U.S. patent application number 13/352903 was filed with the patent office on 2013-01-17 for osteoarthritis biomarkers and uses thereof.
This patent application is currently assigned to GENENEWS, INC.. The applicant listed for this patent is SAMUEL CHAO, ADAM DEMPSEY, CHOONG-CHIN LIEW, WAYNE MARSHALL, THOMAS YAGER, HONGWEI ZHANG. Invention is credited to SAMUEL CHAO, ADAM DEMPSEY, CHOONG-CHIN LIEW, WAYNE MARSHALL, THOMAS YAGER, HONGWEI ZHANG.
Application Number | 20130017965 13/352903 |
Document ID | / |
Family ID | 34138757 |
Filed Date | 2013-01-17 |
United States Patent
Application |
20130017965 |
Kind Code |
A1 |
LIEW; CHOONG-CHIN ; et
al. |
January 17, 2013 |
OSTEOARTHRITIS BIOMARKERS AND USES THEREOF
Abstract
The invention relates to the identification and selection of
novel biomarkers and the identification and selection of novel
biomarker combinations which are differentially expressed in
osteoarthritis and/or in a particular stage of osteoarthritis, as
well as a means of selecting the novel biomarker combinations. The
measurement of expression of the products of the biomarkers and
combinations of biomarkers demonstrates particular advantage in one
or more of the following: (a) diagnosing individuals as having
arthritis, (b) differentiating between two stages of osteoarthritis
(OA) and (c) diagnosing individuals as having a particular stage of
OA. Polynucleotides and proteins which specifically and/or
selectively hybridize to the products of the biomarkers are within
the scope of the invention as are kits containing said
polynucleotides and proteins and the use of said polynucleotides
and proteins. The biomarker products can be used to identify
therapeutic targets for osteoarthritis, and compounds that bind
and/or modulate the gene activity.
Inventors: |
LIEW; CHOONG-CHIN; (TORONTO,
CA) ; YAGER; THOMAS; (MISSISSAUGA, CA) ;
DEMPSEY; ADAM; (TORONTO, CA) ; CHAO; SAMUEL;
(CONCORD, CA) ; ZHANG; HONGWEI; (TORONTO, CA)
; MARSHALL; WAYNE; (SCARBOROUGH, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIEW; CHOONG-CHIN
YAGER; THOMAS
DEMPSEY; ADAM
CHAO; SAMUEL
ZHANG; HONGWEI
MARSHALL; WAYNE |
TORONTO
MISSISSAUGA
TORONTO
CONCORD
TORONTO
SCARBOROUGH |
|
CA
CA
CA
CA
CA
CA |
|
|
Assignee: |
GENENEWS, INC.
RICHMOND HILL
CA
|
Family ID: |
34138757 |
Appl. No.: |
13/352903 |
Filed: |
January 18, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12554141 |
Sep 4, 2009 |
8142998 |
|
|
13352903 |
|
|
|
|
10915680 |
Aug 9, 2004 |
|
|
|
12554141 |
|
|
|
|
60493607 |
Aug 8, 2003 |
|
|
|
60516823 |
Nov 3, 2003 |
|
|
|
Current U.S.
Class: |
506/9 ;
435/6.11 |
Current CPC
Class: |
C12N 2310/15 20130101;
C12N 2310/11 20130101; C12Q 2600/158 20130101; C12Q 1/6883
20130101; C12N 2310/14 20130101; G01N 2800/52 20130101; C12N
2310/12 20130101; C12N 15/113 20130101; G01N 33/564 20130101; G01N
2800/105 20130101 |
Class at
Publication: |
506/9 ;
435/6.11 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 21/64 20060101 G01N021/64; C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-13. (canceled)
14. A method of diagnosing osteoarthritis in a test subject, the
method comprising: for each gene of a set of genes consisting of
alpha glucosidase II alpha subunit (G2AN) and tumor necrosis
factor, alpha-induced protein 6 (TNFAIP6), measuring the RNA
expression level of the gene in a blood sample of the test subject,
wherein measuring the RNA expression level of the gene is done
using a polynucleotide which can specifically and/or selectively
hybridize with RNA encoded by G2AN, or with DNA complementary to
RNA encoded by G2AN.
15. The method of claim 14, further comprising determining a ratio
between the RNA expression level of the gene in the sample of the
test subject and the RNA expression level of the gene in blood of
subjects not having osteoarthritis.
16. The method of claim 15, further comprising measuring the RNA
expression level of the gene in blood samples of a population of
control subjects not having osteoarthritis, thereby obtaining the
RNA expression level of the gene in blood of subjects not having
osteoarthritis.
17. The method of claim 15, further comprising diagnosing the test
subject as having osteoarthritis if the ratio indicates, for G2AN,
that the RNA expression level of the gene in the sample of the test
subject is higher than the RNA expression level of the gene in
blood of subjects not having osteoarthritis, and, for TNFAIP6, that
the RNA expression level of the gene in the sample of the test
subject is lower than the RNA expression level of the gene in blood
of subjects not having osteoarthritis.
18. The method of claim 14, wherein measuring the RNA expression
level of the gene is done by amplification.
19. The method of claim 14, wherein measuring the RNA expression
level of the gene is done using the polynucleotides identified as
SEQ ID NOs: 51, 52, 55 and 56.
20. The method of claim 15, wherein measuring the RNA expression
level of the gene is done using the polynucleotides identified as
SEQ ID NOs: 51, 52, 55 and 56.
21. The method of claim 16, wherein measuring the RNA expression
level of the gene is done using the polynucleotides identified as
SEQ ID NOs: 51, 52, 55 and 56.
22. The method of claim 17, wherein measuring the RNA expression
level of the gene is done using the polynucleotides identified as
SEQ ID NOs: 51, 52, 55 and 56.
23. The method of claim 18, wherein measuring the RNA expression
level of the gene is done using the polynucleotides identified as
SEQ ID NOs: 51, 52, 55 and 56.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 12/554,141, filed Sep. 4, 2009, which is a divisional of U.S.
application Ser. No. 10/915,680, filed Aug. 9, 2004, now abandoned,
which claims priority benefit under 35 U.S.C. .sctn.119 to U.S.
provisional application Ser. No. 60/493,607, filed Aug. 8, 2003 and
U.S. provisional application Ser. No. 60/516,823, filed Nov. 3,
2003, each of which is incorporated herein by reference in its
entirety.
1. FIELD OF THE INVENTION
[0002] The invention relates to the identification and selection of
novel biomarkers and the identification and selection of novel
biomarker combinations which are differentially expressed in
osteoarthritis and/or in a particular stage of osteoarthritis, as
well as a means of selecting the novel biomarker combinations. The
measurement of expression of the products of the biomarkers and
combinations of biomarkers of the invention demonstrates particular
advantage in one or more of the following: (a) diagnosing
individuals as having arthritis, (b) differentiating between two
stages of osteoarthritis (OA) and (c) diagnosing individuals as
having a particular stage of osteoarthritis (OA). As would be
understood, in order to measure the products of biomarkers of the
invention, polynucleotides and proteins which specifically and/or
selectively hybridize to the products of the biomarkers of the
invention are also encompassed within the scope of the invention as
are kits containing said polynucleotides and proteins for use in
(a) diagnosing individuals as having arthritis, (b) differentiating
between two stages of osteoarthritis (OA) and (c) diagnosing
individuals as having a particular stage of osteoarthritis (OA).
Further encompassed by the invention is the use of the
polynucleotides and proteins which specifically and/or selectively
hybridize to the product of the biomarkers of the invention to
monitor disease progression in an individual and to monitor the
efficacy of therapeutic regimens. The invention also provides for
methods of using the products of the biomarkers of the invention in
the identification of novel therapeutic targets for osteoarthritis.
The invention also provides for methods of using the products of
the biomarkers of the invention in the identification of compounds
that bind and/or modulate the activity of the genes of the
invention. The compounds identified via such methods are useful for
the development of assays to study osteoarthritis and
osteoarthritis progression. Further, the compounds identified via
such methods are useful as lead compounds in the development of
prophylactic and therapeutic compositions for the prevention,
treatment, management and/or amelioration of osteoarthritis or a
symptom thereof.
2. BACKGROUND OF THE INVENTION
[0003] Osteoarthritis (OA) is a chronic disease in which the
articular cartilage that lies on the ends of bones that forms the
articulating surface of the joints gradually degenerates over time.
There are many factors that are believed to predispose a patient to
osteoarthritis including genetic susceptibility, obesity,
accidental or athletic trauma, surgery, drugs and heavy physical
demands. Osteoarthritis is thought to be initiated by damage to the
cartilage of joints. The two most common injuries to joints are
sports-related injuries and long term "repetitive use" joint
injuries. Joints most commonly affected by osteoarthritis are the
knees, hips and hands. In most cases, due to the essential
weight-bearing function of the knees and hips, osteoarthritis in
these joints causes much more disability than osteoarthritis of the
hands. As cartilage degeneration progresses, secondary changes
occur in other tissues in and around joints including bone, muscle,
ligaments, menisci and synovium. The net effect of the primary
failure of cartilage tissue and secondary damage to other tissues
is that the patient experiences pain, swelling, weakness and loss
of functional ability in the afflicted joint(s). These symptoms
frequently progress to the point that they have a significant
impact in terms of lost productivity and or quality of life
consequences for the patient.
[0004] Articular cartilage is predominantly composed of
chondrocytes, type II collagen, proteoglycans and water. Articular
cartilage has no blood or nerve supply and chondrocytes are the
only type of cell in this tissue. Chondrocytes are responsible for
manufacturing the type II collagen and proteoglycans that form the
cartilage matrix. This matrix in turn has physical-chemical
properties that allow for saturation of the matrix with water. The
net effect of this structural-functional relationship is that
articular cartilage has exceptional wear characteristics and allows
for almost frictionless movement between the articulating cartilage
surfaces. In the absence of osteoarthritis, articular cartilage
often provides a lifetime of pain-free weight bearing and
unrestricted joint motion even under demanding physical
conditions.
[0005] Like all living tissues, articular cartilage is continually
undergoing a process of renewal in which "old" cells and matrix
components are being removed (catabolic activity) and "new" cells
and molecules are being produced (anabolic activity). Relative to
most tissues, the rate of anabolic/catabolic turnover in articular
cartilage is low. Long-term maintenance of the structural integrity
of mature cartilage relies on the proper balance between matrix
synthesis and degradation. Chondrocytes maintain matrix equilibrium
by responding to chemical and mechanical stimuli from their
environment. Appropriate and effective chondrocyte responses to
these stimuli are essential for cartilage homeostasis. Disruption
of homeostasis through either inadequate anabolic activity or
excessive catabolic activity can result in cartilage degradation
and osteoarthritis (Adams et al., 1995, Nature 377 Suppl:3-174).
Most tissues that are damaged and have increased catabolic activity
are able to mount an increased anabolic response that allows for
tissue healing. Unfortunately, chondrocytes have very limited
ability to up-regulate their anabolic activity and increase the
synthesis of proteoglycan and type II collagen in response to
damage or loss of cartilage matrix.
[0006] Currently there is no known medical treatment to reverse the
effects of this cartilage damage. Rather all current therapies for
osteoarthritis are directed towards treating the symptoms. In
addition, because of the insidious occurence and slow progression
of osteoarthritis, identification of osteoarthritis is often done
at a late stage in disease development rather than early in disease
progression when potential treatments would be more likely to be
effective. As a result further advances in preventing, modifying or
curing the osteoarthritic disease process critically depend on
identification of early diagnostic markers of disease so as to
allow early intervention.
[0007] "Early stage osteoarthritis" is currently very difficult to
diagnose. The physician relies primarily on the patient's history
and physical exam to make the diagnosis of mild osteoarthritis.
X-rays do not show the underlying early changes in articular
cartilage. Currently there are no recognized biochemical markers
used to confirm the diagnosis of early stage osteoarthritis.
Symptoms, such as episodic joint pain, are a common manifestation
of early osteoarthritis. Joints become tender during an episode,
which can last days to weeks and remit spontaneously. These
symptoms, however, often do not correlate well with the
pathological stages of damage to the cartilage. A more reliable
measure of "early stage" osteoarthritis can be obtained by
determining the extent of cartilage damage, however there is
currently no method for measuring cartilage deterioration which is
relatively non-invasive.
[0008] The clinical exam of a joint with "late stage"
osteoarthritis reveals tenderness, joint deformity and a loss of
mobility. Passive joint movement during examination may elicit
crepitus or the grinding of bone-on-bone as the joint moves. X-ray
changes are often profound: the joint space may be obliterated and
misalignment of the joint can be seen. New bone formation
(osteophytes) is prominent. Again, there are no non-invasive
methods which can be used to accurately confirm the diagnosis of
"late stage osteoarthritis".
[0009] Thus there is a need for a simple non-invasive diagnostic
test for detecting the various stages of osteoarthritis, and a
prognostic test that effectively monitors a patient's response to
therapy.
3. SUMMARY OF THE INVENTION
[0010] The invention relates to the identification and selection of
novel biomarkers and the identification and selection of novel
biomarker combinations which are differentially expressed in
osteoarthritis and/or in a particular stage of osteoarthritis, as
well as a means of selecting the novel biomarker combinations. The
measurement of expression of the products of the biomarkers and
combinations of biomarkers of the invention demonstrates particular
advantage in one or more of the following: (a) diagnosing
individuals as having arthritis, (b) differentiating between two
stages of osteoarthritis (OA) and (c) diagnosing individuals as
having a particular stage of osteoarthritis (OA). As would be
understood, in order to measure the products of biomarkers of the
invention, polynucleotides and proteins which specifically and/or
selectively hybridize to the products of the biomarkers of the
invention are also encompassed within the scope of the invention as
are kits containing said polynucleotides and proteins for use in
(a) diagnosing individuals as having arthritis, (b) differentiating
between two stages of osteoarthritis (OA) and (c) diagnosing
individuals as having a particular stage of osteoarthritis (OA).
Further encompassed by the invention is the use of the
polynucleotides and proteins which specifically and/or selectively
hybridize to the product of the biomarkers of the invention to
monitor disease progression in an individual and to monitor the
efficacy of therapeutic regimens. The invention also provides for
the identification of methods of using the products of the
biomarkers of the invention in the identification of novel
therapeutic targets for osteoarthritis. The invention also provides
for the identification of methods of using the products of the
biomarkers of the invention in the identification of compounds that
bind and/or modulate the activity of the genes of the invention.
The compounds identified via such methods are useful for the
development of assays to study osteoarthritis and osteoarthritis
progression. Further, the compounds identified via such methods are
useful as lead compounds in the development of prophylactic and
therapeutic compositions for the prevention, treatment, management
and/or amelioration of osteoarthritis or a symptom thereof.
[0011] In another embodiment, the method of determining whether a
person has OA comprises the steps of (a) isolating total cellular
protein from a test individual; (b) generating monoclonal
antibodies specific for the polypeptides encoded by one or more
biomarkers, or portions thereof, of the invention for use as an
antibody target (c) spotting the antibody targets of step (b) to an
array; and (d) incubating the total cellular protein from a test
individual to said array; and (e) measuring the amount of binding
at each unique location on the array; and (f) using the measured
amount of binding for input into the equation (s) generated by the
mathematical model used to identify the combination in order to
determine a diagnosis as defined by the model.
[0012] In one embodiment, the invention provides for an isolated
biomarker comprising two or more genes selected from the group
consisting of the genes Beta-2-microglobulin, Alpha glucosidase II
alpha subunit, Interleukin 13 receptor alpha 1, Inhibitor of kappa
light polypeptide gene enhancer in B-cells kinase
complex-associated protein, Tumor necrosis factor alpha-induced
protein 6, WD repeat domain 9, Nedd-4-like ubiquitin-protein
ligase, B-cell CLL/lymphoma 6, Complement component 1q subcomponent
receptor 1, Cyclin C, Chromosome 6 open reading frame 151, Heat
shock 90 kDa protein 1 alpha, Laminin gamma 1 and PABP-interacting
protein, Interferon Regulatory Factor 1, Nuclear receptor
coactivator 1, Homo sapiens chloride intracellular channel 4.
ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1),
ATP-binding cassette, sub-family G (WHITE), member 1, as set out in
FIG. 1.
[0013] In one embodiment, the invention provides for an isolated
biomarker consisting essentially of 19 genes as set out in FIG.
1.
[0014] In one embodiment, the invention provides for an isolated
biomarker comprising two or more genes selected from the group
consisting of the genes Tumor necrosis factor alpha-induced protein
6, WD repeat domain 9. Alpha glucosidase II alpha subunit and the
inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein, as set out in FIG. 2.
[0015] In one embodiment, the invention provides for an isolated
biomarker comprising the genes Zinc finger RNA binding protein and
WD repeat domain 9, as set out in FIG. 3.
[0016] In one embodiment, the invention provides for an isolated
biomarker comprising two or more genes selected from the group
consisting of the genes Period 1 (Drosophila). EBNA1 binding
protein 2, Chromosome 6 open reading frame 151 and Laminin gamma 1,
as set out in FIG. 4.
[0017] In one embodiment, the invention provides for an isolated
biomarker comprising one or more polynucleotide sequences from the
5' region of a gene selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1. Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4. ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0018] In one embodiment, the invention provides for an isolated
biomarker comprising one or more polynucleotide sequences from the
3' region of a gene selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit.
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151. Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0019] In one embodiment, the invention provides for an isolated
biomarker comprising one or more polynucleotide sequences from the
internal coding region of a gene selected from the group consisting
of the genes Beta-2-microglobulin. Alpha glucosidase II alpha
subunit. Interleukin 13 receptor alpha 1. Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9. Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1. Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC 1), member 1 (ABCA1). ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0020] In another embodiment, the invention provides for an
isolated biomarker comprising one or more polynucleotide sequences
that are amplified from the 5' region, 3' region or internal coding
region of a gene selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein. Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0021] In another embodiment, the invention provides for an
isolated biomarker comprising one or more polynucleotide sequences
that are expression sequence tags from the 5' region, 3' region or
internal coding region of a gene selected from the group consisting
of the genes Beta-2-microglobulin, Alpha glucosidase II alpha
subunit, Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase. B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151. Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1. Homo sapiens
chloride intracellular channel 4. ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0022] In another embodiment, the invention further provides for an
isolated biomarker comprising the polypeptide sequences encoded by
two or more genes selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1. Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase. B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0023] In another embodiment, the invention further provides for an
isolated biomarker consisting essentially of the polypeptide
sequences encoded by the 19 genes, as set out in FIG. 1.
[0024] In another embodiment, the invention further provides for an
isolated biomarker comprising the polypeptide sequences encoded by
two or more genes selected from the group consisting of the genes
Tumor necrosis factor alpha-induced protein 6, WD repeat domain 9
Alpha glucosidase II alpha subunit and the inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, as set out in FIG. 2.
[0025] In another embodiment, the invention further provides for an
isolated biomarker comprising the polypeptide sequences encoded by
the genes Zinc finger RNA binding protein and WD repeat domain 9,
as set out in FIG. 3.
[0026] In another embodiment, the invention further provides for an
isolated biomarker comprising the polypeptide sequences encoded by
two or more genes selected from the group consisting of the genes
Period 1 (Drosophila), EBNA1 binding protein 2. Chromosome 6 open
reading frame 151 and Laminin gamma 1, as set out in FIG. 4.
[0027] In another embodiment, the invention further provides for an
isolated biomarker comprising the amino terminal polypeptide
sequences encoded by one or more polynucleotide sequences from the
5' region of a gene selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0028] In another embodiment, the invention further provides for an
isolated biomarker comprising the carboxy terminal polypeptide
sequences encoded by one or more polynucleotide sequences from the
3' region of a gene selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9. Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1. Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0029] In another embodiment, the invention further provides for an
isolated biomarker comprising the internal polypeptide region
sequences encoded by one or more polynucleotide sequences from the
internal coding region of a gene selected from the group consisting
of the genes Beta-2-microglobulin, Alpha glucosidase II alpha
subunit, Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9. Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0030] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker comprising two or more genes selected from the
group consisting of the genes Beta-2-microglobulin, Alpha
glucosidase II alpha subunit, Interleukin 13 receptor alpha 1,
Inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein, Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9, Nedd-4-like
ubiquitin-protein ligase, B-cell CLL/lymphoma 6, Complement
component 1q subcomponent receptor 1, Cyclin C, Chromosome 6 open
reading frame 151, Heat shock 90 kDa protein 1 alpha, Laminin gamma
1 and PABP-interacting protein, Interferon Regulatory Factor 1,
Nuclear receptor coactivator 1, Homo sapiens chloride intracellular
channel 4, ATP-binding cassette, sub-family A (ABC1), member 1
(ABCA1), ATP-binding cassette, sub-family (3 (WHITE), member 1, as
set out in FIG. 1.
[0031] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker consisting essentially of 19 genes as set out in
FIG. 1.
[0032] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker comprising two or more genes selected from the
group consisting of the genes Tumor necrosis factor alpha-induced
protein 6, WD repeat domain 9, Alpha glucosidase II alpha subunit
and the inhibitor of kappa light polypeptide gene enhancer in
B-cells kinase complex-associated protein, as set out in FIG.
2.
[0033] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker comprising the genes Zinc finger RNA binding
protein and WD repeat domain 9, as set out in FIG. 3.
[0034] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker comprising two or more genes selected from the
group consisting of the genes Period 1 (Drosophila), EBNA1 binding
protein 2, Chromosome 6 open reading frame 151 and Laminin gamma 1,
as set out in FIG. 4.
[0035] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker comprising one or more polynucleotide sequences
from the 5' region of a gene selected from the group consisting of
the genes Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0036] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker comprising one or more polynucleotide sequences
from the 3' region of a gene selected from the group consisting of
the genes Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein. Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C.
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha. Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1.
