region (Herrera-Estrella et al., Nature 303:209-213) or
the cauliflower mosaic virus .sup.35S RNA promoter (Gardner et al.,
1981, Nucl. Acids Res. 9:2871), and the promoter of the
photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter
elements from yeast or other fingi such as the Gal 4 promoter, the
ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following animal
transcriptional control regions, which exhibit tissue specificity
and have been utilized in transgenic animals: elastase I gene
control region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Omitz et al., 1986, Cold Spring Harbor
Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology
7:425-515); insulin gene control region which is active in
pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region which is active in lymphoid
cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,
1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.
7:1436-1444), mouse mammary tumor virus control region which is
active in testicular, breast, lymphoid and mast cells (Leder et
al., 1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al.,
1987, Science 235:53-58; alpha 1-antitrypsin gene control region
which is active in the liver (Kelsey et al., 1987, Genes and Devel.
1:161-171), beta-globin gene control region which is active in
myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et
al., 1986, Cell 46:89-94; myelin basic protein gene control region
which is active in oligodendrocyte cells in the brain (Readhead et
al., 1987, Cell 48:703-712); myosin light chain-2 gene control
region which is active in skeletal muscle (Sani, 1985, Nature
314:283-286), and gonadotropic releasing hormone gene control
region which is active in the hypothalamus (Mason et al., 1986,
Science 234:1372-1378).
[0522] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
5.18 Pharmaceutical Compositions
[0523] Biologically active compounds identified using the methods
of the invention or a pharmaceutically acceptable salt thereof can
be administered to a patient, preferably a mammal, more preferably
a human, suffering from schizophrenia or bipolar disorder. In a
specific embodiment, a compound or pharmaceutically acceptable salt
thereof is administered to a patient, preferably a mammal, more
preferably a human, suffering from schizophrenia or bipolar
disorder:. In another embodiment, a compound or a pharmaceutically
acceptable salt thereof is administered to a patient, preferably a
mammal, more preferably a human, as a preventative measure against
schizophrenia and/or bipolar disorder. In accordance with these
embodiments, the patient may be a child, an adult or elderly,
wherein a "child" is a subject between the ages of 24 months of age
and 18 years of age, an "adult" is a subject 18 years of age or
older, and "elderly" is a subject 65 years of age or older.
[0524] When administered to a patient, the compound or a
pharmaceutically acceptable salt thereof is preferably administered
as component of a composition that optionally comprises a
pharmaceutically acceptable vehicle. The composition can be
administered orally, or by any other convenient route, for example,
by infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal
mucosa, etc.) and may be administered together with another
biologically active agent. Administration can be systemic or local.
Various delivery systems are known, e.g., encapsulation in
liposomes, microparticles, microcapsules, capsules, etc., and can
be used to administer the compound and pharmaceutically acceptable
salts thereof.
[0525] Methods of administration include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, oral, sublingual, intranasal,
intracerebral, intravaginal, transdermal, rectally, by inhalation,
or topically, particularly to the ears, nose, eyes, or skin. The
mode of administration is left to the discretion of the
practitioner. In most instances, administration will result in the
release of the compound or a pharmaceutically acceptable salt
thereof into the bloodstream.
[0526] In specific embodiments, it may be desirable to administer
the compound or a pharmaceutically acceptable salt thereof locally.
This may be achieved, for example, and not by way of limitation, by
local infusion during surgery, topical application, e.g., in
conjunction with a wound dressing after surgery, by injection, by
means of a catheter, by means of a suppository, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as sialastic membranes, or
fibers. In a specific embodiment, a compound is administered
locally to one or more sections of the brain affected by
schizophrenia and/or bipolar disorder.
[0527] In certain embodiments, it may be desirable to introduce the
compound or a pharmaceutically acceptable salt thereof into the
central nervous system by any suitable route, including
intraventricular, intrathecal and epidural injection.
Intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0528] Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent, or via perfusion in a fluorocarbon or synthetic pulmonary
surfactant. In certain embodiments, the compound and
pharmaceutically acceptable salts thereof can be formulated as a
suppository, with traditional binders and vehicles such as
triglycerides. In another embodiment, the compound and
pharmaceutically acceptable salts thereof can be delivered in a
vesicle, in particular a liposome (see Langer, 1990, Science
249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.).
[0529] In yet another embodiment, the compound and pharmaceutically
acceptable salts thereof can be delivered in a controlled release
system (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra, vol. 2, pp. 115-138 (1984)). Other
controlled-release systems discussed in the review by Langer, 1990,
Science 249:1527-1533 may be used. In one embodiment, a pump may be
used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989,
N. Engl. J. Med. 321:574). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see
also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.
Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet
another embodiment, a controlled-release system can be placed in
proximity of a target RNA of the compound or a pharmaceutically
acceptable salt thereof, thus requiring only a fraction of the
systemic dose.
[0530] The compounds described herein can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the active compound and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifingal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions. The invention includes
methods for preparing pharmaceutical compositions for modulating
the expression or activity of a polypeptide or nucleic acid of
interest. Such methods comprise formulating a pharmaceutically
acceptable carrier with an agent that modulates expression or
activity of a polypeptide or nucleic acid of interest. Such
compositions can further include additional active agents. Thus,
the invention further includes methods for preparing a
pharmaceutical composition by formulating a pharmaceutically
acceptable carrier with an agent that modulates expression or
activity of a polypeptide or nucleic acid of interest and one or
more additional active compounds.
[0531] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Intravenous administration is preferred. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0532] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF; Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0533] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide or antibody)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0534] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
[0535] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0536] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[0537] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0538] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0539] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0540] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0541] For antibodies, the preferred dosage is 0.1 mg/kg to 100
mg/kg of body weight (more preferably, 0.1 to 20 mg/kg, 0.1-10
mg/kg, or 0.1 to to 1.0 mg/kg). If the antibody is to act in the
brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.
Generally, partially human antibodies and fully human antibodies
have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al. (1997, J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14:193).
[0542] In a specific embodiment, an effective amount of protein or
polypeptide (i.e., an effective dosage) ranges from about 0.001 to
30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body
weight, more preferably about 0.1 to 20 mg/kg body weight, and even
more preferably about 0.1 to 1.0 mg/kg, 1 to 10 mg/kg, 2 to 9
mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
[0543] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a protein,
polypeptide, or antibody can include a single treatment or,
preferably, can include a series of treatments.
[0544] In addition to those compounds described above, the present
invention encompasses the use of small molecules that modulate
expression or activity of a nucleic acid or polypeptide of
interest. Non-limiting examples of small molecules include
peptides, peptidomimetics, amino acids, amino acid analogs,
polynucleotides, polynucleotide analogs, nucleotides, nucleotide
analogs, organic or inorganic compounds (i.e,. including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0545] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the ken of the
ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention. Exemplary doses
include milligram or microgram amounts of the small molecule per
kilogram of subject or sample weight (e.g., about 1 microgram per
kilogram to about 500 milligrams per kilogram, about 100 micrograms
per kilogram to about 5 milligrams per kilogram, or about 1
microgram per kilogram to about 50 micrograms per kilogram). It is
furthermore understood that appropriate doses of a small molecule
depend upon the potency of the small molecule with respect to the
expression or activity to be modulated. Such appropriate doses may
be determined using the assays described herein. When one or more
of these small molecules is to be administered to a subject (e.g.,
a human) in order to modulate expression or activity of a
polypeptide or nucleic acid of the invention, a physician,
veterinarian, or researcher may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. In addition, it is
understood that the specific dose level for any particular animal
subject will depend upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degree of expression or activity to
be modulated.
