Note: Descriptions are shown in the official language in which they were submitted.
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LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 4
CONTENANT LES PAGES 1 A 127
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brevets
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VOLUME
THIS IS VOLUME 1 OF 4
CONTAINING PAGES 1 TO 127
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
CA 02574727 2007-01-19
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IS16NUAR&S OF NEURODEGENERATIVE DISEASE
1. ACKNOWLEDGEMENTS
1. This work was supported by grants from the National Institute of Aging
(LEAD
AG09016, RO1 AG1441 1, and AG00107-15) and Alzheimer's Disease Center AG08665
and a
grant from the National Science Foundation (CCR9701911). The U.S. Government
has certain
rights in this invention.
U. CROSS REFERENCE TO RELATED APPLICATIONS
2. This application claims the benefit of priority to U.S. Provisional
Application No.
60/589,318, filed July 19, 2004. U.S. Provisional Application No. 60/589,318
is incorporated by
reference herein in its entirety.
III. BACKGROUND
3. Neurodegenerative diseases affect millions of people, greatly reducing
their quality of
life and, in many, cases, causing death. A relatively common neurodegenerative
disease is
Parkinson's disease, which affects more than half a million Americans each
year. Parkinson's
disease is characterized by slowness of movement (bradykinesia), tremor at
rest, rigidity of the
extremities and neck, stooped posture, minimal facial expressions, problems
swallowing
(dysphagia), and a paucity of associated movements (e.g., arm swinging). Some
patients also
experience dementia associated with such abnormalities of motor function.
Parkinson's disease
is age-dependant and usually has a gradual onset between the ages of 50 and
70, progressing
slowly until death 10 to 20 years later.
4. Alzheimer's disease is another common neurodegenerative disease.
Progression of
Alzheimer's disease is associated with gradual changes of consciousness, loss
of memory,
perception, and orientation as well as loss of personality and intellect. The
prevalence of
Alzheimer's disease increases dramatically with age.
5. Accurate and easy diagnosis of neurodegenerative diseases prior to autopsy
is
challenging. Also, the etiology of many neurodegenerative diseases, such as
Parkinson's and
Alzheimer's disease, is not fully understood. Further, the symptoms associated
with one
neurodegenerative disease are oftentimes similar to the symptoms of other
neurodegenerative
diseases, especially at the early stages of disease. Such difficulties can
cause confusion and
complications with diagnosing and treating patients with such
neurodegenerative diseases. For
example, the changes that take place in the neural fibers of the Alzheimer's
patient are typically
positively diagnosed upon histological analysis of the morphological changes
that take place in
the neurons. It has been shown that gene expression in an Alzheimer's brain
changes and that
CA 02574727 2007-01-19
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#a ~6ed to identify the onset and progression of an Alzheimer's patient.
However, this type of analysis requires obtaining a brain sample from the
patient, and is
therefore, most useful in a post mortem setting.
6. As early diagnosis of neurodegenerative disease such as Parkinson's and
Alzheimer's
can aid in their treatment, a relatively less invasive procedure that is
accurate and easier to
perform is desirable. As such, needed in the art are compositions and methods
for
differentiating, diagnosing, and monitoring neurodegenerative diseases. The
subject matter
disclosed herein addresses these and other needs. For example, disclosed
herein are methods
and compositions for the diagnosis of neurodegenerative diseases such as
Parkinson's and
Alzheimer's that involve sampling of the peripheral blood of the patient,
rather than sampling
neural tissue.
IV. SUMMARY
7. In accordance with the purposes of the disclosed materials, compounds,
compositions, articles, and methods, as embodied and broadly described herein,
the disclosed
subject matter, in one aspect, relates to compounds and compositions and
methods for preparing
and using such compounds and compositions. In a further aspect, the disclosed
subject matter
relates to methods for the diagnosis and prognosis of a neurodegenerative
disease (e.g.,
Parkinson's and Alzheimer's) in a particular subject. In a still further
aspect, the disclosed
subject matter relates to methods of screening for a therapeutic agent for the
treatment of a
neurodegenerative disease. Still fixrther, disclosed herein are methods of
monitoring
neurodegenerative disease progression in a subject. In yet a further aspect,
the disclosed subject
matter relates to methods of monitoring a response to a neurodegenerative
disease treatment in a
subject, methods of identifying a risk for a neurodegenerative disease in a
test subject, and
methods of differentially diagnosing a neurodegenerative disease in a test
subject. Also,
disclosed are diagnostic assays for a neurodegenerative disease and chips,
beads, and arrays that
can be used in the methods disclosed herein. In many examples, the
compositions and methods
disclosed herein involve the use of blood from a subject and the analysis of
gene expression
within the blood cells.
8. The advantages described below will be realized and attained by means of
the
elements and combinations particularly pointed out in the appended claims. It
is to be
understood that both the foregoing general description and the following
detailed description are
exemplary and explanatory only and are not restrictive.
V. BRIEF DESCRIPTION OF THE FIGURES
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t.. ,
~ M~iJg figures, which are incorporated in and constitute a part of this
specification, illustrate several embodiments and together with the
description illustrate the
disclosed compositions and methods.
10. Figure 1A is a plot of canonical variables 1 and 2 for the first study.
Canonical
variables for this plot, and for Figures 1B and 1C, were derived from data for
the cell cycle
related messages: cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3
beta, and protein
kinase C alpha. Wald-Wolfowitz runs test p<0.05 for difference between
Alzheimer's disease
("AD") and control cases ("Con"). The cases included are shown in Table 1.
Table 1:
Dx N Age Gender MMSE CDR Blessed Duration Fam. Hx
AD 8 77.8 3F/5M 21.3 1.3 4.6 4.1 3+/5-
Con 7 76.4 3F/5M 30 0 0 NA 2+/5-
11. Figure 1B is a plot of canonical variables 1 and 2 for the second study.
Canonical
variables for this plot were derived from data for the same cell cycle related
messages as in
Figure 1A. Wald-Wolfowitz runs test p<0.05 for difference between AD and
control cases. The
cases included are shown in Table 2.
Table 2: =
Dx N Age Gender MMSE CDR Blessed Duration Fam. Hx
AD 8 78 5F/3M 19 1.5 3.3 3.4 2+/6-
Con 8 76 5F/3M 30 0 0 NA 0+/8-
12. Figure 1 C is a plot of canonical variables 1 and 2 for the third study.
Canonical
variables for this plot were derived from data for the same cell cycle related
messages as in
Figure 1A. Wald-Wolfowitz runs test p<0.05 for difference between AD and
control cases. The
cases included are shown in Table 3.
Table 3:
Dx N Age Gender MMSE CDR Blessed Duration Fam. Hx
AD 5 77 4F/1M 21 1.2 3.5 3.5 2+/3-
Con 5 62 2F/3M 30 0 0 NA 3+/2-
13. Figure 2A is a plot of canonical variables 1 and 2 for the first study.
Canonical
variables for this plot were derived from data for the inflammatory related
messages: C5, Cl
inhibitor, C5a, complement C3, cyclooxygenase 1, IL17, IL8, LIF, TNF alpha,
and IL10r. Wald-
Wolfowitz runs test p<0.05 for difference between AD and control cases. The
cases included
were the same as for Figure 1A.
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L-4 1kIV J!of canonical variables 1 and 2 for the second study. Canonical
variables for this plot were derived from data for the same inflammatory
related messages as in
Figure 2A. Wald-Wolfowitz runs test p<0.05 for difference between AD and
control cases. The
cases included were the same as for Figure 1B.
15. Figure 2C is a plot of canonical variables 1 and 2 for the third study.
Canonical
variables for this plot were derived from data for the same inflammatory
related messages as in
Figure 2A. Wald-Wolfowitz runs test p<0.05 for difference between AD and
control cases. The
AD and control cases included were the same as for Figure 1 C. Two Parkinson's
disease ("PD")
cases were added here.
16. Figure 3 is a group of plots of the first canonical variable for the
initial set of 8 AD
and 7 control subjects (see Table 1). The transcripts related to cell cycle in
the multivariate
analysis were: cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta,
and protein
kinase C alpha. The transcripts related to inflammatory systems in the
multivariate analysis
were: C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r. The
transcripts related to cell
stress in the multivariate analysis were: Alpha-1 antichymotrypsin, HSP 27,
HSP 90, crystalline,
GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin. In all three
plots Wald-Wolfowitz
runs test p<0.05 for difference between early AD and control cases.
17. Figure 4 is a group of plots of the first canonical variable for the
second set of
subjects (8 AD and 8 controls; see Table 2). The transcripts are the same as
in Figure 3. In all
three plots Wald-Wolfowitz runs test p<0.05 for difference between early AD
and control cases.
18. Figure 5 is a group of plots of the first canonical variable for the third
set of subjects
(5 AD, 5 controls, and 2 PD). The transcripts are the same as in Figure 3. In
all three plots
Wald-Wolfowitz runs test p<0.05 for difference between early AD and control
cases.
19. Figure 6 is a silver stained 2D electrophoresis gel. Difference Image of 2
independent human white blood cell samples showing spots present only in one
sample but not
the other.
20. Figure 7 is a MALDI-TOF mass analysis of isolated differentially expressed
protein.
21. Figure 8 is a series of graphs showing differential expression of
peripheral leukocyte
protein spots from the full cohort of control ("CTL") and Parkinson's disease
subjects.
Computer analysis of a subset of spots from the full cohort of control (n=12)
and PD (n=12)
subjects demonstrates the ability to identify differentially expressed spots
of the leukocyte
proteome.
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12M,-iFWe'q,39S Wi,116tcharts showing the mean baseline characteristics of
study
participants in the experiments described in Example 4.
23. Figures 10A, l OB, l OC, and 10D are four scanned images of two-
dimensional (2D)
protein gels that have been silver-stained and dried. Figure l0A is an image
showing the levels
of proteins expressed in leukocytes obtained from an Alzheimer's patient prior
to treatment with
valproate (VPA). Figure 10B is an image showing proteins expressed in
leukocytes from the
same patient four weeks after initiating VPA treatment. Figures 10C and 10D
are enlarged
versions of the images of Figures 10A and 10B, respectively, after the images
were processed
using protein spot-detection software. Spots labeled #278 in Figures 10C and
10D are the spots
of a differentially expressed protein before VPA treatment and after VPA
treatment.
24. Figure 11 is a chart listing examples of biomarkers identified using the
methods
disclosed herein.
25. Figure 12 is a set of four histograms quantifying the effects of the
indicated VPA
concentrations on expression of four candidate biomarkers in cultured
leukocytes. Three of the
four proteins identified as biomarkers in VPA-treated patients also
demonstrated changes in
expression in response to treatments with increasing doses of VPA.
VI. DETAILED DESCRIPTION
26. Before the present compounds, compositions, articles, devices, and/or
methods are
disclosed and described, it is to be understood that they are not limited to
specific synthetic
methods or specific recombinant biotechnology methods unless otherwise
specified, or to
particular reagents unless otherwise specified, as such may, of course, vary.
It is also to be
understood that the terminology used herein is for the purpose of describing
particular
embodiments only and is not intended to be limiting.
A. Definitions
27. In this specification and in the claims which follow, reference will be
made to a
number of terms which shall be defined to have the following meanings:
28. Throughout the specification and claims the word "comprise" and other
forms of the
word, such as "comprising" and "comprises," means including but not limited
to, and is not
intended to exclude, for example, other additives, components, integers, or
steps.
29. As used in the specification and the claims, the singular forms "a," "an,"
and "the"
include plural referents unless the context clearly dictates otherwise. Thus,
for example,
reference to "a biomarker" includes mixtures of two or more such biomarkers,
reference to "an
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i~ two or more antibodies, reference to "the subject" includes two
or more subjects, and the like.
30. Ranges can be expressed herein as from "about" one particular value,
and/or to
"about" another particular value. When such a range is expressed, another
embodiment includes
from the one particular value and/or to the other particular value. Similarly,
when values are
expressed as approximations, by use of the antecedent "about," it will be
understood that the
particular value forms another embodiment. It will be further understood that
the endpoints of
each of the ranges are significant both in relation to the other endpoint, and
independently of the
other endpoint. It is also understood that there are a number of values
disclosed herein, and that
each value is also herein disclosed as "about" that particular value in
addition to the value itself.
For example, if the value "10" is disclosed, then "about 10" is also
disclosed. It is also
understood that when a value is disclosed that "less than or equal to" the
value, "greater than or
equal to the value" and possible ranges between values are also disclosed, as
appropriately
understood by the skilled artisan. For example, if the value "10" is disclosed
then "less than or
equal to 10"as well as "greater than or equal to 10" is also disclosed. It is
also understood that
throughout the application data is provided in a number of different formats
and that this data
represents endpoints and starting points and ranges for any combination of the
data points. For
example, if a particular data point "10" and a particular data point "15" are
disclosed, it is
understood that greater than, greater than or equal to, less than, less than
or equal to, and equal to
10 and 15 are considered disclosed as well as between 10 and 15.
31. "Optional" or "optionally" means that the subsequently described event or
circumstance may or may not occur, and that the description includes instances
where said event
or circumstance occurs and instances where it does not.
32. "Probes" are molecules capable of interacting with a target nucleic acid,
typically in a
sequence specific manner, for example through hybridization. The hybridization
of nucleic acids
is well understood in the art and discussed herein. Typically a probe can be
made from any
combination of nucleotides or nucleotide derivatives or analogs available in
the art.
33. "Primers" are a subset of probes which are capable of supporting some type
of
enzymatic manipulation and which can hybridize with a target nucleic acid such
that the
enzymatic manipulation can occur. A primer can be made from any combination of
nucleotides
or nucleotide derivatives or analogs available in the art which do not
interfere with the enzymatic
manipulation.
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1i.mnE;te 1'M~a~~~s," "elevates," or "raises" refer to levels above control or
reference levels. The terms can also include the appearance or occurrence of
an event (e.g., a
level above a control or reference level that is zero). The terms "decreases,"
"reduces," or
"lowers" refer to levels below control or reference levels. These terms can
also include the
absence or ablation of an event (e.g., a level of zero when a control or
reference level is not
zero).
35. As used herein, the terms "subject" and "patient" are used interchangeably
and mean
an individual. Thus, "subject" or "patient" can include domesticated animals
(e.g., cats, dogs,
etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory
animals (e.g., mouse,
rabbit, rat, guinea pig, etc.), and birds. "Subject" or "patient" can also
include a mammal, such
as a primate. In one particular aspect, a "subject" or "patient" can be a
human.
36. As used herein, "sample" refers to any biological material obtained from a
subject or
patient. In one aspect, a sample can comprise blood, cerebrospinal fluid
("CSF"), or urine. In
other aspects, a sample can comprise whole blood, plasma, leukocytes enriched
from blood
samples, and cultured cells (e.g., leukocytes from a subject). A sample can
also include a biopsy
or tissue sample including neural tissue. In still other aspects, a sample can
comprise whole cells
and/or a lysate of the cells. Examples of cells include, but are not limited
to, leukocytes such as
neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any
combination thereof. In
another particular aspect, a sample can comprise a leukocyte or substantially
pure population of
leukocytes or a lysate thereof. The term "substantially pure" with respect to
a population of
leukocytes or lysates thereof is intended to refer to a sample that contains
less than about 1%,
less than about 5%, less than about 7%, less than about 10%, less than about
12%, less than
about 15%, less than about 20%, less than about 25%, or less than about 30% of
cells other than
leukocytes, based on the total number of cells in the sample. In a specific
example, a sample can
comprise lymphocytes, a substantially pure population of lymphocytes, or a
lysate of a
substantially pure population of lymphocytes. Optionally, the leukocytes can
be enriched for a
selected type. For example, the leukocyte population can be enriched for
lymphocytes and used
in the methods described herein. Enrichment can be accomplished using cell
sorting techniques
like FACS.
37. By "neurodegenerative disease" is meant any disease characterized by the
dysfunction
and/or death of neurons leading to a loss of neurologic function in the brain,
spinal cord, central
nervous system, and/or peripheral nervous system. Neurodegenerative diseases
can be chronic
or acute. Examples of neurodegenerative diseases include, but are not limited
to, Parkinson's
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~~lteAtias, frontotemporal dementia and Parkinsonism, Alzheimer's
disease, Mild Cognitive Impairment, Diffuse Lewy body disease, Dementia with
Lewy bodies
type, demyelinating diseases such as multiple sclerosis and acute transverse
myelitis,
amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob
disease, AIDs dementia
complex, extrapyramidal and cerebellar disorders such as lesions of the
corticospinal system,
disorders of the basal ganglia, corticobasal ganglionic degeneration,
peripheral neuropathy
(secondary to diabetes or chemotherapy treatment), progressive supranuclear
Palsy, structural
lesions of the cerebellum, spinocerebellar degenerations, such as spinal
ataxia, Friedreich's
ataxia, cerebellar cortical degenerations, multiple systems degenerations
(Mencel, Dejerine-
Thomas, Shi-Drager, and Machado-Joseph), multiple system atrophy, systemic
disorders
(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and
mitochondrial multisystem
disorder), disorders of the motor unit such as neurogenic muscular atrophies
(anterior horn cell
degeneration, infantile spinal muscular atrophy, and juvenile spinal muscular
atrophy), Down's
Syndrome in middle age, subacute sclerosing panencephalitis, Hallervorden-
Spatz disease,
dementia pugilistica, Pick's disease, and the like. Some examples of acute
neurodegenerative
disease are stroke, ischemia, and multiple infarct dementia. Sudden loss of
neurons may also
characterize the brains of patients with epilepsy and those that suffer
hypoglycemic insults and
traumatic injury of the brain, peripheral nerves, or spinal cord.
38. Throughout this application, various publications are referenced. The
disclosures of
these publications in their entireties are hereby incorporated by reference
into this application in
order to more fully describe the state of the art to which this pertains. The
references disclosed
are also individually and specifically incorporated by reference herein for
the material contained
in them that is discussed in the sentence in which the reference is relied
upon.
39. Reference will now be made in detail to specific aspects of the disclosed
materials,
compounds, compositions, articles, devices, and methods, examples of which are
illustrated in
the accompanying Examples and Figures.
B. Compositions and methods
40. Disclosed herein are biomarkers, including methods for identifying and
using
biomarkers. In some specific aspects, the disclosed biomarkers can be used in
(i) methods of
diagnosing neurodegenerative diseases (e.g., Parkinson's disease and
Alzheimer' disease), (ii)
methods of tracking the progression of a neurodegenerative disease, (iii)
methods of monitoring
the response of a subject to a treatment for a neurodegenerative disease, (iv)
methods of
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6:gic--'fd'~Ã'a"i~i~~44generative disease, (v) methods of distinguishing one
neurodegenerative disease from another, and several other methods, as are
disclosed herein.
41. By "biomarker" is meant any assayable characteristic or composition that
can be used
to identify a condition (e.g., a neurodegenerative disease or lack thereof) or
the status of a
condition in a subject or sample. A biomarker can, in some examples disclosed
herein, be a gene
whose expression characteristics can be used to identify a condition or status
of a condition in a
subject or sample. In other examples, a biomarker can be a gene product. By
"gene product" is
meant a transcript, nucleic acid, or protein. Thus, disclosed herein are
biomarkers whose
presence, absence, or relative amount can be used to identify a condition or
status of a condition
in a subject or sample. In one particular example, a biomarker can be a gene
product whose
presence or absence in a subject is characteristic of a subject having or not
having a particular
neurodegenerative disease, having a particular risk for developing a
neurodegenerative disease,
or being at a particular stage of disease. In still another example, a
biomarker can be a gene
product whose increase or decrease indicates a particular neurodegenerative
disease, a particular
risk for developing a neurodegenerative disease, or a particular stage of
disease. In another
example, a biomarker can be a group of various gene products, the presence or
absence of which
is indicative of a subject having or not having a particular neurodegenerative
disease, having a
particular risk for developing a neurodegenerative disease, or being at a
particular stage of
disease. In a further example; a biomarker can be a group of gene products
whose pattern of
increasing and decreasing expression characterizes a particular
neurodegenerative disease or lack
thereof. Still further, a biomarker can be a gene product or group of gene
products whose pattern
of expression is characteristic of the presence or absence of a
neurodegenerative disease, or a
particular prognosis or outcome of a disease. As used herein, a biomarker can
be a surrogate for
other clinical tests. Biomarkers identified herein can be measured to
determine levels,
expression, activity, or to detect variants. As used throughout when detecting
levels of
expression or activity are discussed, it is understood that this could reflect
variants of a given
biomarker. Variants include amino acid or nucleic acid variants or post
translationally modified
variants.
42. Throughout, whenever a protein is discussed, the nucleic acid (e.g.,
transcript) is also
disclosed, unless explicitly stated to the contrary or as would be understood
by one of ordinary
skill in the art based on the context. Similarly, whenever a nucleic acid is
discussed, the protein
is also disclosed. In discussions of gene products herein, proteins, nucleic
acids, and transcripts
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~~.
'~~.eTM collectively, unless explicitly stated to the contrary or as
would be understood by one of ordinary skill in the art based on the context.
43. Also, while a biomarker can be a particular gene product, or a particular
level or
amount of a gene product, a biomarker can also be a particular variable (e.g.,
a first and/or
second canonical variable) obtained when the levels or amounts of such gene
products are
analyzed in a multivariant canonical analysis.
44. In some examples of the disclosed subject matter, biomarkers for a
neurodegenerative
disease such as Alzheimer's or Parkinson's are used to diagnose the disease in
subjects. And
while profiles of message expression by single neurons or homogenates from
postmortem human
brain can be used to distinguish neurodegenerative diseases from control
samples (see e.g.,
Cheetham JE, et al., J. Neurosci. Methods, 1997;77(1):43-48; Chow N, et al.,
Proc. Natl. Acad.
Sci. U.S.A., 1998;95:9620-9625), disclosed herein, in one aspect, are methods
that combine gene
and/or protein profiling and multivariate canonical analysis to differentiate
neurodegenerative
diseases (e.g., mild AD or sporadic PD) from control blood samples.
45. In one example disclosed herein, blood was drawn from patients diagnosed
in an
Alzheimer's Disease Center as having probable AD and from an age and sex
matched control
sample. Messenger RNA was extracted from leukocytes and amplified. The
expression level of
selected messages was then quantified using low density arrays. Multivariate
canonical analyses
differentiated Alzheimer's and control leukocytes. The message species studied
also
differentiated two Parkinson's disease cases from AD and control samples.
These results
illustrate the additional accuracy that may be derived by a biomarker that
makes use of multiple
variables. Of the classes of mRNA species examined, those that best
distinguished AD from
control leukocytes were those involved in the cell cycle and in inflammatory
processes. That
members of these same classes also distinguish AD from control brains is
consistent with
understanding selected phenomena of the brain in Alzheimer's disease without
actually invading
the brain. There are a variety of applications and patents that discuss the
diagnoses of
Alzheimer's disease, including U.S. Application Nos. 60/063,274, filed October
24, 1997,
09/178,170, filed October 23, 1997, 09/770,534, filed January 25, 2001, and
U.S. Published
Application No. 2005-0084875-Al, which are herein incorporated by reference
for material at
least related to diagnoses of Alzheimer's and methods for same, as well as
genes related to the
diagnoses of AD.
46. A similar example was performed with patients diagnosed with Parkinson's
disease,
as is disclosed herein. Specifically, fresh whole blood was drawn from a
patient population that
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4~Y'~AN"k-lontrol patients. Leukocyte protein concentrations were
determined and protein spots that differed in intensity between PD and control
patients were
identified using Progenesis Discovery software (Nonlinear USA, Inc.; Durham,
NC). Difference
measurements were subjected to statistical testing. Protein spots were
identified as either
increasing or decreasing in Parkinson's disease compared with control.
Differentially expressed
spots were isolated and identified. The results show that protein analysis
from peripheral blood
can be used to diagnose neurodegenerative diseases such as PD.
47. The compositions and methods disclosed herein are based on the
identification that
the expression of certain genes in samples (e.g., blood) from a subject with a
neurodegenerative
disease is altered. The disclosed methods typically involve comparing the
expression of certain
genes and sets of genes in the blood of a subject to the expression of the
same gene or sets of
genes in a control sample. It is understood that the control sample, can be a
non-
neurodegenerative diseased subject concurrently run, or it can be a standard
created by assaying
one or more non-neurodegenerative diseased subjects and collecting the
expression data. The
control sample, thus, can be a standard, which is created and used
continuously. For example, a
standard could be created by the expression profiles of non-AD or non-PD cases
disclosed
herein. The standard could include, for example, the average level of
expression of a gene or
particular set of genes in non-neurodegenerative diseased subject or any other
control group.
48. In one particular aspect, disclosed herein are methods of diagnosing a
neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease) in a
subject. The disclosed
methods can comprise assessing a level of expression or activity of one or
more selected
biomarkers (e.g., gene products) in a sample, for example a sample comprising
leukocytes or a
lysate thereof, from the subject to be diagnosed and comparing the level of
expression or activity
of the selected biomarker(s) to a reference standard that indicates the level
of expression or
activity of the selected biomarker(s) in one or more control subjects. In
these methods, a
difference or similarity between the level of expression or activity of the
selected biomarker(s)
and the reference standard can indicate that the subject has or does not have
a particular
neurodegenerative disease, depending on the particular reference standard.
Methods for
assessing and comparing the level of biomarker(s) expression or activity are
disclosed herein.
49. By "diagnose" or other forms of the word such as "diagnosing" and
"diagnosis" is
meant to identify a particular disease. The term also means to distinguish one
particular disease
from another disease or to distinguish one particular disease from the absence
of disease.
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l'Di'igiid4143tii~Alirgin to mean to identify a particular stage of a disease,
to identify the
risk of developing a disease, or to identify a prognosis of a disease.
50. In these particular methods, the subject can be as described herein, for
example, any
individual, such as a human. In one example, the subject is to be diagnosed
for a particular
neurodegenerative disease. The subject to be diagnosed can have symptoms of a
particular
neurodegenerative disease or the subject can be asymptomatic or preclinical
for a particular
neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease).
51. The control subject can be a subject with a particular neurodegenerative
disease (e.g.,
Parkinson's or Alzheimer's disease), at a particular stage of a disease, with
a particular risk of
developing a disease, or without a particular neurodegenerative disease. In
one example, the
subject to be diagnosed and a control subject can be age-matched and/or sex
matched. Thus, by
comparing the level of expression of one or more selected biomarkers from a
subject to the level
of expression of the same biomarker(s) in a control subject (i.e., reference
standard) one can
diagnose the subject for a neurodegenerative disease.
52. A difference or similarity in the level of the biomarker(s) expression or
activity can
be determined by any quantitative or qualitative comparative analysis between
the levels of one
or more selected biomarker(s) in the sample and in the reference standard. For
example, when
the control subject has a particular neurodegenerative disease, then when
using the disclosed
methods, a similarity between the level of the biomarker(s) in the subject and
the control subject
can indicate that the subject to be diagnosed also has the particular
neurodegenerative disease.
In another example, when the control subject has a particular
neurodegenerative disease, then
when using the disclosed methods, a difference between the level of the
biomarker(s) in the
subject and the control subject can indicate that the subject to be diagnosed
does not have the
particular neurodegenerative disease. Alternatively, when the control subject
does not have a
particular neurodegenerative disease, then, in this example, a difference
between the level of the
biomarker(s) in the subject and the control subject can indicate that the
subject to be diagnosed
has the particular neurodegenerative disease. In still another example, when
the control subject
does not have a particular neurodegenerative disease, then, in this example, a
similarity between
the level of the biomarker(s) in the subject and the control subject can
indicate that the subject to
be diagnosed does not have the particular neurodegenerative disease.
53. In a further example, if a selected gene product was identified as a
biomarker by
detecting an increase in expression or activity of the gene product in
subjects diagnosed with a
neurodegenerative disease, for example, Parkinson's or Alzheimer's disease,
then an increase in
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_KpidAkIf QatiQ 4~te'~ ~ei~'erence standard) of the biomarker in a sample from
a subject can
indicate that the subject has the neurodegenerative disease. On the other
hand, if one or more
selected gene products were identified as biomarkers by detecting a decrease
in expression or
activity of the gene products in subjects diagnosed with the neurodegenerative
disease, then a
decrease (relative to the reference standard) in expression of the protein(s)
in a sample from a
subject can indicate that the subject has the neurodegenerative disease.
54. Still further, a combination of biomarkers can be use in the disclosed
methods. For
example, one or more biomarkers can increase and other biomarkers can decrease
relative to the
reference standard and can thus indicate the presence or absence of a
neurodegenerative disease.
1. Assessing levels of expression
a) Sample collection
55. As noted, the methods disclosed herein typically involve collecting a
sample from a
subject. The sample can be of the peripheral blood of a subject, though the
uses of other samples
are contemplated. A blood sample can be collected in any way that allows for
isolation of cells
and, subsequently, gene products from these cells. For example, the blood can
be collected and
via syringe, and then stored at 4 C. Blood samples can be collected in
evacuated tubes
containing, but not limited to, heparin, EDTA or ADC (yellow tube) or any
other anticoagulant.
Another way to collect and store blood can be achieved through the use of
PAXgeneTM Blood
RNA tubes, which allows the stabilization and the storage of whole blood for
up to 5 days at
room temperature. The use of any other compound or chemical enhancing
eukaryotic mRNA
stability can also be used with this procedure. In one specific exanlple, the
pellet can be re-
suspended by vortexing at 4 C in 200 L buffer (20 mM Tris, pH 7.5, 0.5%
Nonidet, 1 mM
EDTA, 1 mM PMSF, 0.1 M NaCl, 1X Sigma Protease Inhibitor, and 1X Sigma
Phosphatase
Inhibitors 1 and 2). The suspension can be kept on ice for 20 minutes with
intermittent
vortexing. After spinning at 15,000 x g for 5 minutes at 4 C, aliquots of
supernatant can be
stored at minus 70 C. The collection of other types of samples, e.g., urine,
CSF, tissue, etc., can
be performed by methods known in the art.
b) Cell collection
56. Typically, once a sample is collected, the various cells contained within
the sample
can be separated. For example, in certain embodiments, the expression pattern
of a gene or set
of genes is assayed within a leukocyte population contained within the sample.
Various cells can
be isolated in any way, as long as the cells are ultimately preserved such
that gene products can
be collected from them. For example, one way of isolating leukocytes is lysing
the erythrocytes
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Wdd~~&W'-ie~~Land then collecting the remaining leukocytes, by centrifugation,
for example. The assay can be applied to subtypes of leukocytes, such as
lymphocytes and their
subclasses, monocytes, and other types of blood cells. In addition, non-blood
cells can be
assayed, such as those from skin, cheek scrapings, muscle, olfactory
epithelium, digestive
system, urinary system, and reproductive system, for example. Lysing can occur
in any way,
including, for example, in RNAse free conditions through the use of ammonium
chloride or
using commercial reagents such as IlVIM[TNOLYSETM and OPTILYSETM (Coulter
International
Corporation; Miami, FL). Alternatively, HISTOPAQUETM (Sigma; Milwaukee, WI)
with or
without Ficoll can be used to centrifug.e anticoagulated blood to separate
leukocytes from
erythrocytes. Another way to achieve this goal is allowing the anticoagulated
blood to sediment
for 1-2 hours at room temperature and collecting the leukocyte fraction.
Another example of
lysing includes centrifuging anticoagulated blood for about 5 minutes at 150-
200 g; and then
removing the buffy coat. Similar methods for lysing are known in the art and
can also be
employed.
57. The leukocytes can also be collected using, for example, leukocyte-
specific markers
for cell sorting. The general leukocyte population can be sorted into
subtypes, for example, such
as B cells, T cells, basophils, eosinophils, neutrophils, monophils,
monocytes, and platelets, and
subtypes of these general categories. Markers include, but are not limited to,
MHC
glycoproteins, integrins, homing receptors, Fc receptors for IgG, IgE, IgM,
IgA, and IgD,
complement receptors for lymphokines, interferons, colony stimulating factors,
receptors for
insulin, receptors for neurotransmitters, chemotactic receptors, membrane
enzymes, and
transport proteins. These can be sorted, for example, by antibodies that
recognize the more than
170 CD antigens.
58. Typically, once the cells, such as leukocytes, are collected the
leukocytes themselves
will be lysed to collect the nucleic acid and/or proteins. These cells can be
lysed in any way.