[0037] In another embodiment, the invention further provides for a
composition comprising a probe that specifically hybridizes to an
isolated biomarker comprising one or more polynucleotide sequences
from the internal coding region of a gene selected from the group
consisting of the genes Beta-2-microglobulin, Alpha glucosidase II
alpha subunit, Interleukin 13 receptor alpha 1, Inhibitor of kappa
light polypeptide gene enhancer in B-cells kinase
complex-associated protein, Tumor necrosis factor alpha-induced
protein 6, WD repeat domain 9. Nedd-4-like ubiquitin-protein
ligase, B-cell CLL/lymphoma 6, Complement component 1q subcomponent
receptor 1, Cyclin C, Chromosome 6 open reading frame 151, Heat
shock 90 kDa protein 1 alpha, Laminin gamma 1 and PABP-interacting
protein, Interferon Regulatory Factor 1, Nuclear receptor
coactivator 1, Homo sapiens chloride intracellular channel 4,
ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1),
ATP-binding cassette, sub-family G (WHITE), member 1, as set out in
FIG. 1.
[0038] In one embodiment, the invention provides for a probe that
is single or double stranded RNA or single or double stranded
DNA.
[0039] In one embodiment, the invention provides for a composition
comprising the gene for the complement component 1q subcomponent
receptor 1.
[0040] In one embodiment, the invention provides for a composition
comprising the gene for chromosome 6 open reading frame 151.
[0041] In one embodiment, the invention provides for a composition
comprising the gene for WD repeat domain 9.
[0042] In one embodiment, the invention provides for a composition
comprising the polypeptide encoded by the gene for the complement
component 1q subcomponent receptor 1.
[0043] In one embodiment, the invention provides for a composition
comprising the polypeptide encoded by the gene for chromosome 6
open reading frame 151.
[0044] In one embodiment, the invention provides for a composition
comprising the polypeptide encoded by the gene for WD repeat domain
9.
[0045] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker comprising the
polypeptide sequences encoded by two or more genes selected from
the group consisting of the genes Beta-2-microglobulin, Alpha
glucosidase II alpha subunit. Interleukin 13 receptor alpha 1,
Inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein, Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9, Nedd-4-like
ubiquitin-protein ligase, B-cell CLL/lymphoma 6, Complement
component 1q subcomponent receptor 1, Cyclin C. Chromosome 6 open
reading frame 151, Heat shock 90 kDa protein 1 alpha, Laminin gamma
1 and PABP-interacting protein, Interferon Regulatory Factor 1,
Nuclear receptor coactivator 1, Homo sapiens chloride intracellular
channel 4. ATP-binding cassette, sub-family A (ABC1), member 1
(ABCA1), ATP-binding cassette, sub-family G (WHITE), member 1, as
set out in FIG. 1.
[0046] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker consisting essentially
of the polypeptide sequences encoded by the 19 genes, as set out in
FIG. 1.
[0047] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker comprising the
polypeptide sequences encoded by two or more genes selected from
the group consisting of the genes Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9 Alpha glucosidase II
alpha subunit and the inhibitor of kappa light polypeptide gene
enhancer in B-cells kinase complex-associated protein, as set out
in FIG. 2.
[0048] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker comprising the
polypeptide sequences encoded by the genes Zinc finger RNA binding
protein and WD repeat domain 9, as set out in FIG. 3.
[0049] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker comprising the
polypeptide sequences encoded by two or more genes selected from
the group consisting of the genes Period 1 (Drosophila), EBNA1
binding protein 2. Chromosome 6 open reading frame 151 and Laminin
gamma 1, as set out in FIG. 4.
[0050] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker comprising the amino
terminal polypeptide sequences encoded by one or more
polynucleotide sequences from the 5' region of a gene selected from
the group consisting of the genes Beta-2-microglobulin, Alpha
glucosidase II alpha subunit, Interleukin 13 receptor alpha 1,
Inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein, Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9, Nedd-4-like
ubiquitin-protein ligase, B-cell CLL/lymphoma 6, Complement
component 1q subcomponent receptor 1, Cyclin C, Chromosome 6 open
reading frame 151, Heat shock 90 kDa protein 1 alpha, Laminin gamma
1 and PABP-interacting protein, Interferon Regulatory Factor 1,
Nuclear receptor coactivator 1, Homo sapiens chloride intracellular
channel 4, ATP-binding cassette, sub-family A (ABC1), member 1
(ABCA1), ATP-binding cassette, sub-family G (WHITE), member 1, as
set out in FIG. 1.
[0051] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker comprising the carboxy
terminal polypeptide sequences encoded by one or more
polynucleotide sequences from the 3' region of a gene selected from
the group consisting of the genes Beta-2-microglobulin. Alpha
glucosidase II alpha subunit. Interleukin 13 receptor alpha 1,
Inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein. Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9. Nedd-4-like
ubiquitin-protein ligase. B-cell CLL/lymphoma 6, Complement
component 1q subcomponent receptor 1, Cyclin C. Chromosome 6 open
reading frame 151, Heat shock 90 kDa protein 1 alpha, Laminin gamma
1 and PABP-interacting protein, Interferon Regulatory Factor 1.
Nuclear receptor coactivator 1, Homo sapiens chloride intracellular
channel 4. ATP-binding cassette, sub-family A (ABC1), member 1
(ABCA1), ATP-binding cassette, sub-family G (WHITE), member 1, as
set out in FIG. 1.
[0052] In one embodiment, the invention provides for a composition
comprising a ligand that specifically binds to a polypeptide
encoded by a gene of an isolated biomarker comprising the internal
polypeptide region sequences encoded by one or more polynucleotide
sequences from the internal coding region of a gene selected from
the group consisting of the genes Beta-2-microglobulin, Alpha
glucosidase II alpha subunit, Interleukin 13 receptor alpha 1,
Inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein, Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9, Nedd-4-like
ubiquitin-protein ligase, B-cell CLL/lymphoma 6, Complement
component 1q subcomponent receptor 1, Cyclin C, Chromosome 6 open
reading frame 151, Heat shock 90 kDa protein 1 alpha, Laminin gamma
1 and PABP-interacting protein, Interferon Regulatory Factor 1,
Nuclear receptor coactivator 1, Homo sapiens chloride intracellular
channel 4, ATP-binding cassette, sub-family A (ABC 1), member 1
(ABCA1), ATP-binding cassette, sub-family G (WHITE), member 1, as
set out in FIG. 1.
[0053] In another embodiment, the invention provides for a ligand
that is a monoclonal antibody.
[0054] In another embodiment, the invention provides for a kit
comprising an isolated biomarker of one or more of the subject
isolated biomarkers described above and packaging means
therefore.
[0055] In another embodiment, the invention provides for a
microarray comprising an isolated biomarker of one or more of the
subject isolated biomarkers, described above, bound to a solid
support.
[0056] In another embodiment, the invention provides for a
microarray comprising ligands bound to a support, where the ligands
specifically bind to one or more of the subject isolated
biomarkers, described above.
[0057] In another embodiment, the ligand is a monoclonal
antibody.
[0058] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising two or more genes selected from
the group consisting of the genes Beta-2-microglobulin, Alpha
glucosidase II alpha subunit, Interleukin 13 receptor alpha 1,
Inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein. Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9. Nedd-4-like
ubiquitin-protein ligase, B-cell CLL/lymphoma 6, Complement
component 1q subcomponent receptor 1, Cyclin C. Chromosome 6 open
reading frame 151, Heat shock 90 kDa protein 1 alpha, Laminin gamma
1 and PABP-interacting protein, Interferon Regulatory Factor 1,
Nuclear receptor coactivator 1. Homo sapiens chloride intracellular
channel 4, ATP-binding cassette, sub-family A (ABC 1), member 1
(ABCA1), ATP-binding cassette, sub-family G (WHITE), member 1, as
set out in FIG. 1, in a blood sample of an individual, and b)
detecting a difference of the level of expression of the biomarker
in the blood sample according to step a) relative to the level of
expression of the same biomarker of a control, where a difference
in expression levels is indicative or predictive of
osteoarthritis.
[0059] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers consisting essentially of 19 genes as set
out in FIG. 1, in a blood sample of an individual, and b) detecting
a difference of the level of expression of the biomarker in the
blood sample according to step a) relative to the level of
expression of the same biomarker of a control, where a difference
in expression levels is indicative or predictive of
osteoarthritis.
[0060] Another aspect of the invention relates to a method of
diagnosing or prognosing mild osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarker comprising two or more genes selected from
the group consisting of the genes Beta-2-microglobulin. Alpha
glucosidase II alpha subunit. Interleukin 13 receptor alpha 1,
Inhibitor of kappa light polypeptide gene enhancer in B-cells
kinase complex-associated protein, Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9, Nedd-4-like
ubiquitin-protein ligase, B-cell CLL/lymphoma 6, Complement
component 1q subcomponent receptor 1, Cyclin C, Chromosome 6 open
reading frame 151, Heat shock 90 kDa protein 1 alpha, Laminin gamma
1 and PABP-interacting protein, Interferon Regulatory Factor 1,
Nuclear receptor coactivator 1, Homo sapiens chloride intracellular
channel 4. ATP-binding cassette, sub-family A (ABC1), member 1
(ABCA1), ATP-binding cassette, sub-family G (WHITE), member 1 as
set out in FIG. 1, in a blood sample of an individual, and b)
detecting a difference of the level of expression of the biomarker
in the blood sample according to step a) relative to the level of
expression of the same biomarker of a control, where a difference
in expression levels is indicative or predictive of mild
osteoarthritis.
[0061] Another aspect of the invention relates to a method of
diagnosing or prognosing mild osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarker consisting essentially of the 19 genes as
set out in FIG. 1 in a blood sample of an individual, and b)
detecting a difference of the level of expression of the biomarker
in the blood sample according to step a) relative to the level of
expression of the same biomarker of a control, where a difference
in expression levels is indicative or predictive of mild
osteoarthritis.
[0062] Another aspect of the invention relates to a method of
diagnosing or prognosing moderate osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising two or more genes selected from
the group consisting of the genes Tumor necrosis factor
alpha-induced protein 6, WD repeat domain 9, Alpha glucosidase II
alpha subunit and the inhibitor of kappa light polypeptide gene
enhancer in B-cells kinase complex-associated protein, as set out
in FIG. 2, in a blood sample of an individual, and b) detecting a
difference of the level of expression of the biomarker in the blood
sample according to step a) relative to the level of expression of
the same biomarker of a control, where a difference in expression
levels is indicative or predictive of moderate osteoarthritis.
[0063] Another aspect of the invention relates to a method of
diagnosing or prognosing moderate from marked osteoarthritis in an
individual, comprising the steps of a) determining the level of
expression of the isolated biomarkers comprising the genes Zinc
finger RNA binding protein and WD repeat domain 9, as set out in
FIG. 3, in a blood sample of an individual, and b) detecting a
difference of the level of expression of the biomarker in the blood
sample according to step a) relative to the level of expression of
the same biomarker in a blood sample of an individual with moderate
osteoarthritis, where a difference in expression levels is
indicative or predictive of marked osteoarthritis.
[0064] Another aspect of the invention relates to a method of
diagnosing or prognosing marked from severe osteoarthritis in an
individual, comprising the steps of a) determining the level of
expression of the isolated biomarkers comprising two or more genes
selected from the group consisting of the genes Period 1
(Drosophila), EBNA1 binding protein 2, Chromosome 6 open reading
frame 151 and Laminin gamma 1, as set out in FIG. 4, in a blood
sample of an individual, and b) detecting a difference of the level
of expression of the biomarker in the blood sample according to
step a) relative to the level of expression of the same biomarker
in a blood sample of an individual with marked osteoarthritis,
where a difference in expression levels is indicative or predictive
of severe osteoarthritis.
[0065] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising the polypeptide sequences
encoded by two or more genes selected from the group consisting of
the genes Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1 q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1, in a blood sample of an
individual, and b) detecting a difference of the level of
expression of the biomarker in the blood sample according to step
a) relative to the level of expression of the same biomarker in a
blood sample of a control, where a difference in expression levels
is indicative or predictive of osteoarthritis.
[0066] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers consisting essentially of the polypeptide
sequences encoded by the 19 genes, as set out in FIG. 1, in a blood
sample of an individual, and b) detecting a difference of the level
of expression of the biomarker in the blood sample according to
step a) relative to the level of expression of the same biomarker
in a blood sample of a control, where a difference in expression
levels is indicative or predictive of osteoarthritis.
[0067] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising the polypeptide sequences
encoded by two or more genes selected from the group consisting of
the genes Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9 Alpha glucosidase II alpha subunit and the inhibitor of
kappa light polypeptide gene enhancer in B-cells kinase
complex-associated protein, as set out in FIG. 2, in a blood sample
of an individual, and b) detecting a difference of the level of
expression of the biomarker in the blood sample according to step
a) relative to the level of expression of the same biomarker in a
blood sample of a control, where a difference in expression levels
is indicative or predictive of osteoarthritis.
[0068] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising the polypeptide sequences
encoded by the genes Zinc finger RNA binding protein and WD repeat
domain 9, as set out in FIG. 3, in a blood sample of an individual,
and b) detecting a difference of the level of expression of the
biomarker in the blood sample according to step a) relative to the
level of expression of the same biomarker in a blood sample of a
control, where a difference in expression levels is indicative or
predictive of osteoarthritis.
[0069] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising the polypeptide sequences
encoded by two or more genes selected from the group consisting of
the genes Period 1 (Drosophila), EBNA1 binding protein 2.
Chromosome 6 open reading frame 151 and Laminin gamma 1, as set out
in FIG. 4, in a blood sample of an individual, and b) detecting a
difference of the level of expression of the biomarker in the blood
sample according to step a) relative to the level of expression of
the same biomarker in a blood sample of a control, where a
difference in expression levels is indicative or predictive of
osteoarthritis.
[0070] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising the amino terminal polypeptide
sequences encoded by one or more polynucleotide sequences from the
5' region of a gene selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151. Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1, in a blood sample of an
individual, and b) detecting a difference of the level of
expression of the biomarker in the blood sample according to step
a) relative to the level of expression of the same biomarker in a
blood sample of a control, where a difference in expression levels
is indicative or predictive of osteoarthritis.
[0071] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising the carboxy terminal polypeptide
sequences encoded by one or more polynucleotide sequences from the
3' region of a gene selected from the group consisting of the genes
Beta-2-microglobulin, Alpha glucosidase II alpha subunit,
Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9. Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1. Cyclin C,
Chromosome 6 open reading frame 151. Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1. Nuclear receptor coactivator 1. Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1). ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1, in a blood sample of an
individual, and b) detecting a difference of the level of
expression of the biomarker in the blood sample according to step
a) relative to the level of expression of the same biomarker in a
blood sample of a control, where a difference in expression levels
is indicative or predictive of osteoarthritis.
[0072] Another aspect of the invention relates to a method of
diagnosing or prognosing osteoarthritis in an individual,
comprising the steps of a) determining the level of expression of
the isolated biomarkers comprising the internal polypeptide
sequences encoded by one or more polynucleotide sequences from the
internal coding region of a gene selected from the group consisting
of the genes Beta-2-microglobulin, Alpha glucosidase II alpha
subunit, Interleukin 13 receptor alpha 1, Inhibitor of kappa light
polypeptide gene enhancer in B-cells kinase complex-associated
protein, Tumor necrosis factor alpha-induced protein 6, WD repeat
domain 9, Nedd-4-like ubiquitin-protein ligase, B-cell CLL/lymphoma
6, Complement component 1q subcomponent receptor 1, Cyclin C,
Chromosome 6 open reading frame 151, Heat shock 90 kDa protein 1
alpha, Laminin gamma 1 and PABP-interacting protein, Interferon
Regulatory Factor 1, Nuclear receptor coactivator 1, Homo sapiens
chloride intracellular channel 4, ATP-binding cassette, sub-family
A (ABC1), member 1 (ABCA1), ATP-binding cassette, sub-family G
(WHITE), member 1, as set out in FIG. 1, in a blood sample of an
individual, and b) detecting a difference of the level of
expression of the biomarker in the blood sample according to step
a) relative to the level of expression of the same biomarker in a
blood sample of a control, where a difference in expression levels
is indicative or predictive of osteoarthritis.
[0073] Another aspect of the invention relates to a method of
diagnosing or prognosing progression of osteoarthritis, comprising
the steps of a) determining the level of expression of the isolated
biomarker as set forth in FIG. 3 in a blood sample of an individual
having moderate osteoarthritis; and b) comparing the level of the
step a) with the level of expression of the corresponding isolated
biomarker in an individual with marked osteoarthritis, where a
difference in the levels indicates progression of osteoarthritis
from moderate to marked osteoarthritis.
[0074] Another aspect of the invention relates to a method of
diagnosing or prognosing progression of osteoarthritis, comprising
the steps of a) determining the level of expression of the isolated
biomarker as set forth in FIG. 4 in a blood sample of an individual
having marked osteoarthritis; and b) comparing the level of the
step a) with the level of expression of the corresponding isolated
biomarker in an individual with severe osteoarthritis, where a
difference in the levels indicates progression of osteoarthritis
from marked to severe osteoarthritis.
[0075] Another aspect of the invention relates to a method of
diagnosing or prognosing progression of osteoarthritis, comprising
the steps of a) determining the level of expression of a biomarker
in a blood sample of an individual having moderate osteoarthritis,
where the biomarker comprises one of the genes disclosed in FIG. 3;
and b) comparing the level of the step a) with the level of
expression of the corresponding isolated biomarker in an individual
with marked osteoarthritis, where a difference in the levels
indicates progression of osteoarthritis from moderate to marked
osteoarthritis.
[0076] Another aspect of the invention relates to a method of
diagnosing or prognosing progression of osteoarthritis, comprising
the steps of a) determining the level of expression of a biomarker
in a blood sample of an individual having marked osteoarthritis,
where the biomarker comprises one of the genes disclosed in FIG. 4;
and b) comparing the level of the step a) with the level of
expression of the corresponding isolated biomarker in an individual
with severe osteoarthritis, where a difference in the levels
indicates progression of osteoarthritis from marked to severe
osteoarthritis.
[0077] Another aspect of the invention relates to a method of
diagnosing or prognosing regression of osteoarthritis, comprising
the steps of a) determining the level of expression of the isolated
biomarker as set forth in FIG. 3 in a blood sample of an individual
having marked osteoarthritis; and b) comparing the level of the
step a) with the level of expression of the corresponding isolated
biomarker in an individual with moderate osteoarthritis, where a
difference in the levels indicates regression of osteoarthritis
from marked to moderate osteoarthritis.
[0078] Another aspect of the invention relates to a method of
diagnosing or prognosing regression of osteoarthritis, comprising
the steps of a) determining the level of expression of the isolated
biomarker as set forth in FIG. 4 in a blood sample of an individual
having severe osteoarthritis; and b) comparing the level of the
step a) with the level of expression of the corresponding isolated
biomarker in an individual with marked osteoarthritis, where a
difference in the levels indicates regression of osteoarthritis
from severe to marked osteoarthritis.
[0079] Another aspect of the invention relates to a method of
diagnosing or prognosing regression of osteoarthritis, comprising
the steps of a) determining the level of expression of a biomarker
in a blood sample of an individual having marked osteoarthritis,
where the biomarker comprises one of the genes disclosed in FIG. 3;
and b) comparing the level of the step a) with the level of
expression of the corresponding isolated biomarker in an individual
with moderate osteoarthritis, where a difference in the levels
indicates regression of osteoarthritis from marked to moderate
osteoarthritis.
[0080] Another aspect of the invention relates to a method of
diagnosing or prognosing regression of osteoarthritis, comprising
the steps of a) determining the level of expression of a biomarker
in a blood sample of an individual having severe osteoarthritis,
where the biomarker comprises one of the genes disclosed in FIG. 4;
and b) comparing the level of the step a) with the level of
expression of the corresponding isolated biomarker in an individual
with marked osteoarthritis, where a difference in the levels
indicates regression of osteoarthritis from severe to marked
osteoarthritis.
[0081] Another aspect of the invention relates to a method for
monitoring efficacy of a drug for treatment of osteoarthritis in a
patient, comprising the steps of a) obtaining a blood sample from a
patient before treatment and a second blood sample from the patient
after the treatment; b) detecting the level of expression of the
isolated biomarker as disclosed in FIG. 3 in the first and second
blood sample; and comparing the level of expression of the
biomarker in the first sample with the second sample, where a
differential expression of the level of expression of the biomarker
in the first sample as compared with the second sample is
indicative of the efficacy of the drug for treatment of
osteoarthritis in the patient.
[0082] Another aspect of the invention relates to a method for
monitoring efficacy of a drug for treatment of osteoarthritis in a
patient, comprising the steps of obtaining a blood sample from a
patient before treatment and a second blood sample from the patient
after the treatment; detecting the level of expression of the
isolated biomarker as disclosed in FIG. 4 in the first and second
blood sample; and comparing the level of expression of the
biomarker in the first sample with the second sample, where a
differential expression of the level of expression of the biomarker
in the first sample as compared with the second sample is
indicative of the efficacy of the drug for treatment of
osteoarthritis in the patient.
[0083] Another aspect of the invention relates to a method for
monitoring efficacy of a drug for treatment of osteoarthritis in a
patient, comprising the steps of obtaining a blood sample from a
patient before treatment and a second blood sample from the patient
after the treatment; detecting the level of expression of a
biomarker in the first and second blood sample, wehrein the
biomarker comprises one of the genes disclosed in FIG. 3; and
comparing the level of expression of the biomarker in the first
sample with the second sample, where a differential expression of
the level of expression of the biomarker in the first sample as
compared with the second sample is indicative of the efficacy of
the drug for treatment of osteoarthritis in the patient.
[0084] Another aspect of the invention relates to a method for
monitoring efficacy of a drug for treatment of osteoarthritis in a
patient, comprising the steps of obtaining a blood sample from a
patient before treatment and a second blood sample from the patient
after the treatment; detecting the level of expression of a
biomarker in the first and second blood sample, wehrein the
biomarker comprises one of the genes disclosed in FIG. 4; and
comparing the level of expression of the biomarker in the first
sample with the second sample, where a differential expression of
the level of expression of the biomarker in the first sample as
compared with the second sample is indicative of the efficacy of
the drug for treatment of osteoarthritis in the patient.