[0546] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
5.18 Kits
[0547] The present invention provides kits for measuring the
expression of the protein and RNA products of at least 1, at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 12, aat least 15, alt
least 20, at least 25, or all or any combination of the biomarkers
of the invention. Such kits comprise materials and reagents
required for measuring the expression of such protein and RNA
products. In specific embodiments, the kits may further comprise
one or more additional reagents employed in the various methods,
such as: (1) reagents for purifying RNA from blood; (2) primers for
generating test nucleic acids; (3) dNTPs and/or rNTPs (either
premixed or separate), optionally with one or more uniquely labeled
dNTPs and/or rNTPs (e.g. biotinylated or Cy3 or Cy5 tagged dNTPs);
(4) post synthesis labeling reagents, such as chemically active
derivatives of fluorescent dyes; (5) enzymes, such as reverse
transcriptases, DNA polymerases, and the like; (6) various buffer
mediums, e.g., hybridization and washing buffers; (7) labeled probe
purification reagents and components, like spin columns, etc.; and
(8) protein purification reagents; (9) signal generation and
detection reagents, e.g., streptavidin-alkaline phosphatase
conjugate, chemifluorescent or chemiluminescent substrate, and the
like. In particular embodiments, the kits comprise prelabeled
quality controlled protein and or RNA isolated from a sample (e.g.,
blood) for use as a control.
[0548] In some embodiments, the kits are RT-PCR kits. In other
embodiments, the kits are nucleic acid arrays and protein arrays.
Such kits according to the subject invention will at least comprise
an array having associated protein or nucleic acid members of the
invention and packaging means therefore. Alternatively the protein
or nucleic acid members of the invention may be prepackaged onto an
array.
[0549] In some embodiments, the kits are Quantitative RT-PCR kits.
In one embodiment, the quantitative RT-PCR kit includes the
following: (a) primers used to amplify each of a combination of
biomarkers of the invention; (b) buffers and enzymes including an
reverse transcripate; (c) one or more thermos table polymerases;
and (d) Sybr.RTM. Green. In a preferred embodiment, the kit of the
invention also includes (a) a reference control RNA and (b) a
spiked control RNA.
[0550] The invention provides kits that are useful for diagnosing
schizophrenia and/or bipolar disorder. For example, in a particular
embodiment of the invention a kit is comprised a forward and
reverse primer wherein the forward and reverse primer are designed
to quantitate expression of all of the species of mRNA
corresponding to each of the biomarkers as identified in Table 2.
In certain embodiments, at least one of the primers is designed to
span an exon junction.
[0551] The invention provides kits that are useful for detecting,
diagnosing, monitoring and prognosing schizophrenia and/or bipolar
disorder based upon the expression of protein or RNA products of at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, or all or any combination of the biomarkers of the
invention in a sample. In certain embodiments, such kits do not
include the materials and reagents for measuring the expression of
a protein or RNA product of a biomarker of the invention that has
been suggested by the prior art to be associated with schizophrenia
and/or bipolar disorder. In other embodiments, such kits include
the materials and reagents for measuring the expression of a
protein or RNA product of a biomarker of the invention that has
been suggested by the prior art to be associated with schizophrenia
and/or bipolar disorder and at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, or more genes other
than the biomarkers of the invention.
[0552] The invention provides kits useful for monitoring the
efficacy of one or more therapies that a subject is undergoing
based upon the expression of a protein or RNA product of at least
1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, or all or any combination of the biomarkers of the
invention in a sample. In certain embodiments, such kits do not
include the materials and reagents for measuring the expression of
a protein or RNA product of a biomarker of the invention that has
been suggested by the prior art to be associated with schizophrenia
and/or bipolar disorder. In other embodiments, such kits include
the materials and reagents for measuring the expression of a
protein or RNA product of a biomarker of the invention that has
been suggested by the prior art to be associated with schizophrenia
and/or bipolar disorder and at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, or more genes other
than the biomarkers of the invention.
[0553] The invention provides kits using for determining whether a
subject will be responsive to a therapy based upon the expression
of a protein or RNA product of at least 1, at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, or all or any
combination of the biomarkers of the invention in a sample. In
certain embodiments, such kits do not include the materials and
reagents for measuring the expression of a protein or RNA product
of a biomarker of the invention that has been suggested by the
prior art to be associated with schizophrenia and/or bipolar
disorder. In other embodiments, such kits include the materials and
reagents for measuring the expression of a protein or RNA product
of a biomarker of the invention that has been suggested by the
prior art to be associated with schizophrenia and/or bipolar
disorder and at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, or more genes other than the
biomarkers of the invention.
[0554] The invention provides kits for measuring the expression of
a RNA product of at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, or all or any combination of the
biomarkers of the invention in a sample. In a specific embodiment,
such kits comprise materials and reagents that are necessary for
measuring the expression of a RNA product of a biomarker of the
invention. For example, a microarray or RT-PCR kit may be produced
for schizophrenia and/or bipolar disorder and contain only those
reagents and materials necessary for measuring the levels of RNA
products of at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, or all or any combination of the
biomarkers of the invention. Alternatively, in some embodiments,
the kits can comprise materials and reagents that are not limited
to those required to measure the levels of RNA products of 1, 2, 3,
4, 5, 6, 7 or all or any combination of the biomarkers of the
invention. For example, a microarray kit may contain reagents and
materials necessary for measuring the levels of RNA products of not
necessarily associated with or indicative of schizophrenia and/or
bipolar disorder, in addition to reagents and materials necessary
for measuring the levels of the RNA products of at least 1, at
least 2, at least 3, at least 4, at least 5, at least 6, at least
7, or all or any combination of the biomarkers of the invention. In
a specific embodiment, a microarray or RT-PCR kit contains reagents
and materials necessary for measuring the levels of RNA products of
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, or all or any combination of the biomarkers of
the invention, and 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,
225, 250, 300, 350, 400, 450, or more genes other than the
biomarkers of the invention, or 1-10, 1-100, 1-150, 1-200, 1-300,
1-400, 1-500, 1-1000, 25-100, 25-200, 25-300, 25-400, 25-500,
25-1000, 100-150, 100-200, 100-300, 100-400, 100-500, 100-1000,
500-1000 other genes than the biomarkers of the invention.
[0555] For nucleic acid micoarray kits, the kits generally comprise
probes attached to a support surface. The probes may be labeled
with a detectable label. In a specific embodiment, the probes are
specific for an exon(s), an intron(s), an exon junction(s), or an
exon-intron junction(s)), of RNA products of 1, 2, 3, 4, 5, 6, 7,
all or any combination of the biomarkers of the invention. The
microarray kits may comprise instructions for performing the assay
and methods for interpreting and analyzing the data resulting from
the performance of the assay. In a specific embodiment, the kits
comprise instructions for diagnosing schizophrenia and/or bipolar
disorder. The kits may also comprise hybridization reagents and/or
reagents necessary for detecting a signal produced when a probe
hybridizes to a target nucleic acid sequence. Generally, the
materials and reagents for the microarray kits are in one or more
containers. Each component of the kit is generally in its own a
suitable container.