Leukocyte nucleic acid can be also collected using 4M guanidinium
isothiocyanate lysis and
cesium chloride ultracentrifugation, for example, or using 4 M guanidine
thiocyanate/ 25 mM
sodium citrate/ 0.5% Sarkosyl / 0.1 M,6-mercaptoethanol to lyse the leukocytes
and then adding
2M NaOAc prior to centrifugation steps and isopropanol precipitation. Further,
nucleic acids
can be collected using hydroxy appetite or they can be collected using
positively charge magnetic
beads. Nucleic acids can also, for example, be precipitated. Total RNA can
also be obtained
using TRiZOLTM reagent (Gibco Life Technologies, Inc.; Rockville, MD), which
is a mono-
phasic solution of phenol and guanidine isothiocyanate. Any other reagent that
maintains the
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t~ ~'; ~ '1'~4611disrupting cells and dissolving cell components can also be
used.
Leukocyte proteins and peptides can also be collected by lysing cells in
"crack buffer" (50 mM
Tris-HCl (pH 6.8), 100 mM DTT, 100 g/ml PMSF, 2% sodium dodecyl sulfate
(SDS), 10%
glycerol, 1 g/ml each of pepstatin A, leupeptin, and aprotinin, and 1 M
sodium orthovanadate),
and sheared with a 22 gauge needle. The protein content of the samples can be
estimated using
the DC protein assay (BioRad). Protein (10-20 g) can be resolved using sodium
dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% SDS.
59. Typically the total gene product (e.g., biomarker) will be first isolated
or collected.
This can be done using any means for collecting nucleic acids and/or proteins.
Once the gene
products are collected, they can be washed, and, for example, eluted, if they
are to be attached to
a type of affinity system, such as magnetic beads. The step of collecting gene
products is
typically done to acquire the mRNA and/or proteins in the sample, and is not
needed if direct
collection of the gene product is used. The gene products can be prepared,
however, in any way
that allows for analysis of gene expression.
c) Preparation of the RNA
60. In some examples, the disclosed methods involve some level of RNA
preparation.
The RNA preparation step is not required to be performed as part of a
contiguous method, but in
certain methods the RNA should be prepared such that it can be hybridized. In
other methods,
the RNA can be used to produce cDNA, which can then be used, for example, as a
template for a
PCR reaction or directly analyzed through, for example, hybridization of a
probe. While in
theory, the RNA preparation step could be performed far removed from the
actual amplification
and quantitation steps (e.g., in another laboratory, or at a much earlier
time), in many
embodiments the RNA isolation and preparation, or amplification of cDNA etc.,
will occur in
conjunction with the amplification and quantitation steps of the methods, such
as PCR or
llybridization; but this is not required.
61. When an RNA preparation step is included in the disclosed methods, the
method of
RNA preparation can be any method of RNA preparation that produces
enzymatically
manipulatable mRNA or analyzable RNA. For example, the RNA can be isolated by
using the
guanidinium isothiocyanate-ultracentrifugation method, the guanidinium and
phenol-chloroform
method, the lithium chloride-SDS-urea method or poly A+ / mRNA from tissue
lysates using
oligo(dT) cellulose method. It is important when isolating the RNA that enough
RNA is
isolated. Furthermore, typically the quantity of RNA obtained can be
determined. For example,
typically at least 0.01 ng or 0.5 ng or 1 ng or 10 ng or 100 ng or 1,000 ng or
10,000 ng or
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As will be discussed herein, during the amplification of
multivariate quantitative PCR it is important that when the amplification is
stopped that the
amplification of each target product remain be at least about 80% or 85% or
90% or 95% the
doubling rate. The number of cycles of PCR that are performed so as to
continue to remain at
about the doubling rate is related to the amount of total RNA that was used in
the cDNA
generation step.
The RNA can be isolated from any desired cell or cell type and from any
organism,
including mammals, such as mouse, rat, rabbit, dog, cat, monkey, and human, as
well as other
non-mammalian animals, such as fish or amphibians, as well as plants and even
prokaryotes,
such as bacteria.
d) RNA expression analysis
62. Once the cells or cell type, such as leukocytes, are collected, the
expressed messenger
RNA contained within these cells can be assayed. This can be done using any of
a number of
means, such as hybridization, Northern blot, RT-PCR, real-time RT-PCR, single
channel
quantitative multiplex RT-PCR, oligo- and/or cDNA arrays or any technology
that can lead to
nucleotide quantification. An example of such an approach can be derived from
automated
instruments based on the use of DNA-chip teclmologies, an example of which can
be found at
Integrated Nano-Technologies LLC, http://www.integratednano.coin/. For
example, one way of
assaying the total mRNA is to collect all of the nucleic acids contained
within the cells, by for
example, lysing the cells, and precipitating the nucleic acids. Messenger RNA
can be collected
in any way, such as using a polyT oligonucleotide, which will specifically
hybridize with the
polyA tail contained on messenger RNA transcripts. This method, however,
relies on the
presence of the 3'-polyA tail, but under certain conditions, this tail may be
degraded. Thus,
another way of directly analyzing all messenger RNA, including fragments, can
be to use
message specific primers with reverse transcriptase to make cDNA. This cDNA
can then be
assayed directly or amplified and assayed by, for example, using quantitative
PCR discussed
herein. It is understood that the ultimate goal is the analysis of gene
expression which can be
accomplished in any way which analyzes the expression of genes and compares
their expression.
It is understood that direct hybridization or other means of identification of
mRNA is
considered, as well as means where the mRNA is manipulated to form, for
example, cDNA
which is directly analyzed through, for example, hybridization or other
identification, or which
itself can be amplified producing, for example, a PCR product, which itself
can be directly
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or otherwise identified or manipulated. As long as the
end goal of identification of gene expression is realized it is contemplated
herein.
63. The analysis of the expression can be through, for example, hybridization
of probes.
For example, probes for the specific genes to be analyzed can be contained on,
for example, a
chip, and the mRNA can be analyzed for binding to the chip. These chips are
typically referred
to as arrays, such as microarrays or macroarrays.
(1) Microarrays
64. An array is an orderly arrangement of samples, providing a medium for
matching
known and unknown DNA samples based on base-pairing rules. Typically the
process of
identifying the unknowns is automated. An array experiment can make use of
common assay,
systems such as microplates or standard blotting membranes and can be created
by hand or make
use of robotics to deposit the sample. In general, arrays are described as
macroarrays or
microarrays, the difference being the size of the sample spots. Macroarrays
contain sample spot
sizes of about 300 m or larger and can be easily imaged by existing gel and
blot scanners. The
sample spot sizes in a microarray can be 300 m or less, but typically less
than 200 m in
diameter and these arrays usually contain thousands of spots. Microarrays
typically require
specialized robotics and/or imaging equipment that are generally constructed
for each unique
application of a microarray. Terminologies that have been used in the
literature to describe this
technology include, but are not limited to, biochip, DNA chip, DNA microarray,
GENECHIP TM
(Affymetrix's high density, oligonucleotide-based DNA array product
(Affymetrix, Inc.; Santa
Clara, CA)), and gene array.
65. DNA microarrays typically are fabricated by high-speed robotics, generally
on glass
or nylon substrates, for which probes with known identity are used to
determine complementary
binding, thus allowing massively parallel gene expression and gene discovery
studies. An
experiment with a single DNA chip can provide information on thousands of
genes
simultaneously. It is herein contemplated that the disclosed microarrays can
be used for any
purpose, including monitoring gene expression, disease diagnosis, gene
discovery, drug
discovery (pharmacogenomics), and toxicological research or toxicogenomics.
66. There are two variants of the DNA microarray technology, in terms of the
property of
arrayed DNA sequence with known identity. The main difference between Type I
and Type II
arrays is that in a Type I array there is typically a single sequence or set
of related sequences,
such as a set of allelic sequences, and in Type II microarrays there are many
different sequences
attached to the surface.
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=x ''.i[ ~,~j:u-~ ~t-Rt A3tcrb "a~-"r~fa~~t=comprise a probe, typically a cDNA
(500 to about 5,000 bases
long) that is immobilized to a solid surface such as glass using robot
spotting and exposed to a
set of targets either separately or in a mixture. This method is traditionally
referred to as a DNA
microarray. With Type I microarrays, localized multiple copies of one or more
polynucleotide
sequences, preferably copies of a single polynucleotide sequence are
immobilized on a plurality
of defined regions of the substrate's surface. A polynucleotide refers to a
chain of nucleotides
ranging typically from 5 to 10,000 nucleotides. These immobilized copies of a
polynucleotide
sequence are suitable for use as probes in hybridization experiments. The
immobilized
sequences are then probed with a number of different samples, typically at
different regions of
the chip, such that samples which contain nucleotides that hybridize to the
immobilized sample
can be identified.
68. To prepare beads coated with immobilized probes, beads are immersed in a
solution
containing the desired probe sequence and then immobilized on the beads by
covalent or
noncovalent means. Alternatively, when the probes are immobilized on rods, a
given probe can
be spotted at defined regions of the rod. Typical dispensers include a
micropipette delivering
solution to the substrate with a robotic system to control the position of the
micropipette with
respect to the substrate. There can be a multiplicity of dispensers so that
reagents can be
delivered to the reaction regions simultaneously. In one embodiment, a
microarray is formed by
using ink-jet technology based on the piezoelectric effect, whereby a narrow
tube containing a
liquid of interest, such as oligonucleotide synthesis reagents, is encircled
by an adapter. An
electric charge sent across the adapter causes the adapter to expand at a
different rate than the
tube and forces a small drop of liquid onto a substrate (see Baldeschweiler,
et al., PCT
publication W095/251116).
69. Samples can be any sample containing polynucleotides (polynucleotide
targets) of
interest and obtained from any bodily fluid (blood, urine, saliva, phlegm,
gastric juices, etc.),
cultured cells, biopsies, or other tissue preparations. DNA or RNA can be
isolated from the
sample according to any of a number of inethods well known to those of skill
in the art. For
example, methods of purification of nucleic acids are described in Laboratory
Techniques in
Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes.
Part I. Theory
and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier (1993). In one
embodiment, total RNA is
isolated using the TRIZOLTM total RNA isolation reagent (Gibco Life
Technologies, Inc.,
Rockville, MD) and mRNA is isolated using oligo d(T) colurnn chromatography or
glass beads.
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j~~e'r' ~i~~~ '~~~~tidi"~t~ ~'WR Aing, the hybridization signals obtained
should reflect accurately
the amounts of control target polynucleotide added to the sample.
70. The plurality of defined regions on the substrate, immobilized
polynulcoeotide, can
be arranged in a variety of formats. For example, the regions may be arranged
perpendicular or
in parallel to the length of the casing. Furthermore, the targets do not have
to be directly bound
to the substrate, but rather can be bound to the substrate through a linker
group. The linker
groups may typically vary from about 6 to 50 atoms long. Suitable linker
groups include
ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on
the substrate
surface react with one of the terminal portions of the linker to bind the
linker to the substrate.
The other terminal portion of the linker is then functionalized for binding
the probes.
71. Sample polynucleotides may be labeled with one or more labeling moieties
to allow
for detection of hybridized probe/target polynucleotide complexes. The
labeling moieties can
include compositions that can be detected by spectroscopic, photochemical,
biochemical,
bioelectronic, immunochemical, electrical, optical or chemical means. The
labeling moieties
include radioisotopes such as 32P, 33P, or 35S, chemiluminescent compounds,
labeled binding
proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers
and dyes,
magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron
transfer donors
and acceptors, biotin, and the like.
72. Labeling can be carried out during an amplification reaction, such as
polymerase
chain reaction and in vitro or in vivo transcription reactions. Alternatively,
the labeling moiety
can be incorporated after hybridization once a probe-target complex is formed.
In one preferred
embodiment, biotin is first incorporated during an amplification step as
described above. After
the hybridization reaction, unbound nucleic acids are rinsed away so that the
only biotin
remaining bound to the substrate is that attached to target polynucleotides
that are hybridized to
the polynucleotide probes. Then, an avidin-conjugated fluorophore, such as
avidin-
phycoerythrin, that binds with high affinity to biotin is added.
73. Hybridization causes a polynucleotide probe and a complementary target to
form a
stable duplex through base pairing. Hybridization methods are well known to
those skilled in
the art, and stringent conditions for hybridization can be defined by salt
concentration,
temperature, and other chemicals and conditions as discussed herein. Varying
additional
parameters, such as hybridization time, the concentration of detergent (sodium
dodecyl sulfate,
SDS) or solvent (formamide), and the inclusion or exclusion of carrier DNA,
are well known to
those skilled in the art. Additional variations on these conditions will be
readily apparent to
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and Berger SL, Methods Enzymol., 1987; 152:399-407;
Kimmel AR, Methods Enzymol., 1987; 152:507-511; Ausubel FM, et al., Short
Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y., 1997; and Sambrook J, et
al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y., 1989).
74. Methods for detecting complex formation are well known to those skilled in
the art.
In one example, the polynucleotide probes are labeled with a fluorescent label
and measurement
of levels and patterns of complex fonnation is accomplished by fluorescence
microscopy, such
as confocal fluorescence microscopy. An argon ion laser excites the
fluorescent label, emissions
are directed to a photomultiplier, and the amount of emitted light detected
and quantitated. The
detected signal should be proportional to the amount of probe/target
polynucleotide complex at
each position of the microarray. The fluorescence microscope can be associated
with a
computer-driven scanner device to generate a quantitative two-dimensional
image of
hybridization intensities. The scanned image is examined to determine the
abundance/expression level of each hybridized target polynucleotide.
75. Tii a differential hybridization experiment, polynucleotide targets from
two or more
different biological samples are labeled with two or more different
fluorescent labels with
different emission wavelengths. Fluorescent signals are detected separately
with different
photomultipliers set to detect specific wavelengths. The relative
abundances/expression levels
of the target polynucleotides in two or more samples are obtained. Typically,
microarray
fluorescence intensities can be normalized to take into account variations in
hybridization
intensities when more than one microarray is used under similar test
conditions. In one example,
individual polynucleotide probe/target complex hybridization intensities are
normalized using
the intensities derived from internal normalization controls contained on each
microarray.
76. Type II microarrays comprise an array of oligonucleotides (e.g., from
about 20 to
about 80-mer oligos) or peptide nucleic acid (PNA) probes that is synthesized
either in situ (on-
chip) or by conventional synthesis followed by on-chip immobilization. The
array is exposed to
labeled sample DNA, hybridized, and the identity/abundance of complementary
sequences is
determined. This method, "historically" called DNA chips, was developed at
Affymetrix, Inc.,
(Santa Clara, CA), which sells its photolithographically fabricated products
under the
GENECHIP trademark.
77. The basic concept behind the use of Type II arrays for gene expression is
simple:
labeled cDNA or cRNA targets derived from the mRNA of an experimental satnple
are
hybridized to nucleic acid probes attached to the solid support. By monitoring
the amount of
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t~lab~l Cie-
location, it is possible to infer the abundance of each mRNA
species represented. Although hybridization has been used for decades to
detect and quantify
nucleic acids, the combination of the miniaturization of the technology and
the large and
growing amounts of sequence information, have enormously expanded the scale at
which gene
expression can be studied.
78. Microarray manufacturing can begin with a 5-inch square quartz wafer.
Initially the
quartz is washed to ensure uniform hydroxylation across its surface. Because
quartz is naturally
hydroxylated, it provides an excellent substrate for the attachment of
chemicals, such as linker
molecules, that are later used to position the probes on the arrays.
79. The wafer is placed in a bath of silane, which reacts with the hydroxyl
groups of the
quartz, and forms a matrix of covalently linked molecules. The distance
between these silane
molecules determines the probes' packing density, allowing arrays to hold over
500,000 probe
locations, or features, within a mere 1.28 square centimeters. Each of these
features harbors
millions of identical DNA molecules. The silane film provides a uniform
hydroxyl density to
initiate probe assembly. Linker molecules, attached to the silane matrix,
provide a surface that
may be spatially activated by light.
80. Probe synthesis occurs in parallel, resulting in the addition of an A, C,
T, or G
nucleotide to multiple growing chains simultaneously. To define which
oligonucleotide chains
will receive a nucleotide in each step, photolithographic masks, carrying 18
to 20 square m
windows that correspond to the dimensions of individual features, are placed
over the coated
wafer. The windows are distributed over the mask based on the desired sequence
of each probe.
When ultraviolet light is shone over the mask in the first step of synthesis,
the exposed linkers
become deprotected and are available for nucleotide coupling.
81. Once the desired features have been activated, a solution containing a
single type of
deoxynucleotide with a removable protection group is flushed over the wafer's
surface. The
nucleotide attaches to the activated linkers, initiating the synthesis
process.
82. Although each position in the sequence of an oligonucleotide can be
occupied by 1 of
4 nucleotides, resulting in an apparent need for 25 x 4, or 100, different
masks per wafer, the
synthesis process can be designed to significantly reduce this requirement.
Algorithms that help
minimize mask usage calculate how to best coordinate probe growth by adjusting
synthesis rates
of individual probes and identifying situations when the same mask can be used
multiple times.
83. Some of the key elements of selection and design are common to the
production of all
microarrays, regardless of their intended application. Strategies to optimize
probe hybridization,
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RWJ' i"ded in the process of probe selection. Hybridization under
particular pH, salt, and temperature conditions can be optimized by taking
into account melting
temperatures and using empirical rules that correlate with desired
hybridization behaviors.
84. To obtain a complete picture of a gene's activity, some probes are
selected from
regions shared by multiple splice or polyadenylation variants. In other cases,
unique probes that
distinguish between variants are favored. Inter-probe distance is also
factored into the selection
process.
85. A different set of strategies is used to select probes for genotyping
arrays that rely on
multiple probes to interrogate individual nucleotides in a sequence. The
identity of a target base
can be deduced using four identical probes that vary only in the target
position, each containing
one of the four possible bases.
86. Alternatively, the presence of a consensus sequence can be tested using
one or two
probes representing specific alleles. To genotype heterozygous or genetically
mixed samples,
arrays with many probes can be created to provide redundant information,
resulting in
unequivocal genotyping. In addition, generic probes can be used in some
applications to
maximize flexibility. Some probe arrays, for example, allow the separation and
analysis of
individual reaction products from complex mixtures, such as those used in some
protocols to
identify single nucleotide polymorphisms (SNPs).
87. In certain examples, the disclosed genes or gene sets whose expression are
related to
the diagnosis of a particular neurodegenerative disease (e.g., Alzheimer's or
Parkinson's) in a
sample (e.g., peripheral blood) are attached. to a microarray. In certain
embodiments, for
example, the chip can be divided into sections each of which contain a variety
of regions and
alleles for one of the genes in the diagnosis set, and another region of the
chip can contain a
variety of regions and alleles for another of the genes in the diagnosis set.
In yet another
example, each region of the chip could contain all of the alleles and regions
of all of the genes in
the set. There are many variations on this type example, which include the use
of all or any
subset of the disclosed neurodegenerative disease diagnostic genes, and it is
understood that any
region of these disclosed genes, from 3 bases to the full-length sequence can
be used as a probe
region for immobilization. Furthermore, any allelic or variant can also be
used.
(2) Multivariate single channel quantitative RT-PCR
88. One particularly useful means for assaying the expression levels is
through the use of
multivariate single channel quantitative RT-PCR. These methods are disclosed
in U.S. Patent
Applications 60/336,095, filed November 30, 2001, 60/397,475, filed July 19,
2002, 10/496,626,
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1229, filed March 12, 2001, and 10/096,710, filed March 12,
2002, which are herein incorporated by reference at least for material related
to methods and
compositions related to multivariate single channel quantitative RT-PCR.
Briefly, the method
utilizes a PCR-based high-throughput method for simultaneously analyzing the
expression of
multiple genes. The method can use minute quantities of starting material and
reach single copy
levels of efficiency, for example, where only a single target nucleic acid was
available, such as a
single copy of transcript from a single target cell. For example, for the
analysis of 20 transcripts
in triplicate for 4 subjects, less than 1 g total RNA per subject is needed.
The disclosed
methods are capable of simultaneously analyzing multiple genes. The disclosed
methods use
gene-specific primers in particular ways. The disclosed methods can quantitate
multiple genes
with the use of a single signal reagent, such as a fluorescent probe.
89. hi general, the method is useful for obtaining quantitative information
about the
expression of many different genes in a sample that can contain as little as a
single cell. Since
the disclosed methods are quantitative, comparisons of the expression patterns
at a quantitative
level between a variety of different cell states or cell types can be
achieved. In general, total
RNA can be isolated from the target sample using any isolation procedure. This
RNA can then
be used to generate first strand copy DNA (cDNA) using any procedure, for
example, using
random primers, oligo-dt primers, or random-oligo-dt primers, which are oligo-
dt primers
coupled on the 3'-end to short stretches of specific sequence covering all
possible combinations,
so the primer primes at the junction between the polyA tract and non-poly A
tract associated
with messenger RNA (mRNA). The cDNA is then used as a template in a PCR
reaction. This
PCR reaction is performed with primer sets, a forward and a reverse primer,
that are specific for
the expressed genes, which are to be tracked. This reaction can contain as
many different primer
sets as desired, but typically would contain between 5 and 100 different sets
of primers, each
specific for a single gene or single isoform (including any specific number
between 5 and 100).
Typically all of the primers will be in about equimolar concentration. After
performing a
number of PCR cycles, for example 15 cycles, such that the DNA is still
amplifying at about
greater than 80% or 85% or 90% or 95% the doubling rate, the PCR is stopped.
Typically, in the
first round of PCR, if quantitative PCR (real time PCR) was performed, one
does not reach the
threshold cycle of amplification. However, the disclosed methods in certain
embodiments can
still work if amplification proceeds for about less than 9, 8, 7, 6, 5, 4, 3,
2, or 1 cycle(s) past the
threshold cycle. The number of cycles in the first round depends on the amount
of starting
materials. For example, 20 cycles can be used for single cell experiments. The
PCR reaction is
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qh''~p~f ~''1~~A~c~ int~i~FriwP'r~e~e~ion tubes for a (new) second round of
PCR. Each of the tubes
contains a fraction of the previous PCR reaction mixture which contains all of
the products
produced from all of the specific primers present in the first PCR mixture. In
the second PCR
mixture, containing the fraction of the first PCR mixture, typically only one
of the specific
primer sets or a new primer set is added, in addition to the universal primer
which has the
molecular beacon attached, and the PCR is performed. Typically this second
round of PCR is
performed using quantitative real time PCR protocols, which, for example, rely
on increases in
fluorescence at each cycle of PCR through (for example, probes that hybridize
to a portion of
one of the amplification probes) the release of fluorescence from a quencher
sequence while the
uniprimer (universal primer) binds to the DNA sequence. Fluorescence
approaches used in real-
time quantitative PCR are typically based on a fluorescent reporter dye such
as SYBR green,
FAM, fluorescein, HEX, TET, TAMRA, etc. and a quencher such as DABSYL, Black
Hole, etc.
When the quencher is separated from the probe during the extension phase of
PCR, the
fluorescence of the reporter can be measured. Systems like Molecular Beacons,
Taqman Probes,
Scorpion Primers or Sunrise Primers and others use this approach to perform
real-time
quantitative PCR. Examples of methods and reagents related to real time probes
can be found in
U.S. Patent Nos. 5,925,517; 6,103,476; 6,150,097, and 6,037,130, which are
incorporated by
reference herein at least for material related to detection methods for
nucleic acids and PCR
methods. In addition to performing the above steps, the generation of a
standard curve for the
primer sets, and typically for each individual primer set, should be made so
that data obtained
from the second round of PCR can be accurately correlated with an absolute
copy number of the
original starting material in the target sample, containing for example, the
target cell or cells.
Each of these steps of the general method, as well as the reagents and
variations of the method,
are discussed in detail herein. A key aspect to understanding the disclosed
methods is the
combination of a first PCR containing the multiple different primer sets in a
batch PCR mixture
in which all target gene products or fragments of gene products are amplified
with a second PCR
panel in which the specific amplification reaction occurs in which a portion
of the batch PCR
mixture is amplified with specific primer sets. Quantitation is typically
achieved by reference to
a standard curve that is generated for the complete primer sets or each
individual primer set.
e) Preparation of protein
90. In some examples, the disclosed methods can involve some level of protein
or
peptide preparation. The protein or peptide preparation step is not required
to be performed as
part of a contiguous method, but in certain methods the protein or peptide
should be prepared
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f if;;,(f
as disclosed herein. While in theory, the protein or peptide
preparation step could be performed far removed from the actual analysis steps
(e.g., in another
laboratory, or at a much earlier time), in many embodiments the protein or
peptide isolation and
preparation, e.g., electrophoresis, will occur in conjunction with the
quantitation steps of the
methods; but this is not required.
91. When a protein or peptide preparation step is included in the disclosed
methods, the
method of protein or peptide preparation can be any method of protein or
peptide preparation
that produces analyzable protein or peptide. For example, the sample cells can
be lysed in
"crack buffer" (50 mM Tris-HCl (pH, 6.8), 100 mM DTT, 100 g/ml PMSF, 2% SDS,
10%
glycerol, 1 g /ml each of pepstatin A, leupeptin, and aprotinin, and 1 m
sodium orthovanadate),
and sheared with a 22 gauge needle. The protein content of the samples can be
estimated using
the DC protein assay (BioRad). Protein (10-20 g) can be resolved using sodium
dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% SDS. Typically
the quantity
of protein or peptide obtained can be determined.
The proteins or peptides can be isolated from any desired cell or cell type
and from any
organism, including mammals, such as mouse, rat, rabbit, dog, cat, monkey, and
human, as well
as other non-mammalian animals, such as fish or amphibians, as well as plants
and even
prokaryotes, such as bacteria.
f) Protein expression analysis
92. In certain embodiments of the disclosed methods, assessing the level of
expression of
one or more proteins or peptides can be performed. The level of protein or
peptide expression
can be assessed in addition to the nucleic acid analysis disclosed herein, or
as an alternative to
the nucleic acid analysis. Assessing a level of expression of one or more
proteins in a sample
can be performed by various techniques known in the art. For example,
assessing the level of
expression can involve analyzing one or more proteins by two-dimensional gel
electrophoresis,
mass spectroscopy (MS), matrix-assisted laser desorption/ionization-time of
fligllt-MS (MALDI-
TOF), surface-enhanced laser desorption ionization-time of flight (SELDI-TOF),
high
performance liquid chromatography (HPLC), fast protein liquid chromatography
(FPLC),
multidimensional liquid chromatography (LC) followed by tandem mass
spectrometry (MS/MS),
protein chip expression analysis, gene chip expression analysis, and laser
densitometry,
including combinations of these techniques. In another example of a technique
for analyzing
protein expression levels, one can assay the amount of mRNA that encodes for a
particular
protein or proteins.
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3- ques an antibody or other agent that selectively binds to a
protein can be used to detect the amount of that protein expressed in a
sample. For example, the
level of expression of a protein can be measured using methods that include,
but are not limited
to, Western blot, immunoprecipitation, enzyme-linked immunosorbent assay
(ELISA),
radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS), or a
combination
thereof. Also, antibodies, aptamers, or other ligands that specifically bind
to a protein can be
affixed to so-called "protein chips" (protein microarrays) and used to measure
the level of
expression of a protein in a sample. In other methods, immunofluorescence
techniques can be
used to visually assess the expression level of a protein iin a sample. In
immunofluorescence
techniques, antibodies that specifically bind to a protein are visualized to
indirectly detect the
presence of a protein on the cell surface of intact leukocytes, and/or
throughout permeabilized
leukocytes.
94. The term "antibodies" is used herein in a broad sense and includes both
polyclonal
and monoclonal antibodies. In addition to intact immunoglobulin molecules,
also included in
the term "antibodies" are fragments or polymers of those immunoglobulin
molecules, and human
or humanized versions of immunoglobulin molecules or fragments thereof.
Monoclonal
antibodies include "chimeric" antibodies in which a portion of the heavy
and/or light chain is
identical with or homologous to corresponding sequences in antibodies derived
from a particular
species or belonging to a particular antibody class or subclass, while the
remainder of the
chain(s) is identical with or homologous to corresponding sequences in
antibodies derived from
another species or belonging to another antibody class or subclass, as well as
fragments of such
antibodies, as long as they exhibit the desired antagonistic activity (See,
U.S. Patent No.
4,816,567 and Morrison, et al., Proc. Natl. Acad. Sci. U.S.A. 1984;81:6851-
6855).
95. In one example, an antibody to alpha synuclein or a conformer thereof can
be used to
identify the level of alpha-synuclein or various conformers thereof.
Specifically, antibodies to
native-alpha synuclein, dopamine-adducted alpha-synuclein, and oligomeric or
aggregated alpha-
synuclein can be used. These antibodies and methods for their preparation
and'isolation are
disclosed in U.S. Patent Application entitled "Alpha-Synuclein Antibodies and
Methods Related
Thereto," filed on July 19, 2005, to Federoff et al.
96. Non-antibody ligands that selectively bind to a protein can also be used
to detect the
presence, the absence, and/or to quantify the expression of a protein. For
example, ligands can
be fluorescently labeled (e.g. conjugated to fluorescent molecule, such as
green fluorescent
protein (GFP)) or ligands can be radiolabeled. Labeled ligands can be
contacted with a sample,
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~'lig~a~i't~~ ~ ~rotein can be assessed. The amount of labeled ligand that
binds
to proteins in the sample is an indication of the amount of a particular
protein present in the
sample. When the protein is a cell surface molecule, a protein ligand can be
contacted to intact
cellular sample to detect the expression level of the protein at the cell
surface. Alternatively, the
integrity of leukocytes in a sample can be compromised by permeabilizing or
lysing the cells,
and subsequently assessing the amount of labeled ligand that binds to proteins
in the sample of
lysed leukocytes.
97. Labels can be directly or indirectly attached to antibodies or non-
antibody ligands.
Direct labeling includes, for example, attaching a label directly to the
antibody or non-antibody
ligand. Indirect labeling includes, for example, attaching a label to a second
or third antibody or
non-antibody ligand.
98. In cases where the level of expression of a protein is regulated at the
genetic level,
expression levels of the protein can be indirectly monitored by detecting the
expression level of
the gene that encodes the protein. Methods suitable for detecting and/or
quantifying genetic
expression that can be used include, but are not limited to, Northern blot,
RNAse protection
analyses, reverse transcription-polymerase chain reaction (RT-PCR), and gene-
chip (e.g.,
nucleotide expression microarray) technologies.
99. Optionally, the level of expression of multiple proteins can be determined
simultaneously or nearly simultaneously. For example, two-dimensional (2D) gel
electrophoresis can be used to simultaneously or nearly simultaneously assess
the expression
level of thousands of proteins in a sample. (See e.g., Vietor and Huber,
Biochim. Biophys.
Acta., 1997;1359:187-99, which is incorporated by reference herein for at
least its teachings of
methods to assess levels of protein expression). In one aspect, the disclosed
methods can
include 2D gel electrophoresis, where a mixture of proteins are prepared from
the sample, e.g.,
by lysing leukocytes and mixing the protein lysate with sample buffer. The
protein mixture can
be loaded onto a gel slab, electrophoresed in two dimensions, and then the gel
slab can be dried.
After resolution by 2D electrophoresis, expression levels of individual
proteins or groups of
proteins can be assessed. Protein levels can be assessed by silver staining or
Coomassie staining.
If the proteins in a sample are labeled, then measuring the amount of label
can be used to assess
the amount of protein.
g) Levels of gene product expression and canonical variables
100. The detection of the levels of expression of the target genes, the genes
disclosed
herein as related to a particular neurodegenerative disease (e.g., Parkinson's
and Alzheimer's)
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the presence of a neurodegenerative disease in a sample (e.g.,
blood) of a subject with the disease, i.e., biomarkers, can occur in any way
as disclosed herein.
In some examples, what is typically required is the detection of nucleic acid
(e.g., transcripts). In
other examples, what is typically required is the detection of proteins. There
are many means for
detection of gene products, such as radioactivity or fluorescence or any other
methods as
disclosed herein. Any means can be used.
101. Typically any data that is collected can be normalized for general
expression
levels in the cell. This can be done in variety of ways, for example, by
comparing all transcripts
to that of 0-actin expression, which is present in all cells. Other methods of
normalization can
be based on approaches other than expression of any single gene. For
quantitative PCR, a
standard curve should be attained for each message assayed. These standard
curves then become
the basis for deriving absolute copy numbers. Internal controls include, but
are not limited to, 0-
actin, GAPDH, tubulin, etc. or external controls such as but not limited to
cytoplasmic or nuclear
LacZ, agamous, or known labeled cRNAs spiked into each hybridization. Using
bio-chip
approaches, data normalization can also be obtained through averaging samples
of interest to the
overall array background.