3.1 DEFINITIONS
[0085] The following definitions are provided for specific terms
which are used in the following written description.
[0086] As used herein, the term "3' end" refers to the end of an
mRNA up to the last 1000 nucleotides or 1/3 of the mRNA, where the
3' terminal nucleotide is that terminal nucleotide of the coding or
untranslated region that adjoins the poly-A tail, if one is
present. That is, the 3' end of an mRNA does not include the poly-A
tail, if one is present. The "3' region" of a gene refers to a
polynucleotide (double-stranded or single-stranded) located within
or at the 3' end of a gene, and includes, but is not limited to,
the 3' untranslated region, if that is present, and the 3' protein
coding region of a gene. The 3' region is not shorter than 8
nucleotides in length and not longer than 1000 nucleotides in
length. Other possible lengths of the 3' region include but are not
limited to 10, 20, 25, 50, 100, 200, 400, and 500 nucleotides.
[0087] As used herein, the term "5' end" refers to the end of an
mRNA up to the first 1000 nucleotides or 1/3 of the mRNA (where the
full length of the mRNA does not include the poly A tail), starting
at the first nucleotide of the mRNA. The "5' region" of a gene
refers to a polynucleotide (double-stranded or single-stranded)
located within or at the 5' end of a gene, and includes, but is not
limited to, the 5' untranslated region, if that is present, and the
5' protein coding region of a gene. The 5' region is not shorter
than 8 nucleotides in length and not longer than 1000 nucleotides
in length. Other possible lengths of the 5' region include but are
not limited to 10, 20, 25, 50, 100, 200, 400, and 500
nucleotides.
[0088] As used herein, the term "amplified", when applied to a
nucleic acid sequence, refers to a process whereby one or more
copies of a particular nucleic acid sequence is generated from a
template nucleic acid, preferably by the method of polymerase chain
reaction (Mullis and Faloona, 1987, Methods Enzymol. 155:335).
"Polymerase chain reaction" or "PCR" refers to an in vitro method
for amplifying a specific nucleic acid template sequence. The PCR
reaction involves a repetitive series of temperature cycles and is
typically performed in a volume of 50-100 .mu.l. The reaction mix
comprises dNTPs (each of the four deoxynucleotides dATP, dCTP,
dGTP, and dTTP), primers, buffers, DNA polymerase, and nucleic acid
template. The PCR reaction comprises providing a set of
polynucleotide primers wherein a first primer contains a sequence
complementary to a region in one strand of the nucleic acid
template sequence and primes the synthesis of a complementary DNA
strand, and a second primer contains a sequence complementary to a
region in a second strand of the target nucleic acid sequence and
primes the synthesis of a complementary DNA strand, and amplifying
the nucleic acid template sequence employing a nucleic acid
polymerase as a template-dependent polymerizing agent under
conditions which are permissive for PCR cycling steps of (i)
annealing of primers required for amplification to a target nucleic
acid sequence contained within the template sequence, (ii)
extending the primers wherein the nucleic acid polymerase
synthesizes a primer extension product. "A set of polynucleotide
primers" or "a set of PCR primers" can comprise two, three, four or
more primers. In one embodiment, an exo-Pfu DNA polymerase is used
to amplify a nucleic acid template in PCR reaction. Other methods
of amplification include, but are not limited to, ligase chain
reaction (LCR), polynucleotide-specific based amplification (NSBA),
or any other method known in the art.
[0089] As used herein, the term "amino terminal" region of a
polypeptide refers to the polypeptide sequences encoded by
polynucleotide sequences (double-stranded or single-stranded)
located within or at the 5' end of a gene, and includes, but is not
limited to, the 5' protein coding region of a gene. As used herein,
the term "amino terminal" region refers to the amino terminal end
of a polypeptide up to the first 300 amino acids or 1/3 of the
polypeptide, starting at the first amino acid of the polypeptide.
The "amino terminal" region of a polypeptide is not shorter than 3
amino acids in length and not longer than 350 amino acids in
length. Other possible lengths of the "amino terminal" region of a
polypeptide include but are not limited to 5, 10, 20, 25, 50, 100
and 200 amino acids.
[0090] As used herein, the term "analog" in the context of
proteinaceous agent (e.g., proteins, polypeptides, peptides, and
antibodies) refers to a proteinaceous agent that possesses a
similar or identical function as a second proteinaceous agent but
does not necessarily comprise a similar or identical amino acid
sequence of the second proteinaceous agent, or possess a similar or
identical structure of the second proteinaceous agent. A
proteinaceous agent that has a similar amino acid sequence refers
to a second proteinaceous agent that satisfies at least one of the
following: (a) a proteinaceous agent having an amino acid sequence
that is at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or at least 99% identical to the amino acid sequence of a
second proteinaceous agent; (b) a proteinaceous agent encoded by a
nucleotide sequence that hybridizes under stringent conditions to a
nucleotide sequence encoding a second proteinaceous agent of at
least 5 contiguous amino acid residues, at least 10 contiguous
amino acid residues, at least 15 contiguous amino acid residues, at
least 20 contiguous amino acid residues, at least 25 contiguous
amino acid residues, at least 40 contiguous amino acid residues, at
least 50 contiguous amino acid residues, at least 60 contiguous
amino residues, at least 70 contiguous amino acid residues, at
least 80 contiguous amino acid residues, at least 90 contiguous
amino acid residues, at least 100 contiguous amino acid residues,
at least 125 contiguous amino acid residues, or at least 150
contiguous amino acid residues; and (c) a proteinaceous agent
encoded by a nucleotide sequence that is at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95% or at least 99% identical
to the nucleotide sequence encoding a second proteinaceous agent. A
proteinaceous agent with similar structure to a second
proteinaceous agent refers to a proteinaceous agent that has a
similar secondary, tertiary or quaternary structure to the second
proteinaceous agent. The structure of a proteinaceous agent can be
determined by methods known to those skilled in the art, including
but not limited to, peptide sequencing, X-ray crystallography,
nuclear magnetic resonance, circular dichroism, and
crystallographic electron microscopy.
[0091] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino acid or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions.times.100%). In one embodiment,
the two sequences are the same length.
[0092] The determination of percent identity between two sequences
can also be accomplished using a mathematical algorithm. A
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.
87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.
Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated
into the NBLAST and XBLAST programs of Altschul et al., 1990, J.
Mol. Biol. 215:403. BLAST nucleotide searches can be performed with
the NBLAST nucleotide program parameters set. e.g., for score=100,
wordlength=12 to obtain nucleotide sequences homologous to a
nucleic acid molecules of the present invention. BLAST protein
searches can be performed with the XBLAST program parameters set,
e.g., to score--50, wordlength=3 to obtain amino acid sequences
homologous to a protein molecule of the present invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., 1997, Nucleic Acids
Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform
an iterated search which detects distant relationships between
molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
of XBLAST and NBLAST) can be used (see, e.g., the NCBI website).
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated
in the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM 120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used.
[0093] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0094] As used herein, the term "analog" in the context of a
non-proteinaceous analog refers to a second organic or inorganic
molecule which possess a similar or identical function as a first
organic or inorganic molecule and is structurally similar to the
first organic or inorganic molecule.
[0095] As used herein, the terms "attaching" and "spotting" refer
to a process of depositing a nucleic acid onto a solid substrate to
form a nucleic acid array such that the nucleic acid is stably
bound to the solid substrate via covalent bonds, hydrogen bonds or
ionic interactions.
[0096] As used herein, the term "biomarker" refers to a gene that
is differentially regulated as between OA and non OA, and/or as
between one or more stages of OA and one or more other stages of
OA.
[0097] As used herein, the term "blood nucleic acid sample" refers
to nucleic acids derived from blood and can include nucleic acids
derived from whole blood, centrifuged lysed blood, serum free whole
blood or fractionated blood including peripheral blood leukocytes
(PBLs) or other fractions of blood as described herein. By whole
blood is meant unfractionated blood, for example, a drop of blood.
By centrifuged lysed blood or `lysed blood` is meant whole blood
that is mixed with lysis buffer and centrifuged as described herein
(see Example 2). By serum free blood is meant whole blood wherein
the serum or plasma is removed by centrifugation as described
herein (see Example 2). Preferably, a blood nucleic acid sample is
whole blood or centrifuged lysed blood and is total RNA, mRNA or is
a nucleic acid corresponding to mRNA, for example, cDNA isolated
from said blood. A nucleic acid sample can also include a PCR
product derived from total RNA, mRNA or cDNA.
[0098] As used herein, the term "carboxy terminal" region of a
polypeptide refers to the polypeptide sequences encoded by
polynucleotide sequences (double-stranded or single-stranded)
located within or at the 3' end of a gene, and includes, but is not
limited to, the 3' protein coding region of a gene. As used herein,
the "carboxy terminal" region refers to the carboxy terminal end of
a polypeptide up to 300 amino acids or 1/3 of the polypeptide from
the last amino acid of the polypeptide. The "3' end" does not
include the polyA tail, if one is present. The "carboxy terminal"
region of a polypeptide is not shorter than 3 amino acids in length
and not longer than 350 amino acids in length. Other possible
lengths of the "carboxy terminal" region of a polypeptide include,
but are not limited to, 5, 10, 20, 25, 50, 100 and 200 amino
acids.
[0099] As used herein, "cartilage" or "articular cartilage" refers
to elastic, translucent connective tissue in mammals, including
human and other species. Cartilage is composed predominantly of
chondrocytes, type II collagen, small amounts of other collagen
types, other noncollagenous proteins, proteoglycans and water, and
is usually surrounded by a perichondrium, made up of fibroblasts,
in a matrix of type I and type II collagen as well as other
proteoglycans. Although most cartilage becomes bone upon
maturation, some cartilage remains in its original form in
locations such as the nose, ears, knees, and other joints. The
cartilage has no blood or nerve supply and chondrocytes are the
only type of cell in this tissue.
[0100] As used herein, the term "cartilage nucleic acid sample"
refers to nucleic acids derived from cartilage. Preferably, a
cartilage nucleic acid sample is total RNA, mRNA or is a nucleic
acid corresponding to RNA, for example, cDNA. A cartilage nucleic
acid sample can also include a PCR product derived from total RNA,
mRNA or cDNA.
[0101] As used herein, "chondrocyte" refers to cells from
cartilage.
[0102] A "coding region" refers to a the region of DNA which is
expressed as RNA RNA.
[0103] As used herein, the terms "compound" and "agent" are used
interchangably.
[0104] As used herein, the term "control" or "control sample" in
the context of diagnosing osteoarthritis, determining the stage of
osteoarthritis, or differentiating between stages of osteoarthritis
refers to one or more samples isolated from an individual or group
of individuals who are classified as not having osteoarthritis (ie
"normal"). The term control or control sample can also refer to the
compilation of data derived from samples of one or more individuals
classified as not having osteoarthritis (ie "normal"). As used
herein, the term "control" in the context of screening for a
prophylactic or therapeutic agent refers to a standard or reference
for an assay or methodology to which other conditions can be
compared.
[0105] As used herein, the term "derivative" in the context of
proteinaceous agent (e.g., proteins, polypeptides, peptides, and
antibodies) refers to a proteinaceous agent that comprises an amino
acid sequence which has been altered by the introduction of amino
acid residue substitutions, deletions, and/or additions. The term
"derivative" as used herein also refers to a proteinaceous agent
which has been modified, i.e., by the covalent attachment of any
type of molecule to the proteinaceous agent. For example, but not
by way of limitation, an antibody may be modified, e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. A
derivative of a proteinaceous agent may be produced by chemical
modifications using techniques known to those of skill in the art,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Further, a derivative of a proteinaceous agent may contain one or
more non-classical amino acids. A derivative of a proteinaceous
agent possesses a similar or identical function as the
proteinaceous agent from which it was derived.
[0106] As used herein, the term "derivative" in the context of a
non-proteinaceous derivative refers to a second organic or
inorganic molecule that is formed based upon the structure of a
first organic or inorganic molecule. A derivative of an organic
molecule includes, but is not limited to, a molecule modified. e.g.
by the addition or deletion of a hydroxyl, methyl, ethyl, carboxyl
or amine group. An organic molecule may also be esterified,
alkylated and/or phosphorylated.
[0107] As used herein "Diagnosis of OA" or "OA diagnosis",
according to one aspect of the invention refers to a process of
determining if an individual is afflicted with OA. In a specific
embodiment, "diagnosis of OA" or "OA diagnosis" refers to a
determination as between two options, that an individual has OA or
that an individual does not have OA. In another specific
embodiment, "diagnosis" refers to a determination as between three
options, an individual has OA, an individual does not have OA, and
it cannot be determined with sufficient degree of certainty whether
an individual has OA or does not have OA. "Diagnosis of whether an
individual has one of two stages of OA" or "differentiating between
stages of OA" refers to a process of determining whether an
individual has one of two stages of OA. (e.g. mild v. control (e.g.
non OA/normal); moderate v. mild; moderate v. severe; marked v.
severe etc. In another specific embodiment, "diagnosis" refers to a
determination as between three options, a diagnosis as to one of
two stages of OA as described above or a third possibility wherein
it cannot be determined with sufficient degree of certainty whether
an individual has either of the two stages referred to. "Diagnosis
of a stage of OA" or "OA staging", according to the invention
refers to a arriving at a decision of whether an individual is
afflicted with a particular stage or grade of OA. In a specific
embodiment, "OA staging" or "diagnosis of a stage of OA" refers to
a determination as between two options, that an individual has one
stage of OA or that an individual does not have OA, or any other
stage of OA. In another specific embodiment, "OA staging" or
"diagnosis of a stage of OA" refers to a determination as between
three options, an individual has a specific stage of OA, an
individual does not OA and does not have any other stage of OA, or
it cannot be determined with sufficient degree of certainty whether
an individual has a specific stage of OA or does not have the
specific stage of OA. As would be understood by a person skilled in
the art, in this context a "sufficient degree of certainty" depends
upon the medical requirements for both the sensitivity and
specificity of the diagnosis. More particularly the sufficient
degree of certaintly includes greater than 50% sensitivity and/or
specificity, greater than 60% sensitivity and/or specificity,
greater than 70% sensitivity and/or specificity, greater than 80%
sensitivity and/or specificity, greater than 90% sensitivity and/or
specificity and 100% sensitivity and/or specificity.
[0108] As used herein, the term "differential expression" refers to
a difference in the level of expression of the RNA and/or protein
products of one or more biomarkers of the invention, as measured by
the amount or level of RNA, including mRNA, and/or one or more
spliced variants of mRNA of the biomarker in one sample as compared
with the level of expression of the same one or more biomarkers of
the invention as measured by the amount or level of RNA, including
mRNA and/or one or more spliced variants of mRNA in a second
sample. "Differentially expressed" can also include a measurement
of the protein, or one or more protein variants encoded by the
biomarker of the invention in a sample or population of samples as
compared with the amount or level of protein expression, including
one or more protein variants of the biomarker or biomarkers of the
invention. Differential expression can be determined as described
herein and as would be understood by a person skilled in the art.
The term "differentially expressed" or "changes in the level of
expression" refers to an increase or decrease in the measurable
expression level of a given biomarker as measured by the amount of
RNA and/or the amount of protein in a sample as compared with the
measurable expression level of a given biomarker a second sample.
The term "differentially expressed" or "changes in the level of
expression" can also refer to an increase or decrease in the
measurable expression level of a given biomarker in a population of
samples as compared with the measurable expression level of a
biomarker in a second population of samples. As used herein,
"differentially expressed" when referring to a single sample can be
measured using the ratio of the level of expression of a given
biomarker in said sample as compared with the mean expression level
of the given biomarker of a control population wherein the ratio is
not equal to 1.0. Differentially expressed can also be used to
include comparing a first population of samples as compared with a
second population of samples or a single sample to a population of
samples using either a ratio of the level of expression or using
p-value. When using p-value, a nucleic acid transcript including
hnRNA and mRNA is identified as being differentially expressed as
between a first and second population when the p-value is less than
0.1. More preferably the p-value is less than 0.05. Even more
preferably the p-value is less than 0.01. More preferably still the
p-value is less than 0.005. Most preferably the p-value is less
than 0.001. When determining differentially expression on the basis
of the ratio of the level of expression, an RNA or protein is
differentially expressed if the ratio of the level of expression in
a first sample as compared with a second sample is greater than or
less than 1.0. For example, a ratio of greater than 1.2, 1.5, 1.7,
2, 3, 4, 10, 20 or a ratio less than 1, for example 0.8, 0.6, 0.4,
0.2, 0.1, 0.05. In another embodiment of the invention a nucleic
acid transcript including hnRNA and mRNA is differentially
expressed if the ratio of the mean of the level of expression of a
first population as compared with the mean level of expression of
the second population is greater than or less than 1.0 For example,
a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 or a ratio
less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1, 0.05. In another
embodiment of the invention a nucleic acid transcript including
hnRNA, and mRNA is differentially expressed if the ratio of its
level of expression in a first sample as compared with the mean of
the second population is greater than or less than 1.0 and includes
for example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10,
20, or a ratio less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1,
0.05. "Differentially increased expression" refers to 1.1 fold, 1.2
fold, 1.4 fold, 1.6 fold, 1.8 fold, or more, relative to a
standard, such as the mean of the expression level of the second
population. "Differentially decreased expression" refers to less
than 1.0 fold, 0.8 fold, 0.6 fold, 0.4 fold, 0.2 fold, 0.1 fold or
less, relative to a standard, such as the mean of the expression
level of the second population.
[0109] As used herein, the term "drug efficacy" refers to the
effectiveness of a drug. "Drug efficacy" is usually measured by the
clinical response of the patient who has been or is being treated
with a drug. A drug is considered to have a high degree of
efficacy, if it achieves desired clinical results, for example, the
reduction of the symptoms of osteoarthritis or the prevention of
osteoarthritis progression as described in the present
specification. The amount of drug absorbed may be used to predict a
patient's response. A general rule is that as the dose of a drug is
increased, a greater effect is seen in the patient until a maximum
desired effect is reached. If more drug is administered after the
maximum point is reached, the side effects will normally
increase.
[0110] As used herein, the term "effective amount" refers to the
amount of a compound which is sufficient to reduce or ameliorate
the progression, severity and/or duration of osteoarthritis or one
or more symptoms thereof, prevent the development, recurrence or
onset of osteoarthritis or one or more symptoms thereof, prevent
the advancement of osteoarthritis or one or more symptoms thereof,
or enhance or improve the prophylactic or therapeutic effect(s) of
another therapy.
[0111] As used herein, the term "fragment" in the context of a
proteinaceous agent refers to a peptide or polypeptide comprising
an amino acid sequence of at least contiguous amino acid residues,
at least 10 contiguous amino acid residues, at least 15 contiguous
amino acid residues, at least 20 contiguous amino acid residues, at
least 25 contiguous amino acid residues, at least 40 contiguous
amino acid residues, at least 50 contiguous amino acid residues, at
least 60 contiguous amino residues, at least 70 contiguous amino
acid residues, at least contiguous 80 amino acid residues, at least
contiguous 90 amino acid residues, at least contiguous 100 amino
acid residues, at least contiguous 125 amino acid residues, at
least 150 contiguous amino acid residues, at least contiguous 175
amino acid residues, at least contiguous 200 amino acid residues,
or at least contiguous 250 amino acid residues of the amino acid
sequence of another polypeptide or a protein. In a specific
embodiment, a fragment of a protein or polypeptide retains at least
one function of the protein or polypeptide. In another embodiment,
a fragment of a protein or polypeptide retains at least two, three,
four, or five functions of the protein or polypeptide. Preferably,
a fragment of an antibody retains the ability to immunospecifically
bind to an antigen.
[0112] As used herein, the term "fusion protein" refers to a
polypeptide that comprises an amino acid sequence of a first
protein or polypeptide or functional fragment, analog or derivative
thereof, and an amino acid sequence of a heterologous protein,
polypeptide, or peptide (i.e., a second protein or polypeptide or
fragment, analog or derivative thereof different than the first
protein or fragment, analog or derivative thereof). In one
embodiment, a fusion protein comprises a prophylactic or
therapeutic agent fused to a heterologous protein, polypeptide or
peptide. In accordance with this embodiment, the heterologous
protein, polypeptide or peptide may or may not be a different type
of prophylactic or therapeutic agent.
[0113] As used herein, the terms "gene expression pattern", "gene
expression profile" and "nucleic acid array expression profile" are
used interchangeably and comprise the pattern of hybridization of a
plurality of target nucleic acid sequences hybridized to a
plurality of nucleic acid probes on an array as compared with a
control.
[0114] As used herein, the terms "hybridizing to" and
"hybridization" refer to the sequence specific non-covalent binding
interactions with a complementary nucleic acid, for example,
interactions between a target nucleic acid sequence and a nucleic
acid member on an array.
[0115] As used herein, the term "immunoglobulin" refers to a
protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. The recognized human
immunoglobulin genes include the kappa, lambda, alpha (IgA1 and
IgA2), gamma (IgG1, IgG2 IgG3, IgG4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 Kd or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH-terminus. Full-length
immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are
similarly encoded by a variable region gene (about 116 amino acids)
and one of the other aforementioned constant region genes, e.g.,
gamma (encoding about 330 amino acids).
[0116] As used herein, the term "in combination" refers to the use
of more than one therapy (e.g., more than one prophylactic agent
and/or therapeutic agent). The use of the term "in combination"
does not restrict the order in which therapies (e.g., prophylactic
and/or therapeutic agents) are administered to a subject. A first
therapy (e.g., a first prophylactic or therapeutic agent) can be
administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with,
or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a
second therapy (e.g., a second prophylactic or therapeutic agent)
to a subject.