[0556] For RT-PCR kits, the kits generally comprise pre-selected
primers specific for particular RNA products (e.g., an exon(s), an
intron(s), an exon junction(s), and an exon-intron junction(s)) of
1, 2, 3, 4, 5, 6, 7, or all or any combination of the biomarkers of
the invention. The RT-PCR kits may also comprise enzymes suitable
for reverse transcribing and/or amplifying nucleic acids (e.g.,
polymerases such as Taq), and deoxynucleotides and buffers needed
for the reaction mixture for reverse transcription and
amplification. The RT-PCR kits may also comprise probes specific
for RNA products of 1, 2, 3, 4, 5, 6, 7, or all or any combination
of the biomarkers of the invention. The probes may or may not be
labeled with a detectable label (e.g., a fluorescent label). Each
component of the RT-PCR kit is generally in its own suitable
container. Thus, these kits generally comprise distinct containers
suitable for each individual reagent, enzyme, primer and probe.
Further, the RT-PCR kits may comprise instructions for performing
the assay and methods for interpreting and analyzing the data
resulting from the performance of the assay. In a specific
embodiment, the kits contain instructions for diagnosing
schizophrenia and/or bipolar disorder.
[0557] In a specific embodiment, the kit is a real-time RT-PCR kit.
Such a kit may comprise a 96 well plate and reagents and materials
necessary for SYBR Green detection. The kit may comprise reagents
and materials so that beta-actin can be used to normalize the
results. The kit may also comprise controls such as water, phospate
buffered saline, and phage MS2 RNA. Further, the kit may comprise
instructions for performing the assay and methods for interpreting
and analyzing the date resulting from the performance of the assay.
In a specific embodiment, the instructions state that the level of
a RNA product of 1, 2, 3, 4, 5, 6, 7, all or any combination of the
biomarkers of the invention should be examined at two
concentrations that differ by, e.g., 5 fold to 10-fold.
[0558] For antibody based kits, the kit can comprise, for example:
(1) a first antibody (which may or may not be attached to a
support) which binds to protein of interest (e.g., a protein
product of 1, 2, 3, 4, 5, 6, 7, all or any combination of the
biomarkers of the invention); and, optionally, (2) a second,
different antibody which binds to either the protein, or the first
antibody and is conjugated to a detectable label (e.g., a
fluorescent label, radioactive isotope or enzyme). The
antibody-based kits may also comprise beads for conducting an
immunoprecipitation. Each component of the antibody-based kits is
generally in its own suitable container. Thus, these kits generally
comprise distinct containers suitable for each antibody. Further,
the antibody-based kits may comprise instructions for performing
the assay and methods for interpreting and analyzing the data
resulting from the performance of the assay. In a specific
embodiment, the kits contain instructions for diagnosing
schizophrenia and/or bipolar disorder.
5.19 SNPs
[0559] A Single Nucleotide Polymorphism (SNP) is a single
nucleotide variation at a specific location in the genome of
different individuals. SNPs are found in both coding and non-coding
regions of genomic DNA. In spite of the paucity of scorable
phenotypes, SNPs are found in large numbers throughout the human
genome (Cooper et al., Hum Genet 69:201-205, 1985). SNPs are stable
genetic variations frequently found in genes, and contribute to the
wide range of phenotypic variations found in organisms. Single
nucleotide polymorphisms (SNPs) can be of predictive value in
identifying many genetic diseases, as well as phenotypic
characteristics. It is known for example that certain SNPs result
in disease-causing mutations such as the SNP correlated with
heritable breast cancer (Cannon-Albright and Skolnick, Semin Oncol
23:1-5, 1996).
[0560] A SNP may be identified in the DNA of an organism by a
number of methods well known to those of skill in the art,
including but not limited to identifying the SNP by DNA sequencing,
by amplifying a PCR product and sequencing the PCR product, by
Oligonucleotide Ligation Assay (OLA), by Doublecode OLA, by Single
Base Extension Assay, by allele specific primer extension, or by
mismatch hybridization.
[0561] The instant invention offers a more focused and efficient
method of screening SNPs to identify those SNPs which are
specifically associated with schizophrenia and/or bipolar disorder
by having identified a selection of genes which are differentially
expressed in blood from individuals having schizophrenia and/or
bipolar disorder. In one aspect of the invention, a selection of
SNPs to be screened are those SNPs found in the genes listed in
Tables 1 and 3. In another aspect of the invention, the SNPs to be
screened are those SNPs listed in FIG. 3. In yet another aspect of
the invention, novel SNPs can be identified in the
disease-associated biomarkers using those methods listed above.
[0562] In particular, this invention focuses on methods for
identifying those SNPs which are associated with schizophrenia
and/or bipolar disorder by screening only those SNPs in the
biomarkers identified herein. Those SNPs which are identified using
the methods disclosed herein will be convenient diagnostic markers.
One preferred aspect of identifying schizophrenia and/or bipolar
disorder associated SNPs encompasses isolating DNA from a sample
such as blood from a population of individuals, some of said
individuals having been diagnosed with schizophrenia and/or bipolar
disorder, some of those individuals not having schizophrenia and/or
bipolar disorder, and screening the genes for the SNPs identified
in FIG. 3 to identify one or more SNPs as diagnostic markers of
schizophrenia and/or bipolar disorder. More specifically a SNP is
considered to be a schizophrenia and/or bipolar disorder associated
snp if those individuals having schizophrenia and/or bipolar
disorder have a different polymorphism at the SNP locus than those
individuals not having schizophrenia and/or bipolar disorder.
Further, a particular SNP is considered to be diagnostic for
schizophrenia and/or bipolar disorder if a particular polymorphism
of the snp is found to present at a statistically significant
higher fequency in those individuals having schizophrenia and/or
bipolar disorder than in those individuals not having schizophrenia
and/or bipolar disorder. Indices of statistitcal significance
include p<0.05, p<0.001, p<0.01, and p<0.10. However
this invention is not limited to identifying schizophrenia and/or
bipolar disorder diagnostic SNPs from FIG. 3, and includes methods
of identifying new SNPs in the schizophrenia and/or bipolar
disorder biomarker genes listed in Tables 1 and 2, and methods of
determining their diagnostic value with respect to schizophrenia
and/or bipolar disorder.
[0563] As would be understood, a preferred sample is blood, but
these methods encompass any samples from which DNA can be obtained
including epithelial cells, buccal cells, hair, saliva, tissue
cells and the like. There are a variety of available methods for
obtaining and storing tissue and/or blood samples. These
alternatives allow tissue and blood samples to be stored and
transported in a form suitable for the recovery of genomic DNA from
the samples for genotype analysis. DNA samples can be collected and
stored on a variety of solid mediums, including Whatmann paper,
Guthrie cards, tubes, swabs, filter paper, slides, or other
containers. When whole blood is collected on filter paper, for
example, it can be dried and stored at room temperature.