102. In certain examples related to hybridization, there can be a stripping
and
reprobing step that increases the specificity of the readings. For example,
samples can be
stripped and reprobed for the T7 promoter.
103. Typically once the expression pattern of a gene product or set of gene
products is
obtained, the expression pattern must be analyzed. The analysis typically
involves performing
some type of statistical analysis of the relative expression levels between
the gene, genes, or
gene sets themselves, as well as the comparison to the control or standard
gene, genes, or gene
sets. Such methods of analysis are disclosed herein.
104. In one example, a level of expression of a biomarker (e.g., a nucleic
acid or
protein gene product) can be subject to a univariant and/or multivariant
canonical analysis to
produce a first and/or second canonical variable. The univariate tests can be
the well known T-
test or the N-test. One N-test that is suitable for use herein is disclosed in
Technical Report
04/01 at http=//www urmc rochester.edulsmd/biostat/people/techreports.html,
which is
incorporated by reference herein at least for its teaching of the statistical
analysis via the N-test.
105. There are a number of methods of multivariate analysis. Any of these may
be
applied. In one example, multivariant analysis can be performed using
commercially available
software, such as SAS/STAT Software, available from SAS Institute, Inc.
(Cary, NC).
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'(9d$ rih ",~Il components analysis can be used, for example. These both deal
with methods of analyzing matrices of data. Typically the canonical variables
consist of a first
canonical and a second canonical variable. The multivariant analysis can be
the essentially non-
parametric test for multiple testing inference. Such multivariate statistical
testing relies on
canonical discriminant analysis. This analysis determines the variables
(messages) that best
distinguish groups and assigns weights to each variable. The first canonical
variable generally
provides the best distinction between groups. The second canonical variable
operates on the
residual variance that remains unaccounted for by canonical variable 1.
Additional iterations are
possible with diminishing effect.
106. Canonical discriminant analysis is equivalent to canonical correlation
analysis
between the quantitative variables and a set of diumny variables coded from
the class variable.
In the following notation the dummy variables can be denoted by y and the
quantitative variables
by x. The total sample covariance matrix for the x and y variables is:
S S,x SXy
SYX Sn,
107. When c is the number of groups, nt is the number of observations in group
t, and
St is the sample covariance matrix for the x variables in group t, the within-
class pooled
covariance matrix for the x variables is
SP = E ~_cF(~,t -1)St
108. The canonical correlations, p;, are the square roots of the eigenvalues,
N. of the
following matrix. The corresponding eigenvectors are v;.
S,pl iz SXYSri 1S'Sp'/z
109. Letting V be the matrix with the eigenvectors v; that correspond to
nonzero
eigenvalues as columns, the raw canonical coefficients are calculated as
follows.
R=Sp'zV
110. The pooled within-class standardized canonical coefficients are:
P = diag(Sp)'ZR
111. And the total sample standardized canonical coefficients are:
T = diag(SXX)"R
112. It may be calculated by any of the following:
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XaZ
X,diag(Sp)'P
,diag(Sxx)Y7
X,
113. For the Multhiariate tests based oi E-1H, where n is the total number of
observations.
E = (n -1)(Syy - SS,,-1SXy)
H = (n -1)SyxS,{,-1SXy
114. The above described multivariant analysis can, as previously noted, be
performed
with commercially available software such SAS/STAT Software, available from
SAS Institute,
Inc. (Cary, NC). In the methods disclosed herein, one can input the levels of
expression from a
set of gene products into such a program. The set of gene products can be any
set of genes, such
as the ones disclosed herein. In some examples, one set of gene products can
correspond to a
control sample or group of controls and another set of gene products can
correspond to a sample
or group of samples with disease. The levels of gene products can be inputted
as absolute or
relative amounts or concentrations. The levels can also be signal intensities,
for example, from
radio- or fluoroanalyses of the gene products. As noted, a result of the
multivariant analysis is a
first and/or second canonical variable.
115. This first and/or second canonical variable produced from the
multivariant
analysis can be used, as is disclosed herein, in the disclosed methods as a
substitute for or in
addition to the level of expression for a biomarker. That is, in some examples
disclosed herein,
the first and/or second canonical variable obtained from analyzing the levels
of expression of one
or more biomarkers from a subject can be compared to a reference standard that
comprises a first
and/or second canonical variable obtained from a multivariant canonical
analysis of levels of
expression of biomarkers from a control or group of control subjects.
116. For example, as shown herein, inultivariant canonical analysis was used
herein as
a diagnostic of Alzheimers disease. For example, when using genes related to
inflammation, the
range for control values for canonical variable 1 can be from about -0.5 to
about -3.1 and the
range for AD can be from about 0 to +4.4. For genes related to cell stress,
the range for control
values for canonical variable 1 can be from about -4.8 to about -0.1 and the
range for AD can be
from about +0.1 to about +4.1. For genes related to the cell cycle/cell death,
the range for
control values for canonical variable 1 can be from about +2.6 to about -3.1
and the range for
AD can be from about +3.0 to about -2.3. Each of these three classes of gene
products
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giia4~~ri crllrol to a degree that is statistically significant by the Wald-
Wolfowitz Runs test, even though there may be some overlap. In terms of
overlap between AD
and control, cell cycle/cell death has the most, inflammation has a one case
overlap and cell
stress shows no overlap.
117. It is noted that the results of the multivariant analysis will, of
course, depend on
the particular input (e.g., the particular gene products, the particular
levels of those gene
products, and the particular sample sets). As such the addition or
substitution of other genes or
the use of different diseases can alter the ranges for canonical variables.
This is illustrated by
comparing Figure 4 and Figure 5, where the canonical variable 1 for cell
stress is different in
Figure 5 due to the inclusion of two PD patients in the control. Dispite
variability in the
particular value of canonical variable 1 and/or 2, distinction of diseases is
still possible, as would
be recognized by one of skill in the art (as is illustrated in Figure 5 for
example).
2. Message class
118. At the core of the disclosed compositions and methods is the analysis of
certain
messages that are correlated with a neurodegenerative disease such as
Parkinson's and
Alzheimer's. In certain embodiments these messages can be single messages, but
typically
classes of messages, and sets of messages will be analyzed because they can
provide more
accurate assessment than for any one of the genes contained within the class
or set by itself.
Table 4 shows exemplary targets that can be analyzed. Multiple gene products,
whether mRNA
or protein, analyzed simultaneously, allows a neurodegenerative disease such
as Parkinson's or
Alzheimer's to be diagnosed.
119. For example, one class of genes that are useful in diagnosing a
neurodegenerative
disease such as Alzheimer's is the class of cell cycle transcripts. These can
include, for example,
cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, and protein
kinase C alpha.
120. In another example, a class of genes that are useful in diagnosing a
neurodegenerative disease such as Alzheimer's is the class of inflammatory
response transcripts.
These can include, for example, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-
alpha, and IL-lOr.
121. In still another example, a class of genes that are useful in diagnosing
a
neurodegenerative disease such as Alzheimer's is the class of cell stress
transcripts. These can
include, for example, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline,
GAPDH, ferritin
H, ferritin L, cox 1, cox 2, and transferrin.
122. In yet another example, any combination or subset of cyclin Dl, cyclin B,
cyclin
Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor,
IL-17r, IL-8,
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i'p"tk1-l antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH,
ferritin H, ferritin L, cox 1, cox 2, and transferrin can be used.
123. In still other examples, other genes related to cell cycle/death,
inflammation, and
stress, as would be know to those of skill in the art, can be used.
124. In another example, a class of genes that are useful in diagnosing a
neurodegenerative disease such as Parkinson's disease include, for example,
HSP60,
Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate
dehydrogenase, ATP-
synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate
mutase 1,
Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory
subunit, Chain A
thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen,
Haptoglobin,
Fibrin beta, actin-interacting protein 1(AIP1), mitogen activated protein
kinase I(MAPKI), actin
or a fragment thereof, glutaraldehyde-3-phosphate dehydrogenase (GAPDH),
transforming
protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B
(ANP32B or
APRIL), peroxiredoxin II, an amyloid precursor protein (APP), a-secretase, 0-
secretase, y-
secretase, Ao peptide, Fe65, Tip60, SERCA, PS 1/2, nectin-la, and non-amyloid
0 component of
senile plaque (NACP/ a-synuclein).
125. There can be any number of gene products in the sets or classes. For
example,
there can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or 100 different genes or gene
products within a set or
class. Furthermore, it is understood that there could be multiple alleles of a
particular gene
which could also make up a set. In certain embodiments, there will be 7 or 8
genes within a set
of transcripts to be used for analysis.
126. In certain embodiments one gene can be used for analysis, such as gene
products
related to alpha-1 antichymotrypsin, crystalline, and cycloogygenase I[.
Table 4: Exemplary targets for neurodegenerative diseases, such as Alzheimer's
and
Parkinson's, which can be analyzed. General classes of these targets are also
provided, though
targets can have functions that impact a variety of cell processes other than
those identified.
SEQ ID Gene Name NCBI Acc No Function Comments Class
NO:
Alpha-l- Inflamin-
1 Alphal-ACT J05176 antichymotrypsin ation / cell
mRNA cycle / stress
Alpha-l- cDNA Inflamm
2 Alphal-ACT T40002 antichymotrypsin, ation / cell
precursor mRNA clone cycle / stress
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~..~ waE w Activates Thiol
3 GAPDH M17851 group during Metabolic
catalysis
cAMP response
4 CREB M34356 element binding Transcrip-
4 factor
protein
Cell specific marker
distinguishes Astrocyte
.5 GFAP J04569 astrocytes from other marker
glial cell during
development of CNS
Developmental-upon
terminal neural
differentiation, Marker of
6 Nestin X65964 nestin is down DNA early
regulated and neurons
replaced by
neurofilaments
Involved in
proteolytical
7 PS 1/S 182 AF416717 processing of APP Proteolytic
and NOTCH, enzyme
regulates epithelial-
cadherin function
Involved in stress
8 HSP27 NM001540 resistance and actin Stress
organization
Molecular
9 HSP90-alpha NM 005348 chaperone, has Stress
ATPase activity by
similarity
Molecular
HSP90-beta NM 007355 chaperone, has Stress
- ATPase activity by
similarity
Contain intermediate
filament proteins N, Neuron
11 NF-M Y00067 M and H involved in DNA structure
maintenance of
neuronal caliber
Contain intermediate
filament proteins N, not Neuron
12 NF-L X05608 M and H involved in complete structure
maintenance of cds, DNA
neuronal caliber
13 C-jun AY217548 Transcription factor DNA Transcrip-
AP-1 tion factor
Tuberin/549 and Titberin/4B2 For these 2 tuberins listed, see Cell
TSC1 and TSC2 below: cycle/death
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P.
Tuberous sclerosis 2
protein: Implicated
as tumor suppressor,
may have function in
vesicular transport,
may also play role in
regulation of cell
growth arrest and in
regulation of
transcription
mediated by steroid
receptors, interaction
of hamartin and mRNA,
tuberin may not Cell
14 TSC2 X75621 facilitate vesicular complete cycle/death
docking, specifically cds
stimulates the
intrinisic GTPase
activity of Ras-
related protein
RAPlA and RAB5,
suggests possible
mechanism for role
in regulating cell
growth, mutations in
tuberin lead to
constitutive
activation of RAP1A
in tumors
Hamartin, tuberous
sclerosis protein 1,
implicated as tumor
suppressor, may
15 TSC1 NM 000368 have function in Cell
- vesicular transport, cycle/death
interaction between
hamartin and tuberin
may facilitate
vesicular docking
16 IAP homo B NM 001166 Apoptotic Cell
- suppressor cycle/death
17 Kinesin light NM182923 May play a role in Molecular
chain 1 - organelle transport motor
Force producing
18 Kinesin light NM022822 protein, may play a clone Molecular
chain 2 - role in organelle motor
transport,
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Intracellular Stress and
inflamm-
molecule, stores inflamm-
19 Ferritin L NM000146 iron in a nontoxic, ation / ROS
soluble, readily (reactive
available form oxygen
s ecies)
Functional molecule
is roughly spherical-
polymeric ferric iron Stress and
20 Ferritin H NM 002032 inflamm-
- core is deposited
into it's central ation / ROS
cavity
Destroys radicals
normally produced
21 SOD-1 NM_000454 within cells, and Stress and
which are toxic to ~~ation
biological systems
22 Alpha tubulin N1VI 006082 Major constituent of Cell
- microtubules structure
Suppresses apoptosis
in variety of cell
systems, regulates
cell death by
controlling
mitochondrial
23 Bcl-2 M13995 membrane Cell death
permeability,
appears to function
in feedback loop
system with
caspases, inhibits
caspase activity
Activation
24 ICE-rel-II U28014 cascade/apoptosis Cell death
execution, cleaves
caspace I
Interleulin 1 beta
converting enzyme,
thiol protease
cleaves IL-1 beta
between an ASP and
an ALA, releasing Cell death /
25 ILIBCE M87507 mature cytokine inflamYna-
which is involved in
tion
a variety of
inflammatory
processes,
specifically inhibited
by cowpox virus
CRMA protein
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... E.._ Accelerates
programmed cell
death by binding
26 BAK NM 001188 to/antagonizing the Cell death
- A suppressor BCL1-
2 or its adenovirus
homolog ElB 19K
protein
Retards apoptosis
27 Bfl-1 NM_004049 induced by IL-3 Cell death
de rivation
GTP-binding nuclear
protein RAN,
involved in
nucleocytoplasmic
28 RAN TC4 N1VI_006325 transport, required Stress
for the import of
proteiui into the
nucleas and for RNA
export
29 Ras-L, TC25 XM 171081 Regulates cellular Stress
- responses
Involved in control
30 Cdk4 AF507942 of cell cycle, cell DNA
division protein
kinase 4
31 Cyclin B1 P14635 Control of cell cycle clones Cell cycle/
in G2/1VI transition only death
Role in growth
regulation,
32 Cyclin Gl NM 004060 associated with Cell cycle/
- G2/M phase arrest in death
response to DNA
damage
Regulates CDK7,
involved in cell
33 Cyclin H NM 001239 cycle control and in Cell cycle/
- RNA transcription death
by RNA polymerase
II
May be involved In
34 Cyclin Al NM 003914 cell cycle at G1/S Cell cycle/
- and G2/M death
transitions
Essential in cell Cell cycle/
35 Cyclin A2 AF518006 cycle at Gl/S and DNA
G2/M transitions death
36 Cyclin E1 AF518727 Control of cell cycle DNA Cell cycle/
at G1/S transition death
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Essential for control
37 Cyclin E2 AF106690 of cell cycle at late Cell cycle/
death
Gl and eary S phase
Role in growth
38 Cyclin G2 NM. 004354 regulation, negative Cell cycle/
- regulation of cell death
cycle progression
Essential for control Cell cycle/
39 Cyclin Dl NM_053056 of cell cycle at Gl/S death
transition
Human heat shock
40 HSP70 M11717 protein (hsp 70) DNA Stress
gene, complete
Heat shock 70 kDa
protein 1, in
cooperation of other
chaperones, HSP70S
stabilize pre-existent
proteins against
aggregation and
mediate the folding
of newly translated
polypeptides,
41 HS71 NM_005345 HSP70S in Stress
mitochondria and
endoplasmic
reticulum provide
driving force for
protein
translocation, are
involved in signal
transduction
pathways with
HSP90
Heat shock-related
70 kDa protein 2,
HSP70S stabilize
pre-existent proteins
42 HS72 NM_021979 against aggregation Stress
and mediate the
folding of newly
translated
ol e tides
43 HS74 NM 002154 Heat shock 70 kDa Stress
rotein 4
44 HS76 X51757 Heat shock 70 kDa DNA Stress
protein 6
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Heat shock 70 kDa DNA,
45 HS77 M11236 gene Stress
protein (fragment)
segment
Regulates the
development of cells
destined to form and
maintain skeleton, Cell
46 c-fos K00650 thought to have role DNA
cycle/death
in signal
transduction, cell
proliferation, and
differentiation
May act as negative Cell
47 Weel X62048 regulator of entry
into G2/M transition cycle/death
48 Fral X16707 Fos-related antigen 1 DNA Cell
cycle/death
Transcriptional
repressor of genes
that require BHLH
49 Hes1 AF264785 protein for Transcrip-
transcription, may tion factor
act as negative
regulator of
myogenesis
see 16 CR genes Stress
crystallin below inflamma-
ion
Alpha crystallin A
chain, may Stress
contribute to the
50 CRAA NM 000394 inflamma-
transparency and
ion
refractive index of
the lens
Rosenthal fiber
protein, Alpha
crystallin B chain, Stress
51 CRAB NM 001885 may contribute to inflamma-
- the transparency and
ion
refractive index of
the lens- Acc#
M24906-CRAB
Alpha crystallin C Stress
52 CRAC NM_014365 chain, protein kinase inflamma-
H11 ion
Breta crystallin B 1, Stress
53 CRB1 U35340 dominant structural inflamma-
components of
vertebrate eye lens ion
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!I ' .N... "~ .. f. ,::!~ ~...(t .. ~ !,::w. ".':::,t ;-.. Beta crystallin B2,
dominant structural Stress
54 CRB2 NM 000496 inflamma-
components of
vertebrate eye lens ion
Beta crystallin B3,
dominant structural Stress
55 CRB3 P26998 inflamma-
components of
vertebrate eye lens ion
Beta crystallin A3, Stress
56 CRBA P05813 dominant structural inflamma-
components of
vertebrate eye lens ion
Beta crystallin A2, Stress
57 CRBB AF166331 dominant structural inflamma-
components of
ion
vertebrate eye lens
Beta crystallin A4, Stress
58 CRBD NM 001886 dominant structural inflamma-
components of
ion
vertebrate eye lens
Beta crystallin S,
Gamma crystallin S, Stress
59 CRBS NM017541 dominant structural inflamma-
components of ion
vertebrate eye lens
Gamma crystallin A,
gamma crystallin 5, Stress
60 CRGA P11844 dominant structural inflamma-
components of ion
vertebrate eye lens
Gamina crystalllin
B, dominant Stress
61 CRGB M19364 structural DNA inflamma-
components of ion
vertebrate eye lens
Gamma crystallin C, Stress
62 CRGC NM 020989 dominant structural inflamma-
components of
ion
vertebrate eye lens
Gamma crystallin D, Stress
63 CRDG NM 006891 doininant structural inflamma-
components of
ion
vertebrate eye lens
Lamda crystallin Stress
64 CRYL NM 015974 homolog, inflatnma-
ion
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P' ' 77,j .::I L.., mu-crystallin
homolog, binds
thyroid hormone,
presumably involved
in regulation of free Stress
65 CRYM NM 001888 intracellular concentration of tri- inflamma-
iodothyronine and ion
access to its nuclear
receptors, expressed
in neural tissue,
muscle and kidney
Required in higher Cell
66 Cdc2 AF512554 cells for entry into S DNA
phase and mitosis cycle/death
67 hTR2-11 M29960 Human steroid unknown
receptor
May have regulatory
role in membrane
interactions during
68 Synaptotagmin NM_005639 trafficking of Synapse
synaptic vesicles at
active zone of
synapse
Believed to interact
69 AP180 AB014556 with cytoplasmic Synapse
tails of membrane
proteins
Promotes survival of
70 BDNF M61176 neuronal populations Cell death
located in CNS or
directly connected
71 bcl-xl U72398 Potent inhibitor of DNA Cell death
cell death
Suppresses
72 bcl-2 P10415 apoptosis, regulates Cell death
cell death, inhibits
caspase activity
Buffers cytosolic hlflamma-
73 calbindin D2 X06661 calcium tion / cell
cycle / death
Destroys radicals
74 SOD-1 NM_000454 normally produced Stress / cell
death
and which are toxic
Reduces low
molecular weight Stress /
75 Glutaredoxin NM002064 disulfides and inflamma-
proteins defends tion
against ROS
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w. !. 7Kinesin aa .;~..,~k r.,,,~ Microtubule-
associated force- Not Molecular
76 H X65873 producing protein, complete
may play a role in cds motor
organelle transport
77 PKC-type beta X07109 type beta I Cell
II cycle/death
Ras family small Cell
78 Rit U71203 GTP binding protein
RIT proliferation
79 Rin U71204 Also see 3 Rin genes Cell death
below
Ras effector protein,
may serve as
80 Rinl L36463 inhibitory modulator Cell death
of neuronal plasticity
in aversive memory
formation
Ras effector protein,
may function as
upstream activator
and/or downstream
81 Rin2 NM_018993 effector for RAB5B, Cell death
may function as
guanine nucleotide
exchange(GEF) of
RAB5B
Potential Ras
effector protein, may
82 Rin3 AL159141 function as guanine Cell death
nucleotide exchange
(GEF)
Tyrosine-preotein
kinase receptor, may
83 Protein tyrosine D50479 be involved in cell Cell death
kinase adhesion processes,
particularly central
nervous system
84 NAIP NM 004536 Prevents motor Cell death
nueron a o tosis
Key role in synaptic
plasticity,synaptogen
esis, excitotoxicity,
NMDA Rec memory aquisition
85 (zetal) L13266 and learning, Synapse
mediates neuronal
functions in
glutamate
neurotransmission
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: (I__, IT Human alpha-actin
86 Alpha actin J05192 (ACTA) mRNA, Cell
structure
complete cds
87 Beta actin X00351 ~volved in various DNA Cell
es of cell motility structure
leads to the
88 Topoisomerase NM003286 conversion of one Cell
I - topological isomer cycle/death
of DNA to another
Makes double-strand
breaks, transient
89 Topoisomerase ~_001067 breakage and Cell
II subsequent rejoining cycle/death
of DNA strands
CDK4 inhibitor p16 see the three CDK Cell
below cycle/death
Cyclin-dependent
kinase 4 inhibitor A,
p 161NK4, interacts
strongly with CDK4
90 CDKN2 L27211 and CDK6, inhibits Cell
its ability to act with cycle/death
cyclins D, could act
as negative regulator
in proliferation of
normal cells
Cyclin-dependent
kinase 4 inhibitor B,
interacts strongly
with CDK4 and Cell
91 CDKN2B U17075 CDK6, potent cycle/death
inhibitor, potential
effector of TBF-beta
induced cell cycle
arrest
Cyclin-dependent
kinase 4 inhibitor D, Cell
92 CDKN2D U49399 interacts strongly cycle/death
with CDK4 and
CDK6
Attach integral
membrane proteins
93 Ankyrin 2, Z26634 to cytoskeletal not Cell
Brain elements, also bind complete structure
to cytoskeletal
proteins
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..t Attach integral
membrane proteins Cell
94 Erythrocyte M28880 to cytoskeletal structure
elements, bind to
cytoskeletal proteins
95 PIG3 AF010309 Cell death
Accelerates
96 BaxA L22473 programmed cell Cell death
death, membrane
isoform alpha
Accelerates
97 BaxB L22474 programmed cell Cell death
death, cytoplasmic
isoform beta
C 1 q see the three C 1 Q Inflamma-
genes below tion
Complement Clq
subcomponent. A
chain (precursor);
associates with
98 C1QA NM015991 coenzymes C1R and Inflamma-
98
1 S to yield C l-the tion
first component of
the serum
complement system
Complement Clq
subcomponent. B
chain (precursor);
associates with not
99 C1QB X03084 coenzymes C1R and complete ~flarruna-
C 1 S to yield C l-the cds tion
first component of
the serum
complement system
Complement Clq
Inflamma-
100 C 1 QC NM_l 72369 subcomponent C Clone tion
100
( recursor
101 C1RF NM 006688 C1Q related factor Inflamma-
- tion
Splicing factor,
arginine/serine-rich
102 SF2 Flag NM_006924 1, pre-mRNA Splicing
splicing factor SF2, factor
P33 subunit, plays a
role in exon skipping
103 MCIF AF273052 CTCL tumor antigen Cell death
se70-2
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May play a role in
post synaptic
104 APP1 rTM005166 function, can Cell death
regulate neurite
outgrowth
May play a role in
the regulation of
hemostasis, may
have inhibitory Organelle
105 APP2 NM_001642 properties towards trafficing
coagulation factors,
may interact with G-
protein signaling
pathways
Possibly involved in
structural functions
as organizing other
106 Synaptophysin P08247 membrane synapse
components or in
targeting vesicles to
plasma membrane
Promotes
107 Tau J03778 microtubule Cell
assembly and structure
stability
Acid protease active
in intracellular
protein break down,
108 Cathepsin D NM_001909 involved in Stress
pathogenesis such as
breast cancer and
Alzheimer's Disease
109 GAP 43 NM 002045 Associated with Cell growth
- nerve owth
Glutathione S-
transferase Mul,
conjugation of
reduced glutathione Stress / cell
110 pGTH4 J03817 to a wide number of death
exogenous and
endogenous
hydrophobic
electrophiles
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Microsomal
glutathione S-
transferase 1,
conjugation of
reduced glutathione
111 pHMGST J03746 to a wide number of Stress / cell
exogenous and death
endogenous
hydrophobic
electrophiles, has
wide substrate
specificity
Arginine/serine rich
splicing factor 10,
sequence-specific
112 Tra2-C2 U61267 RNA-binding Splicing
protein which factor
participates in
control of pre-
mRNA splicing
113 MCD-C2 NM 012470 Transporin-SR unknown
Involved in ATP-
dependent selective
degradation of
proteins,
114 Ubiquitin X1VI 055013 maintenance of Stress / cell
- chromatin structure, death
regulation of gene
expression, stress
response, ribosomal
biogenesis
Putative pre-mRNA
splicing factor RNA
115 Pht6 AB011149 helicase, probable Splicing
ATP-binding RNA factor
Helicase involved in
pre-mRNA splicing
Facilitative glucose
116 pGHT1 P11166 transporter, glucose Stress / cell
transporter type 1, death
erythrocyte/brain
Epidermal growth Stress /
117 CR3 AF251550 factor-like cripto DNA inflamm-
rotein CR3 ation
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.. . ..{ . "!t õd
Implicated in various
adhesive interactions
of monocytes, macs Stress /
118 CR3 alpha NM000632 and granulocytes, as inflamm-
chain well as mediating
uptake of ation
complement-coated
particles
119 C5 rlM 001735 Complement C5 Stress /
precursor inflamm-
ation
Mitochondrial ene See list below:
Component of
mitochondrial
120 MPR-S12 015235 ribosome small unit Cell energy
which comprises a
12S rRNA and 30
distint proteins
28S ribosomal
protein S 16.
Homosapiens
mitochondrial
121 MRP-16 NM 016065 ribosomal protein Cell energy
S16 (MRPS16)
nuclear gene
encoding
mitochondrial
protein, mRNA
Cytochrome C
oxidase polypeptide
I, Homo sapiens
mitochondrial DNA,
16559 bp,
component of
122 COI D38112 respiratory chain that DNA Cell energy
catalyzes the
reduction of oxygen
to water.
Cytochrome C is
subunits 1-3 form
functional core for
enzyme com lex
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Cytochrome C
oxidase polypeptide
II, subunits 1-3 form
fi.inctional core for
123 COII M90100 enzyme coinplex, Cell energy
homosapiens
cyclooxygenase-2
(Cox-2) mRNA,
complete cds
Cytochrome C
oxidase polypeptide
III, subunits 1-3
form functional core
124 COIII J01415 fH~an e complex. DNA Cell energy
mitochondrion,
complete genome,
16569 bp, DNA
circular PRI
H. sapiens
mitochondrial
genome, 16569 bp
DNA circular PRI
mitochondrial gene
includes: 12S, 16S
ribosomal RNA, 22
tRNA, ATPase
subunit 6 8, (NADH
dehydrogenase
125 V00662 subunits 1, 2, 3, 4, DNA Cell energy
4L, 5, 6),
cytochrome b,
(cytochrome c
oxidase subuits I, II,
III) (tranfer RNA:
Arg, Ala, Asn, Asp,
Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys,
Met, Phe, Pro, Ser,
Thr, Trp, Tyr, Val)
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... ,.,.. ,. ~.., , z .. ,,,.. ,..., . .,.. .,.,.,. ,
õ õ
Complement factor
D precursor. Factor
D cleaves factor B
when latter is
Conlplement complexed with Stress /
126 NM_001928 factor C3B, inflamma-
factor D activating the tion / death
C3BBB complex,
which then becomes
the C3 convertase of
alternate pathway
Activation of Cl
complex under
control of Cl Stress /
127 Cl inhibitor M13656 inhibitor; may play inflamma-
important role in tion / death
physiological
pathways
Function not yet Stress /
128 Clusterin NM 001831 clear, associated inflamma-
- with programmed tion / death
cell death
Complement factor
1 precursor,
responsible for
cleaving alpha- Stress /
129 Factor 1 NM 000204 chains of C4B and inflamma-
- C3B in the presence
tion
of cofactors C4-
binding protein and
Factor H
respectively
130 S-protein NM 001264 Expressed in skin
Plays a central role Stress /
C3a in the activation of
131 ~aphylatoxin ~-000064 the complement inflamma-
anaphylatoxin / death
system
Activation of C5 by
a C5 convertase
initiates the
spontaneous Stress /
132 C5a ~ 001735 assembly of the late inflarnma-
anaphylatoxin - complement tion / death
components C5-C9
into the membrane
attack complex
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~ ~ .. = . t Mediates cellular
binding of particles Stress
/
133 Complement Y00816 and immune inflatruna-
receptor typel complexes that have tion / death
activated
complement
Receptor for
complement 3CDd
and for Epstein-Barr Stress /
134 Complement NM001877 virus on human B- inflamma-
receptor type2 - and T-cells, tion / death
participates in B
lymphocytes
activation
May play important Stress /
135 COXl M59979 role in regulating or inflainina-
promoting cell
tion
roliferation
Prostaglandin G/H
synthase 2 precursor,
may have role as Stress /
136 COXII NM 000963 major mediator of inflamma-
- inflamnlation and/or
tion
role for prostanoid
signaling in activity-
de endent plasticity
Mouse ATP-specific succinyl-CoA synthase: see below:
phosphorylation of
137 Q9P2R7 the tricarboxylic acid Cell energy
cycle
Interleukin-I
Interleukin I receptor, type I Stress /
138 NM 000877 precursor, receptor inflamma-
receptor - for IL-1A, IL-1B, IL- tion
1RA
Basic
transcription
139 transcription Q02446
factor
factor SP4
Interleukin 17 Interleukin-17 Stress /
140 NM 014339 receptor precursor inflainma-
receptor
tion
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Pf =K .::F= ~ -- =~:~ = ~'~" ~ ~' ''' !~ ~E Chemotactic factor
attracts neutrophils,
basophils, and T-
cells; involved in Stress /
141 Interleukin 8 NM_000584 neutrophil inflamma-
activation, released tion
in response to
inflammatory
stimulus
Stimulates Stress /
142 Interleukin 15 U14407 proliferation of T- inflamma-
lym hocytes tion
Interleukin 15 Interleukin 15 Stress /
143 receptor U31628 receptor alpha chain inflamma-
recursor tion
Stimulates migratory
response CD4+
lymphocytes,
monocytes, and Stress /
144 Interleukin 16 AF053412 eosinphils; induces inflamma-
T-lymphocyte
tion
expression of
interleukin 2
receptor; ligand for
CD4
Interleukin 10 Receptor for IL-10; Stress /
145 NM001558 binds IL-10 with inflamma-
receptor alpha high affinity tion
Receptor for IL-10
and IL-22, serves as
accessory chain
Interleukin 10 essential for active Stress /
146 inflamma-
receptor beta Z17227 IL-10 receptor inflamma-
complex and to tion
initiate IL- 10-
induced signal
transduction events
Leukemia inhibitory
factor, precursor, has Stress /
147 LIF NM 002309 the capacity to inflamma-
- induce tenninal
tion
differentiation in
leukemic cells
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Can induce cell
death of certain
tumor lines; potent
148 TNF-alpha M10988 pyrogen; under Inflamma-
certain conditions tion / death
can stimulate cell
proliferation; induce
cell differentiation
Controls
proliferation,
149 TGF-Betal NM 000660 differentiation; Growth
regulates actions of factor
many other growth
factors
Interleukin 12 Can act as growth Stress /
150 alpha chain NM_000882 factor for activated T inflamma-
and NK cells tion
Receptor with high
affinity for
TNFSF2/TNF-alpha,
151 TNF-R2 NM_001066 TRAF1/TRAF2 Cell death
complex recruits
apoptotic
suppressors
Tuinor necrosis
factor receptor
family superfamily
member 11B
(precursor), acts as
Osteoclastogene decoy receptor for
152 sis inhibitory NM 002546 RANKI' thereby Cell death
factor - neutralizes its
function in
osteoclastogenesis,
inhibits activation of
osteoclasts and
promotes osteoclast
apoptosis in vivo
Endothelium-
153 Endothelin-2 NM 001956 derived Vasoconstric
vasoconstrictor -tion
peptides
Endothelium-
154 Endothelin-3 NM 000114 derived Vasoconstric
vasoconstrictor -tion
peptides
Non-specific
155 Endothelin M74921 receptor for Cell death
receptor Endothelin 1, 2 and
3
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U 7 -_ r. : u 1 ...[ :' :,! mRNA,
Stress
Cytokine receptor not /
156 CCR5 X68149 binds to BLC coiuplete inflanima-
tion
cds
homo sapiens
Human chromosome 17
157 chromosome 17 AC005837 sequence, clone
sequence hRPK.318_A_15,co
mplete sequence
Human DNA
sequence for clone
RP5-1104E15 on
chromosome 22
q12.3-13.1, contains
MGAT3 gene for
mannosyl (beta-1,4-
)-glycoprotein beta-
1,4-N-
acetylglucosaminyl
transferase, the gene
for a predicted
Human protein, the ATF4
158 chromosome 22 AL022312 gene for activating Cell energy
sequence transcription factor
4(tax-responsive
enhancer element
B67), and 5' end of
CACNAII gene for
voltage-dependent
calcium channel,
alpha II subunit,
contains ESTs,
STSs, GSSs, and 5
putative CpG
islands, complete
sequence
Required for T-cells ni.A' Stress /
159 CD45 Y00062 activation through inflamma-
the antigen receptor c dmplete tion
c
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1 ,! _ ,. 3175i ~ .. Low affinity
immunoglobulin
gamma Fc region
receptor II-C
precursor; involved
Fc gamma R in variety of effector Stress / cell
160 type II C U90938 and regulatory death
functions such as
phagocytosis of
immune complexes
and modulation of
antibody production
by B-cells
161 LOX-1 or OLR- NM002543 Stress / cell
1 - death
Involved in plasma
clearance of
chylomicron
remnants and
activated alpha 2-
macroglobulin, also Inflamma-
162 LRP-1 NM_002332 in the local
tion
metabolism of
complexes between
plasminogen
activators and their
endogenous
irihibitors
Expressed by macs
in chronic mRNA
163 MRP(8) Y00278 inflammations, not Inflamma-
cystic fibrosis complete tion
antigen, calgranulin cds
A
ATP-binding
164 Q9BX80 also: AF352582 cassette transporter Cell energy
MRP8
Is the beta-chain of
major Inflamma-
165 B2 NM 004048 histocompatibility
microglobulin - tion
complex class I
molecules
166 ENA-78 U12709 Involved in DNA Inflamma-
neutro hil activation tion
Plays a critical role Inflamma-
167 CD74 NM 004355 in MHC Class II
tion
antigen processing
168 Ribosomal AA366442 Cell death
rotein S 4
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See also 5 clathrin
169 Clathrin AA361745 genes listed below: synapse
Synaptosomal-
associated protein,
Clathrin coat
assembly protein
AP180, adaptins are
components of
adapter complexes
that link clathrinto Synapse /
170 A180 NM_014841 receptors in coated cell cycle /
vesicles, protein death
complexes are
believed to interact
with cytoplasmic
tails of membrane
proteins, leads to
their selection and
concentration
Clathrin light chain
A, is major protein Synapse /
171 CLCA M20471 of polyhedral coat of cell cycle /
coated pits and death
vesicles
Clathrin light chain
B; major protein of Synapse /
172 CLCB M20469 polyhedral coat of cell cycle /
coated pits and death
vesicles
Clathrin heavy chain
1; major protein of
polyhedral coat of
coated pits and Synapse /
173 CLH1 NM_004859 vesicles, two different adapter link cell cycle /
death
clathrin lattice to
either plasma
membrane or to
trans golgi network
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n~ .,R++:... . õr. . T. . a ua. :. m.,.. .R ..