[0117] As used herein, the term "indicative of disease" refers to
an expression pattern which is diagnostic of disease such that the
expression pattern is found significantly more often in patients
with a disease than in patients without the disease (as determined
using routine statistical methods setting confidence levels at a
minimum of 95%). Preferably, an expression pattern which is
indicative of disease is found in at least 60% of patients who have
the disease and is found in less than 10% of patients who do not
have the disease. More preferably, an expression pattern which is
indicative of disease is found in at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95% or more in
patients who have the disease and is found in less than 10%, less
than 8%, less than 5%, less than 2.5%, or less than 1% of patients
who do not have the disease.
[0118] As used herein, the term "internal coding region" of a gene
refers to a polynucleotide (double-stranded or single-stranded)
located between the 5' region and the 3' region of a gene as
defined herein. The "internal coding region" is not shorter than 8
nucleotides in length and not longer than 1000 nucleotides in
length. Other possible lengths of the "internal coding region"
include but are not limited to 10, 20, 25, 50, 100, 200, 400, and
500 nucleotides. The 5', 3' and internal regions are
non-overlapping and may, but need not be contiguous, and may, but
need not, add up to the full length of the corresponding gene.
[0119] As used herein, the term "internal polypeptide region" of a
polypeptide refers to the polypeptide sequences located between the
amino terminal region and the carboxy terminal region of a
polypeptide, as defined herein. The "internal polypeptide region"
of a polypeptide is not shorter than 3 amino acids in length and
not longer than 350 amino acids in length. Other possible lengths
of the "internal polypeptide region" of a polypeptide include, but
are not limited to, 5, 10, 20, 25, 50, 100 and 200 amino acids.
[0120] The amino terminal, carboxy terminal and internal
polypeptide regions of a polypeptide are non-overlapping and may,
but need not be contiguous, and may, but need not, add up to the
full length of the corresponding polypeptide.
[0121] As used herein, "isolated" or "purified" when used in
reference to a nucleic acid means that a naturally occurring
sequence has been removed from its normal cellular (e.g.,
chromosomal) environment or is synthesized in noon-natural
environment (e.g. artificially synthesized). Thus, an "isolated" or
"purified" sequence may be in a cell-free solution or placed in a
different cellular environment. The term "purified" does not imply
that the sequence is the only nucleotide present, but that it is
essentially free (about 90-95% pure) of non-nucleotide material
naturally associated with it, and thus is distinguished from
isolated chromosomes.
[0122] As used herein, the terms "isolated" and "purified" in the
context of a proteinaceous agent (e.g. a peptide, polypeptide,
protein or antibody) refer to a proteinaceous agent which is
substantially free of cellular material and in some embodiments,
substantially free of heterologous proteinaceous agents (i.e.,
contaminating proteins) from the cell or tissue source from which
it is derived, or substantially free of chemical precursors or
other chemicals when chemically synthesized. The language
"substantially free of cellular material" includes preparations of
a proteinaceous agent in which the proteinaceous agent is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. Thus, a proteinaceous agent that is
substantially free of cellular material includes preparations of a
proteinaceous agent having less than about 30%, 20%, 10%, or 5% (by
dry weight) of heterologous proteinaceous agent (e.g., protein,
polypeptide, peptide, or antibody; also referred to as a
"contaminating protein"). When the proteinaceous agent is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, 10%, or 5% of the volume of the protein preparation. When the
proteinaceous agent is produced by chemical synthesis, it is
preferably substantially free of chemical precursors or other
chemicals, i.e., it is separated from chemical precursors or other
chemicals which are involved in the synthesis of the proteinaceous
agent. Accordingly, such preparations of a proteinaceous agent have
less than about 30%, 20%, 10%, 5% (by dry weight) of chemical
precursors or compounds other than the proteinaceous agent of
interest. Preferably, proteinaceous agents disclosed herein are
isolated.
[0123] As used herein, the term "level of expression" refers to the
measurable quantity of a given nucleic acid or protein as
determined by methods known to a person skilled in the art and as
described herein. In reference to RNA, hnRNA, mRNA or spliced
variants of mRNA corresponding to a biomarker of the invention,
level of expression can be determined by hybridization or more
quantitative measurements such as real-time RT PCR, which includes
use of both SYBR.RTM. green, TaqMan.RTM. and Molecular Beacons
technology can be used.
[0124] As used herein, a "ligand" is a molecule that specifically
binds to a polypeptide encoded by one of the genes of a biomarker
of the invention. A ligand can be a nucleic acid (RNA or DNA),
polypeptide, peptide or chemical compound. A ligand of the
invention can be a peptide ligand, e.g., a scaffold peptide, a
linear peptide, or a cyclic peptide. In a preferred embodiment, the
polypeptide ligand is an antibody. The antibody can be a human
antibody, a chimeric antibody, a recombinant antibody, a humanized
antibody, a monoclonal antibody, or a polyclonal antibody. The
antibody can be an intact immunoglobulin, e.g. an IgA, IgG, IgE,
IgD, IgM or subtypes thereof. The antibody can be conjugated to a
functional moiety (e.g., a compound which has a biological or
chemical function (which may be a second different polypeptide, a
therapeutic drug, a cytotoxic agent, a detectable moiety, or a
solid support. A polypeptide ligand e.g. antibody of the invention
interacts with a polypeptide, encoded by one of the genes of a
biomarker, with high affinity and specificity. For example, the
polypeptide ligand binds to a polypeptide, encoded by one of the
genes of a biomarker, with an affinity constant of at least
10.sup.7 M.sup.-1, preferably, at least 10.sup.8 M.sup.-1, 10.sup.9
or 10.sup.10 M.sup.-1.
[0125] As used herein, the term "majority" refers to a number
representing more than 50% (e.g., 51%, 60%, or 70%, or 80% or 90%
or up to 100%) of the total members of a composition. The term
"majority", when referring to an array, it means more than 50%
(e.g., 51%, 60%, or 70%, or 80% or 90% or up to 100%) of the total
nucleic acid members that are stably associated with the solid
substrate of the array.
[0126] As used herein, the terms "manage", "managing" and
"management" refer to the beneficial effects that a subject derives
from a therapy (e.g., a prophylactic or therapeutic agent) which
does not result in a cure of osteoarthritis. In certain
embodiments, a subject is administered one or more therapies to
"manage" osteoarthritis so as to prevent the progression or
worsening of the osteoarthritis.
[0127] An "mRNA" means an RNA complimentary to a gene; an mRNA
includes a protein coding region and also may include 5' end and 3'
untranslated regions (UTR).
[0128] As used herein, "mRNA integrity" refers to the quality of
mRNA extracts from either cartilage samples or blood samples. mRNA
extracts with good integrity do not appear to be degraded when
examined by methods well known in the art, for example, by RNA
agarose gel electrophoresis (e.g. Ausube) et al., John Weley &
Sons, Inc., 1997, Current Protocols in Molecular Biology).
Preferably, the mRNA samples have good integrity (e.g. less than
10%, preferably less than 5%, and more preferably less than 1% of
the mRNA is degraded) to truly represent the gene expression levels
of the cartilage or blood samples from which they are
extracted.
[0129] As used herein, the terms "non-responsive" and refractory"
describe patients treated with a currently available therapy (e.g.,
prophylactic or therapeutic agent) for osteoarthritis, which is not
clinically adequate to relieve one or more symptoms associated
therewith. Typically, such patients suffer from severe,
persistently active disease and require additional therapy to
ameliorate the symptoms associated with their osteoarthritis.
[0130] As used herein, "normal" refers to an individual or group of
individuals who have not shown any OA symptoms, including joint
pain, and have not been diagnosed with cartilage injury or OA.
Preferably said normal individual(s) is not on medication affecting
OA and has not been diagnosed with any other disease. More
preferably normal individuals have similar sex, age and body mass
index (BMI) as compared with the test samples. "Normal", according
to the invention, also refers to a samples isolated from normal
individuals and includes total RNA or mRNA isolated from normal
individuals. A sample taken from a normal individual can include
RNA isolated from a cartilage tissue sample wherein RNA is isolated
from a whole or a piece of cartilage isolated from cartilage tissue
from an individual who was not diagnosed with OA and does not show
any symptoms of OA at the time of tissue removal. In one embodiment
of the invention, the "normal" cartilage sample is isolated at 14
hours post-mortem and the integrity of mRNA samples extracted is
confirmed. A sample taken from a normal individual can also include
RNA isolated from a blood sample wherein the blood is from an
individual who has not been diagnosed with OA and does not show any
symptoms of OA at the time the blood is isolated.
[0131] As used herein, "nucleic acid(s)" is interchangeable with
the term "polynucleotide(s)" and it generally refers to any
polyribonucleotide or poly-deoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA or any combination
thereof "Nucleic acids" include, without limitation, single- and
double-stranded nucleic acids. As used herein, the term "nucleic
acid(s)" also includes DNAs or RNAs as described above that contain
one or more modified bases. Thus. DNAs or RNAs with backbones
modified for stability or for other reasons are "nucleic acids".
The term "nucleic acids" as it is used herein embraces such
chemically, enzymatically or metabolically modified forms of
nucleic acids, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including for example, simple
and complex cells. A "nucleic acid" or "nucleic acid sequence" may
also include regions of single- or double-stranded RNA or DNA or
any combinations thereof and can include expressed sequence tags
(ESTs) according to some embodiments of the invention. An EST is a
portion of the expressed sequence of a gene (i.e., the "tag" of a
sequence), made by reverse transcribing a region of mRNA so as to
make cDNA.
[0132] As defined herein, a "nucleic acid array" refers a plurality
of unique nucleic acids (or "nucleic acid members") attached to a
support where each of the nucleic acid members is attached to a
support in a unique pre-selected region. In one embodiment, the
nucleic acid probe attached to the surface of the support is DNA.
In a preferred embodiment, the nucleic acid probe attached to the
surface of the support is either cDNA or oligonucleotides. In
another preferred embodiment, the nucleic acid probe attached to
the surface of the support is cDNA synthesized by polymerase chain
reaction (PCR). The term "nucleic acid", as used herein, is
interchangeable with the term "polynucleotide". In another
preferred embodiment, a "nucleic acid array" refers to a plurality
of unique nucleic acids attached to nitrocellulose or other
membranes used in Southern and/or Northern blotting techniques.
[0133] As used herein, a "nucleic acid probe" or a "nucleic acid
marker" or a "nucleic acid member on an array" or "nucleic acid
probe on an array" also includes nucleic acid immobilized on an
array and capable of binding to a nucleic acid member of
complementary sequence through sets of non-covalent bonding
interactions, including complementary base pairing interactions. As
used herein, a nucleic acid probe may include natural (i.e., A, G,
C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In
addition, the bases in nucleic acid probes may be joined by a
linkage other than a phosphodiester bond, so long as it does not
interfere with hybridization (i.e., the nucleic acid probe still
specifically binds to its complementary sequence under standard
stringent or selective hybridization conditions). Thus, nucleic
acid probes may be peptide nucleic acids in which the constituent
bases are joined by peptide bonds rather than phosphodiester
linkages.
[0134] As used herein "nucleic acid target" or "nucleic acid target
marker" is defined as a nucleic acid capable of binding to a
nucleic acid bound to an array of complementary sequence through
sets of non-covalent bonding interactions including complementary
base pairing interactions. The nucleic acid target can either be an
isolated nucleic acid sequence corresponding to a gene or portion
thereof, or the nucleic acid target can be total RNA or mRNA
isolated from a sample. Preferably, the nucleic acid target or
nucleic acid markers are derived from human cartilage, blood, or
synovial fluid extracts. More preferably, the nucleic acid targets
are single- or double-stranded DNA, RNA, or DNA-RNA hybrids, from
human cartilage, blood, or synovial fluid total RNA extracts, and
preferably from mRNA extracts.
[0135] In one embodiment, a conventional nucleic acid array of
`target` sequences bound to the array can be representative of the
entire human genome, e.g. Affymetrix chip, and the isolated
biomarker consisting of or comprising two or more of the 19 genes
described in FIG. 1 or gene probes is applied to the conventional
array.
[0136] In another embodiment, sequences bound to the array can be
an isolated oligonucleotide, cDNA, EST or PCR product corresponding
to a biomarker of the invention total cellular RNA is applied to
the array.
[0137] As used herein, the term "oligonucleotide" is defined as a
molecule comprised of two or more deoxyribonucleotides and/or
ribonucleotides, and preferably more than three. Its exact size
will depend upon many factors which, in turn, depend upon the
ultimate function and use of the oligonucleotide. The
oligonucleotides may be from about 8 to about 1,000 nucleotides
long. Although oliognucleotides of 8 to 100 nucleotides are useful
in the invention, preferred oligonucleotides range from about 8 to
about 15 bases in length, from about 8 to about 20 bases in length,
from about 8 to about 25 bases in length, from about 8 to about 30
bases in length, from about 8 to about 40 bases in length or from
about 8 to about 50 bases in length.
[0138] As used herein, "osteoarthritis" refers to a particular form
of arthritis, and in particular a chronic disease in which the
articular cartilage that lies on the ends of bones that form the
articulating surface of the joints gradually degenerates over time.
Cartilage degeneration can be caused by an imbalanced catabolic
activity (removal of "old" cells and matrix components) and
anabolic activity (production of "new" cells and molecules)
(Westacott et al., 1996. Semin Arthritis Rheum. 25:254-72).
[0139] As used herein, the term "osteoarthritis (OA) staging" or
"osteoarthritis (OA) grading" refers to determining the presence,
the absence, and or the degree of advancement or progression of OA
in an individual. "OA stages" or "OA grades" include "mild OA",
"moderate OA", "marked OA'' and "severe OA" and no OA" as defined
in accordance with the scoring system of Marshall described below,
but are not limited to these stages. For example one could use an
even more refined scoring system of that taught by Marshall wherein
smaller categories are defined (e.g. mild OA'' can be subdivided
into categories as can moderate, marked and severe on the basis of
the Marshall score) In one embodiment, one can utilize traditional
techniques to classify the stages of OA. Many scoring system are
known in the art, and can be used, for example radiography with a
Kellgren-Lawrence score as an alternative means of OA diagnosis and
staging. R would be understood by a person skilled in the art how
to interpret "mild OA", "moderate OA" "marked OA" and "severe OA"
in accordance with other classification systems. In order to
classify cartilage into different disease stages, referably the
scoring system described in Marshall (Marshall W., 1996, The
Journal of Rheumatology, 23:582-584, incorporated by reference) is
used. According to this method, each of the 6 articular surfaces
(patella, femoral trochlea, medial femoral condyle, medial tibial
plateau, lateral femoral condyle and lateral tibial plateau) is
assigned a cartilage grade based on the worst lesion present on
that specific surface. A scoring system is then applied in which
each articular surface receives an OA severity number value that
reflects the cartilage severity grade for that surface. For
example, if the medial femoral condyle has a grade I lesion as its
most severe cartilage damage a value of 1 is assigned. A total
score for the patient is then derived from the sum of the scores on
the 6 articular surfaces. Based on the total score, each patient is
placed into one of 4 OA groups: "mild" (early) is defined as having
a Marshall score of 1-6, "moderate" is defined as having a Marshall
score of 7-12, "marked" is defined as having a Marshall score of
13-18 and "severe" is defined as having a Marshall score of greater
than 18. In another embodiment, one can measure the expression of
the biomarkers of the invention in accordance with the teachings
herein in order to determine the presence and or the degree of
advancement or progression of the disease.
[0140] As used herein, the phrase "pharmaceutically acceptable
salt(s)," includes, but is not limited to, salts of acidic or basic
groups that may be present in compounds identified using the
methods of the present invention. Compounds that are basic in
nature are capable of forming a wide variety of salts with various
inorganic and organic acids. The acids that can be used to prepare
pharmaceutically acceptable acid addition salts of such basic
compounds are those that form non-toxic acid addition salts, i.e.,
salts containing pharmacologically acceptable anions, including but
not limited to sulfuric, citric, maleic, acetic, oxalic,
hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,
bisulfate, phosphate, acid phosphate, isonicotinate, acetate,
lactate, salicylate, citrate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds that
include an amino moiety may form pharmaceutically acceptable salts
with various amino acids, in addition to the acids mentioned above.
Compounds that are acidic in nature are capable of forming base
salts with various pharmacologically acceptable cations. Examples
of such salts include alkali metal or alkaline earth metal salts
and, particularly, calcium, magnesium, sodium lithium, zinc,
potassium, and iron salts.
[0141] As used herein, "polynucleotide" encompasses double-stranded
DNA, single-stranded DNA and double-stranded or single-stranded RNA
of more than 8 nucleotides in length.
[0142] As used herein, "polypeptide sequences encoded by" refers to
the amino acid sequences obtained after translation of the protein
coding region of a gene, as defined herein. The mRNA nucleotide
sequence for each of the 19 genes is identified by its Genbank
Accession number (see FIGS. 1-5) and the corresponding polypeptide
sequence is identified by a Protein Accession number (see FIGS.
1-5). The Genbank Accession numbers identified in FIGS. 1-5 provide
the location of the 5' UTR, protein coding region (CDS) and 3' UTR
within the mRNA nucleotide sequence of each of the 19 genes.
[0143] When a protein or fragment of a protein is used to immunize
a host animal, numerous regions of the protein may induce the
production of antibodies which bind specifically to a given region
or three-dimensional structure on the protein; these regions or
structures are referred to as epitopes or antigenic determinants.
As used herein. "antigenic fragments" refers portions of a
polypeptide that contains one or more epitopes. Epitopes can be
linear, comprising essentially a linear sequence from the antigen,
or conformational, comprising sequences which are genetically
separated by other sequences but come together structurally at the
binding site for the polypeptide ligand. "Antigenic fragments" may
be 5000, 1000, 500, 400, 300, 200, 100, 50 or 25 or 20 or 10 or 5
amino acids in length.
[0144] As used herein, "pre-selected region". "predefined region",
or "unique position" refers to a localized area on a substrate
which is, was, or is intended to be used for the deposit of a
nucleic acid and is otherwise referred to herein in the alternative
as a "selected region" or simply a "region." The pre-selected
region may have any convenient shape, e.g., circular, rectangular,
elliptical, wedge-shaped, etc. In some embodiments, a pre-selected
region is smaller than about 1 cm.sup.2, more preferably less than
1 mm.sup.2, still more preferably less than 0.5 mm.sup.2, and in
some embodiments less than 0.1 mm.sup.2. A nucleic acid member at a
"pre-selected region", "predefined region", or "unique position" is
one whose identity (e.g., sequence) can be determined by virtue of
its position at the region or unique position.
[0145] As used herein, the terms "prevent", "preventing" and
"prevention" referto the prevention of the development, recurrence
or onset of osteoarthritis or one or more symptoms thereof
resulting from the administration of one or more compounds
identified in accordance the methods of the invention or the
administration of a combination of such a compound and another
therapy.
[0146] The term, "primer", as used herein refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product, which
is complementary to a nucleic acid strand, is induced, i.e., in the
presence of nucleotides and an inducing agent such as a DNA
polymerase and at a suitable temperature and pH. The primer may be
either single-stranded or double-stranded and must be sufficiently
long to prime the synthesis of the desired extension product in the
presence of the inducing agent. The exact length of the primer will
depend upon many factors, including temperature, source of primer
and the method used. For example, for diagnostic applications,
depending on the complexity of the target sequence, the
oligonucleotide primer typically contains 15-25 or more
nucleotides, although it may contain fewer nucleotides. The factors
involved in determining the appropriate length of primer are
readily known to one of ordinary skill in the art.
[0147] As used herein, the term "probe" means oligonucleotides and
analogs thereof and refers to a range of chemical species that
recognize polynucleotide target sequences through hydrogen bonding
interactions with the nucleotide bases of the target sequences. The
probe or the target sequences may be single- or double-stranded RNA
or single- or double-stranded DNA or a combination of DNA and RNA
bases. A probe is at least 8 nucleotides in length and less than
the length of a complete gene. A probe may be 10, 20, 30, 50, 75,
100, 150, 200, 250, 400, 500 and up to 2000 nucleotides in length
as long as it is less the full length of the target gene. Probes
can include oligonucleotides modified so as to have a tag which is
detectable by fluorescence, chemiluminescence and the like. The
probe can also be modified so as to have both a detectable tag and
a quencher molecule, for example Taqman.RTM. and Molecular
Beacon.RTM. probes.
[0148] The oligonucleotides and analogs thereof may be RNA or DNA,
or analogs of RNA or DNA, commonly referred to as antisense
oligomers or antisense oligonucleotides. Such RNA or DNA analogs
comprise but are not limited to 2-'O-alkyl sugar modifications,
methylphosphonate, phosphorothiate, phosphorodithioate, formacetal,
3'-thioformacetal, sulfone, sulfamate, and nitroxide backbone
modifications, and analogs wherein the base moieties have been
modified. In addition, analogs of oligomers may be polymers in
which the sugar moiety has been modified or replaced by another
suitable moiety, resulting in polymers which include, but are not
limited to, morpholino analogs and peptide nucleic acid (PNA)
analogs (Egholm, et al. Peptide Nucleic Acids (PNA)-Oligonucleotide
Analogues with an Achiral Peptide Backbone, (1992)).
[0149] Probes may also be mixtures of any of the oligonucleotide
analog types together or in combination with native DNA or RNA. At
the same time, the oligonucleotides and analogs thereof may be used
alone or in combination with one or more additional
oliognucleotides or analogs thereof.
[0150] As used herein, the terms "prophylactic agent" and
"prophylactic agents" refer to any compound(s) which can be used in
the prevention of osteoarthritis. In certain embodiments, the term
"prophylactic agent" refers to a compound identified in the
screening assays described herein. In certain other embodiments,
the term "prophylactic agent" refers to an agent other than a
compound identified in the screening assays described herein which
is known to be useful for, or has been or is currently being used
to prevent or impede the onset, development and/or progression of
osteoarthritis or one or more symptoms thereof.
[0151] As used herein, the phrase "prophylactically effective
amount" refers to the amount of a therapy (e.g., a prophylactic
agent) which is sufficient to result in the prevention of the
development, recurrence or onset of osteoarthritis or one or more
symptoms thereof.