[0564] In another aspect of the invention, schizophrenia and/or
bipolar disorder associated SNPs can be identified from RNA
transcripts of the schizophrenia and/or bipolar disorder biomarker
genes, listed in Tables 1, instead of from genomic DNA. In one
embodiment, RNA is isolated from a sample such as blood, from
individuals with and without the given disease or disorder, and
transcripts encoded by these schizophrenia and/or bipolar disorder
biomarker genes are reversed transcribed into cDNA. The cDNA is
amplified and analyzed to determine the presence of SNPs in the
schizophrenia and/or bipolar disorder biomarker genes. A
schizophrenia and/or bipolar disorder associated snp, can be
identified by then comparing the distribution of each of the SNPs
identified in the schizophrenia and/or bipolar disorder associated
biomarker gene(s) differentially expressed in those individuals
having schizophrenia and/or bipolar disorder, with those of
individuals who do not have schizophrenia and/or bipolar disorder.
In a further variation of this embodiment, instead analyzing cDNA
for the presence of SNPs, the RNA transcripts of the disease
specifc biomarker genes, or their amplified products, are analyzed
for the presence of SNPs.
[0565] Analysis of genomic DNA comprising the schizophrenia and/or
bipolar disorder biomarker genes has the potential to identify SNPs
in the coding region as well as in regulatory regions, the latter
which may contribute to the change in expression levels of the
gene. Analysis of cDNA encoded SNPs has the potential to identify
only SNPs in the coding region of the schizophrenia and/or bipolar
disorder biomarker genes, which may be instrumental in deciphering
protein based mechanisms of schizophrenia and/or bipolar disorder.
Methods of analyzing cDNA encoded SNPs can be carried out by
analyzing the cDNA generated in the rt-PCR reactions described
herein that are used to identify the level of the biomarker in
samples from patients and non patients.
[0566] A schizophrenia and/or bipolar disorder associated SNP may
be identified in the DNA of the schizophrenia and/or bipolar
disorder biomarker genes by a number of methods well known to those
of skill in the art,(see for example U.S. Pat. Nos. 6,221,592 and
5,679,524), including but not limited to identifying the SNP by PCR
or DNA amplification, Oligonucleotide Ligation Assay (OLA)
(Landegren et al., Science 241:1077, 1988), Doublecode OLA,
mismatch hybridization, mass spectrometry, Single Base Extension
Assay, (U.S. Pat. No. 6,638,722), RFLP detection based on
allele-specific restriction-endonuclease cleavage (Kan and Dozy,
Lancet ii:910-912, 1978), hybridization with allele-specific
oligonucleotide probes (Wallace et al., Nucl Acids Res 6:3543-3557,
1978), including immobilized oligonucleotides (Saiki et al., Proc
Natl Acad Sci USA 86:6230-6234, 1989) or oligonucleotide arrays
(Maskos and Southern, Nucl Acids Res 21:2269-2270, 1993),
allele-specific PCR (Newton et al., Nucl Acids Res 17:2503-16,
1989), mismatch-repair detection (MRD) (Faham and Cox, Genome Res
5:474-482,1995), binding of MutS protein (Wagner et al., Nucl Acids
Res 23:3944-3948, 1995), single-strand-conformation-polymorphism
detection (Orita et al., Genomics 5:874-879, 1983), RNAase cleavage
at mismatched base-pairs (Myers et al., Science 230:1242, 1985),
chemical (Cotton et al., Proc Natl Acad Sci USA 85:4397-4401, 1988)
or enzymatic (Youil et al., Proc Natl Acad Sci USA 92:87-91, 1995)
cleavage of heteroduplex DNA, methods based on allele specific
primer extension (Syvanen et al., Genomics 8:684-692, 1990),
genetic bit analysis (GBA) (Nikiforov et al., Nuci Acids Res
22:4167-4175, 1994), and radioactive and/or fluorescent DNA
sequencing using standard procedures well known in the art.
[0567] The instant methods of screening a subset of SNPs to
identify schizophrenia and/or bipolar disorder associated smps in
schizophrenia and/or bipolar disorder biomarker genes also
encompass non-PCR methods of DNA. These methods include ligase
chain reaction ("LCR"), disclosed in European Patent Application
No. 320,308, Qbeta Replicase, described in PCT Patent Application
No. PCT/US87/00880, isothermal amplification methods, Walker et al.
(Nucleic Acids Res 20(7):1691-6, 1992), Strand Displacement
Amplification (SDA) described in U.S. Pat. Nos. 5,712,124,
5,648,211 and 5,455,166, Cyclic Probe Reaction, Transcription-Based
Amplification, including nucleic acid sequence based amplification
(NASBA) and 3SR, Kwoh et al., Proc Natl Acad Sci USA, 86:1173-77,
1989; PCT Patent Application WO 88/10315 et al., 1989, other
amplification methods, as described in British Patent Application
No. GB 2,202,328, and in PCT Patent Application No. PCT/US89/01025,
Davey et al., European Patent Application No. 329,822, Miller et
al., PCT Patent Application WO 89/06700, "race and "one-sided PCR
TM." described in Frohman, In: PCR Protocols: A Guide To Methods
And Applications, Academic Press, N.Y., 1990, methods based on
ligation of two (or more) oligonucleotides in the presence of
nucleic acid having the sequence of the resulting
"di-oligonucleotide, described in Wu et al., Genomics 4:560-569,
1989.
[0568] While it is generally contemplated that the polymerase
employed will be thermostable, non-thermostable polymerases may
also be employed in the context of the present disclosure.
Exemplary polymerases and nucleic acid modifying enzymes that may
be used in the context of the disclosure include the thermostable
DNA Polymerases of OmniBase Sequencing Enzyme, Pfu DNA Polymerase,
Taq DNA Polymerase, Taq DNA Polymerase, Sequencing Grade, TaqBead
Hot Start Polymerase, AmpliTaq Gold, Vent DNA Polymerase, Tub DNA
Polymerase, TaqPlus DNA Polymerase, Tfl DNA Polymerase, Tli DNA
Polymerase, Tth DNA Polymerase; the DNA Polymerases of DNA
Polymerase I, Klenow Fragment, Exonuclease Minus, DNA Polymerase I,
DNA Polymerase I Large (Klenow) Fragment, Terminal Deoxynucleotidyl
Transferase, T7 DNA Polymerase, T4 DNA Polymerase; the Reverse
trancriptases of AMV Reverse Transcriptase and M-MLV Reverse
Transcriptase; T4 DNA ligase and T4 polynucleotide kinase.
[0569] Recognition moieties incorporated into primers, incorporated
into the amplified product during amplification, or attached to
probes are useful in the identification of the amplified molecules.
A number of different labels may be used for this purpose such as,
for example: fluorophores, chromophores, radio-isotopes, enzymatic
tags, antibodies, chemiluminescence, electroluminescence, affinity
labels, etc. One of skill in the art will recognize that these and
other fluorophores not mentioned herein can also be used with
success in this disclosure. Examples of affinity labels include but
are not limited to the following: an antibody, an antibody
fragment, a receptor protein, a hormone, biotin, DNP, or any
polypeptide/protein molecule that binds to an affinity label and
may be used for separation of the amplified gene. Examples of
enzyme tags include enzymes such as urease, alkaline phosphatase,
or peroxidase. Additionally, colorimetric indicator substrates can
be employed to provide a detection means visible to the human eye
or spectrophotometrically, to identify specific hybridization with
complementary nucleic acid-containing samples. All these examples
are generally known in the art and the skilled artisan will
recognize that the present disclosure is not limited to the
examples described above. The following fluorophores are
specifically contemplated to be useful in the present disclosure:
Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665,
BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2,
Cy3, Cy5,6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon
Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green,
Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine, and Texas
Red.