.. . .. ,.
"!i , ... .t .. . ,.., t .., ..
Protein complexes
link clathrin lattice
to either plasma
membrane or to
trans golgi network;
Clathrin heavy chain
2, major protein of
174 CLH2 U41763 polyhedral coat of DNA
coated pits and replication
vesicles, two
different adapter
protein complexes
link clathrin lattice
to either plasma
membrane or to
trans golgi network
Is auxiliary protein
of DNA polymerase
175 PCNA M15796 delta and is involved Cell cycle
in control of
eukaryotic DNA
replication
176 P55cdc NM 001255 CDC20 cell division Cell cycle /
cycle 20 homolog death
Functions as dosage-
dependent inducer in
mitotic control, is a
tyrosine protein
phosphatase required
for progression of
cell cycle, it directly Cell cycle /
177 cdc25A (MPI1) NM_001789 dephosphorylates death
CDC2 and activate
its kinase activity,
also
dephosphorylates
CDK2 in complex
with cyclin E, in
vitro
Functions as dosage-
dependentinducerin
mitotic control, is a
tyrosine protein
178 Cdc25B (MPI2) M81934 phosphatase required Cell cycle /
for progression of death
cell cycle, it directly
dephosphorylates
CDC2 and activate
its kinase activity
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,. e ,,.. :.m . ! ... . ~I a li ... ... !E :Ic ...... .
Apoptotic adapter
molecule recruits
caspase-8 or
caspase-10 to the
activated FAS
(CD95) or TNFR-1
179 FADD NM_003824 receptors, resulting Cell death
aggregate called
DISC-death
inducing signaling
complex- performs
caspase-8 proteolytic
activation
Essential conlponent
of SCF ubiquitin
ligase complex
(serves as adapter to
links F-box protein
to CULl) which Cell cycle
180 Skpl U33760 mediates death
ubiquitination of
proteins involved in
cell cycle
progression, signal
transduction,
transcription
Involved in
activation cascade of
caspases responsible
for apoptosis
execution, recruited
to both FAS and Cell cycle /
181 Mch4 U60519 TNFR-1 I; n FADD death
dependent manner;
cleaves, activates
several caspases and
hydrolyzes some
small molecule
substrates
Participates in Wnt
signaling pathway,
implicated in
hormonal control of
182 GSK-3B NM 002093 several regulatory Cell cycle /
- proteins including death
glycogen synthase,
myb, and
transcription factor
c jun
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li~'' ~w;: 4I~" ~.1~ :t=.!i ~~ f~ ~~ ,,E ~ ' ii ~' !!;,:it f~.,il.. :::i-
.:C.. Involved of
preferential repair of
183 ERCC6 NM 000124 active genes, DNA repair
- presumed DNA or
RNA unwinding
function
May play a role in
terminal
differentiation of Cell
184 SKI X15218 skeletal muscle cells differentia-
but not in tion
determination of
cells to myogenic
lineage
Microtubule
associated protein
RP/EB family
185 EB1: NM 012325 member 1, maybe Cell
involved in structure
microtubule
polymerization and
spindle function
Cytochrome C
186 or NM 004718 oxidase VII-a related Cell energy
protein,
mitochondrial
Phosphorylates
serine- and arginine-
rich (SR) proteins of
spliceosomal
complex, may be
187 CLK3 L29217 constituent of Splicing
network of
regulatory
mechanisms that
enable SR proteins
to control RNA
splicing
Transferrin receptor
protein 1; transferrin
188 Transferrin R NM 003234 receptor is necessary Stress / ROS
- for development of
erythrocytes and
nervous system
Transferrin receptor
protein 2; mediates
189 Transferrin R NM 003227 cellular uptake of Stress / ROS
- transferrin-bound
iron in non-iron
dependent manner
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,ttq
Calcium-activated,
phospholipid-
dependent serine-
and threonine-
specific enzyme,
PKC is activated by
diacylglycreol which Stress / cell
190 PKC-a X52479 in turns
/
phosphorylates a cycle death
range of cellular
proteins; also serves
as the receptor for
phorbol esters, a
class of tumor
promotors
CDK activating
kinase, transcription
factor, CDK's are
191 CAK NM 001799 activated by binding Cell cycle
- to a cyclin, mediate
the progression
through the cell
cycle
Tyrosine kinase of
the non-receptor
type, involved in the
IFN- Inflamma-
192 JAK-1 NM_002227 alpha/beta/gamma
signal pathway, tion / stress
kinase partner for
the interleukin (IL)-2
receptor
Mitogen-activated
193 MAPKAP NM 004635 protein kinase Cell cycle /
- activated protein death
kinase 3
Glutathione S
transferase P,
conjugation of
reduced glutathione
194 GSTP NM_000852 to a wide number of Cell energy
exogenous and
endogenous
homophobic
electrophiles
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1~ . '. ~ ::Sf . ~~:.:~ ..::~t Involved in the
activation cascade of
caspases responsible
for apoptosis
execution, cleaves
195 Apopain U26943 and activates sterol Cell death
regulatory element
binding proteins
(SREBPS), involved
in the cleavage of
Huntingtin
Single stranded
DNA endonuclease
involved in DNA
196 ERCC5 NM 000123 DNA repair
excision repair,
makes the 3' incision
in the repair
ATP-dependent 3'-5'
DNA helicase,
component of the
core-TFIIH basal
transcription factor,
197 ERCC3 NM 000122 involved in DNA repair
- nucleotide incision
repair of DNA, and,
when complexed to
CAK in RNA
transcription by
RNA polymerase II
CCAAT-BP see 9 genes below transcription
Stimulates
transcription of
various genes by
198 CBFA NM 006166 recognizing/binding transcription
- to CCAAT motif in
promotors, CCAAT-
binding transcription
factor subunit A
Stimulates
transcription of
various genes by
199 NFYA M59079 recognizing/binding transcription
to CCAAT motif in
promotors, CCAAT-
binding transcription
factor subunit B
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Stimulates
transcription from
200 CBF NM_005760 the HSP70 Stress /
promotor, CCAAT- transcription
box-binding
transcription factor
DNA binding
protein recognizes
CCAAT homolgy
common to many
promotors and
201 CEBA U34070 enhanced core DNA Transcrip-
homology common tion
to many enhancers,
CCAAT/ enhancer
binding protein
alpha
Important
transcriptional
activator in the
regulation of genes Transcrip-
202 in the tion /
202 CEBB X52560 immune and DNA inflamma-
inflammatory tion
response,
CCAAT/enhancer
binding protein beta
DNA binding
protein recognizes
CCAAT homolgy
common to many
promotors and
enhanced core Transcrip-
203 CEBD NM 005195 homology common tion /
to many enhancers, inflamma-
involved in immune tion
and inflammatory
response,
CCAAT/binding
enhancer protein
delta
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a} %.: ,. L DNA binding
protein recognizes
CCAAT homolgy
common to many
promotors and Transcrip-
204 CEBE U48865 enhanced core DNA tion
homology common
to many enhancers,
CCAAT/enhancer
binding protein
e silon
Transcription factor
binds to enhancer
elemnt PRE-lof IL4 Transcrip-
205 CEBG NM_001806 gene,
tion
CCAAT/enhancer
binding protein
gamma
Probably has role as
repressor of
developmentally
regulated gene
expression,may act Transcrip-
206 CUT1 M74099 by preventing tion / death
binding of
positively-avtiving
factors to promotors,
CCAAT
displacement protein
207 HLHP Id2 NM 002166 DNA-binding Cell cycle /
- protein inhibitor ID2 death
Guanine nucleotide-
binding protein
G(S), alpha subunit
(adenylate cyclase Cell energy I
G-S alpha stimulating G alpha death / many
208 subunit P04895 protein), involved as other
modulators or ftmctions
transducers in
various
transmembrane
signaling systems
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aE T.~. . ::::u : ,w-, .w:;~: . i~;::.. õ i~ ' :s .,, .. Involved in
chromatin
remodeling, part of
complex that opens Transcrip-
209 1NI1 NM_003073 the chromatin to tion
facilitate
transcriptional
machinery to access
their targets
Activates erythroid
210 LCR-F1 U08853 specific, globin gene
expression
Tumor necrosis
factor receptor
superfamily member
16, low affinity Cell owth
211 Low affmity ~_002507 receptor can bind to death
NGF, BDNF, NT-3,
NT-4, mediates cell
survival and cell
death of neural cells
Receptor for EGF, is
212 EGF-R U48722 involed in control of Cell growth
cell growth and
differentiation
Insulin receptor,
213 Insulin R NM 000208 binds insulin, has Cell energy
- tyrosine-protein
kinase activity
Alpha-l-catenin,
cadherin associated
protein, associates
with cytoplasmic
domain of variety of
cadherins,
214 A-Catenin NM 001903 association of Cell cycle /
- catenins to death
cadherons produces
complex linked to
actin filament
network, may play
crucial role in cell
differentiation
Alpha-2-catenin, Cell cycle /
215 NM_004389 alpha catenin related death
rotein
Receptor for
216 Integrin a-3 M59911 fibronectin, laminin, Inflanuna-
collagen, epilegrin, tion / death
thrombospondin
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Integrin alpha-5
precursor, Integrin
alpha-5Beta1 is
Fibronectin R receptor for hiflamma-
217 alpha P08648 fibrinogen and tion / death
fibronectin, it
recognizes the
sequence R-G-D and
its ligands
Cytokine stimulates
growth and
218 GM-CSF M11734 differentiation of Growth
hematopoietic factor
precursor cells from
various lineages
219 Glu-6-P A1250347 Cell energy
isomerase
Chemotactic factor
that attracts
monocytes and
220 MCP-1 M24545 basophils, augments ~flamma-
monocyte anti-tumor tion / death
activity, binds to
CCR2 and CCR4
Heparin binding
221 Pleiotrophin NM 002825 mitogenic protein, Cell cycle
- has neurite extension
activity
Important role in
organization of
cytoskeleton-binds, Cell
222 Thymosin b-10 NM021103 sequesters actin structure
monomers (G actin),
therefore inhibits
actin polyinerization
Highly basic Soares fetal liver
223 protein R94142 spleen 1NFLS homo Unknown
sapiens clone
Required for the
biosynthesis of the
tetrasaccharide
224 XGPT NM 007255 linkage region of Cell energy
- proteoglycans,
specially for small
proteoglycans in
skin fibroblasts
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i..F31 Promotes cell death,
appears to act as a
225 BAD AF031523 link between growth Cell death
factor receptor
signaling and the
a o totic pathways
Involved in
activation cascade of
226 Mch3 U39613 caspases responsible Cell death
for apoptosis
execution
Involved in
activation cascade of
227 Mch6 U56390 caspases responsible Cell death
for apoptosis
execution
228 MPP2 L16783 Putative M phase Cell cycle
ho ho rotein 2
229 Mek2 L11285 Cell cycle
Band 4.1-like
protein 1, (neuronal
protein 4.1), may
230 4.1N Q9H4G0 function to confer Cell
stability and structure
plasticity to neuronal
membrane via
multiple interactions
Gamma enolase, Neuron
231 NSE Full length NM_001975 neuro specific marker
enolase,
Dynein see 9 genes below
Dynein light chain
2A, cytoplasmic,
may be involved in
assembly and motor Cell cycle /
232 DL2A AF161511 function of dynein, organelle
which plays central transport
role in cell division
and intracellular
transport
Dynein light chain
2B, cytoplasmic,
may be involved in
assembly and motor Cell cycle /
233 DL2B NM_130897 function of dynein, organelle
which plays central transport
role in cell division
and intracellular
transport
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E ;i 11.. ..:: .. iE "i Dynein light chain mRNA,
4A, axonemal, force not Cell cycle /
234 DL4A AL035366 generating protein in complete organelle
respiratory cilia cds transport
Ciliary dynein heavy Cell cycle /
chain 9 force
235 DYH9 AF257737 generating protein in organelle
respiratory cilia transport
Dynein light chain 1,
cytoplasinic, maybe
involved in some
dynein-related
intracellular Cell cycle /
236 DYLl N1VI 003746 transport and organelle
- motility, may play a
role in changing or transport
maintaining spatial
distribution of
cytoskeletal
structures
Ciliary dynein heavy Cell cycle /
237 DYHB Q96DT5 chain 11, force organelle
generating protein in
respiratory cilia transport
Dynein heavy chain,
cytosolic;
cytoplasmic dynein
acts as a motor for Cell cycle /
238 DYHC Q14204 intracellular organelle
retrograde motility
of vesicles and transport
organelles along
tubules, dynein has
ATPase activity
Dynein intermediate
chain 1, cytosolic;
intermediate chains
seem to help dynein
bind to dynactin 150
kDa component, Cell cycle /
239 DYI1 AF063228 may play role in organelle
mediating transport
interaction of
cytoplasmic dynein
with membranous
organelles and
kinetochores
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Dynein intermediate
chain 2, cytosolic; Cell cycle /
240 DYI2 MVI 001378 intermediate chains organelle
- seem to help dynein
bind to dynactin 150 transport
kDa component
127. Some additional examples of biomarkers, whose level of expression can be
assessed and compared to a reference standard for example, include human
transformer 2-beta,
hTra2-beta, human SAF-b, Mainclone, pht6, MIF, mainclone interacting factor,
pp 17, ESAF,
hnRNPG, cd2like kinases clkl-4. Further examples of biomarkers include those
listed in Table
5. Specific examples of these biomarkers include, but are not limited to,
HSP60,
Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate
dehydrogenase, ATP-
synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate
mutase 1,
Apoliporotein Al, Superoxide dismutase, RNA-binding protein regulatory
subunit, Chain A
thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen,
Haptoglobin,
Fibrin beta, including combinations thereof. In other examples, suitable
biomarkers include, but
are not limited to, proteins having a molecular weight (MW) of 27,100 and
isoelectric point (pI)
of 7.58, a MW of 25,400 and pI of 6.2, and a MW of 27,600 and pI of 5.92.
Table 5: Identified proteins that differ between Parkinson's disease patients
and control subjects.
Protein MW (molecular weight pI (isoelectric point)
1 66,204 5.67
2 63,131 7.59
3 62,052 5.98
4 59,332 7.42
5 54,879 5.42
6 36,106 7.58
7 32,567 5.10
8 29,785 5.80
9 29,559 7.60
10 26,908 5.65
11 25,546 7.64
12 24,376 6.36
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153 ' :r. 4,3 7 5.82
14 21,719 5.92
15 145,916 5.29
16 42,839 5.42
17 60,376 6.72
18 27,100 7.58
19 25,400 6.2
20 27,600 5.92
128. Other specific examples of biomarkers, whose level of expression can be
assessed
and compared to a reference standard, as described herein, include actin-
interacting protein 1
(AIP1), mitogen activated protein kinase I(MAPKI), actin or a fragment
thereof, annexin Al,
14-3-3 protein epsilon, glutaraldehyde-3-phosphate dehydrogenase (GAPDH),
transforming
protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B
(ANP32B or
APRIL), peroxiredoxin II, an amyloid precursor protein (APP), cx-secretase, 0-
secretase, -y-
secretase, A(3 peptide, Fe65, Tip60, SERCA, PS1/2, nectin-la, or non-amyloid
(3 component of
senile plaque (NACP/ a-synuclein).
3. Comparing levels of expression
129. In some examples of the disclosed methods, when the level of expression
of a
biomarker(s) is assessed (and optionally a first and/or second canonical
variable obtained), the
level (or canonical variable) can be compared with the level of expression of
the biomarker(s)
(or canonical variable obtained therefrom) in a reference standard. By
"reference standard" is
meant the level of expression of a particular biomarker(s) from a sample or
subject lacking a
neurodegenerative disease, at a different stage of a disease, or in the
absence of a particular
variable such as a therapeutic agent. Alternatively, the reference standard
can comprise a known
amount of biomarker. Such a known amount can correlate with an average level
of subjects
lacking a neurodegenerative disease, at a different stage of the disease, or
in the absence of a
particular variable such as a therapeutic agent. A reference standard can also
include the
expression level of one or more biomarkers from one or more different samples
or subjects as
described herein. For example, a reference standard can include an assessment
of the expression
level of one or more biomarkers in a sample from a subject that does not have
a
neurodegenerative disease, is at a different stage of progression of a
neurodegenerative disease,
or has not received treatment for a neurodegenerative disease. Another
exemplary reference
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6'&&. of the expression level of one or more biomarkers in samples
taken from multiple subjects that do not have a neurodegenerative disease, are
at a different stage
of progression of a neurological disease, or have not received treatment for a
neurological
disease.
130. When the reference standard includes the level of expression of one or
more
biomarkers in a sample or subject in the absence of a therapeutic agent, the
control sample or
subject can be the same sample or subject to be tested before or after
treatment with a therapeutic
agent or can be a different sample or subject in the absence of the
therapeutic agent.
Alternatively, a reference standard can be an average expression level
calculated from a number
of subjects without a particular neurodegenerative disease. A reference
standard can also include
a known control level or value known in the art. In one aspect of the methods
disclosed herein,
it can be desirable to age-match a reference standard with the subject
diagnosed with a
neurodegenerative disease. A reference standard can also be a first or second
canonical variable
obtained from a multivariant canonical analysis of levels of expression of a
biomarker(s) from a
control or group of control subjects.
131. In one technique to compare levels of expression of gene products from
two
different samples (e.g., a sample from a subject diagnosed with a
neurodegenerative disease and
a reference standard), each sample can be separately subjected to 2D gel
electrophoresis.
Alternatively, each sample can be differently labeled and both samples can be
loaded onto the
same 2D gel. See e.g., Unlu et al., Electrophoresis, 1997;18:2071-2077, which
is incorporated
by reference herein for at least its teachings of methods to assess and
compare levels of gene
product expression. The same gene product or group of gene products in each
sample can be
identified by the relative position within the pattern of gene products
resolved by 2D
electrophoresis. The expression levels of one or more gene products in a first
sample can then
be compared to the expression level of the saine gene product(s) in the second
sample, thereby
allowing the identification of a gene product or group of gene products that
is expressed
differently between the two samples (e.g., a biomarker). This comparison can
be made for
subjects before and after they are suspected of having a neurodegenerative
disease, before and
after they begin a therapeutic regimen, and over the course of that regimen.
132. In another technique, the expression level of one or more gene products
can be in
a single sample as a percentage of total expressed gene products. This
assessed level of
expression can be compared to a preexisting reference standard, thereby
allowing for the
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~~~nti~icat~~rr eh~ t are differentially expressed in the sample relative to
the
reference standard.
133. Gene products whose expression levels vary from a reference standard can
be
identified by, for example, extracting those gene products from a 2D gel and
employing an
identification technology such as mass spectroscopy (MS), which includes
techniques such as or
matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF).
Accordingly, in
one aspect, disclosed herein are methods of identifying biomarkers relevant to
various stages of a
neurodegenerative disease (e.g., its onset and progression) by examining gene
product
expression in samples (e.g., samples comprising leukocytes or lysates
thereof).
134. Other methods can be used instead of 2D electrophoresis to identify the
level of
gene product expression in a sample and compare that level to a reference
standard, and can be
used in the methods disclosed herein. Some of these methods utilize
spectroscopic techniques
such as surface-enhanced laser desorption ionization-time of flight (SELDI-
TOF). Other
methods rely on chromatographic techniques such as high performance liquid
chromatography
(HPLC), or fast protein liquid chromatography (FPLC). Multidimensional liquid
chromatography (LC) and tandem mass spectrometry (MS/MS) can separate and
identify
multiple peptides. See Link, et al., Nat. Biotechnol., 1999;17:676-82.
Additional
chromatographic methods for identifying multiple proteins are described in
U.S. Patent No.
6,908,740. In still other methods, chips (e.g., arrays of protein binding
antibodies, ligands, or
aptamers) can be used to identify gene products that are expressed differently
in a sample than in
a reference standard. See, e.g., Glokler and Angenendt, J. Chromatogr. B
Analyt. Technol.
Biomed. Life Sci., 2003;797:229-240. These references are incorporated by
reference herein at
least for their teachings of methods to assess and compare gene product
expression levels.
135. When differential gene expression causes the expression of one or more
gene
products to be different in the sample and the reference standard, these one
or more gene
products can be further identified using methods that identify differentially
expressed gene
transcripts, e.g., gene chip (nucleotide expression microarrays) or
differential display
technologies (e.g. differential display kits from Clontech, Palo Alto, CA or
GenHunter,
Nashville, TN). These references are incorporated by reference herein at least
for their teachings
of methods to assess and compare protein expression levels.
136. When comparing the level of expression of a gene product with the
reference
standard an increase in the level of expression of the gene product, as
compared to the reference
standard, can identify the gene product as a biomarker for diagnosing the
neurodegenerative
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aises6, disease. Alternatively, a decrease in the level of
expression of the gene product, as compared to the reference standard, can
also identify the gene
product as a biomarker for diagnosing the neurodegenerative disease. Finally,
a combination of
increased gene products and decreased gene products as compared to a reference
standard can
identify the gene products as biomarkers for diagnosing the neurodegenerative
disease.
137. Biomarkers identified by the disclosed methods can be used in a variety
of other
methods. For example, biomarkers can be used to diagnose a particular
neurodegenerative
disease. In another example, biomarkers can be used monitor the progression of
a disease since
the level of expression of some biomarkers can become more pronounced (or less
pronounced)
as a particular neurodegenerative disease progresses. In yet another example,
a biomarker can be
used to monitor a subject's response to treatment for a disease. These and
other uses are
disclosed herein.
4. Specific methods
138. Disclosed are methods of diagnosing a neurodegenerative disease such as
Alzheimer's or Parkinson's disease comprising collecting a sample (e.g. blood
or leukocytes)
from a subject, assaying the expression of a set of genes in the sample, and
comparing this
expression to a control.
139. Also disclosed are methods of diagnosing a neurodegenerative disease, the
method comprising, collecting a blood sample from a subject, assaying the
expression of a set of
genes in the sample, and comparing this expression to a control.
140. Also disclosed are methods of diagnosing a neurodegenerative disease, the
method coinprising, collecting leukocytes from a subject, assaying the
expression of a set of
genes in the leukocytes, and comparing this expression to a control.
141. Disclosed are methods, wherein the subject has also been diagnosed with a
clinical dementia test, wherein the clinical dementia test is the NINCDS or
DSM-IV test.
142. Disclosed are methods, wherein the control would have a score of above 27
and
an AD patient has a score below 22 in the Mini-Mental Status Examination
(MMSE).
143. Also disclosed are methods, wherein the AD subject would have a score of
above
1.2 or 1.5 in the Clinical Dementia Rating scale (CDR).
144. Disclosed are methods, wherein the control would be determined using the
Blessed Dementia Rating Scale (BDRS).
145. Disclosed are methods of diagnosing a neurodegenerative disease, the
method
comprising collecting peripheral blood sample from a subject, lysing
erythrocytes contained
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NPIr'~naining leukocytes, lysing the leukocytes producing a lysed
sample, collecting total nucleic acids in the lysed sample forming a nucleic
acid sample, isolating
the RNA in the nucleic acid sample, extracting the RNA in the nucleic acid
sample, collecting
the polyA RNA, and identifying the presence of a set of RNA transcripts.
146. Also disclosed are methods of diagnosing a neurodegenerative disease, the
method comprising collecting a sample (e.g., a leukocyte sample) from a
subject, collecting the
mRNA within the sample, hybridizing the mRNA with a collection of nucleic
acids, wherein the
collection of nucleic acids comprises one or more genes found in Table 4, such
as cyclin D1,
cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C
alpha, which are
related to cell cycle, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-
lOr, which are
related to inflammatory systems, and Alpha-1 antichymotrypsin, HSP 27, HSP 90,
crystalline,
GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, which are
related to cell stress,
including any combination thereof.
147. Disclosed are methods of diagnosing a neurodegenerative disease, the
method
comprising collecting peripheral blood sample from a subject, lysing
erythrocytes contained
within this sample, collecting the remaining leukocytes, lysing the leukocytes
producing a lysed
sample, collecting total nucleic acids in the lysed sample forming a nucleic
acid sample, isolating
the RNA in the nucleic acid sample, extracting the RNA in the nucleic acid
sample, collecting
the polyA RNA, and identifying the presence of a set of RNA transcripts.
148. Also disclosed are methods of diagnosing a neurodegenerative disease, the
method comprising collecting a peripheral blood sample from a subject,
collecting leukocytes
from the peripheral blood sample, wherein collecting the leukocytes comprises
lysing
erythrocytes in the peripheral blood sample and centrifuging, lysing the
leukocytes, collecting a
total nucleic acid sample from the lysed leukocytes, wherein the collection of
the nucleic acid
comprises adsorption of the nucleic acids on magnetic beads, collecting a
total RNA sample
from the nucleic acid sample, collecting a polyA mRNA sample from the total
RNA sample,
hybridize the total mRNA sample with a set of diagnostic genes, wherein the
set of diagnostic
genes comprises one or more genes from Table 4, such as cyclin D1, cyclin B,
cyclin G1, weel,
hTR2, CDC25b, GSK3 beta, and protein kinase C alpha, which are related to cell
cycle, C5, Cl
inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, which are related to
inflammatory systems,
and Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H,
ferritin L, cox
1, cox 2, and transferrin, which are related to cell stress, including any
combination thereof,
analyzing which diagnostic genes are hybridized by an mRNA in the mRNA sample.
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õõ, . ...,. .
iused, the data can be normalized by normalizing a
housekeeping gene such as GapDH, cyclophilin, or actin. Other methods include
spiking
samples, normalizing to the average or sum of signal intensity over the whole
array.
a) Method of Screening for a Therapeutic Agent
150. In yet another aspect, disclosed herein are methods for screening for a
therapeutic
agent for the treatment of a neurodegenerative disease (e.g., Parkinson's or
Alzheimer's disease).
The disclosed methods comprise contacting a leukocyte or population of
leukocytes with the
agent to be screened and detecting a level of expression or activity of a
biomarker for the
neurodegenerative disease. Alternatively, instead of leukocytes, neuronal
cells or populations
thereof can be used. In these methods, an increase or decrease in the level of
expression or
activity of the biomarker can indicate a therapeutic agent for the treatment
of the
neurodegenerative disease.
151. In one aspect, the disclosed methods can be utilized to screen agents
that are
nucleic acids, antibodies, polypeptides, or small molecules, including any
therapeutic mixtures
or combinations thereof.
152. Contacting the leukocyte or lysate thereof can be accomplished by any
technique.
For example, the cells or lysate can be submerged or immersed in the agent or
solution
containing the agent. In another example, the cells or lysate can be coated or
sprayed with the
agent or solution containing the agent. In still another example, the cells or
lysate can be
contacted with a medium, such as a culture medium, that contains the agent or
solution
containing the agent. In a further example, the cells or lysate can be infused
with the agent or
solution containing the agent. The particular method of contacting the
leukocytes or lysate
thereof with the agent to be screened will be readily apparent to one of
ordinary skill in the art
and will depend on such factors as the size of the sample, the particular
agent to be screened,
convenience, preference, and the like.
153. The biomarker whose level of expression or activity is detected can be
one or
more genes or proteins that are down-regulated in the neurodegenerative
disease. In this
example, when the agent increases the level of expression or activity of the
gene or protein
biomarkers, this can indicate a therapeutic agent for the treatment of the
particular
neurodegenerative disease. Alternatively, the biomarker can be one or more
genes or proteins
that are up-regulated in the neurodegenerative disease. In this example, a
therapeutic agent for
the treatment of the particular neurodegenerative disease can be indicated
when the agent
decreases the level of expression or activity of the gene or protein
biomarkers. Still further, a
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.., .. ,,.,, ,,,, ~ .,
~u$~g~aed while another biomarker(s) can be down-regulated in the
neurodegenerative disease. In this example, when the agent decreases the level
of expression or
activity of the gene or protein biomarker(s) that is up-regulated in the
neurodegenerative disease
and/or increases the level of expression or activity of the gene or protein
biomarker(s) that is
down regulated in the neurodegenerative disease, this can indicate a
therapeutic agent for the
treatment of the particular neurodegenerative disease.
154. In an additional aspect of the disclosed methods, one can further
determine
whether the therapeutic agent alters the level of expression or activity of
the biomarker in
neurons. In one aspect, the neurons can be dopaminergic neurons.