[0152] As used herein, the terms "protein" and "polypeptide" are
used interchangeably to refer to a chain of amino acids linked
together by peptide bonds. In a specific embodiment, a protein is
composed of less than 200, less than 175, less than 150, less than
125, less than 100, less than 50, less than 45, less than 40, less
than 35, less than 30, less than 25, less than 20, less than 15,
less than 10, or less than 5 amino acids linked together by peptide
bonds. In another embodiment, a protein is composed of at least
200, at least 250, at least 300, at least 350, at least 400, at
least 450, at least 500 or more amino acids linked together by
peptide bonds.
[0153] A "protein coding region" refers to the portion of the mRNA
encoding a polypeptide.
[0154] As used herein the term "polynucleotide which selectively
hybridizes to a product of the Biomarker of the invention" refers
to
[0155] As used herein, "a plurality of" or "a set of" refers to
more than two, for example, 3 or more, 10 or more, 100 or more, or
1000 or more, or 10,000 or more.
[0156] As used herein, the terms "a portion thereof" and "RNA
portion" in context of RNA products of a biomarker of the invention
refer to an RNA transcript comprising a nucleic acid sequence of at
least 6, at least 9, at least 15, at least 18, at least 21, at
least 24, at least 30, at least 60, at least 90, at least 99, or at
least 108, or more nucleotides of a RNA product of a biomarker of
the invention.
[0157] As used herein the term "product of the biomarker of the
invention" refers to the RNA and/or the protein expressed by the
gene corresponding to the biomarker of the invention. The "RNA
product of a biomarker of the invention", includes one or more
products which can include heteronuclear RNA ("hnRNA"), mRNA, and
all or some of the spliced variants of mRNA whose measure of
expression can be used as a biomarker in accordance with the
teachings disclosed herein. The "protein product of a biomarker of
the invention" includes one or more of the products of the
biomarker which can include proteins, protein variants, and any
post-translationally modified proteins.
[0158] As used herein, the term "selectively binds" in the context
of the invention refers to the interaction of a any two of a
peptide, a protein, a polypeptide an antibody, wherein the
interaction is dependent upon the presence of particular structures
on the respective molecules. For example, when the two molecules
are protein molecules, a structure on the first molecule recognizes
and binds to a structure on the second molecule, rather than to
other proteins. "Selective binding", as the term is used herein,
means that a molecule binds to its specific binding partner with a
specificity of at least 70%, at least 80%, at least 90% and most
preferably 100% specificity.
[0159] As used herein "selective hybridization" in the context of
this invention refers to a hybridization which occurs as between a
polynucleotide encompassed by the invention and an RNA or protein
product of the biomarker of the invention wherein the hybridization
is such that the polynucleotide is specific for the RNA or protein
product or products of the biomarker of the invention to the
exclusion of RNA or protein products of other genes in the genome
in question. In a preferred embodiment a polynucleotide which
"selectively hybridizes" is one which hybridizes with a specificity
of greater than 70%, greater than 80%, greater than 90% and most
preferably 100% specificity. As would be understood to a person
skilled in the art, a polynucleotide which "selectively hybridizes"
to the RNA product of a biomarker of the invention can be
determined taking into account the length and composition.
[0160] As used herein, the term "significant match", when referring
to nucleic acid sequences, means that two nucleic acid sequences
exhibit at least 65% identity, at least 70%, at least 75%, at least
80%, at least 85%, and preferably, at least 90% identity, using
comparison methods well known in the art (i.e., Altschul, S. F. et
al., 1997. Nucl. Acids Res., 25:3389-3402; Schaffer. A. A. et al.,
1999, Bioinformatics 15:1000-1011). As used herein, "significant
match" encompasses non-contiguous or scattered identical
nucleotides so long as the sequences exhibit at least 65%, and
preferably, at least 70%, at least 75%, at least 80%, at least 85%,
and preferably, at least 90% identity, when maximally aligned using
alignment methods routine in the art.
[0161] As used herein, the terms "solid substrate" and "solid
support" refers to a material having a rigid or semi-rigid surface.
The terms "substrate" and "support" areused interchangeably herein
with the terms "solid substrate" and "solid support". The solid
support may be biological, non-biological, organic, inorganic, or a
combination of any of these, existing as particles, strands,
precipitates, gels, sheets, tubing, spheres, beads, containers,
capillaries, pads, slices, films, plates, slides, chips, etc.
Often, the substrate is a silicon or glass surface,
(poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,
polycarbonate, a charged membrane, such as nylon 66 or
nitrocellulose, or combinations thereof. In a preferred embodiment,
the solid support is glass. Preferably, at least one surface of the
substrate will be substantially flat. Preferably, the solid support
will contain reactive groups, including, but not limited to,
carboxyl, amino, hydroxyl, thiol, and the like. In one embodiment,
the solid support is optically transparent.
[0162] Solid supports include silica gels, resins, derivatized
plastic films, glass beads, cotton, plastic beads, polystyrene
beads, alumina gels, and polysaccharides. A suitable solid support
may be selected on the basis of desired end use and suitability for
various synthetic protocols. For example, for peptide synthesis, a
solid support can be a resin such as p-methylbenzhydrylamine
(pMBHA) resin (Peptides International, Louisville, Ky.),
polystyrenes (e.g., PAM-resin obtained from Bachem Inc., Peninsula
Laboratories, etc.), including chloromethylpolystyrene,
hydroxymethylpolystyrene and aminomethylpolystyrene,
poly(dimethylacrylamide)-grafted styrene co-divinyl-benzene (e.g.,
POLYHIPE resin, obtained from Aminotech. Canada), polyamide resin
(obtained from Peninsula Laboratories), polystyrene resin grafted
with polyethylene glycol (e.g., TENTAGEL or ARGOGEL, Bayer,
Tubingen, Germany) polydimethylacrylamide resin (obtained from
Milligen/Biosearch, California), or Sepharose (Pharmacia,
Sweden).
[0163] As used herein, the term "specifically binds" refers to the
interaction of two molecules, e.g., a ligand and a protein or
peptide, or an antibody and a protein or peptide wherein the
interaction is dependent upon the presence of particular structures
on the respective molecules. For example, when the two molecules
are protein molecules, a structure on the first molecule recognizes
and binds to a structure on the second molecule, rather than to
proteins in general. "Specific binding", as the term is used
herein, means that a molecule binds its specific binding partner
with at least 2-fold greater affinity, and preferably at least
10-fold, 20-fold, 50-fold, 100-fold or higher affinity than it
binds a non-specific molecule.
[0164] As used herein, "specifically hybridizes", "specific
hybridization" refers to hybridization which occurs when two
nucleic acid sequences are substantially complementary (at least
about 65% complementary over a stretch of at least 14 to 25
nucleotides, preferably at least about 75% complementary, more
preferably at least about 90% complementary). See Kanehisa, M.,
1984. Nucleic acids Res., 12:203, incorporated herein by reference.
As a result, it is expected that a certain degree of mismatch is
tolerated. Such mismatch may be small, such as a mono-, di- or
tri-nucleotide. Alternatively, a region of mismatch can encompass
loops, which are defined as regions in which there exists a
mismatch in an uninterrupted series of four or more nucleotides.
Numerous factors influence the efficiency and selectivity of
hybridization of two nucleic acids, for example, the hybridization
of a nucleic acid member on an array to a target nucleic acid
sequence. These factors include nucleic acid member length,
nucleotide sequence and/or composition, hybridization temperature,
buffer composition and potential for steric hindrance in the region
to which the nucleic acid member is required to hybridize. A
positive correlation exists between the nucleic acid length and
both the efficiency and accuracy with which a nucleic acid will
anneal to a target sequence. In particular, longer sequences have a
higher melting temperature (T.sub.M) than do shorter ones, and are
less likely to be repeated within a given target sequence, thereby
minimizing promiscuous hybridization. Hybridization temperature
varies inversely with nucleic acid member annealing efficiency.
Similarly the concentration of organic solvents, e.g., formamide,
in a hybridization mixture varies inversely with annealing
efficiency, while increases in salt concentration in the
hybridization mixture facilitate annealing. Under stringent
annealing conditions, longer nucleic acids, hybridize more
efficiently than do shorter ones, which are sufficient under more
permissive conditions.
[0165] As used herein, "stably associated" refers to a nucleic acid
that is stably bound to a solid substrate to form an array via
covalent bonds, hydrogen bonds or ionic interactions such that the
nucleic acid retains its unique pre-selected position relative to
all other nucleic acids that are stably associated with an array,
or to all other pre-selected regions on the solid substrate under
conditions in which an array is typically analyzed (i.e., during
one or more steps of hybridization, washes, and/or scanning,
etc.).
[0166] As herein used, the term "standard stringent conditions"
means hybridization will occur only if there is at least 95% and
preferably, at least 97% identity between the sequences, wherein
the region of identity comprises at least 10 nucleotides. In one
embodiment, the sequences hybridize under stringent conditions
following incubation of the sequences overnight at 42.degree. C.,
followed by stringent washes (0.2.times.SSC at 65.degree. C.). The
degree of stringency of washing can be varied by changing the
temperature, pH, ionic strength, divalent cation concentration,
volume and duration of the washing. For example, the stringency of
hybridization may be varied by conducting the hybridization at
varying temperatures below the melting temperatures of the probes.
The melting temperature of the probe may be calculated using the
following formulas:
[0167] For oligonucleotide probes, between 14 and 70 nucleotides in
length, the melting temperature (Tm) in degrees Celcius may be
calculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fraction
G+C)-(600/N) where N is the length of the oligonucleotide.
[0168] For example, the hybridization temperature may be decreased
in increments of 5.degree. C. from 68.degree. C. to 42.degree. C.
in a hybridization buffer having a Na.sup.+ concentration of
approximately 1M. Following hybridization, the filter may be washed
with 2.times.SSC, 0.5% SDS at the temperature of hybridization.
These conditions are considered to be "moderate stringency"
conditions above 50.degree. C. and "low stringency" conditions
below 50.degree. C. A specific example of "moderate stringency"
hybridization conditions is when the above hybridization is
conducted at 55.degree. C. A specific example of "low stringency"
hybridization conditions is when the above hybridization is
conducted at 45.degree. C.
[0169] If the hybridization is carried out in a solution containing
formamide, the melting temperature may be calculated using the
equation Tm=81.5+16.6(log [Na.sup.+])+0.41(fraction G+C)-(0.63%
formamide)-(600/N), where N is the length of the probe.
[0170] For example, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. C. In this case, the concentration of formamide in
the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to
the probe. Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate stringency" conditions above 25%
formamide and "low stringency" conditions below 25% formamide. A
specific example of "moderate stringency" hybridization conditions
is when the above hybridization is conducted at 30% formamide. A
specific example of "low stringency" hybridization conditions is
when the above hybridization is conducted at 10% formamide.
[0171] As used herein, the terms "subject" and "patient" and
"individual" are used interchangeably to refer to an animal (e.g.,
a mammal, a fish, an amphibian, a reptile, a bird and an insect).
In a specific embodiment, a subject is a mammal (e.g., a non-human
mammal and a human). In another embodiment, a subject is a pet
(e.g., a dog, a cat, a guinea pig, a monkey and a bird), a farm
animal (e.g., a horse, a cow, a pig, a goat and a chicken) or a
laboratory animal (e.g., a mouse and a rat). In another embodiment,
a subject is a primate (e.g., a chimpanzee and a human). In a
preferred embodiment, a subject is a human.
[0172] As used herein, the term "synergistic" refers to a
combination of a compound identified using one of the methods
described herein, and another therapy (e.g., agent), which is more
effective than the additive effects of the therapies. Preferably,
such other therapy has been or is currently being to prevent,
treat, manage or ameliorate osteoarthritis or a symptom thereof. A
synergistic effect of a combination of therapies (e.g.,
prophylactic or therapeutic agents) permits the use of lower
dosages of one or more of the therapies and/or less frequent
administration of said therapies to a subject with osteoarthritis.
The ability to utilize lower dosages of a therapy (e.g., a
prophylactic or therapeutic agent) and/or to administer said
therapy less frequently reduces the toxicity associated with the
administration of said agent to a subject without reducing the
efficacy of said therapies in the prevention, treatment, management
or amelioration of osteoarthritis. In addition, a synergistic
effect can result in improved efficacy of therapies (e.g., agents)
in the prevention, treatment, management or amelioration of
osteoarthritis. Finally, a synergistic effect of a combination of
therapies (e.g., prophylactic or therapeutic agents) may avoid or
reduce adverse or unwanted side effects associated with the use of
either therapy alone.
[0173] As used herein, "synovial fluid" refers to fluid secreted
from the "synovial sac" which surrounds each joint. Synovial fluid
serves to protect the joint, lubricate the joint and provide
nourishment to the articular cartilage. Synovial fluid useful
according to the invention contains cells from which RNA can be
isolated according to methods well known in the art as described
herein.
[0174] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any compound(s) which can be used in
the treatment, management or amelioration of osteoarthritis or one
or more symptoms thereof. In a specific emobodiment, the term
"therapeutic agent" refers to a compound that increases or
decreases the expression of a polynucleotide sequence that is
differentially expressed in a chondrocyte from any two of the
following developmental or osteoarthritis disease stages: (a) mild,
(b) moderate, (c) marked and (d) severe, or (e) chondrocyte from a
normal individual, as defined herein. A therapeutic agent according
to the invention also refers to a compound that increases or
decreases the anabolic activity of a chondrocyte. The invention
provides for a "therapeutic agent" that 1) prevents the onset of
osteoarthritis; 2) reduces, delays, or eliminates osteoarthritis
symptoms such as pain, swelling, weakness and loss of functional
ability in the afflicted joints; 3) reduces, delays, or eliminates
cartilage degeneration, and/or enhances chondrocyte metabolic
activity and cell division rates; and/or 4) restores one or more
expression profiles of one or more disease-indicative nucleic acids
of a patient to a profile more similar to that of a normal
individual when administered to a patient. In certain embodiments,
the term "therapeutic agent" refers to a compound identified in the
screening assays described herein. In other embodiments, the term
"therapeutic agent" refers to an agent other than a compound
identified in the screening assays described herein which is known
to be useful for, or has been or is currently being used to treat,
manage or ameliorate osteoarthritis or one or more symptoms
thereof.
[0175] As used herein, the term "therapeutically effective amount"
refers to that amount of a therapy (e.g., a therapeutic agent)
sufficient to result in the amelioration of osteoarthritis or one
or more symptoms thereof, prevent advancement of osteoarthritis,
cause regression of osteoarthritis, or to enhance or improve the
therapeutic effect(s) of another therapy (e.g., therapeutic agent).
In a specific embodiment, a therapeutically effective amount refers
to the amount of a therapy (e.g., a therapeutic agent) that reduces
joint pain or swelling of the joint. Preferably, a therapeutically
effective of a therapy (e.g. a therapeutic agent) reduces the
swelling of the joint by at least 5%, preferably at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 100% relative to
a control such as phosphate buffered saline ("PBS").
[0176] As used herein, the terms "treat", "treatment" and
"treating" refer to the reduction or amelioration of the
progression, severity and/or duration of osteoarthritis or one or
more symptoms thereof resulting from the administration of one or
more compounds identified in accordance the methods of the
invention, or a combination of one or more compounds identified in
accordance with the invention and another therapy.
[0177] As used herein, the term "up regulated" or "increased level
of expression" in the context of this invention refers to a
sequence corresponding to a gene which is expressed wherein the
measure of the quantity of the sequence demonstrates an increased
level of expression of the gene, as can be determined using array
analysis or other similar analysis, in cartilage or blood isolated
from an individual having osteoarthritis or an identified disease
state of osteoarthritis as determined by osteoarthritis staging as
compared with the same gene in cartilage or blood isolated from
normal individuals or from an individual with a different
identified disease state of osteoarthritis as determined by
osteoarthritis staging. An "increased level of expression"
according to the present invention, is an increase in expression of
at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%,
80%, 90% or more, or greater than 1-fold, 2-fold, 3-fold, 4-fold,
5-fold, 10-fold, 50-fold, 100-fold or more as measured, for
example, by the intensity of hybridization according to methods of
the present invention. For example, up regulated sequences includes
sequences having an increased level of expression in cartilage or
blood isolated from individuals characterized as having mild,
moderate, marked or severe OA as compared with cartilage isolated
from normal individuals. Up regulated sequences can also include
sequences having an increased level of expression in cartilage or
blood isolated from individuals characterized as having one stage
of osteoarthritis as compared to another stage of osteoarthritis
(e.g. marked OA v. severe OA).
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0178] The objects and features of the invention can be better
understood with reference to the following detailed description and
drawings.
[0179] FIG. 1 is a figure showing the polynucleotide and
polypeptide sequences of 19 genes that are differentially regulated
in a population of mild OA individuals when compared with the same
genes in a population of normal individuals.
[0180] FIG. 2 is a figure showing the polynucleotide and
polypeptide sequences of 4 genes that are differentially regulated
in a population of individuals with moderate OA as compared with a
population of normal individuals.
[0181] FIG. 3 is a figure showing the polynucleotide and
polypeptide sequences of 2 genes that are differentially regulated
in a population of OA individuals with moderate OA as compared with
a population of individuals having marked OA.
[0182] FIG. 4 is a figure showing the polynucleotide and
polypeptide sequences of 4 genes that are differentially regulated
in a population of individuals with marked OA as compared with a
population of individuals with severe OA.
[0183] FIG. 5 shows the differential Alpha glucosidase II alpha
subunit (G2AN) gene expression in blood samples from patients with
mild vs moderate OA using QRT-PCR analysis.
[0184] FIG. 6 shows the differential Tumor necrosis factor,
alpha-induced protein 6 (TNFAIP6) gene expression in blood samples
from patients with mild vs moderate OA using SYBR.RTM. Green
QRT-PCR and TaqMan.RTM. analysis.
[0185] FIG. 7 shows the differential Period homolog 1 (Drosophila)
(PER1) gene expression in blood samples from patients with marked
vs severe OA using QRT-PCR analysis.
[0186] FIG. 8 shows the differential Zinc finger RNA binding
protein (ZFR) gene expression in blood samples from patients with
moderate vs marked OA using Taqman.RTM. QRT-PCR analysis.
[0187] FIG. 9 is a figure showing differential ATP-binding
cassette, sub-family A, member 1 (ABCA1) and ATP-binding cassette,
sub-family G (WHITE), member 1 (ABCG1) gene expression in blood
samples from patients with mild vs marked OA using QRT-PCR
analysis.
[0188] FIG. 10 is a figure showing differential Interferon
Regulatory Factor 1 (IRF1) gene expression in blood samples from
patients with mild vs marked OA using QRT-PCR analysis.
[0189] FIG. 1I is a figure showing differential Nuclear Receptor
Co-Activator 1 (NRCOA1) and Chloride Intracellular Channel 4
(CLIC4) gene expression in blood samples from patients with mild vs
marked OA using QRT-PCR analysis.
[0190] Table 1 is a table showing, in one embodiment, the genes of
the invention and in particular identifying the genes on the basis
of their locus link ID.
[0191] Table 2 is a table showing, specific embodiments of the
genes of the invention and in the reference accession numbers
corresponding mRNA transcripts and proteins corresponding to the
genes of the invention.
5. DETAILED DESCRIPTION OF THE INVENTION
[0192] The practice of the present invention employs in part
conventional techniques of molecular biology, microbiology and
recombinant DNA techniques, which are within the skill of the art.
Such techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Second Edition; Oligonucleotide Synthesis (M.J.
Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Harnes & S.
J. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B.
Perbal, 1984); and a series, Methods in Enzymology (Academic Press,
Inc.); Short Protocols In Molecular Biology, (Ausubel et al., ed.,
1995). All patents, patent applications, and publications mentioned
herein, both supra and infra, are hereby incorporated by reference
in their entireties.
[0193] The invention as disclosed herein identifies biomarkers and
biomarker combinations useful in diagnosing OA and/or in diagnosing
stages of OA. In order to use these biomarkers, the invention
teaches the identification of the products of these biomarkers
including the RNA products and the protein products. The invention
further discloses the oligonucleotides, cDNA, DNA, RNA, PCR
products, synthetic DNA, synthetic RNA, or other combinations of
naturally occurring of modified nucleotides that specifically
and/or selectively hybridize to the RNA products of the biomarkers
of the invention. The invention further discloses proteins,
peptides, antibodies, ligands that specifically and/or selectively
hybridize to the protein products of the biomarkers of the
invention. The measuring of the expression of the RNA product of
the biomarkers and combination of biomarkers of the invention, can
be done by using those polynucleotides which are specific and/or
selective for the RNA products of the biomarkers of the invention
to quantitate the expression of the RNA product. In a specific
embodiment of the invention, the polynucleotides which are specific
and/or selective for the RNA products are probes or primers. In one
embodiment, these polynucleotides are in the form of a nucleic acid
probe which can be hybridized to a manufactured array. In another
embodiment, commercial arrays can be used to measure the expression
of the RNA product and the invention teaches which combination of
genes to analyze. In another embodiment, the polynucleotides which
are specific and/or selective for the RNA products of the
biomarkers of the invention are used in the form of probes and
primers in techniques such as quantitative real-time RT PCR, using
for example SYBR.RTM.Green, or using TaqMan.RTM. or Molecular
Beacon techniques, where the polynucleotides used are used in the
form of a forward primer, a reverse primer, a TaqMan labelled probe
or a Molecular Beacon labelled probe. In one specific embodiment,
the results generated from measuring the level of expression of the
RNA products of the invention can be input into the model of the
invention which is used to identify the combinations of biomarkers
to determine a diagnosis as defined by the model. In a preferred
embodiment, the same method is used to generate the expression data
used to generate the mathematical model as is used to diagnose the
test individual.