[0570] In the context of the present disclosure, it is specifically
contemplated that the DNA amplification products of the disclosed
methods may be analyzed using DNA chips or microarrays in order to
detect SNPs. The amplified DNA products may then be passed over a
DNA chip or microarray encompassing oligonucleotide or
polynucleotide probes. The ability or inability of the amplified
DNA to hybridize to the microarray or DNA chip will facilitate the
characterization of the SNPs present in the biomiarker genes
encoding the transcripts present in the sample.
[0571] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention.
5.19 EXAMPLES
Example 1
[0572] RNA Isolation from Lysed Blood
[0573] 10 ml whole blood is obtained in a Vacutainer and spun at
2,000 rpm for 5 min at 4.degree. C. and the plasma layer removed.
Lysis Buffer is added to blood sample in a ratio of 3 parts Lysis
Buffer to 1 part blood (Lysis Buffer (IL) 0.6 g EDTA; 1.0 g
KHCO.sub.2, 8.2 g NH.sub.4Cl adjusted to pH 7.4 (using NaOH)).
Sample is mixed and placed on ice for 5-10 minutes until
transparent. Lysed sample is centrifuged at 1000 rpm for 10 minutes
at 4.degree. C., and supernatant is aspirated. Pellet is
resuspended in 5 ml Lysis Buffer, and centrifuged again at 1000 rpm
for 10 minutes at 4.degree. C. Pelleted cells are homogenized using
TRIzol.RTM. (D (GIBCO/BRL) in a ratio of approximately 6 ml of
TRIzol.RTM. for every 10 ml of the original blood sample and
vortexed well. Samples are left for 5 minutes at room temperature.
RNA is extracted using 1.2 ml of chloroform per 1 ml of
TRIzol.RTM.. Sample is centrifuged at 12,000.times.g for 5 minutes
at 4.degree. C. and upper layer is collected. To upper layer,
isopropanol is added in ratio of 0.5 ml per 1 ml of TRIzol.RTM..
Sample is left overnight at -20.degree. C. or for one hour at
-20.degree. C. RNA is pelleted in accordance with known methods,
RNA pellet air dried, and pellet resuspended in DEPC treated
ddH.sub.2O. RNA samples can also be stored in 75% ethanol where the
samples are stable at room temperature for transportation.
Example 2
[0574] From Whole Blood
[0575] 100 ul whole blood is obtained in a microcentrifuge tube and
spun at 2,000 rpm (800 g) for 5 min at 4.degree. C. and the
supernatant removed. Pelleted cells are homogenized using TRIzol
(GIBCO/BRL) in a ratio of approximately 6 .mu.l of TRIzol for every
10 .mu.l of the original blood sample and vortexed well. Samples
are left for 5 minutes at room temperature. RNA is extracted using
12 .mu.l of chloroform per 10 .mu.l of TRIzol. Sample is
centrifuged at 12,000.times.g for 5 minutes at 4.degree. C. and
upper layer is collected. To upper layer, isopropanol is added in
ratio of 5 .mu.l per 10 .mu.l of TRIzol. Sample is left overnight
at -20.degree. C. or for one hour at -20.degree. C. RNA is pelleted
in accordance with known methods, RNA pellet air dried, and pellet
resuspended in DEPC treated ddH.sub.2O. RNA samples can also be
stored in 75% ethanol where the samples are stable at room
temperature for transportation.
[0576] From Centrifuged Lysed Blood
[0577] 10 ml whole blood is obtained in a Vacutainer and spun at
2,000 rpm (800 g) for 5 min at 4.degree. C. and the plasma layer
removed. Lysis Buffer is added to blood sample in a ratio of 3
parts Lysis Buffer to 1 part blood (Lysis Buffer (IL) 0.6 g EDTA;
1.0 g KHCO2, 8.2 g NH4Cl adjusted to pH 7.4 (using NaOH)). Sample
is mixed and placed on ice for 5-10 minutes until transparent.
Lysed sample is centrifuged at 1000 rpm for 10 minutes at 4.degree.
C., and supernatant is aspirated. Pellet is resuspended in 5 ml
Lysis Buffer, and centrifuged again at 1000 rpm for 10 minutes at
4.degree. C. Pelleted cells are homogenized using TRIzol
(GIBCO/BRL) in a ratio of approximately 6 ml of TRIzol for every 10
ml of the original blood sample and vortexed well. Samples are left
for 5 minutes at room temperature. RNA is extracted using 1.2 ml of
chloroform per 1 ml of TRIzol. Sample is centrifuged at
12,000.times.g for 5 minutes at 4.degree. C. and upper layer is
collected. To upper layer, isopropanol is added in ratio of 0.5 ml
per 1 ml of TRIzol. Sample is left overnight at -20.degree. C. or
for one hour at -20.degree. C. RNA is pelleted in accordance with
known methods, RNA pellet air dried, and pellet resuspended in DEPC
treated ddH.sub.2O. RNA samples can also be stored in 75% ethanol
where the samples are stable at room temperature for
transportation.
[0578] From Serum Free Whole Blood
[0579] 10 ml whole blood is obtained in a Vacutainer and spun at
2,000 rpm (800 g) for 5 min at 4.degree. C. and the plasma layer
removed. Pelleted cells are homogenized using TRIzol (GIBCO/BRL) in
a ratio of approximately 6 ml of TRIzol for every 10 ml of the
original blood sample and vortexed well. Samples are left for 5
minutes at room temperature. RNA is extracted using 1.2 ml of
chloroform per 1 ml of TRIzol. Sample is centrifuged at
12,000.times.g for 5 minutes at 4.degree. C. and upper layer is
collected. To upper layer, isopropanol is added in ratio of 0.5 ml
per 1 ml of TRIzol. Sample is left overnight at -20.degree. C. or
for one hour at -20.degree. C. RNA is pelleted in accordance with
known methods, RNA pellet air dried, and pellet resuspended in DEPC
treated ddH.sub.2O. RNA samples can also be stored in 75% ethanol
where the samples are stable at room temperature for
transportation.
Example 3
[0580] Target Nucleic Acid Preparation and Hybridization
[0581] Preparation of Fluorescent DNA Probe from mRNA
[0582] Fluorescently labeled target nucleic acid samples of RNA are
prepared for analysis with an array of the invention.
[0583] 1 .mu.g Oligo-dT primers are annealed to 10 ug of total RNA
isolated from blood from patient diagnosed with schizophrenia
and/or bipolar disorder or suspected of having schizophrenia and/or
bipolar disorder in a total volume of 10 ul, by heating to
70.degree. C. for 10 min, and cooled on ice. The mRNA is reverse
transcribed by incubating the sample at 42.degree. C. for 40 min in
a 25 ll volume containing a final concentration of 50 mM Tris-HCl
(pH 8.3), 75 mM KCl, 3 mM MgC12, 25 mM DTT, 25 mM unlabeled dNTPs,
400 units of Superscript II (200 U/uL, Gibco BRL), and 15 mM of Cy3
or Cy5 (Amersham). The reaction is stopped by the addition of 2.5
.mu.l of 55500 mM EDTA and 5 .mu.l of 1M NaOH, and incubation at
65.degree. C. for 10 min. The reaction mixture is neutralized by
addition of 12.5 .mu.l of 1M Tris HCl (pH7.6).