155. In yet another aspect, the disclosed methods can further comprise
determining
whether the therapeutic agent prevents the development of or slows the
progression of a
neurodegenerative disease in an animal model of the disease. For example, when
the
neurodegenerative disease is Parkinson's disease, suitable animal models
include, but are not
limited to, a MPTP model, a 6-OHDA model, a paraquat model, or a rotenone
model.
b) Method of Monitoring Neurodegenerative Disease Progression
156. In still another aspect, disclosed herein are methods of monitoring
neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease)
progression in a subject.
The disclosed methods comprise comparing a level of expression or activity of
a biomarker for a
neurodegenerative disease in a sample comprising leukocytes or a lysate
thereof obtained from
the subject at multiple time points.
157. Also, disclosed herein are methods of monitoring a response to a
neurodegenerative disease (e.g., Parkinson's disease) treatment in a subject.
The disclosed
methods can comprise comparing a level of expression or activity of a
biomarker for the
neurodegenerative disease in a sample comprising leukocytes or a lysate
thereof obtained from
the subject at multiple time points during treatment of the subject.
158. In these methods, the subject can be as disclosed above (e.g., human).
Also, the
subject can be asymptomatic or preclinical for neurodegenerative disease at
one or more of the
multiple time points. In another example, the subject has not received
treatment for the
neurodegenerative disease at one or more of the multiple time points.
159. By "treatment" is meant any medical intervention that the subject
received or
undergoes for the purpose of curing, preventing, or alleviating the disease.
Treatment can
include, but is not limited to, pharmacological therapy (e.g., the
administration of
pharmaceuticals), nutritional therapy (e.g., the administration of vitamins,
hormones,
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... ,, i
: rlaitra~~~bi~~~Lg~ tra~~z~il~'i'6~t' ; or supplements, or the alteration of
diet), physical therapy,
surgical treatment, non-pharmacological therapy, behavioral modification, and
the like.
Optionally, the subject receives treatment for a neurodegenerative disease at
one or more of the
multiple time points. Optionally, the subject is treated with a
neuroprotective agent at or before
one of the inultiple time points. Optionally, the subject is treated with a
dopamine agonist (e.g.,
levodopa) at one or more of the multiple time points. In another specific
example, the subject is
treated with a neuroprotective agent at one or more of the multiple time
points.
160. Examples of neuroprotective agents which can be used to treat a subject
include,
but are not limited to, an acetylcholinesterase inhibitor, a glutamatergic
receptor antagonist,
kinase inhibitors, HDAC inhibitors, anti-flammatory agents, divalproex sodium,
or any
combination thereof. Examples of other neuroprotective agents can include, but
are not limited
to, Obidoxime Chloride; Pralidoxime Chloride; Pralidoxime Iodide; Pralidoxime
Mesylate,
Alverinc Citrate; Anisotropine Methylbromide; Atropine; Atropine Oxide
Hydrochloride;
Atropine Sulfate; Belladonna; Benapryzine Hydrochloride; Benzetimide
Hydrochloride;
Benzilonium Bromide; Biperiden; Biperiden Hydrochloride; Biperiden Lactate;
Clidinium
Bromide; Cyclopentolate Hydrochloride; Dexetimide;.Dicyclomine Hydrochloride;
Dihexyverine Hydrochloride; Domazoline Fumarate; Elantrine; Elucaine;
Ethybenztropine;
Eucatropine Hydrochloride; Glycopyrrolate; Heteronium Bromide; Homatropine
Hydrobromide;
Homatropine Methylbromide; Hyoscyamine; Hyoscyamine Hydrobromide; Hyoscyamine
Sulfate; Isopropamide Iodide; Mepenzolate Bromide; Methylatropine Nitrate;
Metoquizine;
Oxybutynin Chloride; Parapenzolate Bromide; Pentapiperium Methylsulfate;
Phencarbamide;
Poldine Methylsulfate; Proglumide; Propantheline Bromide; Propenzolate
Hydrochloride;
Scopolamine Hydrobromide; Tematropium Methylsulfate; Tiquinamide
Hydrochloride;
Tofenacin Hydrochloride; Toquizine; Triampyzine Sulfate; Trihexyphenidyl
Hydrochloride;
Tropicamide. Further examples include, but are not limited to, Albutoin;
Ameltolide; Atolide;
Buramate; Carbamazepine; Cinromide; Citenamide; Clonazepam; Cyheptamide;
Dezinamide;
Dimethadione; Divalproex Sodium; Eterobarb; Ethosuximide; Ethotoin; Flurazepam
Hydrochloride; Fluzinamide; Fosphenytoin Sodium; Gabapentin; Ilepcimide;
Lamotrigine;
Magnesium Sulfate; Mephenytoin; Mephobarbital; Methetoin; Methsuximide;
Milacemide
Hydrochloride; Nabazenil; Nafimidone Hydrochloride; Nitrazeparn; Phenacemide;
Phenobarbital; Phenobarbital Sodium; Phensuximide; Phenytoin; Phenytoin
Sodium; Primidone;
Progabide; Ralitoline; Remacemide Hydrochloride; Ropizine; Sabeluzole;
Stiripentol;
Sulthiame; Thiopental Sodium; Tiletamine Hydrochloride; Topirarnate;
Trimethadione;
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;-%l~~bhf&[ S:6Hju&j:7-VTd''~~kcid; Vigabatrin; Zoniclezole Hydrochloride;
Zonisamide. Still
other examples of anti-imflammatory agents include, but are not limited to,
Alclofenac;
Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal;
Amcinafide;
Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen;
Apazone;
Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;
Bromelains;
Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;
Clobetasol
Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
Cormethasone Acetate;
Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone
Dipropionate;
Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone
Sodium;
Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;
Endrysone; Enlimomab;
Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;
Fenbufen;
Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone;
Fluazacort; Flufena.mic
Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin
Butyl;
Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone
Propionate;
Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone
Acetate; Ibufenac;
Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin;
Indomethacin
Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac;
Isoxicam;
Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate;
Meclofenamate
Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine;
Meseclazone; Methylprednisolone Suleptanate; Momiflumate; Nabumetone;
Naproxen;
Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin;
Oxaprozin;
Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium;
Phenbutazone
Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam
Olamine;
Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole;
Proxazole Citrate;
Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride;
Seclazone;
Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;
Talosalate; Tebufelone;
Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;
Tixocortol
Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; or
Zomepirac
Sodium.
161. In other examples, the subject can be treated with a non-pharmacological
treatment, i.e., treatments that do not primarily involve drugs. Examples of
such non-
pharmacological drugs include, but are not limited to, brain stimulation,
which is typically used
in PD, ventricular shunt and transposition of omentum, which has been used in
AD. In still other
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~
f- e~anri0l6 9 yii~i~~~ulij~'t ca[~ f~~ t~eated by behavioral modification.
Still further examples of
treatments involve gene therapy, transplants, and stem cells.
162. In the disclosed methods, a level of biomarker(s) expression or activity
assessed
at one point in time can be the same as the level assessed at another point in
time. This can
indicate that the particular neurodegenerative disease has not changed (e.g.,
the disease has not
gotten worse or better). In another example, a level of biomarker(s)
expression or activity at an
earlier point in time can be more or less than the level at a later point in
time. This can indicate
that the neurodegenerative disease is progressing. If a biomarker's level of
expression has been
previously shown to increase from an earlier point in time to a later point in
time and to correlate
with (a) worsening or (b) improvement of the symptoms of a disease, then a
lower amount of
biomarker present in the earlier sample relative to the later sample can be
considered to be an
indication that the subject's condition is (a) worsening or (b) improving,
respectively. On the
other hand, if a biomarker's level of expression has been shown to decrease
from an earlier point
in time to a later point in time and to correlate with (a) worsening or (b)
improvement of the
symptoms of a disease, then a higher amount of biomarker present in the
earlier sample relative
to the later sample can be an indication that the subject's condition is (a)
worsening or (b)
improving, respectively. In another example, a combination of biomarkers,
where some
biomarkers in the combination increase from an earlier point in time to a
later point in time
during disease progression and other biomarkers decrease, can be used.
163. Also, the level of a biomarker can be correlated with a worsening or an
improveinent in one or more symptoms of a neurodegenerative disease in
response to the
therapy. Gene product(s) whose expression levels are different between a
sample taken prior to
treatment or at an earlier point in time during treatment and a sample taken
at a later point in
time during treatment or after treatment can identify a biomarker for the
response of a subject to
a treatment for a neurodegenerative disease.
164. In these methods, a difference in a level of expression or activity of a
biomarker
between various samples can be indicative of the subject's responsiveness to
the administered
treatment for the neurodegenerative disease. If a biomarker's expression has
been previously
shown to increase in subjects that (a) respond or (b) fail to respond to the
treatment for a
neurodegenerative disease, then a larger amount of biomarker in a later sample
relative to an
earlier sample can be an indication that the subject is (a) responding or (b)
not responding,
respectively, to the treatment. Alternatively, if a biomarker's expression has
been previously
shown to decrease in subjects that (a) respond or (b) fail to respond to a
treatment for a
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It : aur'ougiarlvaf'dis~~a~'e;=~~h~n a smaller amount of biomarker in a later
sample relative to an
earlier sample can be considered to be an indication that the subject is (a)
responding or (b) not
responding, respectively to the treatment. Alternatively, if a combination of
biomarkers are used
and one or more biomarkers have been previously shown to decrease in subjects
that (a) respond
or (b) fail to respond to a treatment for a neurodegenerative disease and one
or more other
biomarkers have been previously shown to increase, then a change in the amount
of biomarkers
in the combination of biomarkers from a later sample relative to an earlier
sample can be
considered to be an indication that the subject is (a) responding or (b) not
responding,
respectively to the treatment.
c) Method of Identifying a Risk for a Neurodegenerative Disease in a
Subject
165. In yet a further aspect, disclosed herein are methods for identifying a
risk for a
neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease) in a test
subject. The
disclosed methods comprise determining a level of expression or activity of a
biomarker for a
neurodegenerative disease from a sample obtained from the test subject,
wherein the sample
comprises leukocytes or a lysate thereof; and correlating the level of
expression or activity level
of the biomarker determined for the test subject with the levels for a
reference subject. The
method can further comprise determining the level of the biomarker(s) from a
population of
reference subjects diagnosed with the neurodegenerative disease and/or from a
population of
reference subjects without the neurodegenerative disease. In the disclosed
methods, a correlation
between levels for a reference population without the neurodegenerative
disease and the levels
for the test subject can identify a low risk for the particular
neurodegenerative disease in the test
subject. Also, a correlation between the levels determined for the reference
population with the
neurodegenerative disease and the levels for the test subject can identify a
high risk for the
neurodegenerative disease in the test subject.
166. By "correlation" is meant any relationship between data. For example, a
correlation can be determined through a statistical analysis of the levels of
biomarker expression
or activity (e.g., standard deviation, degree of confidence, etc.). A
correlation can also be an
empirical determination based on the levels of biomarker expression or
activity.
167. Gene product data (e.g., transcript and/or proteomic data) can be
subjected to the
following statistical analyses. To account for technical variability, each
subject sample can be
run in triplicate on 2D gels (e.g., 52 subjects x 3 gels equals 156 gels).
Averaged spot intensities
can then be used for further analysis. Gender, basic clinical diagnosis, and
clinical indices can
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ih'tllepot~~ohIs data. Primary comparisons using simple univariate statistical
methods such as two-sample t-tests can proceed to identify those proteins
whose expression in
leukocytes are different between disease subjects and the control group.
Proteomic data from 2D
gel electrophoresis and MALDI-TOF mass spectroscopy can be first analyzed
using the
statistical tools, including t-tests and ANOVAs, contained within the
Progenesis Workstation
Image Analysis and Informatics software program (Nonlinear USA, Inc.; Durham,
NC). In cases
of heavy dependence in these data a step-down multivariate resampling
algorithm can be used to
address the multiplicity of tests, as disclosed in Troendle, A permutational
step-up method of
testing multiple outcomes, Biometrics, 1996;52:846-859, which is incorporated
by reference
herein at least for its teachings of statistical methods.
168. In some aspects, multivariate statistical methods can be more appropriate
and
powerf-ul in all classifications and associations being considered in such
studies. Canonical
discriminant analysis can be performed to use the profiling of all gene
products together for the
disclosed methods. Logistic discrimination between groups based on
multivariate observations
can be used since it generally out-performs the normal-theory-based linear
discriminant analysis
(see McLachlan, Discriminant analysis and statistical pattern recognition,
Wiley, New York,
1992, which is incorporated by reference herein at least for its teaching of
statistical methods).
To identify those proteins associated to behavioral indices, linear models and
generalized linear
models (e.g., those disclosed in Nelder and McCullagh, Generalized Linear
Models, CRC Press,
Boca Raton, FL, 1999, which is incorporated by reference herein at least for
its teaching of linear
models) can be fitted to profile proteins differentially expressed in the
neurodegenerative disease
and non-neurodegenerative disease leukocytes and to identify those proteins
whose expression
changes are related to the severity of disease. These models can also take in
consideration the
confounding issues of some clinical factors. Missing values can be handled as
suggested by
Little and Little (Applications of Modern Missing Data Methods, CRC Press,
Boca Raton, FL,
2002, which is incorporated by reference herein at least for its teaching of
statistical methods).
In cases of unexpected complication of data analysis, the skill artisan can
pursue appropriate
statistical methods and even develop and program statistical methods to serve
these specific
aims, including the use of nonparametric empirical Bayesian analysis proposed
by Efron and
Tibshirani, Empirical bayes methods and false discovery rates for microarrays,
Genet Epidemiol.
2002;23:70-86, which is incorporated by reference herein at least for its
teaching of statistical
methods.
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Hfteihod of Distinguishing One Neurodegenerative Disease from
Another Disease
169. In another aspect, disclosed herein are methods of differentially
diagnosing a
neurodegenerative disease, (e.g., Parkinson's or Alzheimer's disease) in a
test subject. The
disclosed methods comprise assessing a level of expression or activity of one
or more selected
biomarker(s) in a sample comprising leukocytes or a lysate thereof from the
test subject and
comparing the level of expression or activity of the selected biomarker(s) to
a reference standard
that indicates the level of expression or activity of the selected
biomarker(s) in one or more
populations of neuropathologic control subjects with one or more
neuropathological control
diseases. In the disclosed methods, a difference or similarity between the
level of expression or
activity of the selected biomarker(s) and the reference standard can indicate
a differential
diagnosis of one neurodegenerative as compared to the neuropathological
control diseases.
170. By "differentially diagnosing" is meant to identify the presence of one
particular
disease in a subject and/or identify the absence of another disease in a
subject. The phrase also
means to distinguish one particular disease from another disease or from the
absence of a
disease. "Differentially diagnose" is also used herein to mean to identify a
particular stage of
one disease, to identify the risk of developing a particular disease, or to
identify a prognosis of a
particular disease.
171. By "neuropathologic control subject" is meant a subject (e.g., human) or
group of
subjects that have one or more neurodegenerative diseases, as described
herein. For example,
the neuropathologic control subject can be one or more subjects with
Alzheimer's disease,
frontal-temporal dementia, mild cognitive impairment, and Parkinson's disease,
plus disorders
that comprise additional symptoms (e.g., multiple system atrophy, corticobasal
ganglionic
degeneration, Parkinson's disease with Alzheimer's). The neuropathologic
control subject can
also be a subject or group of subjects that does not have a particular
disease. Still further,
neuropathologic control subject can also be a subject or group of subjects
that has a particular
risk or predisposition of developing a disease.
172. In these methods, the test subject and the reference populations can be
age or sex
matched or both.
e) Methods of Identifying a Biomarker
173. Biomarkers for a neurodegenerative disease, such as Parkinson's or
Alzheimer's
disease, can be identified by the methods disclosed herein. In one aspect,
methods for
identifying a biomarker for a particular neurodegenerative disease can
comprise assessing a level
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qf o~t~i~ir~1&6~~flgene products in a sample comprising leukocytes or a lysate
thereof
from at least one subject (e. g., . human) diagnosed with the
neurodegenerative disease, and
comparing the level of expression of the gene products to a reference
standard. In this method,
an increase or decrease in the level of expression of the gene products, as
compared to the
reference standard, can identify the gene products as biomarkers for the
particular
neurodegenerative disease.
174. In some cases, the same biomarkers can be used for diagnosing, monitoring
disease progression, and/or monitoring the response of a subject to a therapy
for a disease, are
the same biomarker. The altered expression of the biomarker (under the
conditions of the two or
more different uses) can reflect the same fundamental biochemical and
metabolic pathways that
underlie the pathology of the particular neurodegenerative disease. For
example, the same
biological pathways can cause both a gene product to be expressed at a
particular level in a
healthy subject that is free of the neurodegenerative disease and the same
gene product to be
expressed at a similar level in a in a subject responding to a treatment for
the disease.
175. Furthermore, because the altered expression of certain biomarkers can be
a due to
changes in biochemical/inetabolic pathways that underlie the pathology of a
disease, these
biomarkers also represent therapeutic targets. If a change in expression of a
biomarker causes
one or more symptoms of a neurodegenerative disease, then the biomarker can be
a therapeutic
target. Therapeutic targets can be used in methods for discovering compounds
that modulate the
expression or activity of one or more candidate biomarkers and/or improve one
or more
symptoms of the neurodegenerative disease (i.e., candidate therapeutic
agents).
5. Clinical features of Alzheimer's
176. One hundred years ago Alois Alzheimer described the major behavioral and
neuropathological features of the neurodegenerative disorder bearing his name.
AD is
characterized clinically/behaviorally by progressive impairment of memory and
cognition.
Neuropathological and neurobiological changes associated with this slow
progression of clinical
symptoms include accumulation of amyloid plaques and neurofibrillary tangles
(NFTs) (Gearing
M, et al., The Consortium to Establish a Registry for Alzheimer's Disease
(CERAD). Part X.
Neuropathology confirmation of the clinical diagnosis of Alzheimer's disease.
Neurology.
1995;45(3 Pt 1):461-466) gliosis (Unger JW, Microscopy Res. Technique,
1998;43:24-28),
reduced dendritic plasticity relative to normal aged (Buell and Coleman,
Science,
1979;206(4420):854-856; Flood DG, et al., Brain Research, 1985;345(2):366-368;
Flood DG, et
al., Brain Research, 1987;402(2):205-216), and reduced density of neurons
(Coleman PD, et al.,
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[ W6iiro~=~~ilm~~:t-t~~~' ~g~,1~9~~8 /,19(6):521-545; TerryRD, et al., Annals
of Neurology,
1987;21:530-539; West MJ, et al., Lancet, 1994;344:769-772) and synapses
(Scheff SW, et al.,
Neurobiology of Aging, 1990;11(1):29-37).
6. Gene expression in Alzheimer's
177. Studies of altered gene expression in Alzheimer's disease brain tissue
have shown
a general reduction of message level estimated at about 35% (Doebler JA, et
al., J.
Neuropathology & Experimental Neurology, 1987;46(l):28-39), (Griffin WS, et
al., Alzheimer
Disease & Associated Disorders, 1990;4(2):69-78), (Harrison PJ, et al.,
Psychological Medicine,
1991;21:855-866). Against this background of a general reduction of mRNA,
selected studies
have demonstrated increased as well as decreased expression of a wide variety
of genes. Some
gene classes affected in Alzheimer's disease are expressed in a neuron
specific manner. These
especially include decreased expression of selected genes that are related to
synaptic structure
and function and the neuronal cytoskeleton (Ginsberg SD, et al., Annals of
Neurology,
2000;48(1):77-87; Yao P, et al., J. Neuroscience, 1998;18(7):2399-2411). Other
classes of genes
whose expression is altered in AD include those related to the cell cycle
(Arendt T,
Neurobiology of Aging, 2000;21(6):783-796; Husseman JW, et al., Neurobiology
of Aging,
2000;21(6):815-828; Nagy Z, et al., Neurobiology of Aging, 2000;21(6):761-769;
Vincent I, et
al., J. Neuroscience, 1997;17:3588-3598) and inflammatory/stress responses
(for a review, see
Akiyama H, et al., Neurobiology of Aging, 2000;21(3):383-421). These gene
classes are
expressed in a variety of cell types that reside outside the nervous system
including leukocytes
(Wakutani Y, et al., Dementia, 1995;6(6):301-305), monocytes (Jung SS, et al.,
Neurobiology of
Aging, 1999;20(3):249-257), and epitllelial cells (Schmitz A, et al.,
Histochemistry & Cell
Biology, 2002;117(2):171-180) as well as other cell types.
178. Multivariate analysis of profiles of expression of multiple gene products
(messages) by single neurons or homogenates from postmortem human brain can be
used to
distinguish neurodegenerative disease (e.g., Parkinson's and Alzheimer's
disease) from control
samples (Cheetham JE, et al., J. Neuroscience Methods, 1997;77(l),:43-48, Chow
N, et al., Proc.
Natl. Acad. Sci. U.S.A., 1998;95:9620-9625).
179. As disclosed herein, data relating to using multiple genes to diagnose of
neurodegenerative disease (e.g., Parkinson's and Alzheimer's disease) is
obtained from samples
such as peripheral blood and blood leukocytes. For example, varying sets of
genes are used and
genes related to the inflammatory response as well as the cell cycle are
predicative. Blood was
drawn from patients diagnosed in our Alzheimer's Disease Center as having
probable (mild) AD
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ed control sample. Message was extracted from peripheral blood
leukocytes and amplified (Eberwine J, et al., PNAS U.S.A. 1992;89(7):3010-
3014). The
expression level of selected messages was then quantified. Multivariate
statistical analyses
differentiated Alzheimer's and control white blood cells. It was found that
expression levels of
genes related to the cell cycle and to inflammatory responses distinguish
blood samples of AD
cases from samples from non-demented control cases. These specific gene sets
and classes were
also shown to be classes of genes that are also differentially expressed in AD
brain. This study
was repeated three times with 3 different sets of cases.
180. As disclosed herein, the expression of genes related to the cell cycle
and to
inflammatory responses is affected in peripheral leukocytes from AD cases, a
finding that
parallels altered expression of these gene classes in the AD brain. There are
two major
conclusions to be reached regarding the data presented here: (1) expression
profiles of multiple
genes are effective at distinguishing mild AD (average CDR 1.2-1.5) from non-
demented control
cases and, (2) the gene classes described as distinguishing AD from control
peripheral blood
samples are similar to gene classes whose expression has been shown to be
altered in the brain in
AD. This is consistent with the concept of AD as a systemic disease or a
disease with major
systemic consequences.
C. Compositions
181. Disclosed are the components to be used to prepare the disclosed
compositions as
well as the compositions themselves to be used within the methods disclosed
herein. These and
other materials are disclosed herein, and it is understood that when
combinations, subsets,
interactions, groups, etc. of these materials are disclosed that while
specific reference of each
various individual and collective combinations and permutation of these
compounds may not be
explicitly disclosed, each is specifically contemplated and described herein.
For example, if a
particular method of diagnosing a neurodegenerative disease is disclosed and
discussed and a
number of modifications that can be made to a number of molecules including
the method of
diagnosing a neurodegenerative disease are discussed, specifically
contemplated is each and
every combination and permutation of the method of diagnosing the
neurodegenerative disease
and the modifications that are possible unless specifically indicated to the
contrary. Thus, if a
class of molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if each is not
individually
recited each is individually and collectively contemplated meaning
combinations, A-E, A-F, B-
D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset
or combination
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I-ORTUs, for example, the sub-group of A-E, B-F, and C-E would be
considered disclosed. This concept applies to all aspects of this application
including, but not
limited to, steps in methods of making and using the disclosed compositions.
Thus, if there are a
variety of additional steps that can be performed it is understood that each
of these additional
steps can be perfonned with any specific embodiment or combination of
embodiments of the
disclosed methods.
1. Sequence similarities
182. It is understood that as discussed herein the use of the terms homology
and
identity mean the same thing as similarity. Thus, for example, if the use of
the word homology
is used between two non-natural sequences it is understood that this is not
necessarily indicating
an evolutionary relationship between these two sequences, but rather is
looking at the similarity
or relatedness between their nucleic acid sequences. Many of the methods for
determining
homology between two evolutionarily related molecules are routinely applied to
any two or more
nucleic acids or proteins for the purpose of measuring sequence similarity
regardless of whether
they are evolutionarily related or not.
183. In general, it is understood that one way to define any known variants
and
derivatives or those that might arise, of the disclosed genes and proteins
herein, is through
defining the variants and derivatives in terms of homology to specific known
sequences. This
identity of particular sequences disclosed herein is also discussed elsewhere
herein. In general,
variants of genes and proteins herein disclosed typically have at least, about
70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99
percent homology to the stated sequence or the native sequence. Those of skill
in the art readily
understand how to determine the homology of two proteins or nucleic acids,
such as genes. For
example, the homology can be calculated after aligning the two sequences so
that the homology
is at its highest level. '
184. Another way of calculating homology can be performed by published
algorithms.
Optimal alignment of sequences for comparison may be conducted by the local
homology
algorithm of Smith and Waterman, Adv. Appl. Math, 1981;2:482, by the homology
alignment
algorithm of Needleman and Wunsch, J. Mol. Biol., 1970;48:443, by the search
for similarity
method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 1988;85:2444, by
computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison,
WI), or by
inspection.
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&VOel.of homology can be obtained for nucleic acids by for example the
algorithms disclosed in Zuker M, Science, 1989;244:48-52; Jaeger, et al.,
Proc. Natl. Acad. Sci.
U.S.A., 1989;86:7706-7710; Jaeger, et al., Methods Enzymol., 1989;183:281-306,
which are
herein incorporated by reference for at least material related to nucleic acid
alignment. It is
understood that any of the methods typically can be used and that in certain
instances the results
of these various methods may differ, but the skilled artisan understands if
identity is found with
at least one of these methods, the sequences would be said to have the stated
identity, and be
disclosed herein.
186. For example, as used herein, a sequence recited as having a particular
percent
homology to another sequence refers to sequences that have the recited
homology as calculated
by any one or more of the calculation methods described above: For example, a
first sequence
has 80 percent homology, as defined herein, to a second sequence if the first
sequence is
calculated to have 80 percent homology to the second sequence using the Zuker
calculation
method even if the first sequence does not have 80 percent homology to the
second sequence as
calculated by any of the other calculation methods. As another example, a
first sequence has 80
percent homology, as defined herein, to a second sequence if the first
sequence is calculated to
have 80 percent homology to the second sequence using both the Zuker
calculation method and
the Pearson and Lipman calculation method even if the first sequence does not
have 80 percent
homology to the second sequence as calculated by the Smith and Waterman
calculation method,
the Needleman and Wunsch calculation method, the Jaeger calculation methods,
or any of the
other calculation methods. As yet another example, a first sequence has 80
percent homology, as
defined herein, to a second sequence if the first sequence is calculated to
have 80 percent
homology to the second sequence using each of calculation methods (although,
in practice, the
different calculation methods will often result in different calculated
homology percentages).
2. Hybridization/selective hybridization
187. The term hybridization typically means a sequence driven interaction
between at
least two nucleic acid molecules, such as a primer or a probe and a gene.
Sequence driven
interaction means an interaction that occurs between two nucleotides or
nucleotide analogs or
nucleotide derivatives in a nucleotide specific manner. For example, G
interacting with C or A
interacting with T are sequence driven interactions. Typically sequence driven
interactions occur
on the Watson-Crick face or Hoogsteen face of the nucleotide. The
hybridization of two nucleic
acids is affected by a number of conditions and parameters known to those of
skill in the art. For
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ct~nc~rit~ 1Is, pH, and temperature of the reaction all affect whether two
nucleic acid molecules will hybridize.
188. Parameters for selective hybridization between two nucleic acid molecules
are
well known to those of skill in the art. For example, in some embodiments
selective
hybridization conditions can be defined as stringent hybridization conditions.
For example,
stringency of hybridization is controlled by both temperature and salt
concentration of either or
both of the hybridization and washing steps. For example, the conditions of
hybridization to
achieve selective hybridization may involve hybridization in high ionic
strength solution (6X
SSC or 6X SSPE) at a temperature that is about 12-25 C below the T. (the
melting temperature
at which half of the molecules dissociate from their hybridization partners)
followed by washing
at a combination of temperature and salt concentration chosen so that the
washing temperature is
about 5 C to about 20 C below the Tm. The temperature and salt conditions are
readily
determined empirically in preliminary experiments in which samples of
reference DNA
immobilized on filters are hybridized to a labeled nucleic acid of interest
and then washed under
conditions of different stringencies. Hybridization temperatures are typically
higher for DNA-
RNA and RNA-RNA hybridizations. The conditions can be used as described above
to achieve
stringency, or as is known in the art. (Sambrook, et al., Molecular Cloning: A
Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989; Kunkel, et
al. Methods Enzymol., 1987;154:367, which is herein incorporated by reference
for material at
least related to hybridization of nucleic acids). A preferable stringent
hybridization condition for
a DNA:DNA hybridization can be at about 68 C (in aqueous solution) in 6X SSC
or 6X SSPE
followed by washing at 68 C. Stringency of hybridization and washing, if
desired, can be
reduced accordingly as the degree of complementarity desired is decreased, and
further,
depending upon the G-C or A-T richness of any area wherein variability is
searched for.
Likewise, stringency of hybridization and washing, if desired, can be
increased accordingly as
homology desired is increased, and further, depending upon the G-C or A-T
richness of any area
wherein high homology is desired, all as known in the art.
189. Another way to define selective hybridization is by looking at the amount
(percentage) of one of the nucleic acids bound to the other nucleic acid. For
example, in some
embodiments selective hybridization conditions would be when at least about,
60, 65, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98,
99, 100 percent of the limiting nucleic acid is bound to the non-limiting
nucleic acid. Typically,
the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This
type of assay can
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re~~per1'e1W-iRaWtofi'iWns where both the limiting and non-limiting primer are
for
example, 10 fold or 100 fold or 1000 fold below their kd, or where only one of
the nucleic acid
molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic
acid molecules are
above their kd.
190. Another way to define selective hybridization is by looking at the
percentage of
primer that gets enzymatically manipulated under conditions where
hybridization is required to
promote the desired enzymatic manipulation. For example, in some embodiments
selective
hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73,
74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100 percent of the
primer is enzymatically manipulated under conditions which promote the
enzymatic
manipulation, for example if the enzymatic manipulation is DNA extension, then
selective
hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73,
74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100 percent of the
primer molecules are extended. Preferred conditions also include those
suggested by the
manufacturer or indicated in the art as being appropriate for the enzyme
performing the
manipulation.
191. Just as with homology, it is understood that there are a variety of
methods herein
disclosed for determining the level of hybridization between two nucleic acid
molecules. It is
understood that these methods and conditions may provide different percentages
of hybridization
between two nucleic acid molecules, but unless otherwise indicated meeting the
parameters of
any of the methods would be sufficient. For example if 80% hybridization was
required and as
long as hybridization occurs within the required parameters in any one of
these methods it is
considered disclosed herein.
192. It is understood that those of skill in the art understand that if a
composition or
method meets any one of these criteria for determining hybridization either
collectively or singly
it is a composition or method that is disclosed herein.
3. Nucleic acids
193. There are a variety of molecules disclosed herein that are nucleic acid
based,
including, for example, the nucleic acids that encode, for example, any of the
genes disclosed
herein as being associated with the onset or progression of a
neurodegenerative disease (e.g.,
Parkinson's and Alzheimer's disease), as well as any other proteins disclosed
herein, as well as
various functional nucleic acids. The disclosed nucleic acids are made up of
for example,
nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting
examples of these and
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It is understood that, for example, when a vector is
expressed in a cell, the expressed mRNA will typically be made up of A, C, G,
and U. Likewise,
it is understood that if, for example, an antisense molecule is introduced
into a cell or cell
environment through for example exogenous delivery it is advantageous that the
antisense
molecule be made up of nucleotide analogs that reduce the degradation of the
antisense molecule
in the cellular environment.
a) Nucleotides and related molecules
194. A nucleotide is a molecule that contains a base moiety, a sugar moiety
and a
phosphate moiety. Nucleotides can be linked together through their phosphate
moieties and
sugar moieties creating an internucleoside linkage. The base moiety of a
nucleotide can be
adenine-9-yl (A), cytosine-l-yl (C), guanine-9-yl (G), uracil,l-yl (U), and
thymin-1-yl (T). The
sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate
moiety of a nucleotide
is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'-
AMP (3'-
adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
195. A nucleotide analog is a nucleotide which contains some type of
modification to
either the base, sugar, or phosphate moieties. Modifications to nucleotides
are well known in the
art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine,
xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the
sugar or phosphate
moieties.