[0194] The invention further contemplates the use of proteins or
polypeptides as disclosed herein and would be known by a person
skilled in the art to measure the protein products of the
biomarkers of the invention. Techniques known to persons skilled in
the art (for example, techniques such as Western Blotting,
Immunoprecipitation protein microarray analysis and the like) can
then be used to measure the level of protein products corresponding
to the biomarkers of the invention. As would be understood to a
person skilled in the art, the measure of the level of expression
of the protein products of the biomarkers of the invention requires
a protein which specifically or selectively binds to one or more of
the protein products corresponding to each biomarker of the
invention. Data representative of the level of expression of the
protein products of the biomarker of the invention can then be
input into the model generated to identify the combination in order
to determine a diagnosis as defined by the model. In a preferred
embodiment, the same method is used to generate the expression data
used to generate the mathematical model as is used to diagnose the
test individual.
[0195] 5.1 Samples for Use in the Invention
[0196] Unless otherwise indicated herein, any tissue sample (e.g.,
a cartilage, synovial fluid or blood sample) or cell sample (e.g.,
chondrocyte sample or a blood cell sample) obtained from any
subject may be used in accordance with the methods of the
invention. Examples of subjects from which such a sample may be
obtained and utilized in accordance with the methods of the
invention include, but are not limited to, asymptomatic subjects,
subjects manifesting or exhibiting 1, 2, 3, 4 or more symptoms of
osteoarthritis, subjects clinically diagnosed as having
osteoarthritis, subjects predisposed to osteoarthritis (e.g.,
subjects with a family history of osteoarthritis, subjects with a
genetic predisposition to osteoarthritis, and subjects that lead a
lifestyle that predisposes them to osteoarthritis or increases the
likelihood of contracting osteoarthritis), subjects suspected of
having osteoarthritis, subjects undergoing therapy for
osteoarthritis, subjects with osteoarthritis and at least one other
condition (e.g., subjects with 2, 3, 4, 5 or more conditions),
subjects not undergoing therapy for osteoarthritis, subjects
determined by a medical practitioner (e.g., a physician) to be
healthy or osteoarthritis-free (i.e., normal), subjects that have
been cured of osteoarthritis, subjects that are managing their
osteoarthritis, and subjects that have not been diagnosed with
osteoarthritis. In a specific embodiment, the subjects from which a
sample may be obtained and utilized have osteoarthritis of the
hands, feet, spine, knee, hip and/or wrist.
[0197] In another embodiment, the subjects from which a sample may
be obtained and utilized have mild, marked, moderate or severe
osteoarthritis. In a further embodiment, the subject from which a
sample may be obtained is a test individual wherein it is unknown
whether the person has osteoarthritis, and/or it is unknown what
stage of osteoarthritis the test individual has.
[0198] In order to classify an individual according to disease
state, a scoring system based on cartilage may be used, whereby
subjective decisions by the arthroscopist are minimized. An example
of a scoring system which defines disease states described herein
is that of Marshall, 1996. The Journal of Rheumatology 23:582-584,
incorporated herein by reference. According to this method, each of
the 6 articular surfaces (patella, femoral trochlea, medial femoral
condyle, medial tibial plateau, lateral femoral condyle and lateral
tibial plateau) is assigned a cartilage grade based on the worst
lesion present on that specific surface. A scoring system is then
applied in which each articular surface receives an osteoarthritis
severity number value that reflects the cartilage severity grade
for that surface, as described in Table 3.
TABLE-US-00001 TABLE 3 Articular Cartilage Grading System Grade
Articular Cartilage Points 0 Normal 0 I Surface intact-softening,
edema 1 II Surface-disrupted-partial thickness lesions (no 2
extension to bone) III Full thickness lesions-extensions to intact
bone 3 IV Bone erosion or eburnation 4
[0199] For example, if the medial femoral condyle has a grade I
lesion as its most severe cartilage damage, a value of 1 is
assigned. A total score for the patient is then derived from the
sum of the scores of the 6 articular surfaces. Based on the total
score, each patient is placed into one of 4 osteoarthritis groups:
mild (1-6), moderate (7-12), marked (13-18) and severe
(>18).
[0200] In certain embodiments, the sample obtained from a subject
is a cartilage sample (including a sample of cells from cartilage).
In other embodiments, the sample obtained from a subject is a
synovial fluid sample (including a sample of cells from synovial
fluid). In yet other embodiments, the sample obtained from a
subject is a blood sample (including a sample of cells from
blood).
[0201] 5.1.1 Cartilage
[0202] In one aspect, a cartilage sample is obtained from a fetus
using methods known in the art. The chondrocytes of fetal cartilage
have a higher level of metabolic activity and cell division rates
as compared to chondrocytes from cartilage from either a normal
adult or from an individual diagnosed with any stage of
osteoarthritis (mild, moderate, marked and severe).
[0203] In another aspect, a cartilage sample is obtained from a
normal individual who is alive or is obtained from cartilage tissue
less than 14 hours post mortem, according to methods known in the
art and described below. Normal articular cartilage from human
adults are obtained using any known method. In a specific
embodiment, cartilage is obtained from individuals undergoing
arthroscopy or total knee replacements and samples are stored in
liquid nitrogen until needed. Typically, truly normal cartilage
cannot generally be sampled from live donors due to ethical
considerations. Thus, preferably, normal cartilage samples are
obtained from deceased donors, within a fourteen-hour post-mortem
window after cessation of perfusion to the sampled joint, to
minimize the degradation of RNA observed beyond the window. In
other embodiments, the "normal" tissue is obtained less than 14
hours post-mortem, such as 13, 12, 11, 10, 9, 8, 6, 4, 2, or 1 hour
post-mortem. Preferably, the normal cartilage is obtained less than
12 hours post-mortem.
[0204] In another aspect, cartilage is obtained from a subject
diagnosed with one of the following disease stages of
osteoarthritis: mild, marked, moderate or severe. Human cartilage
samples from osteoarthritic individuals are obtained using any
known method. Preferably, the cartilage is obtained from
individuals undergoing arthroscopy or total knee replacements and
samples are stored in liquid nitrogen until needed. In a specific
embodiment, a minimum of 0.05 g of cartilage sample is isolated to
obtain 2 .mu.g total RNA extract for the construction of a cDNA
library. In another embodiment, a minimum of 0.025 g cartilage
sample is isolated to obtain 1 .mu.g total RNA extract to use as a
target sample for a microarray. A cartilage sample that is useful
according to the invention is in an amount that is sufficient for
the detection of one or more nucleic acid sequences or amino acid
sequences according to the invention.
[0205] The cartilage collected is optionally but preferably stored
at refrigerated temperatures, such 4.degree. C., prior to use in
accordance with the methods of the invention. In some embodiments,
a portion of the cartilage sample is used in accordance with the
methods of the invention at a first instance of time whereas one or
more remaining portions of the sample is stored for a period of
time for later use. This period of time can be an hour or more, a
day or more, a week or more, a month or more, a year or more, or
indefinitely. For long term storage, storage methods well known in
the art, such as storage at cryo temperatures (e.g., below
-60.degree. C.) can be used. In some embodiments, in addition to
storage of the cartilage or instead of storage of the cartilage,
isolated nucleic acid or protein are stored for a period of time
(e.g., an hour or more, a day or more, a week or more, a month or
more, a year or more, or indefinitely) for later use.
[0206] In some embodiments of the present invention, chrondrocytes
present in the cartilage are separated using techniques known in
the art and used in accordance with the methods of the invention.
Chondrocytes may be obtained from a subject having any of the
following developmental or disease stages: fetal, normal, mild,
osteoarthritic, moderate osteoarthritic, marked osteoarthritic or
severe osteoarthritic. Chondrocytes can be frozen by standard
techniques prior to use in the present methods.
[0207] 5.1.2 Synovial Fluid
[0208] In one aspect, a sample of synovial fluid is obtained from a
subject according to methods well known in the art. For example,
arthrocentesis may be performed. During arthrocentesis, a sterile
needle is used to remove synovial fluid from a joint. Synovial
fluid may be collected from a knee, elbow, wrist, finger, hip,
spine or any other joint using arthrocentesis. In a specific
embodiment, synovial fluid is collected from the joint affected or
suspected to be affected by osteoarthritis. Synovial fluid may be
obtained from a subject having any of the following developmental
or disease stages: fetal, normal, mild, osteoarthritic, moderate
osteoarthritic, marked osteoarthritic or severe osteoarthritic.
[0209] A synovial fluid sample that is useful according to the
invention is in an amount that is sufficient for the detection of
one or more nucleic acid or amino acid sequences according to the
invention. In a specific embodiment, a synovial fluid sample useful
according to the invention is in an amount ranging from 0.1 ml to
20 ml, 0.1 ml to 15 ml, 0.1 ml to 10 ml, 0.1 ml to 5 ml, 0.1 to 2
ml, 0.5 ml to 20 ml, 0.5 ml to 15 ml, 0.5 ml to 10 ml, 0.5 ml to 5
ml, or 0.5 ml to 2 ml. In another embodiment, a synovial fluid
sample useful according to the invention is 0.1 ml or more, 0.5 ml
or more, 1 ml or more, 2 ml or more, 3 ml or more, 4 ml or more, 5
ml or more, 6 ml or more, 7 ml or more, 8 ml or more, 9 ml or more,
10 ml or more, 11 ml or more, 12 ml or more, 13 ml or more, 14 ml
or more, 15 ml or more, 16 ml or more, 17 ml or more, 18 ml or
more, 19 ml or more, or 20 ml or more.
[0210] The synovial fluid collected is optionally but preferably
stored at refrigerated temperatures, such 4.degree. C., prior to
use in accordance with the methods of the invention. In some
embodiments, a portion of the synovial fluid sample is used in
accordance with the methods of the invention at a first instance of
time whereas one or more remaining portions of the sample is stored
for a period of time for later use. This period of time can be an
hour or more, a day or more, a week or more, a month or more, a
year or more, or indefinitely. For long term storage, storage
methods well known in the art, such as storage at cryo temperatures
(e.g., below -60.degree. C.) can be used. In some embodiments, in
addition to storage of the synovial fluid or instead of storage of
the synovial fluid, isolated nucleic acid or protein are stored for
a period of time (e.g., an hour or more, a day or more, a week or
more, a month or more, a year or more, or indefinitely) for later
use.
[0211] In some embodiments of the present invention, cells present
in the synovial fluid are separated using techniques known in the
art and used in accordance with the methods of the invention.
Generally, the following cells are found in synovial fluid:
lymphocytes (B and T lymphocytes), monocytes, neurtophils,
synoviocytes and macrophages. In synovial fluid from patients with
a pathological condition, such as osteoarthritis, the following
cells may also be found: chondrocytes, osteoblasts and osteoclasts.
Such cells may be isolated and used in accordance with the methods
of the invention. In a specific embodiment, lymphocytes (B and T
lymphocytes) are isolated from the synovial fluid sample and used
in accordance with the methods of the invention. In another
embodiment, monocytes or neutrophils are isolated from the synovial
fluid sample and used in accordance with the methods of the
invention. Cells isolated from the synovial fluid can be frozen by
standard techniques prior to use in the present methods.
[0212] 5.1.3 Blood
[0213] In one aspect of the invention, a sample of blood is
obtained from a subject according to methods well known in the art.
A sample of blood may be obtained from a subject having any of the
following developmental or disease stages: fetal, normal, mild,
osteoarthritic, moderate osteoarthritic, marked osteoarthritic or
severe osteoarthritic or from a test individual where it is unknown
whether the individual has osteoarthritis, or has a stage of
osteoarthritos. In some embodiments, a drop of blood is collected
from a simple pin prick made in the skin of a subject. In such
embodiments, this drop of blood collected from a pin prick is all
that is needed. Blood may be drawn from a subject from any part of
the body (e.g., a finger, a hand, a wrist, an arm, a leg, a foot,
an ankle, a stomach, and a neck) using techniques known to one of
skill in the art, in particular methods of phlebotomy known in the
art. In a specific embodiment, venous blood is obtained from a
subject and utilized in accordance with the methods of the
invention. In another embodiment, arterial blood is obtained and
utilized in accordance with the methods of the invention. The
composition of venous blood varies according to the metabolic needs
of the area of the body it is servicing. In contrast, the
composition of arterial blood is consistent throughout the body.
For routine blood tests, venous blood is generally used.
[0214] Venous blood can be obtained from the basilic vein, cephalic
vein, or median vein. Arterial blood can be obtained from the
radial artery, brachial artery or femoral artery. A vacuum tube, a
syringe or a butterfly may be used to draw the blood. Typically,
the puncture site is cleaned, a tourniquet is applied approximately
3-4 inches above the puncture site, a needle is inserted at about a
15-45 degree angle, and if using a vacuum tube, the tube is pushed
into the needle holder as soon as the needle penetrates the wall of
the vein. When finished collecting the blood, the needle is removed
and pressure is maintained on the puncture site. Usually, heparin
or another type of anticoagulant is in the tube or vial that the
blood is collected in so that the blood does not clot. When
collecting arterial blood, anesthetics can be administered prior to
collection.
[0215] The amount of blood collected will vary depending upon the
site of collection, the amount required for a method of the
invention, and the comfort of the subject. However, an advantage of
one embodiment of the present invention is that the amount of blood
required to implement the methods of the present invention can be
so small that more invasive procedures are not required to obtain
the sample. For example, in some embodiments, all that is required
is a drop of blood. This drop of blood can be obtained, for
example, from a simple pinprick. In some embodiments, any amount of
blood is collected that is sufficient to detect the expression of
one, two, three, four, five, ten or more genes listed in Table 1.
As such, in some embodiments, the amount of blood that is collected
is 1 .mu.l or less, 0.5 .mu.l or less, 0.1 .mu.l or less, or 0.01
.mu.l or less. However, the present invention is not limited to
such embodiments. In some embodiments more blood is available and
in some embodiments, more blood can be used to effect the methods
of the present invention. As such, in various specific embodiments,
0.001 ml, 0.005 ml, 0.01 ml, 0.05 ml, 0.1 ml, 0.15 ml, 0.2 ml, 0.25
ml, 0.5 ml, 0.75 ml, 1 ml, 1.5 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml,
15 ml or more of blood is collected from a subject. In another
embodiment, 0.001 ml to 15 ml, 0.01 ml to 10 ml, 0.1 ml to 10 ml,
0.1 ml to 5 ml, 1 to 5 ml of blood is collected from a subject.
[0216] In some embodiments of the present invention, blood is
stored within a K3/EDTA tube. In another embodiment, one can
utilize tubes for storing blood which contain stabilizing agents
such as disclosed in U.S. Pat. No. 6,617,170 (which is incorporated
herein by reference). In another embodiment the PAXgene.TM. blood
RNA system:provided by PreAnalytiX, a Qiagen/BD company may be used
to collect blood. In yet another embodiment, the Tempus.TM. blood
RNA collection tubes, offered by Applied Biosystems may be used.
Tempus.TM. collection tubes provide a closed evacuated plastic tube
containing RNA stabilizing reagent for whole blood collection.
[0217] The collected blood collected is optionally but preferably
stored at refrigerated temperatures, such 4.degree. C. prior to use
in accordance with the methods of the invention. In some
embodiments, a portion of the blood sample is used in accordance
with the invention at a first instance of time whereas one or more
remaining portions of the blood sample is stored for a period of
time for later use. This period of time can be an hour or more, a
day or more, a week or more, a month or more, a year or more, or
indefinitely. For long term storage, storage methods well known in
the art, such as storage at cryo temperatures (e.g. below
-60.degree. C.) can be used. In some embodiments, in addition to
storage of the blood or instead of storage of the blood, isolated
nucleic acid or proteins are stored for a period of time for later
use. Storage of such molecular markers can be for an hour or more,
a day or more, a week or more, a month or more, a year or more, or
indefinitely.
[0218] In one aspect, whole blood is obtained from a normal
individual or from an individual diagnosed with, or suspected of
having osteoarthritis according the methods of phlebotomy well
known in the art. Whole blood includes blood which can be used
directly, and includes blood wherein the serum or plasma has been
removed and the RNA or mRNA from the remaining blood sample has
been isolated in accordance with methods well known in the art
(e.g. using, preferably, gentle centrifugation at 300 to
800.times.g for 5 to 10 minutes). In a specific embodiment, whole
blood (i.e., unseparated blood) obtained from a subject is mixed
with lysing buffer (e.g., Lysis Buffer (1 L): 0.6 g EDTA; 1.0 g
KHCO.sub.2, 8.2 g NH.sub.4Cl adjusted to pH 7.4 (using NaOH)), the
sample is centrifuged and the cell pellet retained, and RNA or mRNA
extracted in accordance with methods known in the art ("lysed
blood") (see for example Sambrook et al.). The use of whole blood
is preferred since it avoids the costly and time-consuming need to
separate out the cell types within the blood (Kimoto, 1998. Mol.
Gen. Genet 258:233-239; Chelly J et al., 1989, Proc. Nat. Acad.
Sci. USA 86:2617-2621; Chelly J et al., 1988. Nature
333:858-860).
[0219] In some embodiments of the present invention, whole blood
collected from a subject is fractionated (i.e., separated into
components). In specific embodiments of the present invention,
blood cells are separated from whole blood collected from a subject
using techniques known in the art. For example, blood collected
from a subject can be subjected to Ficoll-Hypaque (Pharmacia)
gradient centrifugation. Such centrifugation separates erythrocytes
(red blood cells) from various types of nucleated cells and from
plasma. In particular, Ficoll-Hypaque gradient centrifugation is
useful to isolate peripheral blood leukocytes (PBLs) which can be
used in accordance with the methods of the invention.
[0220] By way of example but not limitation, macrophages can be
obtained as follows. Mononuclear cells are isolated from peripheral
blood of a subject, by syringe removal of blood followed by
Ficoll-Hypaque gradient centrifugation. Tissue culture dishes are
pre-coated with the subject's own serum or with AB+ human serum and
incubated at 37.degree. C. for one hour. Non-adherent cells are
removed by pipetting. Cold (4.degree. C.) 1 mM EDTA in
phosphate-buffered saline is added to the adherent cells left in
the dish and the dishes are left at room temperature for fifteen
minutes. The cells are harvested, washed with RPMI buffer and
suspended in RPMI buffer. Increased numbers of macrophages can be
obtained by incubating at 37.degree. C. with macrophage-colony
stimulating factor (M-CSF). Antibodies against macrophage specific
surface markers, such as Mac-1, can be labeled by conjugation of an
affinity compound to such molecules to facilitate detection and
separation of macrophages. Affinity compounds that can be used
include but are not limited to biotin, photobiotin, fluorescein
isothiocyante (FITC), or phycoerythrin (PE), or other compounds
known in the art. Cells retaining labeled antibodies are then
separated from cells that do not bind such antibodies by techniques
known in the art such as, but not limited to, various cell sorting
methods, affinity chromatography, and panning.
[0221] Blood cells can be sorted using a using a fluorescence
activated cell sorter (FACS). Fluorescence activated cell sorting
(FACS) is a known method for separating particles, including cells,
based on the fluorescent properties of the particles. See, for
example, Kamarch, 1987, Methods Enzymol 151:150-165. Laser
excitation of fluorescent moieties in the individual particles
results in a small electrical charge allowing electromagnetic
separation of positive and negative particles from a mixture. An
antibody or ligand used to detect a blood cell antigenic
determinant present on the cell surface of particular blood cells
is labeled with a fluorochrome, such as FITC or phycoerythrin. The
cells are incubated with the fluorescently labeled antibody or
ligand for a time period sufficient to allow the labeled antibody
or ligand to bind to cells. The cells are processed through the
cell sorter, allowing separation of the cells of interest from
other cells. FACS sorted particles can be directly deposited into
individual wells of microtiter plates to facilitate separation.
[0222] Magnetic beads can be also used to separate blood cells in
some embodiments of the present invention. For example, blood cells
can be sorted using a using a magnetic activated cell sorting
(MACS) technique, a method for separating particles based on their
ability to bind magnetic beads (0.5-100 m diameter). A variety of
useful modifications can be performed on the magnetic microspheres,
including covalent addition of an antibody which specifically
recognizes a cell-solid phase surface molecule or hapten. A
magnetic field is then applied, to physically manipulate the
selected beads. In a specific embodiment, antibodies to a blood
cell surface marker are coupled to magnetic beads. The beads are
then mixed with the blood cell culture to allow binding. Cells are
then passed through a magnetic field to separate out cells having
the blood cell surface markers of interest. These cells can then be
isolated.
[0223] In some embodiments, the surface of a culture dish may be
coated with antibodies, and used to separate blood cells by a
method called panning. Separate dishes can be coated with antibody
specific to particular blood cells. Cells can be added first to a
dish coated with blood cell specific antibodies of interest. After
thorough rinsing, the cells left bound to the dish will be cells
that express the blood cell markers of interest. Examples of cell
surface antigenic determinants or markers include, but are not
limited to, CD2 for T lymphocytes and natural killer cells. CD3 for
T lymphocytes, CD 11a for leukocytes. CD28 for T lymphocytes, CD19
for B lymphocytes. CD20 for B lymphocytes. CD21 for B lymphocytes.
CD22 for B lymphocytes, CD23 for B lymphocytes. CD29 for
leukocytes, CD14 for monocytes, CD41 for platelets, CD61 for
platelets, CD66 for granulocytes. CD67 for granulocytes and CD68
for monocytes and macrophages.
[0224] Whole blood can be separated into cells types such as
leukocytes, platelets, erythrocytes, etc. and such cell types can
be used in accordance with the methods of the invention. Leukocytes
can be further separated into granulocytes and agranulocytes using
standard techniques and such cells can be used in accordance with
the methods of the invention. Granulocytes can be separated into
cell types such as neutrophils, eosinophils, and basophils using
standard techniques and such cells can be used in accordance with
the methods of the invention. Agranulocytes can be separated into
lymphocytes (e.g., T lymphocytes and B lymphocytes) and monocytes
using standard techniques and such cells can be used in accordance
with the methods of the invention. T lymphocytes can be separated
from B lymphocytes and helper T cells separated from cytotoxic T
cells using standard techniques and such cells can be used in
accordance with the methods of the invention. Separated blood cells
(e.g., leukocytes) can be frozen by standard techniques prior to
use in the present methods.