[0584] The labeled target nucleic acid sample is purified by
centrifugation in a Centricon-30 micro-concentrator (Amicon). If
two different target nucleic acid samples (e.g., two samples
derived from different patients) are being analyzed and compared by
hybridization to the same array, each target nucleic acid sample is
labeled with a different fluorescent label (e.g., Cy3 and Cy5) and
separately concentrated. The separately concentrated target nucleic
acid samples (Cy3 and Cy5 labeled) are combined into a fresh
centricon, washed with 500 .mu.l TE, and concentrated again to a
volume of less than 7 .mu.l. 11 .mu.L of 10 .mu.g/.mu.l polyA RNA
(Sigma, #P9403) and 1 .mu.l of 10 .mu.g/ul tRNA (Gibco-BRL,
#15401-011) is added and the volume is adjusted to 9.5 .mu.l with
distilled water. For final target nucleic acid preparation 2.1
.mu.l 20XSSC (1.5M NaCl, 150 mM NaCitrate (pH8.0)) and 0.35 .mu.l
10% SDS is added.
[0585] Hybridization
[0586] Labeled nucleic acid is denatured by heating for 2 min at
100.degree. C., and incubated at 37.degree. C. for 20-30 min before
being placed on a nucleic acid array under a 22 mm.times.22 mm
glass cover slip. Hybridization is carried out at 65.degree. C. for
14 to 18 hours in a custom slide chamber with humidity maintained
by a small reservoir of 3.times.SSC. The array is washed by
submersion and agitation for 2-5 min in 2.times.SSC with 0.1% SDS,
followed by 1.times.SSC, and 0.1.times.SSC. Finally, the array is
dried by centrifugation for 2 min in a slide rack in a Beckman GS-6
tabletop centrifuge in Microplus carriers at 650 RPM for 2 min.
Example 4
[0587] Real Time RT PCR
[0588] Real time RT PCR was performed on the genes as disclosed in
Table 1 using the SYBR.RTM. Green Kit from Qiagen (Product Number
204143). The experimental results of these genes are shown in
Example 6 below.
[0589] Either a one step (reverse transcription and PCR combined)
or a two step (reverse transcription first and then subsequent PCR)
can be used. In the case of the two step protocol, reverse
transcription was first performed using the High-Capacity cDNA
Archive Kit from Applied Biosystems (Product number 4322171) and
following the protocol utilized therein.
[0590] More specifically purified RNA as described previously
herein was incubated with Reverse Transcriptase buffer, dNTPs,
Random primers and Reverse transcriptase and incubated for
25.degree. C. for 10 minutes and subsequently for 37.degree. C. for
two hours and the resulting mixture utilized as the starting
product for quantitative PCR.
[0591] cDNA resulting from reverse transcription was incubated with
the QuantiTect SYBR.RTM. Green PCR Master Mix as provided and no
adjustments were made for magnesium concentration.
Uracil-N-Glycosylase was not added. 5 .mu.M of both forward primer
and reverse primer specific to the genes of the invention were
added and the reaction was incubated and monitored in accordance
with the standard protocol utilizing the ABI PRISM 7700/ABI GeneAmp
5700/iCycler/DNA Engine Opticon.
8TABLE 8 Primers used in the performance of Real Time RT PCR
Forward Primer Reverse Primer Gene Primer Position Primer Position
ADSS CTGCGTTGGCACT 1361 GACTTCTTGGTTT 1476 TACCAAGTT GCTGGGA SEQ ID
NO 1 SEQ ID NO 2 APOBEC3B CTCAGATACCTGA 619 CGCTCCACCTCAT 719
TCCAGACAC AGCACAAGT ATGGA SEQ ID NO 4 SEQ ID NO 3 ATM TGTGGATGGCATG
8931 GAAGGACCTCTAC 9046 GGCATTA AATGGTTAA SEQ ID NO 5 CAGAG SEQ ID
NO 6 CLC GCCAGATAAGTAC 364 ATCTCTCCACACT 463 CAGGTAATGG TGCACCA SEQ
ID NO 7 SEQ ID NO 8 CTBP1 ATCACAGGCCGGA 1150 ATTGAGCTCAGGG 1174
TCCCAGA TGCACGA SEQ ID NO 9 SEQ ID NO 10 CXCL1 ACCGAAGTCATAG 293
GTTGGATTTGTCA 400 CCACACT CTGTTCAGC SEQ ID NO 11 SEQ ID NO 12 DATF1
AGCAGAAGTCTAG 1575 GCCTCTATCACAG 1677 CGAAGACCAAG GCTGGAA SEQ ID NO
13 SEQ ID NO 14 S100A9 TTTGGGACAGAGT 25 CCAGCTTCACAGA 124 GCAAGACGA
GTATTGGTGGA SEQ ID NO 15 SEQ ID NO 16
Example 5
[0592] TaqMan.RTM.
[0593] Quantitative real time RT PCR can also be performed using
the QuantiTect.TM. Probe RT-PCR system from Qiagen (Product Number
204343) in conjunction with a TaqMan.RTM. dual labelled probe and
primers corresponding to the gene of interest. The TaqMan.RTM.
probe and primers can be ordered from Applied Biosystems
Assays-On-Demand.TM..
[0594] The dual labelled probe contains both a fluorophore and a
quencher molecule. The proximity of the fluorescent reporter with
the quencher prevents the reporter from fluorescing, but during the
PCR extension step, the 5'-3' exonuclease activity of the Taq DNA
polymerase releases the fluorophore which allows it to fluoresce.
As such, the amount of fluorescence correlates with the amount of
PCR product generated.
Example 6
[0595] Statistical Analysis of Real Time PCR Results
[0596] Real Time PCR was performed to analyze potential biomarkers
from blood samples isolated from individuals categorized as normal
(not having schizophrenia or bipolar disorder), having
schizophrenia or having bipolar disorder. T-tests and or Mann
Whitney tests were utilized on age and sex matched sample sets of
approximately 16-25 in size. FIG. 1 shows analysis for the eight
biomarkers on total RNA isolated from centrifuged lysed blood for
each biomarkers ability to differentiate as between: (a)
schizophrenia and non-schizophrenia (b) bipolar disorder and
non-bipolar disorder and (c) schizophrenia and bipolar disorder.
Biomarkers which demonstrated an ability to differentiate with a p
value of less than 0.05 are shaded.