196. Nucleotide substitutes are molecules having similar functional properties
to
nucleotides, but which do not contain a phosphate moiety, such as peptide
nucleic acid (PNA).
Nucleotide substitutes are molecules that will recognize nucleic acids in a
Watson-Crick or
Hoogsteen manner, but which are linked together through a moiety other than a
phosphate
moiety. Nucleotide substitutes are able to confonn to a double helix type
structure when
interacting with the appropriate target nucleic acid.
197. It is also possible to link other types of molecules (conjugates) to
nucleotides or
nucleotide analogs to enhance for example, cellular uptake. Conjugates can be
chemically
linked to the nucleotide or nucleotide analogs. Such conjugates include but
are not limited to
lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.
Acad. Sci. USA,
1989,86, 6553-6556).
198. A Watson-Crick interaction is at least one interaction with the Watson-
Crick face
of a nucleotide, nucleotide analog, or nucleoticle substitute. The Watson-
Crick face of a
nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl,
and C6 positions of a
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hie1d'btide; C~ii%Iotide analog, or nucleotide substitute and the C2, N3, C4
positions
of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
199. A Hoogsteen interaction is the interaction that takes place on the
Hoogsteen face
of a nucleotide or nucleotide analog, which is exposed in the major groove of
duplex DNA. The
Hoogsteen face includes the N7 position and reactive groups (NH2 or 0) at the
C6 position of
purine nucleotides.
(1) Primers and probes
200. Disclosed are compositions including primers and probes, which are
capable of
interacting with the genes disclosed herein. In certain embodiments the
primers are used to
support DNA amplification reactions. Typically the primers will be capable of
being extended
in a sequence specific manner. Extension of a primer in a sequence specific
manner includes
any methods wherein the sequence and/or composition of the nucleic acid
molecule to which the
primer is hybridized or otherwise associated directs or influences the
composition or sequence of
the product produced by the extension of the primer. Extension of the primer
in a sequence
specific manner therefore includes, but is not limited to, PCR, DNA
sequencing, DNA
extension, DNA polymerization, RNA transcription, or reverse transcription.
Techniques and
conditions that amplify the primer in a sequence specific manner are
preferred. In certain
embodiments the primers are used for the DNA amplification reactions, such as
PCR or direct
sequencing. It is understood that in certain embodiments the primers can also
be extended using
non-enzymatic techniques, where for example, the nucleotides or
oligonucleotides used to
extend the primer are modified such that they will chemically react to extend
the primer in a
sequence specific manner. Typically the disclosed primers hybridize with the
nucleic acid or
region of the nucleic acid or they hybridize with the complement of the
nucleic acid or
compleinent of a region of the nucleic acid.
201. The size of the primers or probes for interaction witli the transcripts
listed in
Table 4, such as transcripts related to cell cycle such as cyclin Dl, cyclin
B, cyclin Gl, wee1,
hTR2, CDC25b, GSK3 beta, and protein kinase C alpha, transcripts related to
inflammatory
systems such as C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r,
and transcripts
related to cell stress such as Alpha-1 antichymotrypsin, HSP 27, HSP 90,
crystalline, GAPDH,
ferritin H, ferritin L, cox 1, cox 2, and transferrin, and transcripts of
proteins listed in Tables 5
and 6. In certain embodiments, the primers or probes can be any size that
supports the desired
enzymatic manipulation of the primer, such as DNA amplification or the simple
hybridization of
the probe or primer. A typical primer or probe for the genes listed in Table
4, such as cyclin D 1,
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~-ac~11n~~;"~~~~'~~'~';K~~*2, CDC25b, GSK3 beta, and protein kinase C alpha,
which are
related to cell cycle, C5, C1 inhibitor, IL-17r, IL-8, L1F, TNF-alpha, and IL-
lOr, which are
related to inflammatory systems, and Alpha-1 antichymotrypsin, HSP 27, HSP 90,
crystalline,
GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, which are
related to cell stress, and
genes of proteins listed in Tables 5 and 6, would be at least 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,
375, 400, 425, 450,
475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750,
2000, 2250,
2500, 2750, 3000, 3500, or 4000 nucleotides long.
202. In other embodiments a primer or probe for the genes listed in Table 4,
such as
cyclin D1, cyclin B, cyclin Gl, wee1, hTR2, CDC25b, GSK3 beta, and protein
kinase C alpha,
which are related to cell cycle, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-
alpha, and IL-lOr, which
are related to inflammatory systems, and Alpha-1 antichymotrypsin, HSP 27, HSP
90,
crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin,
which are related to cell
stress, and genes of proteins listed in Tables 5 and 6, can be less than or
equal to 6, 7, 8, 9, 10,
11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250,
275, 300, 325, 350,
375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
1000, 1250, 1500,
1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
203. In certain embodiments the primers and probes are designed such that they
are
outside primers whose nearest point of interaction with the genes found in
Table 4, such as
cyclin Dl, cyclin B, cyclin G1, weel, hTR2, CDC25b, GSK3 beta, and protein
kinase C alpha,
which are related to cell cycle, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-
alpha, and IL-lOr, which
are related to inflammatory systenls, and Alpha-1 antichymotrypsin, HSP 27,
HSP 90,
crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin,
which are related to cell
stress, and genes of proteins listed in Tables 5 and 6, is within 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89,
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99, 100, 125, 150, 175, or 200 nucleotides of the outermost
defining nucleotide of the genes listed in Table 4, such as cyclin D1, cyclin
B, cyclin Gl, wee 1,
hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-
8, LIF, TNF-
alpha, and 1L-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline,
GAPDH, ferritin H,
ferritin L, cox 1, cox 2, and transferrin, region or complement of the genes
listed in Table 4, such
as cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein
kinase C alpha,
C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r, Alpha-1
antichymotrypsin, HSP 27,
HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and
transferrin, and genes of
proteins listed in Tables 5 and 6.
204. In certain embodiments the primers and probes are designed such that they
are
outside primers whose nearest point of interaction with the genes listed in
Table 4, such as cyclin
Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, Cl
inliibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1
antichymotrypsin, HSP 27, HSP 90,
crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and
genes of proteins
listed in Tables 5 and 6, is at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 125, 150, 175, or 200 nucleotides away from the outermost
defining nucleotide of
the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin G1, weel,
hTR2, CDC25b, GSK3
beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha,
and IL-lOr, Alpha-1
antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L,
cox 1, cox 2, and
transferrin, region or complement of the genes listed in Table 4, such as
cyclin D1, cyclin B,
cyclin G1, wee1, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, C1
inhibitor, IL-17r,
IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90,
crystalline,
GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of
proteins listed in
Tables 5 and 6.
205. The primers for the genes listed in Table 4, such as cyclin D1, cyclin B,
cyclin
Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5. Cl inhibitor,
IL-17r, IL-8,
LIF, TNF-alpha, and IL-l Or, Alpha-1 antichymotrypsin, HSP 27, HSP 90,
crystalline, GAPDH,
ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins
listed in Tables 5 and 6,
typically will be used to produce an amplified DNA product that contains a
specific region of the
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16:16ize of the product will be such that the size can be accurately
determined to within 1, or 2, or 3 nucleotides.
206. In certain embodiments this product is at least 20, 21, 22, 23, 24, 25,
26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 125, 150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650,
700, 750, 800, 850,
900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000
nucleotides
long.
207. In other embodiments the product is less than or equal to 20, 21, 22, 23,
24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100, 125, 150,
175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550,
600, 650, 700, 750,
800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000,
3500, or 4000
nucleotides long.
(2) Functional Nucleic Acids
208. Functional nucleic acids are nucleic acid molecules that have a specific
function,
such as binding a target molecule or catalyzing a specific reaction.
Functional nucleic acid
molecules can be divided into the following categories, which are not meant to
be limiting. For
example, functional nucleic acids include antisense molecules, aptamers,
ribozymes, triplex
forming molecules, and external guide sequences. The functional nucleic acid
molecules can act
as affectors, inhibitors, modulators, and stimulators of a specific activity
possessed by a target
molecule, or the functional nucleic acid molecules can possess a de novo
activity independent of
any other molecules.
209. Functional nucleic acid molecules can interact with any macromolecule,
such as
DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids
can interact
with the mRNA of the genes listed in Table 4, such as cyclin D1, cyclin B,
cyclin G1, wee 1,
hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, C1 inhibitor, IL-17r, IL-
8, LIF, TNF-
alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline,
GAPDH, ferritin H,
ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in
Tables 5 and 6, or the
genomic DNA of the genes listed in Table 4, such as cyclin D1, cyclin B,
cyclin Gi, weel,
hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-
8, LIF, TNF-
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1al~~ia U&-~~~~~f~r;"'"~''alchymotrypsin, HSP 27, HSP 90, crystalline, GAPDH,
ferritin H,
ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in
Tables 5 and 6, or they can
interact with the polypeptide product of the genes listed in Table 4, such as
cyclin D1, cyclin B,
cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl
inhibitor, IL-17r,
IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90,
crystalline,
GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and the proteins
listed in Tables 5
and 6. Often functional nucleic acids are designed to interact with other
nucleic acids based on
sequence homology between the target molecule and the functional nucleic acid
molecule. In
other situations, the specific recognition between the functional nucleic acid
molecule and the
target molecule is not based on sequence homology between the functional
nucleic acid
molecule and the target molecule, but rather is based on the formation of
tertiary structure that
allows specific recognition to take place.
210. Antisense molecules are designed to interact with a target nucleic acid
molecule
through either canonical or non-canonical base pairing. The interaction of the
antisense
molecule and the target molecule is designed to promote the destruction of the
target molecule
through, for example, RNAseH mediated RNA-DNA hybrid degradation.
Alternatively the
antisense molecule is designed to interrupt a processing function that
normally would take place
on the target molecule, such as transcription or replication. Antisense
molecules can be designed
based on the sequence of the target molecule. Numerous methods for
optimization of antisense
efficiency by finding the most accessible regions of the target molecule
exist. Exemplary
methods would be in vitro selection experiments and DNA modification studies
using DMS and
DEPC. It is preferred that antisense molecules bind the target molecule with a
dissociation
constant (kd) less than or equal to 10-6, 10-8, 10-10, or 10-12. A
representative sample of methods
and techniques which aid in the design and use of antisense molecules can be
found in the
following non-limiting list of U.S. Patents: 5,135,917, 5,294,533, 5,627,158,
5,641,754,
5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590,
5,990,088,
5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042,
6,025,198,
6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.
211. Aptamers are molecules that interact with a target molecule, preferably
in a
specific way. Typically aptamers are small nucleic acids ranging from 15-50
bases in length that
fold into defined secondary and tertiary structures, such as stem-loops or G-
quartets. Aptamers
can bind small molecules, such as ATP (U.S. Patent No. 5,631,146) and
theophiline (U.S. Patent
No. 5,580,737), as well as large molecules, such as reverse transcriptase
(United States patent
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(44.S. Patent No. 5,543,293). Aptamers can bind very tightly with
kds from the target molecule of less than 10-12 M. It is preferred that the
aptamers bind the target
molecule with a kd less than 10-6, 10, 10-", or 10"12. Aptamers can bind the
target molecule
with a very high degree of specificity. For example, aptamers have been
isolated that have
greater than a 10000 fold difference in binding affinities between the target
molecule and another
molecule that differ at only a single position on the molecule (U.S. Patent
No. 5,543,293). It is
preferred that the aptamer have a kd with the target molecule at least 10,
100, 1000, 10,000, or
100,000 fold lower than the ka with a background binding molecule. It is
preferred when doing
the comparison for a polypeptide for example, that the background molecule be
a different
polypeptide. For example, when determining the specificity of aptamers of the
genes listed in
Table 4, such as cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3
beta, protein
kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr,
Alpha-1
antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L,
cox 1, cox 2, and
transferrin, or genes of proteins listed in Tables 5 and 6, the background
protein could be serum
albumin. Representative examples of how to make and use aptamers to bind a
variety of '
different target molecules can be found in the following non-limiting list of
U.S. Patent Nos.
5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721,
5,846,713,
5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020,
6,013,443,
6,020,130, 6,028,186, 6,030,776, and 6,051,698.
212. Ribozymes are nucleic acid molecules that are capable of catalyzing a
chemical
reaction, either intramolecularly or intermolecularly. Ribozymes are thus
catalytic nucleic acid.
It is preferred that the ribozymes catalyze intermolecular reactions. There
are a number of
different types of ribozymes that catalyze nuclease or nucleic acid polymerase
type reactions
which are based on ribozymes found in natural systems, such as hammerhead
ribozymes, (for
example, but not limited to, the following U.S. Patent Nos. 5,334,711,
5,436,330, 5,616,466,
5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288,
5,891,683,
5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig
and Sproat,
WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin
ribozymes (for example, but not limited to the following U.S. Patent Nos.
5,631,115, 5,646,031,
5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and
tetrahymena
ribozymes (for exarnple, but not limited to the following U.S. Patent Nos.
5,595,873 and
5,652,107). There are also a number of ribozymes that are not found in natural
systems, but
which have been engineered to catalyze specific reactions de novo (for
example, but not limited
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=tb'tli~ . .... eos. 5,580,967, 5,688,670, 5,807,718, and 5,910,408).
Preferred
ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA
substrates.
Ribozymes typically cleave nucleic acid substrates through recognition and
binding of the target
substrate with subsequent cleavage. This recognition is often based mostly on
canonical or non-
canonical base pair interactions. This property makes ribozymes particularly
good candidates for
target specific cleavage of nucleic acids because recognition of the target
substrate is based on
the target substrates sequence. Representative examples of how to make and use
ribozymes to
catalyze a variety of different reactions can be found in the following non-
limiting list of U.S.
Patent Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253,
5,877,021,
5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.
213. Triplex forming functional nucleic acid molecules are molecules that can
interact
with either double-stranded or single-stranded nucleic acid. When triplex
molecules interact
with a target region, a structure called a triplex is formed, in which there
are three strands of
DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-
pairing. Triplex
molecules are preferred because they can bind target regions with high
affinity and specificity. It
is preferred that the triplex forming molecules bind the target molecule with
a kd less than 10"6,
10-8, 10-10, or 10-12. Representative examples of how to make and use triplex
forming molecules
to bind a variety of different target molecules can be found in the following
non-limiting list of
U.S. Patent Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773,
5,834,185, 5,869,246,
5,874,566, and 5,962,426.
214. External guide sequences (EGSs) are molecules that bind a target nucleic
acid
molecule forming a complex, and this complex is recognized by RNAse P, which
cleaves the
target molecule. EGSs can be designed to specifically target a RNA molecule of
choice. RNAse
P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can
be recruited to
cleave virtually any RNA sequence by using an EGS that causes the target
RNA:EGS complex
to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and
Altman, Science,
1990;238:407-409).
215. Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be
utilized to
cleave desired targets within eukaryotic cells. (Yuan, et al., Proc. Natl.
Acad. Sci. U.S.A.,
1992;89:8006-8010; WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman,
EMBO
J., 1995;14:159-168, and Carrara, et al., Proc. Natl. Acad. Sci. U.S.A.,
1995;92:2627-2631).
Representative examples of how to make and use EGS molecules to facilitate
cleavage of a
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11~7zeqaal~~e~~rit be found in the following non-limiting list of U.S.
Patents:
5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.
4. Peptides
216. Also disclosed herein are compositions that are amino acid based, such as
proteins, peptides, and polypeptides. By "protein," "peptide," or
"polypeptide" is meant an
amino-acid based polymer, including variants, derivatives, and modifications,
as described
herein and as are well understood by those of skill in the art. Amino-acid
sequence
modifications typically fall into one or more of three classes:
substitutional, insertional, or
deletional variants. Insertions include amino and/or carboxyl terminal fusions
as well as
intrasequence insertions of single or multiple amino acid residues. Insertions
ordinarily will be
smaller insertions than those of amino or carboxyl terminal fusions, for
example, on the order of
one to four residues. Deletions are characterized by the removal of one or
more amino acid
residues from the protein sequence. Typically, no more than about from 2 to 6
residues are
deleted at any one site within the protein molecule. Amino acid substitutions
are typically of
single residues, but can occur at a number of different locations at once;
insertions usually will
be on the order of about from 1 to 10 amino-acid residues; and deletions will
range about from 1
to 30 residues. Substitutions, deletions, insertions or any combination
thereof can be present in
the proteins disclosed herein. The tenns "protein," "peptide," and
"polypeptide" are used
interchangeably herein.
217. Exemplary proteins that can be used in the methods disclosed herein
include
HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate
dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 protein epsilon,
Prohibitin,
Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding
protein
regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein
RAP1B, Tumor
rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein
1(AIP1), mitogen activated
protein kinase I(MAPY-T), actin or a fragment thereof, glutaraldehyde-3-
phosphate
dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear
phosphoprotein 32 family member B (ANP32B or APRIL), peroxiredoxin II, an
amyloid
precursor protein (APP), an cx secretase, a,6-secretase, a-y-secretase, an A,6
peptide, Fe65, Tip60,
SERCA, PS 1/2, nectin-1 a, and non-amyloid 0 component of senile plaque (NACP/
a-synuclein).
5. Variants
218. It is understood that there are numerous variants and alleles of the
genes used
herein for analysis of neurodegenerative diseases, such as the genes listed in
Table 4, such as
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Cy. cI~~~ .,--weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5,
Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1
antichymotrypsin, HSP 27, HSP
90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin.
These variants and
alleles can be used to detect neurodegenerative diseases (e.g., Parkinson's
and Alzheimer's) as
disclosed herein. As discussed herein there are numerous variants of the gene
products from the
genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gl, wee 1, hTR2,
CDC25b, GSK3
beta, protein kinase C alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha,
and IL-lOr, Alpha-1
antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L,
cox 1, cox 2, and
transferrin, that are known and herein contemplated. Typically these variants
will manifest
themselves in changes in the related nucleic acid or gene, and thus, variants
of the disclosed
diagnostic and prognostic genes, such as the genes listed in Table 4 (e.g.,
cyclin D1, cyclin B,
cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl
inhibitor, IL-17r,
IL-8, LIF, TNF-alpha, and IL-l Or, Alpha-1 antichymotrypsin, HSP 27, HSP 90,
crystalline,
GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin) produced, by for
example, as alleles
or strain differences, are disclosed. Protein and nucleic acid variants and
derivatives and alleles
are well understood to those of skill in the art and in can involve amino acid
sequence
modifications. It is understood that modifications in the methods or
compositions can be
accomplished to deal with, for example particular alleles.
6. Sequences
219. There are a variety of sequences related to the, for example, genes
listed in Table
4, such as cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta,
protein kinase C
alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r, Alpha-1
antichymotrypsin,
HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and
transferrin, and
Table 5, such as HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease,
Glucose-6-
phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 protein
epsilon,
Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase,
RNA-binding
protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related
protein RAP1B,
Tumor rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein
1(AIP1), mitogen
activated protein kinase I(1VIAPKI), actin or a fragment thereof,
glutaraldehyde-3-phosphate
dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear
phosphoprotein 32 family member B (ANP32B or APRIL), peroxiredoxin II, an
amyloid
precursor protein (APP), an a-secretase, a,6-secretase, ay-secretase, an A,13
peptide, Fe65, Tip60,
SERCA, PS1/2, nectin-la, or non-amyloid 0 component of senile plaque (NACP/ a-
synuclein),
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~ ~. ...1..,. i1~F ti:~a:,' rÃr It ".. iIn ,. il, ~~as ~vell as~~~~ ot4~~r ~
tei~ slosed herein that are disclosed on Genbank, and these sequences
and others are herein incorporated by reference in their entireties as well as
for individual
subsequences contained therein.
220. A variety of sequences are provided herein and these and others can be
found in
Genbank, at www.pubmed.gov. Those of skill in the art understand how to
resolve sequence
discrepancies and differences and to adjust the compositions and methods
relating to a particular
sequence to other related sequences. Primers and/or probes can be designed for
any sequence
given the information disclosed herein and known in the art.
7. Alternative embodiments
221. It is understood that post-transcriptional as well as post-translational
processes
can take place with any of the variants presented here. Disclosed are
technologies that can
measure such post-transcriptional or post translational processes such as and
not limited to post-
transcriptional silencing.
8. Methods of Validating Biomarkers
222. Biomarkers for a neurodegenerative disease can be validated in a variety
of ways.
For example, the expression of a biomarker can be assessed in one or more
subjects diagnosed
with a neurodegenerative and in one or more subjects who do not have the
neurodegenerative
disease. A biomarker whose expression varies between the two groups can be a
validated
biomarker. The larger the two groups of subjects are, the more reliable the
validation. The
expression level of a biomarker can also be assessed in a model system for
neurodegenerative
disease. For example, the level of expression can be tested in leukocyte-
containing samples of
an animal model for a neurodegenerative disease. Expression of a biomarker (or
its homolog)
can be assessed in an animal model of a neurodegenerative disease and in a
control group. A
biomarker, or homolog thereof, whose expression varies between the model
animals and the
control animals can be a validated biomarker.
9. Solid Supports
223. Disclosed herein are solid supports (including, stable and mobile forms)
wherein
at least one address is a biomarker or ligand as disclosed herein. Also
disclosed are solid
supports wherein at least one address is the sequences, portion of the
sequences, or variant of the
sequences set forth in any of the nucleic acid sequences or peptide sequences
disclosed herein or
a ligand for said sequences. Disclosed are chips where at least one address is
the sequences or
part of the sequences set forth in any of the nucleic acid sequences disclosed
herein or a nucleic
acid that hybridizes thereto. Also disclosed are chips where at least one
address is the sequences
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ghh in any of the peptide sequences disclosed herein or a ligand for
said sequence.
224. Also disclosed are chips where at least one address is a variant of the
sequences
or part of the sequences set forth in any of the nucleic acid sequences
disclosed herein or a
nucleic acid that hybridizes to said nucleic acid variant. Also disclosed are
chips where at least
one address is a variant of the sequences or portion of sequences set forth in
any of the peptide
sequences disclosed herein or a ligand that binds to said variant peptide.
225. Solid supports include stable supports like slides, chips, microarrays,
and
nanoarrays comprising any of the biomarkers or antibodies or non-antibody
ligands for the
biomarkers disclosed herein. Solid supports also include mobile supports like
beads comprising
any of the biomarkers or antibodies or non-antibody ligands for the biomarkers
disclosed herein.
10. Computer readable mediums
226. It is understood that the disclosed nucleic acids and proteins can be
represented as
a sequence consisting of the nucleotides or amino acids. There are a variety
of ways to display
these sequences, for example the nucleotide guanosine can be represented by G
or g. Likewise
the amino acid valine can be represented by Val or V. Those of skill in the
art understand how
to display and express any nucleic acid or protein sequence in any of the
variety of ways that
exist, each of which is considered herein disclosed. Specifically contemplated
herein is the
display of these sequences on computer readable mediums, such as, commercially
available
floppy disks, tapes, chips, hard drives, compact disks, and video disks, or
other computer
'readable mediums. Also disclosed are the binary code representations of the
disclosed
sequences. Those of skill in the art understand what computer readable
mediums. Thus,
computer readable mediums on which the nucleic acids or protein sequences are
recorded,
stored, or saved.
227. Disclosed are computer readable mediums comprising the sequences and
information regarding the sequences set forth herein.
11. Kits
228. Disclosed herein are kits that are drawn to reagents that can be used in
practicing
the methods disclosed herein. The kits can include any reagent or combination
of reagent
discussed herein or that would be understood to be required or beneficial in
the practice of the
disclosed methods. For example, the kits could include primers to perform the
amplification
reactions discussed in certain embodiments of the methods, as well as the
buffers and enzymes
required to use the primers as intended. In other examples, the kits could
include one or more of
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~ ~ i~,.. lõ
~Eu~h~~ . ~,,., ._ ~_... ,. ..,..ar~d~ 1 ~~,~
~t
... .blo~at~~~s~~9r= 'Yit s~'~tlf~c~~sed herein, as well as the buffers,
labels, enzyme5, secondary or
tertiary antibodies, etc. required to use the biomarkers or ligands as
intended. In a further
exarnple, disclosed is a kit for diagnosing a subject for a neurodegenerative
disease (e.g.,
Parkinson's or Alzheimer's), comprising one or more of the oligonucleotides
set forth in Table
4.
12. Diagnostic Assays
229. Also disclosed are diagnostic assays for neurodegenerative diseases. The
disclosed assays comprise contacting a sample comprising a leukocyte or a
lysate thereof with
one or more antibodies or fragments thereof for a biomarker for a
neurodegenerative disease.
Antibodies for the disclosed biomarkers can be made my methods known in the
art and as
disclosed herein.
D. Methods of making the compositions
230. The compositions disclosed herein and the compositions necessary to
perform the
disclosed methods can be made using any method known to those of skill in the
art for that
particular reagent or compound unless otherwise specifically noted.
1. Nucleic acid synthesis
231. For example, the nucleic acids, such as, the oligonucleotides to be used
as primers
can be made using standard chemical synthesis methods or can be produced using
enzymatic
methods or any other known method. Such methods can range from standard
enzymatic
digestion followed by nucleotide fragment isolation (see for example, Sambrook
et al.,
Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods,
for example, by the
cyanoethyl phosphoramidite method using a Milligen or Beckman System 1Plus DNA
synthesizer (for example, Model 8700 automated synthesizer of Milligen-
Biosearch, Burlington,
MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides
are also
described by Ikuta et al., Ann. Rev. Biochem., 1984;53:323-356,
(phosphotriester and phosphite-
triester methods), and Narang, et al., Methods Enzyinol., 1980;65:610-620,
(phosphotriester
method). Protein nucleic acid molecules can be made using known methods such
as those
described by Nielsen, et al., Bioconjug. Chem., 1994;5:3-7.
2. Peptide synthesis
232. One method of producing the disclosed proteins is to link two or more
peptides or
polypeptides together by protein chemistry techniques. For example, peptides
or polypeptides
can be chemically synthesized using currently available laboratory equipment
using either Fmoc
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or Boc (tert butyloxycarbonoyl) chemistry (Applied Biosystems,
Inc., Foster City, CA). One skilled in the art can readily appreciate that a
peptide or polypeptide
corresponding to the disclosed proteins, for example, can be synthesized by
standard chemical
reactions. For example, a peptide or polypeptide can be synthesized and not
cleaved from its
synthesis resin whereas the other fragment of a peptide or protein can be
synthesized and
subsequently cleaved from the resin, thereby exposing a terminal group which
is functionally
blocked on the other fragment. By peptide condensation reactions, these two
fragments can be
covalently joined via a peptide bond at their carboxyl and amino termini,
respectively, to form an
antibody, or fragment thereof. (Grant, Synthetic Peptides: A User Guide. WH
Freeman and Co.,
N.Y., 1992; Bodansky and Trost, Ed. Principles of Peptide Synthesis. Springer-
Verlag Inc.,
N.Y., 1993, which are herein incorporated by reference at least for material
related to peptide
synthesis).
233. Alternatively, the peptide or polypeptide is independently synthesized in
vivo as
described herein. For example, advances in recombinant glycoprotein production
methods,
which allow more cost effective production of human glycoproteins by colonies
of transgenic
rabbits (www.bioprotein.com) or by yeast strains carrying human N-
glycosylation system
enzymes (Hamilton, et al., Science, 2003;301:1244-6; Gerngross, Nature
Biotechnology,
2004;22:1409) can be used.
234. Once isolated, independent peptides or polypeptides may be linked, if
needed, to
form a peptide or fragment thereof via similar peptide condensation reactions.
For example,
enzymatic ligation of cloned or synthetic peptide segments allow relatively
short peptide
fragments to be joined to produce larger peptide fragments, polypeptides or
whole protein
domains (Abrahmsen, et al., Biochemistry, 1991;30:4151). Alternatively, native
chemical
ligation of synthetic peptides can be utilized to synthetically construct
large peptides or
polypeptides from shorter peptide fragments. This method consists of a two
step chemical
reaction (Dawson, et al., Science, 1994; 266:776-9). The first step is the
chemoselective reaction
of an unprotected synthetic peptide thioester with another unprotected peptide
segment
containing an amino-terminal Cys residue to give a thioester-linked
intermediate as the initial
covalent product. Without a change in the reaction conditions, this
intermediate undergoes
spontaneous, rapid intramolecular reaction to form a native peptide bond at
the ligation site
(Baggiolini, et al., FEBS Lett. 1992;307:97-101; Clark-Lewis, et al., J. Biol.
Chem.,
1994;269:16075; Clark-Lewis, et al., Biochemistry, 1991;30:3128; Rajarathnam,
et al.,
Biochemistry, 1994;33:6623-30).
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fWNSf~ ET 11.1.
1lterlia~'iIV 4~;=~Wprotected peptide segments are chemically linked where the
bond
formed between the peptide segments as a result of the chemical ligation is an
unnatural (non
peptide) bond (Schnolzer, et al., Science, 1992;256:221). This technique has
been used to
synthesize analogs of protein domains as well as large amounts of relatively
pure proteins with
full biological activity (deLisle Milton, et al., Techniques in Protein
Chemistry IV. Academic
Press, New York, N.Y., pp. 257-67, 1992).
3. Antibodies
236. The disclosed antibodies can be made using any procedure which produces
antibodies. For example, disclosed monoclonal antibodies can be prepared using
hybridoma
methods, such as those described by Kohler and Milstein, Nature, 1975;256:495.
In a hybridoma
method, a mouse or other appropriate host animal is typically immunized with
an immunizing
agent to elicit lymphocytes that produce or are capable of producing
antibodies that will
specifically bind to the immunizing agent. Alternatively, the lymphocytes may
be immunized in
vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein.
237. The monoclonal antibodies may also be made by recombinant DNA methods,
such as those described in U.S. Patent No. 4,816,567. DNA encoding the
disclosed monoclonal
antibodies can be readily isolated and sequenced using conventional procedures
(e.g., by using
oligonucleotide probes that are capable of binding specifically to genes
encoding the heavy and
light chains of murine antibodies). Libraries of antibodies or active antibody
fragments can also
be generated and screened using phage display techniques, e.g., as described
in U.S. Patents Nos.
5,804,440 and 6,096,441, which are incorporated by reference herein at least
for their teachings
of antibody preparation.
238. In vitro methods are also suitable for preparing monovalent antibodies.
Digestion
of antibodies to produce fragments thereof, particularly, Fab fragments, can
be accomplished
using routine techniques known in the art. For instance, digestion can be
performed using
papain. Examples of papain digestion are described in WO 94/29348 and U.S.
Patent No.
4,342,566, which are incorporated by reference herein at least for their
teachings of antibody
preparation. Papain digestion of antibodies typically produces two identical
antigen binding
fragments, called Fab fragments, each with a single antigen binding site, and
a residual Fc
fragment. Pepsin treatment yields a fragment that has two antigen combining
sites and is still
capable of cross-linking antigen.
239. The fragments, whether attached to other sequences or not, can also
include
insertions, deletions, substitutions, or other selected modifications of
particular regions or
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~f 'VW~t~'~L:I~rovided the activity of the antibody or antibody fragment is
not
significantly altered or impaired compared to the non-modified antibody or
antibody fragment.
These modifications can provide for some additional property, such as to
remove/add amino
acids capable of disulfide bonding, to increase its bio-longevity, to alter
its secretory
characteristics, etc. In any case, the antibody or antibody fragment must
possess a bioactive
property, such as specific binding to its cognate antigen. Functional or
active regions of the
antibody or antibody fragment may be identified by mutagenesis of a specific
region of the
protein, followed by expression and testing of the expressed polypeptide. Such
methods are
readily apparent to a skilled practitioner in the art and can include site-
specific mutagenesis of
the nucleic acid encoding the antibody or antibody fragment. (Zoller, Curr.
Opin. Biotechnol.,
1992;3:348-354, which is incorporated by reference herein at least for its
teachings of antibody
preparation).
240. The disclosed human antibodies can be prepared using any technique.
Examples
of techniques for human monoclonal antibody production include those described
by Cole, et al.
(Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by
Boemer, et al.
(J. Immunol., 1991;147(1):86 95), which are incorporated by reference herein
at least for their
teachings of antibody preparation. Human antibodies (and fragments thereof)
can also be
produced using phage display libraries (Hoogenboom, et al., J. Mol. Biol.,
1991;227:381; Marks,
et al., J. Mol. Biol. 1991;222:581, which are incorporated by reference herein
at least for their
teachings of antibody preparation). The disclosed human antibodies can also be
obtained from
transgenic animals. For example, transgenic, mutant mice that are capable of
producing a full
repertoire of human antibodies, in response to immunization, have been
described (see, e.g.,
Jakobovits, et al., Proc. Natl. Acad. Sci. U.S.A., 1993;90:2551-5; Jakobovits,
et al., Nature,
1993;362:255-8; Bruggermann, et al., Year in Immunol., 1993;7:33, which are
incorporated by
reference herein at least for their teachings of antibody preparation).