[0225] A blood sample that is useful according to the invention is
in an amount that is sufficient for the detection of one or more
nucleic acid or amino acid sequences according to the invention. In
a specific embodiment, a blood sample useful according to the
invention is in an amount ranging from 1 .mu.l to 100 ml,
preferably 10 .mu.l to 50 ml, more preferably 10 .mu.l to 25 ml and
most preferably 10 .mu.l to 1 ml.
[0226] 5.1.4 RNA Preparation
[0227] In one aspect of the invention, RNA is isolated from an
individual in order to measure the RNA products of the biomarkers
of the invention. RNA is isolated from cartilage samples from
various disease or developmental stages as described herein.
Samples can be from a single patient or can be pooled from multiple
patients.
[0228] In another aspect. RNA is isolated directly from synovial
fluid of persons with various disease or developmental stages of
osteoarthritis as described herein. Samples can be from a single
patient or can be pooled from multiple patients.
[0229] In another aspect, RNA is isolated directly from blood
samples of persons with various disease or developmental stages of
osteoarthritis as described herein. Samples can be from a single
patient or can be pooled from multiple patients.
[0230] Total RNA is extracted from the cartilage samples according
to methods well known in the art. In one embodiment, RNA is
purified from cartilage tissue according to the following method.
Following removal of a tissue of interest from an individual or
patient, the tissue is quick frozen in liquid nitrogen, to prevent
degradation of RNA. Upon the addition of a volume of tissue
guanidinium solution, tissue samples are ground in a tissuemizer
with two or three 10-second bursts. To prepare tissue guanidinium
solution (1 L) 590.8 g guanidinium isothiocyanate is dissolved in
approximately 400 ml DEPC-treated H.sub.2O. 25 ml of 2 M Tris-Cl,
pH 7.5 (0.05 M final) and 20 ml Na.sub.2EDTA (0.01 M final) is
added, the solution is stirred overnight, the volume is adjusted to
950 ml, and 50 ml 2-ME is added.
[0231] Homogenized tissue samples are subjected to centrifugation
for 10 min at 12,000.times.g at 12.degree. C. The resulting
supernatant is incubated for 2 min at 65.degree. C. in the presence
of 0.1 volume of 20% Sarkosyl, layered over 9 ml of a 5.7M CsCl
solution (0.1 g CsCl/ml), and separated by centrifugation overnight
at 113,000.times.g at 22.degree. C. After careful removal of the
supernatant, the tube is inverted and drained. The bottom of the
tube (containing the RNA pellet) is placed in a 50 ml plastic tube
and incubated overnight (or longer) at 4.degree. C. in the presence
of 3 ml tissue resuspension buffer (5 mM EDTA, 0.5% (v/v) Sarkosyl,
5% (v/v) 2-ME) to allow complete resuspension of the RNA pellet.
The resulting RNA solution is extracted sequentially with 25:24:1
phenol/chloroform/isoamyl alcohol, followed by 24:1
chloroform/isoamyl alcohol, precipitated by the addition of 3 M
sodium acetate, pH 5.2, and 2.5 volumes of 100% ethanol, and
resuspended in DEPC water (Chirgw in et al., 1979. Biochemistry,
18:5294).
[0232] Alternatively, RNA is isolated from cartilage tissue
according to the following single step protocol. The tissue of
interest is prepared by homogenization in a glass teflon
homogenizer in 1 ml denaturing solution (4M guanidinium
thiosulfate, 25 mM sodium citrate, pH 7.0, 0.1M 2-ME, 0.5% (w/v)
N-laurylsarkosine) per 100 mg tissue. Following transfer of the
homogenate to a 5-ml polypropylene tube, 0.1 ml of 2 M sodium
acetate, pH 4, 1 ml water-saturated phenol, and 0.2 ml of 49:1
chloroform/isoamyl alcohol are added sequentially. The sample is
mixed after the addition of each component, and incubated for 15
min at 0-4.degree. C. after all components have been added. The
sample is separated by centrifugation for 20 min at 10,000.times.g,
4.degree. C. precipitated by the addition of 1 ml of 100%
isopropanol, incubated for 30 minutes at -20.degree. C. and
pelleted by centrifugation for 10 minutes at 10,000.times.g,
4.degree. C. The resulting RNA pellet is dissolved in 0.3 ml
denaturing solution, transferred to a microfuge tube, precipitated
by the addition of 0.3 ml of 100% isopropanol for 30 minutes at
-20.degree. C., and centrifuged for 10 minutes at 10,000.times.g at
4.degree. C. The RNA pellet is washed in 70% ethanol, dried, and
resuspended in 100-200 .mu.l DEPC-treated water or DEPC-treated
0.5% SDS (Chomczynski and Sacchi, 1987, Anal. Biochem.,
162:156).
[0233] Preferably, the cartilage samples are finely powdered under
liquid nitrogen and total RNA is extracted using TRIzol.RTM.
reagent (GIBCO/BRL).
[0234] Alternatively, RNA is isolated from blood by the following
protocol. Lysis Buffer is added to blood sample in a ratio of 3
parts Lysis Buffer to 1 part blood (Lysis Buffer (IL) 0.6 g EDTA;
1.0 g KHCO.sub.2, 8.2 g NH.sub.4Cl adjusted to pH 7.4 (using
NaOH)). Sample is mixed and placed on ice for 5-10 minutes until
transparent. Lysed sample is centrifuged at 1000 rpm for 10 minutes
at 4.degree. C. and supernatant is aspirated. Pellet is resuspended
in 5 ml Lysis Buffer, and centrifuged again at 1000 rpm for 10
minutes at 4.degree. C. Pelleted cells are homogenized using
TRIzol.RTM. (GIBCO/BRL) in a ratio of approximately 6 ml of
TRIzol.RTM. for every 10 ml of the original blood sample and
vortexed well. Samples are left for 5 minutes at room temperature.
RNA is extracted using 1.2 ml of chloroform per 1 ml of
TRIzol.RTM.. Sample is centrifuged at 12,000.times.g for 5 minutes
at 4.degree. C. and upper layer is collected. To upper layer,
isopropanol is added in ratio of 0.5 ml per 1 ml of TRIzol.RTM..
Sample is left overnight at -20.degree. C. or for one hour at
-20.degree. C. RNA is pelleted in accordance with known methods.
RNA pellet air dried, and pellet resuspended in DEPC treated
ddH.sub.2O. RNA samples can also be stored in 75% ethanol where the
samples are stable at room temperature for transportation.
[0235] Alternatively. RNA is isolated from synovial fluid using
TRIzol.RTM. reagent (GIBCO/BRL) as above.
[0236] Purity and integrity of RNA is assessed by absorbance at
260/280 nm and agarose gel electrophoresis followed by inspection
under ultraviolet light.
[0237] 5.2 Biomarkers of the Invention
[0238] In one embodiment, the invention provides biomarkers and
biomarker combinations wherein the measure of the level of
expression of the product or products of said biomarkers is
indicative of the existence of osteoarthritis. In another
embodiment, the invention provides biomarkers and biomarker
combinations, wherein the measure of the level of expression of the
product or products of said biomarkers can be used to diagnose
whether an individual has one of two stages of osteoarthritis. In
yet another embodiment, the invention provides biomarkers and
biomarker combinations, wherein the measure of the level of
expression of the product or products of said biomarkers can be
used to diagnose an individual as having a specific stage of
osteoarthritis as compared with any other stage of
osteoarthritis.
[0239] Table 1 provides a list of the gene names and the associated
locus link ID for the biomarkers of the invention wherein the
measure of the level of expression of the biomarkers, either
individually, or in combination can be used to diagnose an
individual as having osteoarthritis, diagnose whether an individual
has one of two stages of osteoarthritis or diagnose an individual
as having a specific stage of osteoarthritis. As would be
understood by a person skilled in the art, the locus link ID can be
used to determine the sequence of all the RNA transcripts and all
of the proteins which correspond to the biomarkers of the
invention. Table 2 provides, in one embodiment of the invention,
sequences of the RNA transcripts whose sequences can be used to
measure the level of expression of the biomarkers of the invention
using those techniques known to a person skilled in the art. Table
2 also provides, in one embodiment of the invention, sequences of
the proteins which can be used to measure the level of expression
of the biomarkers of the invention. The invention thus encompasses
the use of those methods known to a person skilled in the art to
measure the expression of these biomarkers and combinations of
biomarkers for each of the purposes outlined above.
[0240] 5.3 Combinations of Biomarkers
[0241] In one embodiment, combinations of biomarkers of the present
invention includes any combination of 2, 3, 4, 5, 6, 7, 8, 10, 15,
20, 30, 40 or all of the biomarkers listed in Table 1. In another
embodiment of the invention, combinations of biomarkers of the
present invention include any combination of the biomarkers
selected from the list in FIG. 1 the measurement of expression of
the products of which can be used for diagnosing whether an
individual has mild osteoarthritis or does not have osteoarthritis.
In another embodiment of the invention, combinations of biomarkers
of the present invention include any combination of the biomarkers
selected from the list in FIG. 2 the measurement of expression of
the products of which can be used in diagnosing whether an
individual has moderate osteoarthritis or does not have
osteoarthritis. In another embodiment of the invention,
combinations of biomarkers of the present invention include any
combination of the biomarkers selected in FIG. 3, the measurement
of expression of the products of which can be used for use in
diagnosing whether an individual has moderate osteoarthritis or has
mild osteoarthritis. In another embodiment of the invention,
combinations of biomarkers of the present invention include any
combination of the biomarkers selected from the list in FIG. 4 the
measurement of expression of the products of which can be used for
diagnosing whether an individual has marked osteoarthritis or has
severe osteoarthritis.
[0242] For instance, the number of possible combinations of a
subset m of n genes is described in Feller, Intro to Probability
Theory, Third Edition, volume 1, 1968, ed. J. Wiley, using the
general formula:
m!/(n)!(m-n)!
[0243] In one embodiment, where n is 2 and in is 19, there are:
19 ! 2 ! ( 19 - 2 ) ! = 19 .times. 18 .times. 17 .times. 16 .times.
15 .times. 14 .times. 13 .times. 12 .times. 11 .times. 10 .times. 9
.times. 8 .times. 7 .times. 6 .times. 5 .times. 4 .times. 3 .times.
2 .times. 1 ( 2 .times. 1 ) ( 19 .times. 18 .times. 17 .times. 16
.times. 15 .times. 14 .times. 13 .times. 12 .times. 11 .times. 10
.times. 9 .times. 8 .times. 7 .times. 6 .times. 5 .times. 4 .times.
3 .times. 2 .times. 1 ) = 1.21610 17 7.1110 14 = 171
##EQU00001##
unique two-gene combinations. The measurement of the gene
expression of each of these two-gene combinations can independently
be used to determine whether a patient has osteoarthritis. In
another specific embodiment in which in is 19 and n is three, there
are 19!/3!(19-3)! unique three-gene combinations. Each of these
unique three-gene combinations can independently serve as a model
for determining whether a patient has osteoarthritis.
[0244] 5.4 Particularly Useful Combinations of Biomarkers
[0245] Although all of the combinations of the biomarkers of the
invention are useful for diagnosing OA, the invention further
provides a means of selecting those combinations of biomarkers
particularly useful for each of the following (a) diagnosing
individuals as having osteoarthritis, (b) differentiating between
two stages of osteoarthritis (OA) and (c) diagnosing individuals as
having a particular stage of osteoarthritis (OA). The invention
further provides a method of evaluating the combinations identified
for each of the utilities described above.
[0246] In order to identify useful combinations of biomarkers a
mathematical model of the invention is used to test each of the
possible combinations of the biomarkers of the invention for each
combinations ability to separate as between the two (e.g. binary
models such as logistic regression) or more (e.g. models such as
neural networks) phenotypic traits of a training population used
for input into the model. The phenotypic traits of the training
population used for input into the model are phenotypic traits for
use in (a) diagnosing as OA; (b) diagnosed as having mild OA (c)
diagnosed as having moderate OA (d) diagnosed as having severe OA
(e) does not have OA. The phenotypic traits of the training
population used for input into the mathematical model, and the
model used, will determine the utility of the combinations
generated by the model as a diagnostic for one of the following a)
diagnosing individuals as having osteoarthritis, (b)
differentiating between two stages of osteoarthritis (OA) and (c)
diagnosing individuals as having a particular stage of
osteoarthritis (OA). The result of the choice of phenotypic traits
of the training population used for entry into the mathematical
model is described more thoroughly below.
[0247] The mathematical model generated can be subsequently
evaluated by determining the ability of the model to correctly call
each individual for one of the two (or more) phenotypic traits of
the population used for input into the model. In a preferred
embodiment, the individuals of the training population used to
derive the model are different from the individuals of the training
population used to test the model. As would be understood by a
person skilled in the art, this allows one to predict the ability
of the combinations as to their ability to properly characterize an
individual whose phenotypic characterization is unknown.
[0248] The data which is input into the mathematical model can be
any data which is representative of the expression level of the
product of the biomarkers being evaluated. Mathematical models
useful in accordance with the invention include those using both
supervised or unsupervised learning. In a preferred embodiment of
the invention, the mathematical model chosen uses supervised
learning in conjunction with a "training population" evaluate each
of the possible combination of biomarkers of the invention. In one
embodiment of the invention, the mathematical model used is
selected from the following: a regression model, a logistic
regression model, a neural network, a clustering model, principal
component analysis, nearest neighbour classifier analysis, linear
discriminant analysis, quadratic discriminant analysis, a support
vector machine, a decision tree, a genetic algorithm, classifier
optimization using bagging, classifier optimization using boosting,
classifier optimization using the Random Subspace Method, a
projection pursuit, and weighted voting. In a preferred embodiment,
a logistic regression model is used. In another preferred
embodiment, a neural network model is used.
[0249] The resulting mathematical model can be used to diagnosis an
unknown or test individual for the phenotypic trait used for input
into the model. In a preferred embodiment, the diagnosis result
from equations generated by logistic regression to answer one of
the following questions: (a) does an individual have
osteoarthritis, (b) which of two stages of osteoarthritis does an
individual have (wherein not having osteoarthritis is considered a
stage of OA) and (c) does an individual have a specific stage of
osteoarthritis. In yet another embodiment of the invention, the
answer to any of the questions above may be an answer of non
determinable.
[0250] In one preferred embodiment of the invention, each model is
evaluated for its ability to properly characterize each individual
of the training population using those methods known to a person
skilled in the art. For example one can evaluate the model using
cross validation Leave One out Cross Validation, n-fold cross
validation, jackknife analysis using standard statistical methods
and disclosed. In an even more preferred embodiment of the
invention, each model is evaluated for its ability to properly
characterize those individuals of the training population which
were not used to izenerate the model.
[0251] In one embodiment, the method used to evaluate the model for
its ability to properly characterize each individual of the
training population is a method which evaluates the models
sensitivity (TPF, true positive fraction) and 1-specificity (TNF,
true negative fraction). In a preferred embodiment, the method used
to test the model is Receiver Operating Characteristic ("ROC")
which provides several parameters to evaluate both the sensitivity
and specificity of the diagnostic result of the equation generated.
In a particularly preferred embodiment, the ROC area (area under
the curve) is used to evaluate the equations. In a preferred
embodiment, an ROC area greater than 0.5, 0.6, 0.7, 0.8, 0.9 is
preferred. A perfect ROC area score of 1.0 on the other hand
indicates with both 100% sensitivity and 100% specificity.
[0252] As would be understood by a person skilled in the art, the
utility of the combinations and equations determined by a
mathematical model will depend upon the phenotypes of the
populations used to generate the data for input into the model.
Examples of specific embodiments are described more thoroughly
herein.
[0253] 5.5 Populations for Input into the Mathematical Models
[0254] In some embodiments, the reference or training population
includes between 10 and 30 subjects. In another embodiment the
training population contains between 30-50 subjects. In still other
embodiments, the reference population includes two or more
populations each containing between 50 and 100, 100 and 500,
between 500 and 1000, or more than 1000 subjects.
[0255] The mathematical model used will also determine how many
phenotypic traits can be contained within the reference population.
For example some models (binary mathematical models) can only
identify biomarker combinations useful to differentiate as between
two phenotypic trait (e.g. logistic regression). In other models,
biomarker combinations which are useful to differentiate as between
three or more phenotypic traits can be used (e.g. neural networks).
In another embodiment of the invention, one can use combinations of
binary decisions using binary models to differentiate multiple
groups.
[0256] 5.6 Populations to Identify Biomarkers for Diagnosis of
Osteoarthritis for Input into Binary Mathematical Models
[0257] For example, in order to identify those biomarkers which are
useful in diagnosing an individual as having osteoarthritis, or not
having osteoarthritis, data reflective of the level of expression
of all of the mRNA products of the biomarkers of Table 1 are used
from a population of individuals having osteoarthritis, and a
second population of individuals not having osteoarthritis are
used. For purposes of characterizing the populations as having or
not having osteoarthritis, any traditional method of OA diagnosis
can be used. In a preferred embodiment, the scoring method of
Marshall as described herein is used. In one embodiment of the
invention, a population of individuals considered to have
osteoarthritis includes any individual who is scored as having mild
OA, moderate OA, marked OA or severe OA. Similarly population of
individuals not having OA are chosen according to similar methods.
In a preferred embodiment, the phenotypic characteristics of the
two populations used in the training set are similar but for having
or not having OA. In another preferred embodiment, the two
populations are at least age, sex and BMI (body mass index)
matched.
[0258] In another embodiment, in order to identify those biomarkers
which are useful in diagnosing an individual as having
osteoarthritis, or not having osteoarthritis, data reflective of
the level of expression of one or more of the mRNA products of the
biomarkers of Table 1 resulting from a population of individuals
having osteoarthritis, and the second population of individuals
does not have osteoarthritis are used.
[0259] In another embodiment, in order to identify those biomarkers
which are useful in diagnosing an individual as having
osteoarthritis, or not having osteoarthritis, data reflective of
the level of expression one or more of the mRNA products of the
biomarkers of Table 1 as noted in Table 2 resulting from a
population of individuals having osteoarthritis, and a second
population of individuals not having osteoarthritis are used.
[0260] In another embodiment, in order to identify those biomarkers
which are useful in diagnosing an individual as having
osteoarthritis, or not having osteoarthritis, data reflective of
the level of expression of all of the RNA products of the
biomarkers of Table 1 which are expressed in blood resulting from a
population of individuals having osteoarthritis, and a second
population of individuals not having osteoarthritis are used.
[0261] 5.7 Populations to Identify Biomarkers for Differentiation
as Between Stages of Osteoarthritis Using Binary Mathematical
Models
[0262] In one embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having mild osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of all
of the mRNA products of the biomarkers of Table 1 resulting from a
population of individuals identified by any traditional method of
OA diagnosis as having mild osteoarthritis, and the a second
population of individuals not having osteoarthritis are used.
[0263] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having mild osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of one
or more of the mRNA products of the biomarkers of Table 1 from a
population of individuals identified by any traditional method of
OA diagnosis as having mild osteoarthritis, and the second
population of individuals not having osteoarthritis are used.
[0264] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having mild osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of the
mRNA products from the biomarkers of Table 1 as disclosed in Table
2 from a population of individuals identified by any traditional
method of OA diagnosis as having mild osteoarthritis, and the
second population of individuals not having osteoarthritis are
used.
[0265] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having mild osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of the
mRNA products of the biomarkers of Table 1 which are expressed in
blood resulting from a population of individuals identified by any
traditional method of OA diagnosis as having mild osteoarthritis,
and the second population of individuals not having osteoarthritis
are used.
[0266] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having mild osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of all
of the mRNA products of the biomarkers of FIG. 1 resulting from a
population of individuals identified by any traditional method of
OA diagnosis as having mild osteoarthritis, and the second
population of individuals not having osteoarthritis are used.
[0267] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having moderate osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of all
of the product of the mRNA of the biomarkers of Table 1 resulting
from a population of individuals identified by any traditional
method of OA diagnosis as having moderate osteoarthritis, and the
second population of individuals not having osteoarthritis are
used.
[0268] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having moderate osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of one
or more of the mRNA products of the biomarkers of Table 1 from a
population of individuals identified by any traditional method of
OA diagnosis as having moderate osteoarthritis, and the second
population of individuals not having osteoarthritis are used.
[0269] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having moderate osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of the
mRNA products of the biomarkers of Table 1 which are expressed in
blood resulting from a population of individuals identified by any
traditional method of OA diagnosis as having moderate
osteoarthritis, and the second population of individuals not having
osteoarthritis are used.
[0270] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having moderate osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of the
mRNA products of the biomarkers of Table 1 as reflected in Table 2
resulting from a population of individuals identified by any
traditional method of OA diagnosis as having moderate
osteoarthritis, and the second population of individuals not having
osteoarthritis are used.
[0271] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in diagnosing an
individual as having moderate osteoarthritis, or not having
osteoarthritis, data reflective of the level of expression of the
mRNA products of the biomarkers of FIG. 2 resulting from a
population of individuals identified by any traditional method of
OA diagnosis as having moderate osteoarthritis, and the second
population of individuals not having osteoarthritis are used.
[0272] Similarly, combinations of biomarkers useful to
differentiate between individuals as having marked OA or no OA;
having severe OA or no OA; mild OA or moderate OA; mild OA or
marked OA, mild OA or severe OA; moderate OA or marked OA; moderate
OA or severe OA; and marked OA or severe OA can be identified using
similar methods to those outlined above.
[0273] 5.8 Populations to Identify Biomarkers for Diagnosing Stage
Specific OA for Input into Binary Mathematical Models
[0274] In one embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in identifying
whether an individual has mild OA as compared to any other stage of
OA (ie identifying biomarkers to diagnose the stage of an
individual's OA) data reflective of the level of expression of the
mRNA products of each of the biomarkers of Table 1 resulting from a
population of individuals identified by any traditional method of
OA diagnosis as having mild osteoarthritis, and the second
population of individuals which is a combination of individuals
having moderate OA, individuals having marked OA, individuals
having severe OA, and individuals not having OA are used.
Preferably individuals as between both populations are matched for
age, sex and BMI.