Example 7
[0597] Identification of Combinations of Biomarkers Using Logistic
Regression
[0598] First strand cDNA synthesis was performed on a Perkin-Elmer
DNA Thermal Cycler using the ABI High Capacity cDNA Archive Kit
(Cat #4322171). Quantitation of specific cDNA was achieved with the
Qiagen Quantitect SYBR.RTM. Green PCR Kit (Cat #204143) with
confirmation of desired product using agarose gel. In each sample,
the expression level of a target gene was quantified by its
Ct-value, the concentration-dependent PCR cycle number at which the
amplicon becomes distinguishable over background. Each Ct-value was
related to an internal standard by subtracting the Ct value of
beta-actin as a housekeeping gene. To obtain increased
discriminating ability, logistic regression was used to generate
linear combinations of A Ct for (a) schizophrenia v. control (b)
bipolar disorder v. control and (c) schizophrenia v. bipolar
disorder. The diagnostic accuracy of the combinations (ie the
probably of true vs. false positive and true v. false negative
calls) was quantitatively evaluated by ROC (Receiver Operating
Characteristic) curve analysis using MedCalc.RTM., (MedCalc;
Mariakerke, Belgium); XLSTAT.RTM. (AddinSoft; Paris, France) and
our own software. ROC analysis of the classifiers generated using
logistic regression were analyzed. Examples of classifiers for (a)
schizophrenia v. control with an ROC of >0.9 are shown in FIG.
5. Examples of classifiers for (b) bipolar disorder v. control with
an ROC of >0.9 are shown in FIG. 6. Examples of classifiers for
(c) bipolar disorder v. schizophrenia with an ROC of >0.9 are
shown in FIG. 7.
Example 8
[0599] Diagnosis of Individual Utilizing Classifiers of the
Invention
[0600] Classifiers of the invention can be generated as described
in Example 8 above or using other mathematical models as described
herein. For example one of the classifiers identified for
differentiating between schizophrenia and non-schizophrenia as
shown in FIG. 5 is written as follows:
X=-27.66-1.2*APOBEC3B+4.69*ADSS-4.04*ATM-1.66*CLC+4.68*CTBP1-1.58*CXCL1+3.-
18*DATF1-2.7*S100A9(ROC0.96)
[0601] In order to utilize the classifier for diagnosis of
schizophrenia, the .DELTA.Ct of an test individual for the
following genes ABOBEC3B, ADSS, ATM, CLC, CTBP1 CXCL1, DATF1 and
S100A9 are measured, for example, as outlined in Example 8 and
substituted into the equation above.
[0602] The test individual is considered to diagnose as a control
(not having schizophrenia) when x<0. Similarly the test
individual is considered to diagnose as having schizophrenia when
x>0. As would be understood by a person skilled in the art, in
the equation
X=+27.66+1.2*APOBEC3B-4.69*ADSS+4.04*ATM+1.66*CLC-4.68*CTBP1+1.58*CXCL1-3-
.18*DATF1+2.7*S100A9a test individual is diagnosed as control when
x>0 and diagnosed as having schizophrenia when x<0.
[0603] One of the classifiers identified for differentiating
between bipolar disorder and non-bipolar disorder as shown in FIG.
6 is written as follows:
X=45.84-1.13*APOBEC3B-5.01*ADSS+0.75*CLC+1.07*CXCL1-5.13*S100A9(ROC0.948)
[0604] One of the classifiers identified for differentiating
between bipolar disorder and schizophrenia as shown in FIG. 7 is
written as follows:
X=0.2+6.13*ADSS-5.58*ATM-2.69*CXCL1+3.87*DATF1+2.48*S100A9(ROC0.972)
Example 9
[0605] Diagnosis of Individuals Utilizing Biomarker Combinations of
the Invention
[0606] This example demonstrates the use of the biomarkers
combinations of the invention to diagnose schizophrenia.
Measurement of the RNA and/or protein products of the five
biomarkers identified, for example in the classifier of Example 10
ABOBEC3, ATM, CLC, CTBP1 and DATF1 can be used to diagnose an
individual as having schizophrenia or not having schizophrenia.
Level of expression of the RNA and/or Protein products of the
combination of biomarkers identified can be measured for a
population of individuals having schizophrenia and a population of
individuals not having schizophrenia and a new classifier generated
to allow the diagnosis of an unknown individual as having or not
having schizophrenia.
Example 10
[0607] Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Schizophrenia as Compared with Gene Expression
Profiles from Normal Individuals Using the Biomarkers of Table
1
[0608] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0609] Blood samples are taken from patients who are clinically
diagnosed with schizophrenia as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0610] Total mRNA from a drop of blood is taken from each patient
is first isolated using TRIzol.RTM. reagent (GIBCO) and
fluorescently labeled probes for each blood sample are then
generated, denatured and hybridized to a microarray containing full
length cDNA sequences for each of the genes as described in Table
1. Detection of specific hybridization to the array is then
measured by scanning with a GMS Scanner 418 and processing of the
experimental data with Scanalyzer software (Michael Eisen, Stanford
University), followed by GeneSpring software (Silicon Genetics, CA)
analysis. Differential expression of the 8 genes in blood samples
from patients with schizophrenia as compared to non-schizophrenic
patients is determined by statistical analysis using the t-test
(Glantz S A. Primer of Biostatistics. 5th ed. New York, USA:
McGraw-Hill Medical Publishing Division, 2002).
Example 11
[0611] Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Schizophrenia as Compared with Gene Expression
Profiles from Healthy Individuals Using the 5' Regions of the Genes
Described in Table 1
[0612] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0613] Blood samples are taken from patients who are clinically
diagnosed with schizophreniar as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0614] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzol.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing DNA sequences of 25
nucleotides in length corresponding to the 5' region of each of the
genes as described in Table 1. Detection of specific hybridization
to the array is then measured by scanning with a GMS Scanner 418
and processing of the experimental data with Scanalyzer software
(Michael Eisen, Stanford University), followed by GeneSpring
software (Silicon Genetics, CA) analysis. Differential expression
of the genes in blood samples from patients with schizophrenia as
compared to healthy patients is determined by statistical analysis
using the t test (Glantz S A. Primer of Biostatistics. 5th ed. New
York, USA: McGraw-Hill Medical Publishing Division, 2002).
Differential expression of each of the genes described in Table 1
is diagnostic for schizophrenia.
Example 12
[0615] Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Schizophrenia as Compared with Gene Expression
Profiles from Non Schizophrenic Individuals Using the 3' Regions of
The Genes Described in Table 1
[0616] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0617] Blood samples are taken from patients who were clinically
diagnosed with schizophrenia as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0618] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzol.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing DNA sequences of 50
nucleotides in length corresponding to the 3' region of each of the
mRNA as described in Table 1. Detection of specific hybridization
to the array is then measured by scanning with a GMS Scanner 418
and processing of the experimental data with Scanalyzer software
(Michael Eisen, Stanford University), followed by GeneSpring
software (Silicon Genetics, CA) analysis. Differential expression
of the enes in blood samples from patients with schizophrenia as
compared to healthy patients is determined by statistical analysis
using the t-test (Glantz S A. Primer of Biostatistics. 5th ed. New
York, USA: McGraw-Hill Medical Publishing Division, 2002).
Differential expression of each of the mRNA described in Table 1 is
diagnostic for schizophrenia.
Example 13
[0619] Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Schizophrenia as Compared with Gene Expression
Profiles from Healthy Individuals Using the Internal Coding Regions
Of the Genes Described in Table 1
[0620] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0621] Blood samples are taken from patients who are clinically
diagnosed with schizophrenia as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0622] Total mRNA from a drop of blood taken from each patient wis
as first isolated using TRIzol.RTM. reagent (GIBCO) and
fluorescently labeled probes for each blood sample are then
generated, denatured and hybridized to a microarray containing DNA
sequences of 70 nucleotides in length corresponding to the internal
coding region of each of the genes as described in Table 1.