241. Methods for huma.nizing non-human antibodies are well known in the art.
For
example, humanized antibodies can be generated according to the methods of
Winter and co-
workers (Jones, et al., Nature, 1986;321:522-5, Riechmann, et al., Nature,
1988;332:323-7,
Verhoeyen et al., Science 1988;239:1534-6), by substituting rodent CDRs or CDR
sequences for
the corresponding sequences of a human antibody. Methods that can be used to
produce
humanized antibodies are also described in U.S. Patent Nos. 4,816,567,
565,332, 5,721,367,
5,837,243, 5,939,598, 6,130,364, and 6,180,377, wliich are incorporated by
reference herein at
least for their teachings of antibody preparation.
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..~.~ ~~o16Q!"s for making the compositions
242. Disclosed are processes for making the compositions as well as making the
intermediates leading to the compositions. For example, disclosed are nucleic
acids and proteins
in SEQ IDNOs:l-257. There are a variety of methods that can be used for making
these
compositions, such as synthetic chemical methods and standard molecular
biology methods. It is
understood that the methods of making these and the other disclosed
compositions are
specifically disclosed.
243. Disclosed are nucleic acid molecules produced by the process comprising
linking
in an operative way a nucleic acid comprising the sequence set forth in Table
4, such as cyclin
D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, Cl
inhibitor, IL-17r, IL-g, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin,
HSP 27, HSP 90,
crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and
genes of proteins
listed in Tables 5 and 6, and a sequence controlling the expression of the
nucleic acid.
244. Also disclosed are nucleic acid molecules produced by the process
comprising
linking in an operative way a nucleic acid molecule comprising a sequence
having 80% identity
to a sequence set forth in Table 4, and a sequence controlling the expression
of the nucleic acid.
245. Disclosed are nucleic acid molecules produced by the process comprising
linking
in an operative way a nucleic acid molecule comprising a sequence that
hybridizes under
stringent hybridization conditions to a sequence set forth in Table 4 and a
sequence controlling
the expression of the nucleic acid.
246. Disclosed are nucleic acid molecules produced by the process comprising
linking
in an operative way a nucleic acid molecule comprising a sequence encoding a
protein such as
HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate
dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin,
Phospoglycerate
mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein
regulatory subunit,
Chain A thioredoxin peroxidase B, RAS-related protein RAP 1B, Tumor rejection
antigen,
Haptoglobin, Fibrin beta, actin-interacting protein 1(AIP1), mitogen activated
protein kinase I
(MAPKI), actin or a fragment thereof, glutaraldehyde-3-phosphate dehydrogenase
(GAPDH),
transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32
family member B
(ANP32B or APRIL), peroxiredoxin II, an arnyloid precursor protein (APP), a-
secretase, ,6-
secretase, y-secretase, Ao peptide, Fe65, Tip60, SERCA, PS 1/2, nectin-la, and
non-amyloid
component of senile plaque (NACP/ a-synuclein), or proteins set forth in
Tables 5 or 6, or
proteins of genes and a sequence controlling an expression of the nucleic acid
molecule.
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+ E
'"~1;: id ~Ijis~l~ 'i ~'~ii~~Cleic acid molecules produced by the process
comprising linking
in an operative way a nucleic acid molecule comprising a sequence encoding a
protein having
80% identity to a protein such as HSP60, Dihydrolipoamide dehydrogenase, ER-60
protease,
Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3
epsilon,
Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase,
RNA-binding
protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related
protein RAP1B,
Tumor rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein
1(AIPl), mitogen
activated protein kinase I(MAPKI), actin or a fragment thereof, glutaraldehyde-
3-phosphate
dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear
phosphoprotein 32 family member B (ANP32B or APRIL), peroxiredoxin II, an
amyloid
precursor protein (APP), a-secretase, (3-secretase, 7-secretase, A(3 peptide,
Fe65, Tip60, SERCA,
PS1/2, nectin-la, and non-amyloid 0 component of senile plaque (NACP/ a-
synuclein), or a
protein set forth in Tables 5 or 6, and a sequence controlling an expression
of the nucleic acid
molecule.
248. Disclosed are nucleic acids produced by the process comprising linking in
an
operative way a nucleic acid molecule comprising a sequence encoding a peptide
having 80%
identity to a protein set forth in Tables 5 or 6, wherein any change from the
Table 5 or 6 are
conservative changes and a sequence controlling an expression of the nucleic
acid molecule.
249. Disclosed are cells produced by the process of transforming the cell with
any of
the disclosed nucleic acids. Disclosed are cells produced by the process of
transforming the cell
with any of the non-naturally occurring disclosed nucleic acids.
250. Disclosed are any of the disclosed peptides produced by the process of
expressing
any of the disclosed nucleic acids. Disclosed are any of the non-naturally
occurring disclosed
peptides produced by the process of expressing any of the disclosed nucleic
acids. Disclosed are
any of the disclosed peptides produced by the process of expressing any of the
non-naturally
disclosed nucleic acids.
251. Disclosed are animals produced by the process of transfecting a cell
within the
animal with any of the nucleic acid molecules disclosed herein. Disclosed are
animals produced
by the process of transfecting a cell within the animal any of the nucleic
acid molecules
disclosed herein, wherein the animal is a mammal. Also disclosed are animals
produced by the
process of transfecting a cell within the animal any of the nucleic acid
molecules disclosed
herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.
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[I::JAd df ~~~bf~6't~JE animals produced by the process of adding to the
animal any of
the cells disclosed herein.
E. Methods of using the compositions
1. Methods of using the compositions as research tools
253. The disclosed compositions can be used in a variety of ways as research
tools.
For example, the disclosed compositions, such as SEQ ID NOs: 1-257 can be used
to study the
effects of various therapies on a neurodegenerative disease.
254. The compositions can be used for example as targets in conibinatorial
chemistry
protocols or other screening protocols to isolate molecules that possess
desired functional
properties related to a neurodegenerative disease (e.g., Alzheimer's and
Parkinson's disease).
255. The disclosed compositions can also be used diagnostic tools related to
neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
256. The disclosed compositions can be used as discussed herein as either
reagents in
micro arrays or as reagents to probe or analyze existing microarrays. The
disclosed
compositions can be used in any known method for isolating or identifying
single nucleotide
polymorphisms. The compositions can also be used in any known method of
screening assays,
related to chip/micro arrays. The compositions can also be used in any known
way of using the
computer readable embodiments of the disclosed compositions, for example, to
study relatedness
or to perform molecular modeling analysis related to the disclosed
compositions.
VII. EXAMPLES
257. The following examples are put forth so as to provide those of ordinary
skill in
the art with a complete disclosure and description of how the compounds,
compositions, articles,
devices and/or methods claimed herein are made and evaluated, and are intended
to be purely
exemplary and are not intended to limit the disclosure. Efforts have been made
to ensure
accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some
errors and
deviations should be accounted for. Unless indicated otherwise, parts are
parts by weight,
temperature is in C or is at ambient temperature, and pressure is at or near
atmospheric.
A. Example 1 Molecular distinction of Alzheimer's disease from analysis of
leukocyte RNA
1. Methods:
a) Patient Recruitment
258. Patients were recruited through the Geriatric Neurology and Psychiatry
Clinic at
Monroe Community Hospital. Following entrance into this study with informed
consent,
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1~nf~rrn~ti~oi a'bat~'c' 1o~ cical dementia tests was gathered from patients.
Blood samples
were stored at 4 C until processed for RNA isolation (less than 12 hours).
259. Data presented here were obtained from three independent samples from
three
different groups of people. RNA from each of these samples was extracted;
hybridized and
analyzed at different times. Sample 1 consisted of 8 AD and 7 control cases.
Sample 2
consisted of 8 AD and 8 control cases. Sample 3 consisted of 5 AD, 4 control
and 2 PD cases.
Sample characteristics are summarized in Tables 1-3.
2. Subjects
260. AD subjects included in the study were diagnosed with probable or
possible AD
on the basis of NINCDS (McKhann G, et al., Neurology, 1984;34(7):939-944) and
DSM IV
criteria for AD. Examination by a neurologist was performed to confirm
diagnosis and to
measure disease severity. Disease severity was assessed using the Mini-Mental
Status
Examination (MMSE) (Folstein MF, et al., J. Psychiatric Res., 1975;12(3):189-
198), the Clinical
Dementia Rating scale (CDR) (Hughes CP, et al., British J. Psychiatry,
1982;140:566-572), and
the Blessed Dementia Rating Scale (BDRS) (Blessed G, et al., British J.
Psychiatry,
1968;114(512):797-811). Control subjects included in the study scored above 27
on the MMSE,
wliile AD cases scored below 22. The average CDR of AD cases ranged from 1.2
to 1.5. Any
subject with a history of bleeding diathesis or coagulopathy was excluded.
3. Isolation of RNA from whole blood samples
261. To extract polyA-RNA from leukocytes, an inRNA isolation kit for blood
was
utilized (Roche). In brief, erythrocytes were selectively lysed and leukocytes
were collected by
centrifugation. The leukocytes were then lysed and the total nucleic acids
were collected by non-
specific adsorption to magnetic glass beads and magnetic separation. Following
a series of
washes and elution of the nucleic acids from the magnetic glass beads, the
mRNA was
specifically captured by the use of biotin-labeled oligo(dT) and streptavidin-
coated magnetic
particles. After removal of other nucleic acids (DNA, rRNA, tRNA) by washing,
mRNA
samples were collected and stored at minus 80 C until later use. The
concentration and purity of
samples were checked by OD260i280. It is understood that any RNA isolation
procedure could be
used.
4. Construction and hybridization of cDNA arrays
262. The cDNA clones used are listed in Table 4. The dbEST database of the
National
Center for Biotechnology Information was searched for relevant 3'-cDNA clones
and the clones
were either purchased from distributors or gifts from various laboratories.
The cDNA clones
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'' ~~=us~d i~..~~~s~4~~t~t~~'vi~~~~~~~~~i~i~~is gifts from many investigators:
CREB, tuberin, nestin, cyclin Dl
and GAD from Jim Eberwine; BDNF, bcl-2, bcl-xs, bcl-xl, calbindin, and SOD-1
from Denise
Figlewicz; hTR2 from Chawnshang Chang, GAP-43, APP, and PS 1 from Rachel Neve;
P19 from
Bert Vogelstein; synaptotagmin I from T. Sudhof; GTH, pGTH4, and pHMGST from
Dr. Davi;
ubiquitin from Dr. Roharaker; cdc2 from Inez Vincent; and SF2flag, Pht6, and
tra2-C2 from
Stefan Stamm. The rest of the cDNA clones were dbEST clones from distributors
of I.M.A.G.E.
Consortium cDNA clones.
263. All of the cDNA clones were sequenced to confirm their identity. One
microgram
of each linearized cDNA was denatured in 0.2 N NaOH/0.2 mM EDTA at 37 C for 30
minutes.
The sample volume was neutralized with 0.3 M NaOAc, pH 4.5. The cDNAs were
immediately
printed as previously described (Chow N, et al., Proc. Natl. Acad. Sci. U.S.A.
1998;95:9620-
9625) on a nylon membrane (Micron Separations) using a 96 pin replicator
(Nalge Nunc) with
each cDNA spotted four times. The membranes were prehybridized at 42 C in
hybridization
solution (50% fonnamide/5X SSPE/5X Denhardt's solution/0. 1% SDS/ 10% dextran
sulfate/50,
g/ml denatured salmon sperm DNA/100 g/ml tRNA) for 3 hours before adding the
RNA
probes. After oveniight incubation at 42 C, blots were washed in 2X SSC/0.1%
SDS at 55 C
for 1 hour, 2X SSC/0.1% SDS/10 g/ml RNAse A at 37 C for 1 hour, and 2X
SSC/0.1% SDS at
37 C for 1 hour. Membranes were then exposed to a storage phosphor screen.
5. Data acquisition and analysis
264. Hybridization intensity of each dot was detected by laser densitometric
scanning
(Phosphoimager, Molecular Dynamics). Values (counts) for each spot obtained by
phosphoimager analysis were corrected using local background. The amount of
cDNA deposited
on each spot in the array was quantified by stripping and reprobing the
membrane with an
oligonucleotide specific for the T7 promoter present in all vectors. These
data provided a
correction for potential spot-to-spot differences in deposition of cDNA on the
membrane. To
ensure accurate comparisons across arrays, signals were normalized using the
average of all
markers (cDNAs) in an array for each RNA sample. The resulting standardized
data were
analyzed by canonical analysis. This analysis determines the variables
(messages) that best
distinguish groups and assigns weights to each variable. As is true for
multivariate analyses, the
number of variables must not exceed the number of cases (Kshirsager AM,
Multivariate
Analysis, Dekker M, New York, N.Y., 1972). The first canonical variable
provides the best
distinction between groups. The second canonical variable operates on the
residual variance that
remains unaccounted for by canonical variable 1. Additional iterations are
possible with
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If'~'''~iiri~i~isl~i~'=~~~~~-'"T-N-iffi~~ihalal significance of the separation
between AD and control cases
resulting from canonical analysis was assessed by the Wald-Wolfowitz runs test
(Siegel S,
Nonparametric Statistics, McGraw-Hill, New York, N.Y., 1-312, 1956).
B. Results
1. Message classes that distinguish AD from control blood samples.
265. Since valid results from multivariate canonical analyses require that the
number
of variables (messages in this case) be less than the number of cases
(subjects) used (Kshirsager
AM, Multivariate Analysis, Dekker M, New York, N.Y., 1972) messages were
formed into
subsets of 7 or 8 messages out of the total of 64 messages studied. Only two
out of the 5 subsets
of messages examined across a113 samples produced consistent separation of AD
from control
cases. These two subsets were messages related to the cell cycle and messages
related to
inflammatory responses. Figures 2A, 2B, and 2C plot canonical variable 1 vs.
canonical variable
2 for those messages related to the cell cycle. Figures 2A, 2B, and 2C plot
canonical variable 1
vs. canonical variable 2 for those messages related to the inflammatory
responses.
266. These 6 plots demonstrate that generally distinction of disease
categories can be
achieved with little overlap between groups. Note that the categorization of
41 (43 including 2
Parkinson's disease cases) cases on the basis of leukocyte expression of cell
cycle messages
agreed with the clinical classification in 38/41 (40/43 counting PD) of the
cases with 3 control
cases in the AD space and no AD cases in the control space. Genes related to
the inflammatory
system placed no control cases in the AD space and one AD case in the control
space. The
inflammatory genes correctly distinguished the 2 PD cases from both control
and AD spaces.
267. It should be noted that a very small number of individual messages among
those
we sampled yielded statistically significant (or close to significant)
differences between AD and
control cases. These were alpha-1 antichymotrypsin, crystallin and
cyclooxygenase II.
However, they were not sufficient in themselves to distinguish AD from control
without
significant overlap.
268. Similar plots of canonical variables for other sets of messages were not
as
successful in distinguishing AD from control leukocyte message profiles (data
not shown). This
provides evidence that the positive results illustrated in Figures 1 and 2 are
specific to the two
gene sets presented in Figures 1 and 2.
269. There are two major conclusions regarding the data presented here: (1)
expression profiles of multiple genes are effective at distinguishing mild AD
(average CDR 1.2-
1.5) from non-demented control cases and, (2) the gene classes we describe as
distinguishing AD
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1~5Walfs&nples are similar to gene classes whose expression has been
shown to be altered in the brain in AD. Blood cells can be used to conduct
basic research on the
molecular mechanisms of AD. Data resulting from such studies can lead to
design of therapeutic
molecules. Additionally blood cells can used to monitor therapeutic efficacy
in clinical trials of
therapeutic agents as well as the efficacy of treatment of individual
patients.
2. Expression profiles of multiple genes are effective at distinguishing
Alzheimer's disease
270. The most comprehensive clinical diagnosis of AD is a complex, expensive
process involving many assessments (McKhann G, et al., Clinical diagnosis of
Alzheimer's
disease: report of the NINCDS-ADRDA Work Group under the auspices of
Department of
Health and Human Services Task Force on Alzheimer's Disease. Neurology,
1984;34(7):939-
944). Even in expert hands the diagnosis is considered provisional, subject to
neuropathological
confirmation at autopsy. In AD centers, the clinical diagnosis reaches an
autopsy confirmation
rate of 85-90% (Gearing M, et al., Neurology, 1995;45(3 Pt 1):461-466). In
less expert hands,
the rate of autopsy confirmed diagnosis is lower. The expense and difficulties
of diagnosing AD
have led to a search for an easily sampled and successful biomarker of
disease. Some of the
biomarkers that have been described include those that are invasive (e.g.,
spinal tap (Davidson P,
et al., J. Neural Transmission-General Section, 1997;104(6-7):711-720)) or
require expensive
equipment and expertise (Killiany RJ, et al., Neurology, 2002;58(8):1188-
1196). Although these
procedures are extremely useful in investigative studies, they do not offer
promise for routine,
large scale diagnostic use. Other tests have been described that draw on
easily obtained
peripheral samples from, for example, blood cells (Nagy Z, et al.,
Neuroscience Letters,
2002;317(2):81-84), (Padovani A, et al., Archives of Neurology, 2002;59:71-
75), skin (Ikeda K,
et al., Dementia & Geriatric Cognitive Disorders, 2000; 11(5):245-25 0) and
urine (Pratico D, et
al., Archives of Neurology, 2002;59:972-976). Additional studies have
described tests for AD
that utilize responses to pharmacological intervention (Scinto LF, et al.,
Neurology,
1999;52(3):673-674). Many of these studies directed at distinguishing AD from
control samples
on the basis of peripheral tissues have been successful at yielding
statistically significant
differences between AD and control samples. However, the clinical utility of
the tests described
in these studies has been limited by significant overlap between AD and
control samples. The
disclosed data indicate that this overlap may be diminished by making use of
multiple variables.
271. Expression profiling in brain has shown that multivariate analysis of the
expression of multiple messages can distinguish AD from control brain (Chow N,
et al., Proc.
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31 ]4;' 5:9620-9625). More recently, multivariate analysis of expression
of multiple genes has been successful in distinguishing malignant from non-
malignant samples
of tissues other than brain (Welsh JB, et al., Proc. Nat. Acad. Sci. U.S.A.,
2001;98(3):1176-
1181). In the data presented herein, applying analysis to quantitative data on
expression levels of
multiple message species extracted from peripheral blood leukocytes has
yielded promising
separation of AD from control samples. In addition, two samples from PD cases
have been
distinguished from both AD and control samples, suggesting that the
distinction being made
between AD and control samples is distinguishing properties other than those
of general
neurodegeneration.
272. The data do however "misclassify" a very few number of cases. The nature
of the
misclassification is that 3 out of 19 cases that were clinically defmed as non-
demented yielded
values that fell among the AD cases. The data herein is consistent with these
cases being
"preclinical" cases in which there exists some degree of AD pathology. Such a
suggestion is
consistent with the Braaks' data demonstrating that brains of as many as 20%
of persons in their
20s exhibited AD pathology. (Braak H, et al., Neurobiology of Aging,
1997;18(4):351-357).
These data provide a compelling argument for the existence of an extended
preclinical stage of
AD during which there is frank pathology.
273. In addition to these control cases that gave values consistent with AD,
there was
one clinically defined AD case (out of 21) that fell among the control cases.
A search of the
existing clinical and pathological data for this case has not revealed
distinguishing characteristics
that would rationally separate this from the other AD cases in our sample.
274. Other sets of genes selected either randomly or on the basis of other
properties
(e.g., growth factors) do not distinguish AD from control samples to the
extent that is possible
with the gene sets related to the cell cycle and to the inflammatory system
(data not shown).
This suggests that the disclosed finding of segregation on the basis of
selected gene sets is not an
artifact of the methods of analysis we employed.
275. The data presented here suggest that the gene sets disclosed herein have
can be
clinically useful and, as discussed herein, also suggest a relationslZip
between molecular events
in brain and in peripheral blood cells.
3. Gene classes that distinguish AD brain also distinguish AD blood samples.
276. It is notable that the classes of gene products shown herein to
distinguish AD
from control peripheral blood leukocytes are also among those classes of gene
products that have
altered expression in AD brain. Certainly, selected neuron specific gene
products that are
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9~fE~ct6cfL Jn related to the synapse (Callahan LM, et al., J.
Neuropathology & Experimental Neurology, 2002;61(5):384-395; Yao P, et al.,
Neurobiology of
Disease, 2003;12:97-109), are not to be expected to play a role in leukocytes.
However, there
are gene products that are not neuron specific that have also been shown to
have altered
expression in AD brain. Of particular interest here are molecules related to
the cell cycle
(Arendt T, Neurobiology of Aging, 2000;21(6):783-796; Vincent I, et al., J.
Neurosci.,
1997;17:3588-3598; Nagy Z, et al., Neuroscience Letters, 2002;317(2):81-84;
Harris PL, et al.,
Neurobiology of Aging, 2000;21(6):837-841; Wu Q, et al., Neurobiology of
Aging,
2000;21(6):797-806; Zhu, et al., Neurobiology of Aging, 2000;21(6):837-841)
and molecules
related to inflammatory systems (Akiyama H, et al., Neurobiology of Aging,
2000;21(3):383-421
and Bamberger ME, and Landreth GE, Microscopy Research & Technique,
2001;54(2):59-70).
In fact, a recent suminary of the inflammatory system in AD brain states "A
virtual textbook of
inflammatory mediators has been observed in the Alzheimer's disease brain over
the last 15
years" (Akiyama H, et al., Neurobiology of Aging, 2000;21(3):383-421). Indeed,
studies of AD
tissues other than brain have demonstrated altered expression of cell cycle
(Nagy Z, et al.,
Neuroscience Letters, 2002;317(2):81-84; Stieler JT, et al., NeuroReport,
2001;12(18):3969-
3972) as well as inflammatory systein (Scali C, et al., Neurobiology of Aging,
2002;23(4):523-
530; De Luigi A, et al., Mechanisms of Ageing & Development, 2001;122(16):1985-
1995;
Kusdra L., et al., Immunobiology, 2000;202(1):26-33; Lombardi VR, et al., J.
Neuroimmunology, 1999;97(1-2):163-171; Remarque EJ, et al., Experimental
Gerontology,
2001;36:171-176) genes in several tissues, including blood cells (Nagy Z, et
al.,. Neuroscience
Letters, 2002;317(2):81-84; Scali C, et al., Neurobiology of Aging,
2002;23(4):523-530) and
epithelial cells (Schmitz A, et al., Histochemistry & Cell Biology,
2002;117(2):171-180).
277. The above studies do not prove that selected common changes in gene
expression
in both peripheral and brain cells may be consistent with the concept of a
common, systemic
disease mechanism or common links between blood and brain. (Webster S, et al.,
Biochemical
& Biophysical Research Communications, 1995;217:869-875; Kimberly WT, et al.,
J. Biol.
Chem., 2001;276(43):40288-40292; Cao X, et al., Science, 2001;293(5527):115-
120).
278. The data suggest that disordered expression of cell cycle and
inflammatory genes
may play a central role in the pathophysiology of AD in many cell types, both
within and outside
the nervous system. Additional neuropathophysiological aspects of AD may be
consequences of
these pivotal events, including tau phosphorylation by kinases also related to
the cell cycle
(Busciglio J, et al., Neuron, 1995;14(4):879-888; Ferreira A, et al.,
Molecular & Cellular
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"I~Tei~rosc~~~ri~#es;~ 7t;0~~'1F~' -J~4; Greenberg SM, et al., Proc. Nat.
Acad. Sci. U.S.A.,
1994;91(15):7104-7108) and consequent cytoskeletal disruption, a variant of
programmed cell
death as a response to the inability of post mitotic neurons to successfully
complete the cell cycle
as well as cell death as a response to inflammatory challenge.
279. RNA profiling of the expression of multiple genes by peripheral
leukocytes
followed by canonical discriminant analysis can be used both as a biological
tool for the analysis
of molecular alterations in disease, as well as a tool for differentiation
between Alzheimer's and
control patients. These methods also suggest a potential to differentiate
numerous other
diseases. It is not the significant difference between individual genes
(although a few genes have
significant correlations) that provides a clear discrimination between
patients with a specific
disease and others, but rather an analysis based on weighted sums of many
genes.
280. The finding that gene expression by peripheral blood leukocytes is
affected in AD
reinforces the concept of AD as a systemic disease. Furthermore, that the cell
cycle and
inflammatory gene classes affected in peripheral leukocytes are similar to
gene classes affected
in brain in AD is a commonality that suggests and is consistent with parallel
molecular
mechanisms of AD in brain and blood cells, perhaps initiated by APP peptides.
C. Example 2 Inflammatory, cell cycle, and stress transcripts and molecular
distinction of Alzheimer's disease from peripheral blood leukocytes
1. Methods:
a) Patient Recruitment
281. Patients were recruited through the Geriatric Neurology and Psychiatry
Clinic at
Monroe Community Hospital. Following entrance into this study with inÃormed
consent,
information on a battery of clinical dementia tests was gathered from
patients.
282. Data presented here were obtained from three independent sets of samples
from
three different groups of people. Leukocyte RNA from each of these samples was
extracted,
hybridized to custom cDNA arrays and analyzed at different times. Sample 1
consisted of 8
early AD and 7 control cases. Sample 2 consisted of 8 new early AD and 8 new
control cases.
Sample 3 consisted of 5 new early AD, 4 new control and 2 (new) Parkinson's
disease (without
dementia) cases. A total of 21 early AD, 19 control, and 2 PD cases were
investigated.
2. Subjects
283. AD subjects included in the study were diagnosed with probable or
possible AD
on the basis of NINCDS (McKhann G, et al., Neurology, 1984;34(7):939-944) and
DSM IV
criteria for AD. Examination by a neurologist was performed to confirm
diagnosis and to
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~I~D7 ~a~ ~ , ,t,.. ..
r~ea~urE ~c~~s~~fs~ ~e veti~ ~t e severity was assessed using the Mini-Mental
Status
Examination (MMSE; Folstein et al., J. Psychiatric Res., 1975;12(3):189-198),
the Clinical
Dementia Rating scale (CDR; Hughes et al., British J. Psychiatry, 1982;
140:566-572), and the
Blessed Dementia Rating Scale (BDRS; Blessed et al., British J. Psychiatry,
1968;114(512):797-
811). Control subjects included in the study scored above 27 on the MMSE,
while AD cases
scored below 22. The mean CDR of AD cases in each of the three samples ranged
from 1.2 to
1.5. Since these were not autopsy confirmed cases, the assignment of each case
to a specified
disease category relies on the accuracy of the clinical diagnosis. Any subject
with a history of
bleeding diathesis or coagulopathy was excluded. Blood samples were drawn by a
phlebotomist
and stored at 4 C until processed for RNA isolation (less than 8 hours).
3. Isolations of RNA from whole blood samples
284. To extract polyA-RNA from leukocytes, an inRNA isolation kit for blood
was
utilized (Roche). In brief, erythrocytes were selectively lysed and leukocytes
were collected by
centrifugation. The leukocytes were then lysed and the total nucleic acids
were collected by non-
specific adsorption to magnetic glass beads and magnetic separation. Following
a series of
washes and elution of the nucleic acids from the magnetic glass beads, the
inRNA was captured
by the use of biotin-labeled oligo(dT) and streptavidin-coated magnetic
particles. After removal
of other nucleic acids (DNA, rRNA, tRNA) by washing, mRNA samples were
collected and
stored at minus 80 degree Celsius until later use. The concentration and
purity of samples were
checked by OD260i280= It is understood that any RNA isolation procedure could
be used.
4. Construction of cDNA arrays
285. The cDNA clones represented in the arrays were selected based on previous
microarray studies (e.g., Chow, et al., Proc. Natl. Acad. Sci. USA.
1998;95:9620-9625) and a
subset of those of interest in the field of AD research. The dbEST database of
the National
Center for Biotechnology Information was searched for relevant 3' cDNA clones.
The cDNA
clones used in this study were gifts from many investigators or were from
distributors of
I.M.A.G.E. Consortium cDNA clones. 172 transcripts were represented in the
arrays.
286. All of the cDNA clones were sequenced in house to confirm their identity.
One
microgram of each linearized cDNA was denatured in 0.2 N NaOH/0.2 mM EDTA at
37 C for
30 minutes. The sample volume was neutralized with 0.3 M NaOAc, pH 4.5. The
cDNAs were
immediately printed as previously described (Chow, et al., Proc. Natl. Acad.
Sci. USA.
1998;95:9620-9625) on a nylon membrane (Micron Separations) using a 96 pin
replicator (Nalge
Nunc) with each cDNA spotted four times. The membranes were prehybridized at
42 C in
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Ml- ~u2cNri10641;='forinamide/5X SSPE/5X Denhardt's solution/0. 1% SDS/ 10%
~' '' gtlo
dextran sulfate/50, g/ml denatured salmon sperm DNA/100 g/ml tRNA) for 3
hours before
adding the RNA probes. After overnight incubation at 42 C, blots were washed
in 2X
SSC/0.1% SDS at 55 C for 1 hour, 2X SSC/0.1% SDS/10 g/ml RNAse A at 37 C for
1 hour,
and 2X SSC/0.1% SDS at 37 C for 1 hour. Membranes were then exposed to a
storage
phosphor screen.
5. Data acquisition and analysis
287. Hybridization intensity of each dot was detected by laser densitometric
scanning
(Phosphoimager, Molecular Dynamics). Values (counts) for each spot obtained by
phosphoimager analysis were corrected using local background. The amount of
cDNA deposited
on each spot in the array was quantified by stripping and reprobing the
membrane with an
oligonucleotide specific for the T7 promoter present in all vectors. These
data provided a
correction for potential spot-to-spot differences in deposition of cDNA on the
membrane. To
ensure accurate comparisons across arrays, signals were normalized using the
average of all
markers (cDNAs) in an array for each RNA sample. The resulting standardized
data were
analyzed by two univariate tests and one multivariate test. The univariate
tests were the t-test
and the N-test. The latter N-test is a newly devised, essentially non-
parametric test for multiple
testing inference. (Technical Report 04/01 at
http://www.urmc.rochester.edu/smd/biostat/people/techreports.html).
Multivariate statistical
testing relied on canonical discriminant analysis. The multivariant analysis
was performed on
SAS/STATTM software from SAS Institute, Inc. (Cary, NC). This analysis
determines the
variables (messages) that best distinguish groups and assigns weights to each
variable. The first
canonical variable provides the best distinction between groups. The second
canonical variable
operates on the residual variance that remains unaccounted for by canonical
variable 1.
Additional iterations are possible with diminishing effect.
D. Results
288. Since valid results from multivariate canonical analyses requires that
the number
of variables analyzed (messages in this case) be less than the number of cases
(subjects) used
(Kshirsager, AM. Multivariate Analysis. New York: Dekker, M., 1972) messages
were formed
into 5 subsets of 7 or 8 messages out of the total of 172 transcripts studied.
Three of the subsets
formed were based on hypotheses that specified systems known to be affected in
AD brain
would also be affected in AD leukocytes. These three sets of transcripts were
those related to
either cell stress (especially oxidative stress), inflammatory system, or cell
cycle/apoptosis. Two
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~~==~t~Et ~~~I~{~E ~~~~e b ~~t~riie composed of transcripts that approached
significance in the t-test
and one composed of transcripts chosen at random as a control for spurious
results from the
analytical methods used. These transcript sets were then utilized to analyze
the data from three
independent samples of early AD and control subjects. The statistical
significance of the
separation between AD and control cases resulting from canonical analysis was
assessed by the
Wald-Wolfowitz runs test (Siegel S, Nonparametric Statistics, McGraw-Hill, New
York, N.Y.
1956:1-312).
289. Univariate analysis by t-test revealed three transcripts to be
statistically
significantly different between AD and control samples. These were: alpha-1
antichymotrypsin,
crystallin and cyclooxygenase II. Univariate analysis by N-test analysis
revealed one transcript
to approach statistically significant difference between AD and control. This
was ERCC6. None
of the transcripts revealed by either of these univariate statistics were
sufficient in themselves to
distinguish early AD from control without significant overlap.
290. Multivariate canonical discriminant analysis was, however, able to
deliver
excellent distinction between peripheral blood leukocytes of early Alzheimer's
disease and
control subjects. The first study established that canonical discriminant
analysis of the sets of
transcripts related to cell stress, inflammatory system and cell
cycle/apoptosis was able to
distinguish AD from control samples with minimal overlap. Each point in each
plot represents a
composite "score" [canonical variable 1] for one individual. This score was
determined by
multivariate canonical discriminant analysis of the 8-10 transcripts selected
to represent cell
stress (e.g., Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH,
ferritin H, ferritin
L, cox 1, cox 2, and transferrin) (see Fig. 3A), inflannnation (e.g., C5, Cl
inhibitor, IL-17r, IL-8,
LIF, TNF-alpha, and IL-10r) (see Fig. 3B), or cell cycle (e.g., cyclin Dl,
cyclin B, cyclin Gl,
weel, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha) (see Fig. 3C). Two
transcript
sets, chosen on the basis of t-tests of significance or selected randomly, did
not cleanly
distinguish early AD fiom control cases (data not shown).
291. A repetition with new, non-overlapping sets of subjects confirmed that
transcripts
sets related to cell stress, inflammatory system and cell cycle/apoptosis were
again able to
distinguish early AD from control cases (Figure 4). Subsequently, a new,
additional, set of
subjects was recruited, leukocytes collected, mRNA extracted and hybridized to
new arrays.
This replicate again confirmed that transcript sets related to cell stress,
inflammatory system and
cell cycle/apoptosis were able to distinguish early AD from control cases
(Figure 5).
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MM ea~~ A&' -study the other two transcript sets, chosen on the basis of t-
test
significance and selected randomly did not cleanly distinguish early AD from
control cases.
These 9 plots of Figures 3-5 demonstrate that generally excellent distinction
of disease
categories can be achieved with little overlap between groups (The
differentiation of early AD
from control is significant beyond the p<0.01 level (Wald-Wolfowitz runs
test). Note that the
analysis distinguished 2 Parkinson's disease cases from early AD and control
cases.
1. Gene classes that distinguish AD in brain also distinguish AD blood
samples
293. Like Example 1, the classes of gene products shown here to distinguish AD
from
control peripheral blood leukocytes are also among those classes of gene
products that are
known to have altered expression in AD brain. The three classes of transcripts
investigated do
not appear to have performed equally well. Although transcripts related to
cell stress and
inflammatory system were quite consistent in their distinction of disease
state, cell cycle
transcripts were less consistent. Nevertheless, the data suggest that
multivariate analysis of
expression profiles of peripheral cells can be used in the study of molecular
phenomena of
Alzheim.er's disease.
294. Selected neuropathological/neurobiological aspects of AD can be
consequences
of altered expression of cell cycle, cell stress, and inflamination events,
all of which are pivotal
in the life of a cell. As one example, activation of cell cycle kinases by A
beta leads to tau
ph.osphorylation (Busciglio et al., Neuron. 1995;14(4):879-888; Greenberg et
al., Proc. Nat.
Acad. Sci. U.S.A., 1994;91(15):7104-7108), which produces the cytoskeletal
disassembly
required by cell division. Cytoskeleton disassembly then would have
consequences for
maintenance of neuronal processes, transport and the support of synapses.
295. The data presented here suggest that the gene classes used can be become
clinically useful in a variety of ways including diagnosis (perhaps even
early, preclinical or
antecedent, diagnosis) and monitoring therapeutic efficacy. Furthermore, the
evidence that
similar classes of gene products are affected in both brain and blood allows
that peripheral blood
cells can serve as surrogates for brain cells in the study of selected
fundamental molecular and
cell biological mechanisms of Alzheimer's or neurodegenerative diseases in
general. The data
presented here indicate that in AD and PD similar systems are affected in both
brain and blood
does not require that they behave perfectly equivalently in both classes of
cells.
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i ~"~is'o~ ~ 1ray Data
296. The meanings that may be extracted from array data are dependent on the
array
specifics and the analysis methods used. The combination of targeted arrays
and analytical
methods used was designed to test the hypothesis that selected molecular
systems related to
fundamental cell biology known to be affected in AD brain would also be found
to be affected in
peripheral blood leukocytes. Focused was specifically on transcripts related
to inflammatory
systems, cellular stress, and cell cycle/apoptosis. The targeted arrays used
herein were,
therefore, designed to include multiple probes related to each of these three
systems. Rather than
emphasize analytical methods designed to test AD/control differences of
individual transcripts, a
method of analysis that was able to test differences between AD and control
profiles of gene
expression by making simultaneous use of multiple transcripts was used. The
classical method
of canonical discriminant analysis was selected. This method makes use of
knowledge of the
existence of two (or more) groups to find the set of variables (transcripts)
that best differentiates
the groups. The analysis assigns a weight to each transcript, and these
weights are used to
calculate a "score" (e.g., canonical variable) for each person. The analysis
is as described
hereinbefore and was performed with the SAS/STATTM software. It is this score
that is
presented in Figures 3-5.
297. The value of multivariate statistical methods in distinguishing effects
of disease
on profiles of gene expression has been demonstrated in a variety of studies,
including
distinguishing both rion-brain (ovarian cancer, Welsh et al., Proc. Nat. Acad.
Sci. U.S.A.,
2001;98(3):1176-1181), as well as brain diseases (Tang et al., 2004). This
latter study
differentiated three neurological conditions (tuberous sclerosis complex 2,
neurofibromatosis
type 1, and Down's syndrome) on the basis of expression profiles of
transcripts derived from
blood samples. They state "Each disease was associated with a unique gene
expression pattern
in blood that can be accurately distinguished by a classifier." The present
data emphasize that
differing expression levels of individual genes are often not as powerful in
discriminating
disease state as an analysis based on weighted sums of many gene products. The
interpretation
of array data continues to be a developing enterprise.
3. Mechanism of Common Effects on Blood and Brain Cells
298. The fact that gene expression by peripheral blood leukocytes is altered
in
Alzheimer's disease in ways that are suggestive of events in brain can be
explained by
communication between peripheral blood elements and the brain (e.g. Hickey WF,
et al., 1991;
see Hickey, 1999 for review). Furthermore, the permeability of the blood-brain
barrier to such
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''&ci 'f ~~a~~=b~ri~~s~tov~'ri"~o be~~eiihanced by a variety of factors,
including A beta peptides (e.g.
~
Farkas IG, et al., 2003) and selected A beta peptides have been shown to be
increased in the
peripheral circulation in AD (see Irizarry MC, 2004, for review). It is not
necessary to rely on
blood-brain cominunication alone as a source of A beta peptides in the blood
since blood cells
themselves, especially platelets, express APP, BACE and other components
differentially in AD
(e.g. Colciaghi et al., 2004; Baskin et al., 2000; Li et al., 1999; Rosenberg
et al., 1997). This
suggests that mechanisms other than (or in addition to) blood-brain
communication may play
important roles in modulating AD effects on peripheral cells. Although the
presence of A beta
peptides in the circulatory system may be pertinent, other peripheral cells
outside the
vasculature, including fibroblasts, express components of the APP system and
show differences
between AD and control samples (Emiliani et al., 2003; Ikeda et al., 2000;
Zhao et al., 2002;
Scali et al., 2002; see Etcheberrigaray et al., 1996; Gibson et al., 1996 and
Gibson and Huang,
2002, for reviews). These data are consistent with the concept of AD as a
systemic disease
whose major clinical manifestations arise from its effects on the brain.
299. As disclosed herein, multivariate analyses of transcripts related to
inflammation,
cell stress and cell cycle/apoptosis expressed by peripheral blood leukocytes
are able to
distinguish early AD from control cases. Several other conclusions can be
made: (1) The
classes of transcripts shown herein to be able to distinguish AD from control
leukocytes are
similar to classes of transcripts known to be affected in the brain in AD. (2)
These data may
have implications for the early (perhaps preclinical, antecedent) diagnosis of
AD and for
monitoring disease progression and therapeutic efficacy.
E. Example 3 molecular distinction of Parkinson's disease from peripheral
blood
leukocytes
300. Parkinson's disease protein analysis identified a patient population
which
included 13 Parkinson's disease (PD) patients and 9 age-matched control
patients. Fresh whole
blood was drawn, red blood cells lysed and leukocytes harvested. Leukocyte
protein
concentrations were determined and protein integrity determined by SDS-PAGE
electrophoresis
followed by Coomassie blue staining. Equal amounts of leukocyte protein
lysates were
subjected to 2D-gel electrophoresis. Gels were silver stained, dried and
scanned with a laser
densitometer followed by limited computerized comparisons (Figure 6). Protein
spots that differ
in intensity between PD and control patients were identified using Progenesis
Discovery
software (Nonlinear USA, Inc.; Durham, NC). Difference measurements subjected
to statistical
testing. Seventeen spots were identified as either increasing or decreasing in
Parkinson's disease
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1;n~p~r~a 3''fi~A ~~btitiEa~~a~~~r Differentially expressed spots were punched
from duplicate
Coomassie blue stained gels and subject to in-gel trypsin digestion. Isolated
proteins were
identified following MALDI-TOF mass spectrometry followed by comparison with
public
protein databases (Table 6 and Figure 6).
Table 6: Provisionally identified proteins that differ between Parkinson's
disease patients and
control subjects.
SEQ ID NO: MW pI Accession Number Mass Spec ID
241 66,204 5.67 N1V1 002147 HSP60
242 63,131 7.59 AAH18648 Dihydrolipoamide
dehydrogenase
243 62,052 5.98 JC5704 ER-60 protease
244 59,332 7.42 AAH00337 Glucose-6-phosphate
dehydrogenase
245 54,879 5.42 NP001677 ATP-synthase beta chain
246 36,106 7.58 NP_000691 Annexin I
247 32,567 5.10 NP 006752 14-3-3 epsilon
248 29,785 5.80 NP_002625 Prohibitin
249 29,559 7.60 AAH62302 Phospoglycerate mutase 1
250 26,908 5.65 AAA51747 Apoliporotein AI
251 25,546 7.64 1MSD A Superoxide dismutase
252 24,376 6.36 NP_009193 RNA-binding protein regulatory
subunit
253 23,327 5.82 1QMV A Chain A, thioredoxin peroxidase
B
254 21,719 5.92 NP_056461 RAS-related protein RAP1B
255 145,916 5.29 NP003290 Tumor rejection antigen
256 42,839 5.42 AAC27432 Haptoglobin
257 60,376 6.72 P02675 Fibrin beta
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iftnpi~'~ ~f'~6lli~ication of Differentially Expressed Biomarkers in Patients
Undergoing Treatment for Alzheimer's Disease
301. A study was carried out to identify valproate-responsive proteins. 15
patients
with mild to moderate Alzheimer's disease were examined before (baseline) and
following four
weeks of divalproex sodium treatment. Figure 9 shows some characteristics of
the patients that
participated in the study. Peripheral blood samples were collected into 10 ml
lavender top tubes
using standard venipuncture at baseline and four weeks following initiation of
treatment.
Samples were processed for leukocyte proteins by selective lysis of red blood
cells, followed by
centrifugation,of the sample, retention of the leukocyte pellet, and storage
at minus 80 C.
302. Prior to two-dimensional (2D) gel analysis, samples were quickly thawed
on ice
and lysed using a standard solubilization buffer. Protein concentration was
determined and
protein integrity assayed by SDS-PAGE electrophoresis followed by Coomassie-
blue staining.
Equal amounts of leukocyte protein lysates were subjected to 2D gel
electrophoresis and gels
were silver-stained, dried, and scanned with a laser densitometer followed by
limited
computerized comparisons. See Figure 10. Differentially expressed protein
spots were
identified using the Progenesis Discovery software and statistical testing
(from Nonlinear
Dynamics, Durham, NC). Nine spots were identified as either increasing or
decreasing in
patients on valproate therapy. Differentially expressed spots were punched
from duplicate
Coomassie-blue stained gels, subjected to in-gel trypsin digestion, and
identified using MALDI-
TOF mass spectrometry (see Figure 11) and comparisons with public protein
databases. The
nine biomarkers identified are listed and described in Figure 12. These nine
biomarkers include
actin-interacting protein 1(A1P 1), mitogen activated protein kinase
I(1VIAPKI), actin or a
fragment thereof, annexin Al, 14-3-3 protein epsilon, glutaraldehyde-3-
phosphate
dehydrogenase (GAPDH), transfonning protein RhoA, acidic leucine-rich nuclear
phosphoprotein 32 family member B (ANP32B or APRIL), or peroxiredoxin II.
303. These results show that biological leukocyte-containing biological
samples can be
used to identify biomarkers that are useful for monitoring a patient's
response to treatment for
Alzheimer's disease.
G. Example 5 Confirmation of Differentially Expressed Proteins Using in vitro
Leukocytes.
304. Three of the nine targets were validated using human leukocytes cultured
in the
presence of valproate at two days in vitro. Briefly, human leukocytes were
cultured from non-
demented (control) subjects and subjected to varying concentrations of
valproate (0-5 mM)
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egrziii~r~tifaEt'~~ire:-='I;eukocyte proteins were subjected to SUS-PAGE
electrophoresis and Western blotting using antibodies to the targets. For
three of the proteins,
Valproate treatment recapitulated the change observed in the initial 2D gel
study (Figure 12).
305. Expression levels of annexin Al and APRIL in cultured leukocytes
decreased in a
dose-dependent manner in response to Valproate treatment. These results are
consistent with the
observed decrease of annexin Al and APRIL expression in patients treated with
valproate
(VPA). Again, consistent with the results of Example 4, expression levels of
peroxiredoxin II
increased in cultured Leukocytes in a dose-dependent manner in response to VPA
treatment,
with expression peaking in response to approximately 1.0 mM of VPA. Of the
biomarkers
tested, only actin expression in cultured leukocytes failed to track the
change in expression
patterns observed in VPA-treated patients.
306. These results use indicate that the expression patterns (of at lest some,
and
perhaps most) of the candidate biomarkers identified in VPA-treated
Alzheimer's patients were
not artifactual, since they correlated with the expression pattern of the
biomarkers in cultured
cells, as assessed using a different quantitative technique.
307. It will be apparent to those skilled in the art that various
modifications and
variations can be made in the present invention without departing from the
scope or spirit of the
invention. Other embodiments of the invention will be apparent to those
skilled in the art from
consideration of the specification and practice of the invention disclosed
herein. It is intended
that the specification and examples be considered as exemplary only, with a
true scope and spirit
of the invention being indicated by the following claims.
VIII. References
Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole GM. Cooper NR.
Eikelenboom
P. Emmerling M. Fiebich BL. Finch CE. Frautschy S. Griffin WS. Hampel H. Hull
M. Landreth
G. Lue L. Mrak R. Mackenzie IR. McGeer PL. O'Banion MK. Pachter J. Pasinetti
G. Plata-
Salaman C. Rogers J. Rydel R.Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van
Muiswinkel
FL. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T..
Inflammation
and Alzheimer's disease. Neurobiology of Aging. 2000;21(3):383-421.
Arendt T. Alzheimer's disease as a loss of differentiation control in a subset
of neurons
that retain immature features in the adult brain. Neurobiology of Aging.
2000;21(6):783-796.
Ariga T. Kiso M. Hasegawa A. Miyatake T. Gangliosides inhibit the release of
interleukin-lbeta in amyloid beta-protein-treated human monocytic cells.
Journal of Molecular
Neuroscience. 2001;17(3):371-377.
- 121 -
CA 02574727 2007-01-19
WO 2006/020269 PCT/US2005/025491
f i!'., t" tC.. ...,...f} ~ Il:. !1
~=$'ib~~r~e~ ME: ~ MGE. Microglial interaction with beta-amyloid: implications
for
the pathogenesis of Alzheimer's disease. Microscopy Research & Technique.
2001;54(2):59-70.
Baron P. Galimberti D. Meda L. Scarpini E. Conti G. Cogiamanian F. Scarlato G.
Production of IL-6 by human myoblasts stimulated with Abeta: relevance in the
pathogenesis of
IBM. Neurology. 2001;57(9):1561-1565.
Blessed G. Tomlinson BE. Roth M. The association between quantitative measures
of
dementia and of senile change in the cerebral grey matter of elderly subjects.
British Journal of
Psychiatry. 1968;114(512):797-811.
Braak H. Braak E. Frequency of stages of Alzheimer-related lesions in
different age
categories. Neurobiology of Aging. 1997;18(4):351-357.
Buell SJ. Coleman PD. Dendritic growth in the aged human brain and failure of
growth
in senile dementia. Science. 1979;206(4420):854-856.
Busciglio J. Lorenzo A. Yeh J. Yankner BA. beta-amyloid fibrils induce tau
phosphorylation and loss of microtubule binding. Neuron. 1995;14(4):879-888.
Callahan LM. Selski DJ. Martzen MR. Cheetham JE. Coleman PD. Preliminary
evidence: decreased GAP-43 message in tangle-bearing neurons relative to
adjacent tangle-free
neurons in Alzheimer's disease parahippocampal gyrus. Neurobiology of Aging.
1994;15(3):381-
386.
Callahan LM. Vaules WA. Coleman PD. Progressive reduction of synaptophysin
message in single neurons in Alzheimer disease. Journal of Neuropathology &
Experimental
Neurology. 2002;61(5):3 84-395.
Cao X. Sudhof TC. A transcriptionally [correction of transcriptively] active
complex of
APP with Fe65 and histone acetyltransferase Tip60. Science. 2001;293(5527):115-
120.
Cheetham JE. Coleman PD. Chow N. Isolation of single immunohistochemically
identified whole neuronal cell bodies from post-mortem human brain for
simultaneous analysis
of inultiple gene expression. Journal of Neuroscience Methods. 1997;77(1):43-
48.
Chow, N., Cox, C., Callahan, L.M., Weimer, J.M., Guo, L. and Coleman, P.D.
Expression profiles of multiple genes in single neurons of Alzheimer's
disease. Proc. Natl. Acad.
Sci. USA. 1998;95:9620-9625.
Coleman PD. Flood DG. Neuron numbers and dendritic extent in normal aging and
Alzheimer's disease. Neurobiology of Aging. 1987;8(6):521-545.
Davidsson P. Elffnan R. Blennow K. A new procedure for detecting brain-
specific
proteins in cerebrospinal fluid. Journal of Neural Transmission - General
Section. 1997;104(6-
- 122 -
CA 02574727 2007-01-19
WO 2006/020269 PCT/US2005/025491
De Luigi A. Fragiacomo C. Lucca U. Quadri P. Tettamanti M. Grazia De Simoni M.
Inflammatory markers in Alzheimer's disease and multi-infarct dementia.
Mechanisms of Ageing
& Development. 2001;122(16):1985-1995
Doebler JA. Markesbery WR. Anthony A. Rhoads RE. Neuronal RNA in relation to
neuronal loss and neurofibrillary pathology in the hippocampus in Alzheimer's
disease. Journal
of Neuropathology & Experimental Neurology. 1987;46(1):28-39.
Eberwine J. Yeh H. Miyashiro K. Cao Y. Nair S. Finnell R. Zettel M. Coleman P
United
States of America. 1992;89(7):3010-3014.
Eckert A. Cotman CW. Zerfass R. Hennerici M. Muller WE. Lymphocytes as cell
model
to study apoptosis in Alzheimer's disease: vulnerability to programmed cell
death appears to be
altered. Journal of Neural Transmission. Supplementum. 54:259-67, 1998.
Ferreira A. Lu Q. Orecchio L. Kosik KS. Selective phosphorylation of adult tau
isoforms
in mature hippocampal neurons exposed to fibrillar A beta. Molecular &
Cellular Neurosciences.
1997;9(3):220-234.
Flood DG. Buell SJ. Defiore CH. Horwitz GJ. Coleman PD. Age-related dendritic
growth in dentate gyrus of human brain is followed by regression in the
'oldest old'. Brain
Research. 1985;345(2):366-368.
Flood DG. Buell SJ. Horwitz GJ. Coleman PD. Dendritic extent in human dentate
gyrus
granule cells in normal aging and senile dementia. Brain Research.
1987;402(2):205-216.
Folstein MF. Folstein SE and McHugh P. Mini-Mental state - practical method
for
grading cognitive state of patients for clinician. Journal of Psychiatric
Research. 1975;12(3):189-
198.
Gao Y. Pimplikar SW. The gamma -secretase-cleaved C-tenninal fragment of
amyloid
precursor protein mediates signaling to the nucleus. Proceedings of the
National Academy of
Sciences of the United States of America. 2001;98(26):14979-14984.
Gearing M. Mirra SS. Hedreen JC. Sumi SM. Hansen LA. Heyman A. The Consortium
to Establish a Registry for Alzheimer's Disease (CERAD). Part X.
Neuropathology confirmation
of the clinical diagnosis of Alzheimer's disease. Neurology. 1995;45(3 Pt
1):461-466.
Ginsberg SD. Hemby SE. Lee VM. Eberwine JH. Trojanowski JQ. Expression profile
of
transcripts in Alzheimer's disease tangle-bearing CAl neurons. Annals of
Neurology.
2000;48(1):77-87.
Greenberg SM. Koo EH. Selkoe DJ. Qiu WQ. Kosik KS. Secreted beta-amyloid
- 123 -
CA 02574727 2007-01-19
WO 2006/020269 PCT/US2005/025491
hu~~'tE~~'' u"i~~gen-activated protein kinase and enhances tau
phosphorylation.
Proceedings of the National Academy of Sciences of the United States of
America.
1994;91(15):7104-7108.
Griffin WS. Ling C. White CL 3rd. Morrison-Bogorad M. Polyadenylated messenger
RNA in paired helical filament-immunoreactive neurons in Alzheimer disease.
Alzheimer
Disease & Associated Disorders. 1990;4(2):69-78.
Harris PL. Zhu X. Pamies C. Rottkanlp CA. Ghanbari HA. McShea A. Feng Y.
Ferris
DK. Smith MA. Neuronal polo-like kinase in Alzheimer disease indicates cell
cycle changes.
Neurobiology of Aging. 2000;21(6):837-841.
Harrison PJ, Barton, A.J., Najlerahim, A., McDonald, B. and Pearson, R.C.
Regional and
neuronal reductions of polyadenylated messenger RNA in Alzheimer's disease.
Psychological
Medicine. 1991;21:855-866.
Hatanpaa K, Brady. DR, Stoll J et al. Neuronal activity and early
neurofibrillary tangles in
Alzheimer's disease. Ann. Neurol. 1996;40:411-420.
Hoffinann J. Twiesselmann C. Kummer MP. Romagnoli P. Herzog V. A possible role
for
the Alzheimer ainyloid precursor protein in the regulationof epidermal basal
cell proliferation.
European Journal of Cell Biology. 2000;79(12):905-914.
Hughes CP. Berg L. Danziger WL. Coben LA. Martin RL. A new clinical scale for
the
staging of dementia. British Journal of Psychiatry. 1982;140:566-572.
Husseman JW. Nochlin D. Vincent I. Mitotic activation: a convergent mechanism
for a
cohort of neurodegenerative diseases. Neurobiology of Aging. 2000;21(6):815-
828.
Ikeda K. Urakami K. Arai H. Wada K. Wakutani Y. Ji Y. Adachi Y. Okada A. Kowa
H.
Sasaki H. Ohno K. Ohtsuka Y. Ishikawa Y. Nakashima K. The expression of
presenilin 1 mRNA
in skin fibroblasts and brains from sporadic Alzheimer's disease. Dementia &
Geriatric
Cognitive Disorders. 2000;11(5):245-250.
Jung SS. Gauthier S. Cashman NR. Beta-amyloid precursor protein is detectable
on
monocytes and is increased in Alzheimer's disease. Neurobiology of Aging.
1999;20(3):249-
257.
Killiany RJ. Hyman BT. Gomez-Isla T. Moss MB. Kikinis R. Jolesz F. Tanzi R.
Jones K.
Albert MS. MRI measures of entorhinal cortex vs hippocampus in preclinical AD.
Neurology.
2002;58(8):1188-1196.
Kimberly WT. Zheng JB. Guenette SY. Selkoe DJ. The intracellular domain of the
beta-
amyloid precursor protein is stabilized by Fe65 and translocates to the
nucleus in a notch-like
- 124 -
CA 02574727 2007-01-19
WO 2006/020269 PCT/US2005/025491
Qa66rà =I'~'i~'al~oi cal,themistry. 2001;276(43):40288-40292.
Kinoshita A. Whelan CM. Berezovska 0. Hyman BT. The gamma secretase-generated
carboxyl-terminal domain of the amyloid precursor protein induces apoptosis
via Tip60 in H4
cells. Journal of Biological Chemistry. 2002;277(32):28530-28536.
Kshirsager AM. Multivariate Analysis. New York: Dekker, M., 1972
Kuo YM. Emmerling MR. Lampert HC. Hempelman SR. Kokjohn TA. Woods AS.
Cotter RJ. Roher AE. High levels of circulating Abeta42 are sequestered by
plasma proteins in
Alzheimer's disease. Biochemical & Biophysical Research Communications.
257(3):787-91,
1999.
Kusdra L. Rempel H. Yaffe K. Pulliam L. Elevation of CD69+
monocyte/macrophages in
patients with Alzheimer's disease. Tmmunobiology. 2000;202(1):26-33.
Leissring MA. Murphy MP. Mead TR. Akbari Y. Sugarman MC. Jannatipour M.
Anliker
B. Muller U. Saftig P. De Strooper B. Wolfe MS. Golde TE. LaFerla FM. A
physiologic
signaling role for the gamma-secretase-derived intracellular fragment of APP.
Proceedings of the
National Academy of Sciences of the United States of America. 2002;99:4697-
4702.
Li QX. Fuller SJ. Beyreuther K. Masters CL. The amyloid precursor protein of
Alzheimer disease in human brain and blood. Journal of Leukocyte Biology.
1999;66(4):567-
574.
Lombardi VR. Garcia M. Rey L. Cacabelos R. Characterization of cytokine
production,
screening of lymphocyte subset patterns and in vitro apoptosis in healthy and
Alzheimer's
Disease (AD) individuals. Journal of Neuroimmunology. 1 9 i 9;97(1-2):163-171.
McKhann G. Drachman D. Folstein M. Katzman R. Price D. Stadlan EM. Clinical
diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under
the
auspices of Department of Health and Human Services Task Force on Alzheimer's
Disease.
Neurology. 1984;34(7):939-944.
Meda L. Baron P. Prat E. Scarpini E. Scarlato G. CasI satella MA. Rossi F.
Proinflammatory profile of cytokine production by human monocytes and murine
microglia
stimulated with beta-amyloid[25-35]. Journal of Neuroimmunology. 1999;93(1-
2):45-52.
Nagy Z. Cell cycle regulatory failure in neurones: causes and consequences.
Neurobiology of Aging. 2000;21(6):761-769.
Nagy Z. Combrinck M. Budge M. McShane R. Cell cycle kinesis in lymphocytes in
the
diagnosis of Alzheimer's disease. Neuroscience Letters. 2002;317(2):81-84.
Padovani A, Borroni B, Colciaghi F, al. e. Abnormalities in the pattern of
platelet
- 125 -
CA 02574727 2007-01-19
WO 2006/020269 PCT/US2005/025491
in patients with mild cognitive impairment and Alzheimer's
disease. Archives of Neurology. 2002;59:71-75.
Paris D. Town T. Mori T. Parker TA. Humphrey J. Mullan M. Soluble beta-amyloid
peptides mediate vasoactivity via activation of a pro-inflammatory pathway.
Neurobiology of
Aging. 2000;21(2):183-197.
Pietrzik CU. Hoffinann J. Stober K. Chen CY. Bauer C. Otero DA. Roch JM.
Herzog V.
From differentiation to proliferation: the secretory amyloid precursor protein
as a local mediator
of growth in thyroid epithelial cells. Proceedings of the National Academy of
Sciences of the
United States of America. 1998;95(4):1770-1775.
Pratico D. Clark CM. Liun F. Rokach J. Lee VY. Trojanowski JQ. Increase in
brain
oxidative stress in mild cognitive impairment: A possible predictor of
Alzheimer disease.
Archives of Neurology. 2002;59:972-976.
Remarque EJ. Bollen EL. Weverling-Rijnsburger AW. Laterveer JC. Blauw GJ.
Westendorp RG. Patients with Alzheimer's disease display a pro-inflammatory
phenotype.
Experimental Gerontology. 2001;36:171-176.
Scali C. Prosperi C. Bracco L. Piccini C. Baronti R. Ginestroni A. Sorbi S.
Pepeu G.
Casamenti F. Neutrophils CD l lb and fibroblasts PGE(2) are elevated in
Alzheimer's disease.
Neurobiology of Aging. 2002;23(4):523-530.
. Scheff SW. DeKosky ST. Price DA. Quantitative assessment of cortical
synaptic density
in Alzheimer's disease. Neurobiology of Aging. 1990;11(1):29-37.
Schmitz A. Tikkanen R. Kirfel G. Herzog V. The biological role of the
Alzheimer
amyloid precursor protein in epithelial cells. [Review]. Histochemistry & Cell
Biology.
2002;117(2):171-180.
Scinto LF. Rentz DM. Potter H. Daffner KR. Pupil assay and Alzheimer's
disease: a
critical analysis. Neurology. 1999;52(3):673-674.
Siegel S. Nonparametric Statistics. New York: McGraw-Hill, 1956:1-312.
Stieler JT. Lederer C. Bruckner MK. Wolf H. Holzer M. Gertz HJ. Arendt T.
Iinpairment
of mitogenic activation of peripheral blood lymphocytes in Alzheimer's
disease. NeuroReport.
2001; 12(18):3969-3972.
Terry RD, DeTeresa R, Hansen LA. Neocortical cell counts in normal human adult
aging.
Annals of Neurology. 1987;21:530-539.
Trieb K. Ransmayr G. Sgonc R. Lassmann H. Grubeck-Loebenstein B. APP peptides
stimulate lymphocyte proliferation in normals, but not in patients with
Alzheimer's disease.
- 126 -
CA 02574727 2007-01-19
WO 2006/020269 PCT/US2005/025491
9}b"1f~~4):541-5'47.
Unger JW. Glial reaction in aging and Alzheimer's disease. Microscopy Res.
Technique.
1998;43:24-28.
Urcelay E. Ibarreta D. Parrilla R. Ayuso MS. Martin-Requero A. Enhanced
proliferation
of lymphoblasts from patients with Alzheimer dementia associated with
calmodulin-dependent
activation of the na+/H+ exchanger. Neurobiology of Disease. 2001;8(2):289-
298.
Vincent I, Jicha, G., Rosado, M. and Dickson, Q.W. Aberrant expression of
mitotic
Cdc2/cyclin B 1 kinase in degenerating neurons of Alzheimer's disease brain.
J. Neurosci.
1997;17:3588-3598.
Wakutani Y. Urakami K. Shimomura T. Takahashi K. Heat shock protein 70 mRNA
levels in mononuclear blood cells from patients with dementia of the Alzheimer
type. Dementia.
1995;6(6):301-305.
Webster S, C. G, J. R. Multivalent binding of complement protein C 1 Q to the
amyloid
beta-peptide (A beta) promotes the nucleation. Biochemical & Biophysical
Research
Communications. 1995;217:869-875.
Welsh JB. Zarrinkar PP. Sapinoso LM. Kern SG. Behling CA. Monk BJ. Lockhart
DJ.
Burger RA. Hampton GM. Analysis of gene expression profiles in normal and
neoplastic ovarian
tissue samples identifies candidate molecular markers of epithelial ovarian
cancer. Proceedings
of the National Academy of Sciences of the United States of America.
2001;98(3):1176-1181.
West MJ, Coleman PD, Flood DG, Troncoso JC. Differences in the pattern of
hippocampal neuronal loss in normal aging and Alzheimer's disease. Lancet.
1994;344:769-772
Wu Q. Combs C. Cannady SB. Geldmacher DS. Hemtp K. Beta-amyloid activated
microglia induce cell cycling and cell death in cultured cortical neurons.
Neurobiology of Aging.
2000;21(6):797-806.
Yao P, Tao, P., Zhu, M., Pyun, E., Brooks, A., Therianos, S., Meyers, V. and
Coleman,
PD. Defects in expression of genes related to synaptic vesicle trafficking in
frontal cortex of
Alzheimer's disease. Neurobiology of Disease, 12, 97-109, 2003.Yao P, and PD
Coleman.
Reduction of 0-linked N-acetylglucosamine-modified assembly protein-3 in
Alzheimer's
disease. Journal of Neuroscience. 1998;18(7):2399-2411.
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