Detection of specific hybridization to the array is then measured
by scanning with a GMS Scanner 418 and processing of the
experimental data with Scanalyzer software (Michael Eisen, Stanford
University), followed by GeneSpring software (Silicon Genetics, CA)
analysis. Differential expression of the genes in blood samples
from patients with schizophrenia as compared to healthy patients is
then determined by statistical analysis using the t-test (Glantz S
A. Primer of Biostatistics. 5th ed. New York, USA: McGraw-Hill
Medical Publishing Division, 2002). Differential expression of each
of the mRNA described in Table 1 is diagnostic for
schizophrenia.
Example 14
[0623] Analysis of Blood Samples from Individuals Having
Schizophrenia as Compared with Blood Samples from Healthy
Individuals Using Monoclonal Antibodies Directed to the
Polypeptides Encoded by the Genes Described in Table 1
[0624] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0625] Blood samples are taken from patients who are clinically
diagnosed with schizophrenia as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0626] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract. The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to full length
polypeptides encoded by the genes described in Table 1. Detection
of specific binding to the array is then measured by scanning with
a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen, Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the genes in blood samples from patients
with schizophrenia as compared to healthy patients is determined by
statistical analysis using the Wilcox Mann Whitney rank sum test
(Glantz S A. Primer of Biostatistics. 5th ed. New York, USA:
McGraw-Hill Medical Publishing Division, 2002). Differential
expression of each of the genes described in Table 1 is diagnostic
for schizophrenia.
Example 15
[0627] Analysis of Blood Samples from Individuals Having
Schizophrenia as Compared with Blood Samples from Healthy
Individuals Using Monoclonal Antibodies Directed to the Amino
Terminal Region of Polypeptides Encoded by the 5' Regions of the
Genes Described in Table 1
[0628] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0629] Blood samples are taken from patients who are clinically
diagnosed with schizophrenia as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0630] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to amino terminal
regions of polypeptides encoded by the 5' regions of the genes
described in in Table 1. Detection of specific binding to the array
is then measured by scanning with a GMS Scanner 418 and processing
of the experimental data with Scanalyzer software (Michael Eisen,
Stanford University), followed by GeneSpring software (Silicon
Genetics, CA) analysis. Differential expression of the 3 genes in
blood samples from patients with schizophrenia as compared to
healthy patients is determined by statistical analysis using the
Wilcox Mann Whitney rank sum test (Glantz S A. Primer of
Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
genes described in Table 1 is diagnostic for schizophrenia.
Example 16
[0631] Analysis of Blood Samples from Individuals Having
Schizophrenia as Compared with Blood Samples from Healthy
Individuals Using Monoclonal Antibodies Directed to the Carboxy
Terminal Region of Polypeptides Encoded by the 3' Regions of the
Genes Described in Table 1.
[0632] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0633] Blood samples are taken from patients who were clinically
diagnosed with schizophrenia as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0634] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to the carboxy terminal
regions of polypeptides encoded by the 3' regions of the genes
described in in Table 1. Detection of specific binding to the array
is then measured by scanning with a GMS Scanner 418 and processing
of the experimental data with Scanalyzer software (Michael Eisen,
Stanford University), followed by GeneSpring software (Silicon
Genetics, CA) analysis. Differential expression of the genes in
blood samples from patients with schizophrenia as compared to
healthy patients is determined by statistical analysis using the
Wilcox Mann Whitney rank sum test (Glantz S A. Primer of
Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
genes described in in Table 1 is diagnostic for schizophrenia.
Example 17
[0635] Analysis of Blood Samples from Individuals Having
Schizophrenia as Compared with Blood Samples from Healthy
Individuals Using Antibodies Directed to the Internal Polypeptide
Region of Polypeptides Encoded by the Internal Coding Region of the
Genes Described in Table 1.
[0636] This example demonstrates the use of the claimed invention
to diagnose schizophrenia by detecting differential gene expression
in blood samples taken from patients with schizophrenia as compared
to blood samples taken from healthy patients.
[0637] Blood samples are taken from patients who were clinically
diagnosed with schizophrenia as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by schizophrenia. In each case, the diagnosis of
schizophrenia is corroborated by a skilled Board certified
physician.
[0638] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract. The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to internal polypeptide
regions of polypeptides encoded by the internal coding regions of
the genes described in Table 1. Detection of specific binding to
the array is then measured by scanning with a GMS Scanner 418 and
processing of the experimental data with Scanalyzer software
(Michael Eisen, Stanford University), followed by GeneSpring
software (Silicon Genetics, CA) analysis. Differential expression
of the genes in blood samples from patients with schizophrenia as
compared to healthy patients is determined by statistical analysis
using the Wilcox Mann Whitney rank sum test (Glantz S A. Primer of
Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
genes described in Table 1 is diagnostic for schizophrenia.
[0639] Variations, modifications, and other implementations of what
is described herein will occur to those of ordinary skill in the
art without departing from the spirit and scope of the invention.
The references provided below are incorporated herein by reference
in their entireties. All patents, patent applications, and
published references cited herein are hereby incorporated by
reference in their entirety.
[0640] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims. Those
skilled in the art will recognise that other embodiments and
configurations known in the art would be within the spirit and
scope of the present invention.
Sequence CWU 1
1
16 1 22 DNA artificial Forward Primer for ADSS 1 ctgcgttggc
acttaccaag tt 22 2 20 DNA artificial Reverse Primer for ADSS 2
gacttcttgg tttgctggga 20 3 27 DNA artificial Forward Primer for
APOBEC3B 3 ctcagatacc tgatggatcc agacaca 27 4 22 DNA artificial
Reverse Primer for APOBE3C 4 cgctccacct catagcacaa gt 22 5 20 DNA
artificial Forward Primer for ATM 5 tgtggatggc atgggcatta 20 6 27
DNA artificial Reverse Primer for ATM 6 gaaggacctc tacaatggtt
aacagag 27 7 23 DNA artificial Forward Primer for CLC 7 gccagataag
taccaggtaa tgg 23 8 20 DNA artificial Reverse Primer for CLC 8
atctctccac acttgcacca 20 9 20 DNA artificial Forward Primer for
CTBP1 9 atcacaggcc ggatcccaga 20 10 20 DNA artificial Reverse
Primer for CTBP1 10 attgagctca gggtgcacga 20 11 20 DNA artificial
Forward Primer for CXCL1 11 accgaagtca tagccacact 20 12 22 DNA
artificial Reverse Primer for CXCL1 12 gttggatttg tcactgttca gc 22
13 24 DNA artificial Forward Primer for PATF1 13 agcagaagtc
tagcgaagac caag 24 14 20 DNA artificial Reverse Primer for PATF1 14
gcctctatca caggctggaa 20 15 22 DNA artificial Forward Primer for
S100A9 15 tttgggacag agtgcaagac ga 22 16 24 DNA artificial Reverse
Primer for S100A9 16 ccagcttcac agagtattgg tgga 24
* * * * *