[0275] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in identifying
whether an individual has mild OA as compared to any other stage of
OA (ie identifying biomarkers to diagnose an individual with a
specific stage of OA) data reflective of the level of expression of
each of the possible variants of biomarkers of Table 1 resulting
from a population of individuals identified by any traditional
method of OA diagnosis as having mild osteoarthritis, and the
second population of individuals which is a combination of
individuals having moderate OA, individuals having marked OA,
individuals having severe OA, and individuals not having OA are
used. Preferably individuals as between both populations are
matched for age, sex and BMI.
[0276] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in identifying
whether an individual has mild OA as compared to any other stage of
OA (ie identifying biomarkers to diagnose an individual with a
specific stage of OA) data reflective of the level of expression
each of the possible variants of biomarkers of Table 1 as selected
from the list provided in Table 2 resulting from a population of
individuals identified by any traditional method of OA diagnosis as
having mild osteoarthritis, and the second population of
individuals which is a combination of individuals having moderate
OA, individuals having marked OA, individuals having severe OA, and
individuals not having OA are used. Preferably individuals as
between both populations are matched for age, sex and BMI.
[0277] In another embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in identifying
whether an individual has mild OA as compared to any other stage of
OA (ie identifying biomarkers to diagnose an individual with a
specific stage of OA) data reflective of the level of expression of
those variants of biomarkers of Table 1 expressed in blood
resulting from a population of individuals identified by any
traditional method of OA diagnosis as having mild osteoarthritis,
and the second population of individuals which is a combination of
individuals having moderate OA, individuals having marked OA,
individuals having severe OA, and individuals not having OA are
used. Preferably individuals as between both populations are
matched for age, sex and BMI.
[0278] Similarly, combinations of biomarkers useful to
differentiate between individuals as having moderate OA as compared
with any other stage of OA; marked OA as compared with any other
stage of OA; and severe OA as compared with any other stage of OA
can be identified using data from populations using similar methods
to those outlined above.
[0279] 5.9 Populations to Identify Biomarkers for Diagnosing Stage
Specific OA for Input into Non Binary Mathematical Models
[0280] In one embodiment of the invention, in order to identify
those combinations of biomarkers which are useful in identifying
whether an individual has mild OA as compared to any other stage of
OA, data reflective of the level of expression of each of the
biomarkers of Table 1 resulting from the following populations of
individuals are used (a) individuals having mild OA (b) individuals
having moderate OA (c) individuals having marked OA (d) individuals
having severe OA. The populations are made up of individuals
identified by any traditional method of OA diagnosis but preferably
the scoring method of Marshall (supra) is used. Preferably
individuals as between the populations are matched for age, sex and
BMI.
[0281] Similarly, combinations of biomarkers useful to characterize
an individual as having moderate OA as compared with any other
stage of OA; marked OA as compared with any other stage of OA; and
severe OA as compared with any other stage of OA can be identified
using similar methods to those outlined above.
[0282] 5.10 Regression Models
[0283] In some embodiments the expression data for some or all of
the possible combination of biomarkers identified in the present
invention are used in a regression model, preferably a logistic
regression model. Such a regression model will determine one or
more equations for each possible combination of biomarkers, each
equation providing a coefficient for each of the biomarkers
represented by the model.
[0284] In general, the multiple regression equation of interest can
be written
Y+.alpha.+.beta..sub.1X.sub.1+.beta..sub.2X.sub.2+ . . .
+.beta..sub.kX.sub.k+.epsilon.
where Y, the dependent variable, is presence (when Y is positive)
or absence (when Y is negative) of the biological feature (e.g.,
absence/presence/stage of osteoarthritis) associated with the first
subgroup. This model says that the dependent variable Y depends on
k explanatory variables (the measured characteristic values for the
k select genes from subjects in the first and second subgroups in
the training data set), plus an error term that encompasses various
unspecified omitted factors. In the above-identified model, the
parameter .beta..sub.1 gauges the effect of the first explanatory
variable X.sub.1 on the dependent variable Y, holding the other
explanatory variables constant. Similarly, .beta..sub.2 gives the
effect of the explanatory variable X.sub.2 on Y, holding the
remaining explanatory variables constant.
[0285] The logistic regression model is a non-linear transformation
of the linear regression. The logistic regression model is termed
the "logit" model and can be expressed as
ln[p/(1-p)]=.alpha..beta..sub.1X.sub.1+.beta..sub.2X.sub.2+ . . .
+.beta..sub.kX.sub.k+.epsilon. or
[p/(1-p)]=exp.sup..alpha.exp.sup..beta..sup.1.sup.X.sup.1exp.sup..beta..-
sup.2.sup.X.sup.2.times. . . . .times.exp.sup..epsilon.
where,
[0286] ln is the natural logarithm, log.sup.exp, where exp=2.71828
. . .
[0287] p is the probability that the event Y occurs, p(Y=1),
[0288] p/(1-p) is the "odds ratio",
[0289] ln[p/(1-p)] is the log odds ratio, or "logit", and
[0290] all other components of the model are the same as the
general regression equation described above. It will be appreciated
by those of skill in the art that the term for .alpha. and
.epsilon. can be folded into the same constant. Indeed, in
preferred embodiments, a single term is used to represent .alpha.
and .epsilon.. The "logistic" distribution is an S-shaped
distribution function. The logit distribution constrains the
estimated probabilities (p) to lie between 0 and 1.
[0291] In some embodiments of the present invention, the logistic
regression model is fit by maximum likelihood estimation (MLE). In
other words, the coefficients (e.g., .alpha., .beta..sub.1,
.delta..sub.2 . . . ) are determined by maximum likelihood. A
likelihood is a conditional probability (e.g., P(Y|X), the
probability of Y given X). The likelihood function (L) measures the
probability of observing the particular set of dependent variable
values (Y.sub.1, Y.sub.2, . . . , Y.sub.n) that occur in the sample
data set. It is written as the probability of the product of the
dependent variables:
L=Prob(Y.sub.1*Y.sub.2***Y.sub.n)
The higher the likelihood function, the higher the probability of
observing the Ys in the sample. MLE involves finding the
coefficients (.alpha., .beta..sub.1, .beta..sub.2 . . . ) that
makes the log of the likelihood function (LL<0) as large as
possible or -2 times the log of the likelihood function (-2 LL) as
small as possible. In MLE, some initial estimates of the parameters
.alpha., .beta..sub.1, .beta..sub.2 . . . are made. Then the
likelihood of the data given these parameter estimates is computed.
The parameter estimates are improved the likelihood of the data is
recalculated. This process is repeated until the parameter
estimates do not change much (for example, a change of less than
0.01 or 0.001 in the probability). Examples of logistic regression
and fitting logistic logistic regression models are found in
Hastie, The Elements of Statistical Learning, Springer, N.Y., 2001,
pp. 95-100 which is incorporated herein in its entirety.
[0292] 5.11 Neural Networks
[0293] In another embodiment, the expression measured for each of
the biomarkers of the present invention can be used to train a
neural network. A neural network is a two-stage regression or
classification model. A neural network has a layered structure that
includes a layer of input units (and the bias) connected by a layer
of weights to a layer of output units. For regression, the layer of
output units typically includes just one output unit. However,
neural networks can handle multiple quantitative responses in a
seamless fashion. As such a neural network can be applied to allow
identification of biomarkers which differentiate as between more
than two populations. In one specific example, a neural network can
be trained using expression data from the biomarkers in Table 1 to
identify those combinations of biomarkers which are specific for a
stage of osteoarthritis (e.g mild osteoarthritis) as compared with
any other stage of osteoarthritis wherein not having osteoarthritis
can be considered a stage of osteoarthritis. As a result, the
trained neural network can be used to directly identify combination
of biomarkers useful as stage specific biomarkers. In some
embodiments, the back-propagation neural network (see, for example
Abdi, 1994, "A neural network primer", J. Biol System. 2, 247-283)
containing a single hidden layer of ten neurons (ten hidden units)
found in EasyNN-Plus version 4.0 g software package (Neural Planner
Software Inc.) is used.
[0294] Neural networks are described in Duda et al., 2001, Pattern
Classification, Second Edition, John Wiley & Sons, Inc. New
York; and Hastie et al., 2001. The Elements of Statistical
Learning, Springer-Verlag, New York which is incorporated herein in
its entirety.
[0295] 5.12 Other Mathematical Models
[0296] The pattern classification and statistical techniques
described above are merely examples of the types of models that can
be used to construct a model for OA classification, for example
clustering as described on pages 211-256 of Duda and Hart. Pattern
Classification and Scene Analysis, 1973, John Wiley & Sons.
Inc., New York, incorporated herein by reference in its entirety;
Principal component analysis, (see for Jolliffe, 1986, Principal
Component Analysis, Springer. New York, incorporated herein by
reference); nearest neighbour classifier analysis, (see for example
Duda, Pattern Classification, Second Edition, 2001, John Wiley
& Sons, Inc; and Hastie, 2001, The Elements of Statistical
Learning, Springer, New York); linear discriminant analysis, (see
for example Duda, Pattern Classification, Second Edition, 2001,
John Wiley & Sons, Inc; and Hastie, 2001, The Elements of
Statistical Learning, Springer, New York; Venables & Ripley,
1997, Modern Applied Statistics with s-plus, Springer. New York);
Support Vector Machines (see, for example, Cristianini and
Shawe-Taylor, 2000, An Introduction to Support Vector Machines,
Cambridge University Press, Cambridge, Boser et al., 1992, "A
training algorithm for optimal margin classifiers, in Proceedings
of the 5.sup.th Annual ACM Workshop on Computational Learning
Theory, ACM Press, Pittsburgh, Pa., pp. 142-152; Vapnik, 1998,
Statistical Learning Theory, Wiley, New York, incorporated herein
by reference.)
[0297] 5.13 Products of the Biomarkers of the Invention
[0298] As would be understood by a person skilled in the art, the
identification of one or more of a combination of biomarkers which
are differentially expressed during OA and or stages of OA allows
the diagnosis of OA and stages of OA by measurement of the
expression of the products of the biomarkers (gene) in an
individual.
[0299] The products of each of the biomarkers of the invention
includes both RNA and protein. RNA products of the biomarkers of
the invention include populations of hnRNA, mRNA, and one or more
spliced variants of mRNA. To practice the invention, measurement of
one or more of the populations of the RNA products of the
biomarkers of the invention can be used for purposes of diagnosis.
More particularly, measurement of those populations of RNA products
of the biomarkers which are differentially expressed during OA, or
during a stage of OA are useful for purposes of diagnosis and are
encompassed herein.
[0300] In one embodiment of the invention, the RNA products of the
biomarkers of the invention which are measured is the population of
RNA products including the hnRNA, the mRNA, and all of the spliced
variants of the mRNA. In another embodiment, the RNA products of
the biomarkers of the invention which are measured is the
population of mRNA. In another embodiment of the invention the RNA
products of the biomarkers of the invention which are measured is
the population of mRNA which is expressed in blood. In yet another
embodiment of the invention, RNA products of the biomarkers of the
invention which are measured is the population of one or more
spliced variants of the mRNA. In yet another embodiment of the
invention, RNA products of the biomarkers of the invention which
are measured is the population of one or more spliced variants of
the mRNA which are expressed in blood. In yet another embodiment of
the invention, RNA products of the biomarkers of the invention are
those products which are listed in Table 2.
[0301] Protein products of the biomarkers of the invention are also
included within the scope of the invention and include the entire
population of protein arising from a biomarker of the invention. As
would be understood by a person skilled in the art, the entire
population of proteins arising from a biomarker of the invention
include proteins, protein variants arising from spliced mRNA
variants, and post translationally modified proteins. To practice
the invention, measurement of one or more of the populations of the
protein products of the biomarkers of the invention can be used for
purposes of diagnosis. More particularly, measurement of those
populations of protein products of the biomarkers which are
differentially expressed during OA, or during a stage of OA are
useful for purposes of diagnosis and are encompassed herein.
[0302] In one embodiment of the invention the protein products of
the biomarkers of the invention which are measured is the entire
population of protein products translated from the RNA products of
the biomarkers of the invention. In another embodiment, the protein
products of the biomarkers of the invention are those protein
products which are expressed in blood. In yet another embodiment of
the invention, the protein products of the biomarkers of the
invention are one or more of the protein products translated from
one or more of the mRNA spliced variants. In yet another embodiment
of the invention, the protein products of the biomarkers of the
invention are one or more of the protein products translated from
one or more of the mRNA spliced variants expressed in blood. In yet
another embodiment of the invention, protein products of the
biomarkers of the invention are those products which are listed in
Table 2.
[0303] 5.14 Use of the Combinations Identified to Diagnose
[0304] The invention teaches the ability to identify useful
combinations of biomarkers for the purpose of diagnosing OA,
differentiating as between stages of OA and diagnosing a particular
stage of OA. Data representative of the RNA or protein products of
the biomarkers of the invention is input into a model of the
invention so as to identify particularly useful combinations. The
diagnostic purpose for which the combination is used is dependant
upon the phenotypic traits of the input population as described
herein. The combinations identified can be used with traditional
techniques for measuring the level of expression of the RNA and
protein products of the combinations to determine whether the
pattern of expression as between the individual tested and one or
more individuals from a control population (wherein the control
population would include subpopulations with phenotypic subtraits
as defined by the populations input into the model and can include
data representative of the control population). In a preferred
embodiment, one would use the model generated so as to diagnose an
individual, e.g., by the measure of the level of expression of the
RNA products of the biomarkers of the invention in a test
individual for input into the model generated to identify the
combination to determine a diagnosis as defined by the model. In a
preferred embodiment, the same method is used to generate the
expression data used to generate the mathematical model as is used
to diagnose the test individual.
[0305] 5.15 Use of the Combinations Identified to Monitor
Progression or Regression of OA
[0306] The invention teaches the ability to identify useful
combinations of biomarkers for the purposes of diagnosing an
individual as having a particular stage of OA. Having identified
combinations which diagnose an individual with a particular stage
of OA (wherein the stage can be any stage in accordance with known
staging methods and includes subclassifications of the Marshall
method wherein the stages are defined by the Marshall score. In one
embodiment, there are five stages including (a) normal, (b) mild
(c) moderate (d) marked (e) severe. It would be understood by a
person skilled in the art that combinations which are diagnostic
for a specific stage are useful in determining whether an
individual has progressed or regressed with regards to the severity
of their OA, for example, in response to treatment. For example, an
individual can be diagnosed as having a particular stage of OA
prior to treatment using one or more of the combinations identified
as diagnosing a particular stage of OA. Subsequent to treatment the
individual could again be diagnosed using one or more combinations
identified as diagnosing a particular stage of OA. In the event
that the individual can no longer be identified the stage prior to
treatment, this may in itself suggest treatment is effective. In
addition, the treatment may lead to regression of the stage of OA
such that the individual now is diagnosed with a lesser degree of
OA, or the treatment may have progressed the disease such that the
individual is now diagnosed with a more severe stage of OA. As
such, one or more of the combinations identified as specific to
diagnosing a stage of OA is useful so as to monitor progression or
regression OA or so as to monitor response to treatment.
[0307] 5.16 Polynucleotides Used to Measure the Products of the
Biomarkers of the Invention
[0308] As a means of measuring the expression of the RNA products
of the biomarkers of the invention, one can use one or more of the
following as would be understood by a person skilled in the art in
combination with one or more methods to measure RNA expression in a
sample of the invention: oligonucleotides, cDNA, DNA, RNA, PCR
products, synthetic DNA, synthetic RNA, or other combinations of
naturally occurring of modified nucleotides which specifically
hybridize to one or more of the RNA products of the biomarker of
the invention. In another specific embodiment, the
oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA,
synthetic RNA, or other combinations of naturally occurring of
modified nucleotides oligonucleotides which selectively hybridize
to one or more of the RNA products of the biomarker of the
invention are used. In a preferred embodiment, the
oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA,
synthetic RNA, or other combinations of naturally occurring of
modified nucleotides oligonucleotides which both specifically and
selectively hybridize to one or more of the RNA products of the
biomarker of the invention are used.
[0309] In one embodiment of the invention, the polynucleotide used
to measure the RNA products of the invention can be used as nucleic
acid members. Nucleic acid members can be stably associated with a
solid support to comprise an array according to one aspect of the
invention. The length of a nucleic acid member can range from 8 to
1000 nucleotides in length and are chosen so as to be specific for
the RNA products of the biomarkers of the invention. In one
embodiment, these members are selective for RNA products of the
biomarkers of the invention. In yet another embodiment these
members are selective for the mRNA products of the biomarkers of
the invention. In a preferred embodiment, these members are
selective for all of the variants of the mRNA products of the
biomarkers of the invention. In yet another preferred embodiment,
these members are selective for one or more variants of the mRNA
products of the biomarkers of the invention. The nucleic acid
members may be single or double stranded, and/or may be
oligonucleotides or PCR fragments amplified from cDNA. Preferably
oligonucleotides are approximately 20-30 nucleotides in length.
ESTs are preferably 100 to 600 nucleotides in length. It will be
understood to a person skilled in the art that one can utilize
portions of the expressed regions of the biomarkers of the
invention as a probe on the array. More particularly
oligonucleotides complementary to the genes of the invention and or
cDNA or ESTs derived from the genes of the invention are useful.
For oligonucleotide based arrays, the selection of oligonucleotides
corresponding to the gene of interest which are useful as probes is
well understood in the art. More particularly it is important to
choose regions which will permit hybridization to the target
nucleic acids. Factors such as the Tm of the oligonucleotide, the
percent GC content, the degree of secondary structure and the
length of nucleic acid are important factors. See for example U.S.
Pat. No. 6,551,784.
[0310] 5.17 Techniques to Measure the RNA Products of the
Biomarkers of the Invention Array Hybridization
[0311] In some embodiments of the invention the polynucleotides
capable of specifically and/or selectively hybridizing to RNA
products of the biomarkers of the invention can be spotted onto an
array for use in the invention. In one embodiment, the array
consists of sequences of between 10-1000 nucleotides in length
capable of hybridizing to one or more of the products of each of
the biomarkers of the invention as disclosed in Table 1.
[0312] The target nucleic acid samples that are hybridized to and
analyzed with an array of the invention are preferably from human
cartilage, blood or synovial fluid. A limitation for this procedure
lies in the amount of RNA available for use as a target nucleic
acid sample. Preferably, at least 1 microgram of total RNA is
obtained for use according to this invention. Lesser quantities of
RNA can be used in combination with PCR and primers directed to the
mRNA subspecies (e.g. poly T oligonucleotides).
Construction of a Nucleic Acid Array
[0313] In the subject methods, an array of nucleic acid members
stably associated with the surface of a substantially solid support
is contacted with a sample comprising target nucleic acids under
hybridization conditions sufficient to produce a hybridization
pattern of complementary nucleic acid members/target complexes in
which one or more complementary nucleic acid members at unique
positions on the array specifically hybridize to target nucleic
acids. The identity of target nucleic acids which hybridize can be
determined with reference to location of nucleic acid members on
the array.
[0314] The nucleic acid members may be produced using established
techniques such as polymerase chain reaction (PCR) and reverse
transcription (RT). These methods are similar to those currently
known in the art (see e.g. PCR Strategies, Michael A. Innis
(Editor), et al. (1995) and PCR: Introduction to Biotechniques
Series, C. R. Newton, A. Graham (1997)). Amplified nucleic acids
are purified by methods well known in the art (e.g., column
purification or alcohol precipitation). A nucleic acid is
considered pure when it has been isolated so as to be substantially
free of primers and incomplete products produced during the
synthesis of the desired nucleic acid. Preferably, a purified
nucleic acid will also be substantially free of contaminants which
may hinder or otherwise mask the specific binding activity of the
molecule.
[0315] An array, according to one aspect of the invention,
comprises a plurality of nucleic acids attached to one surface of a
solid support at a density exceeding 20 different nucleic
acids/cm.sup.2, wherein each of the nucleic acids is attached to
the surface of the solid support in a non-identical pre-selected
region (e.g. a microarray). Each associated sample on the array
comprises a nucleic acid composition, of known identity, usually of
known sequence, as described in greater detail below. Any
conceivable substrate may be employed in the invention.
[0316] In one embodiment, the nucleic acid attached to the surface
of the solid support is DNA. In a preferred embodiment, the nucleic
acid attached to the surface of the solid support is cDNA or RNA.
In another preferred embodiment, the nucleic acid attached to the
surface of the solid support is cDNA synthesized by polymerase
chain reaction (PCR). Preferably, a nucleic acid member in the
array, according to the invention, is at least 10, 25 or 50
nucleotides in length. In one embodiment, a nucleic acid member is
at least 150 nucleotides in length. Preferably, a nucleic acid
member is less than 1000 nucleotides in length. More preferably, a
nucleic acid member is less than 500 nucleotides in length.
[0317] In the arrays of the invention, the nucleic acid
compositions are stably associated with the surface of a support,
where the support may be a flexible or rigid solid support. By
"stably associated" is meant that each nucleic acid member
maintains a unique position relative to the solid support under
hybridization and washing conditions. As such, the samples are
non-covalently or covalently stably associated with the support
surface. Examples of non-covalent association include non-specific
adsorption, binding based on electrostatic interactions (e.g., ion
pair interactions), hydrophobic interactions, hydrogen bonding
interactions, specific binding through a specific binding pair
member covalently attached to the support surface, and the like.
Examples of covalent binding include covalent bonds formed between
the nucleic acids and a functional group present on the surface of
the rigid support (e.g., --OH), where the functional group may be
naturally occurring or present as a member of an introduced linking
group, as described in greater detail below
[0318] The amount of nucleic acid present in each composition will
be sufficient to provide for adequate hybridization and detection
of target nucleic acid sequences during the assay in which the
array is employed. Generally, the amount of each nucleic acid
member stably associated with the solid support of the array is at
least about 0.001 ng, preferably at least about 0.02 ng and more
preferably at least about 0.05 ng, where the amount may be as high
as 1000 ng or higher, but will usually not exceed about 20 ng.
Preferably multiple samples corresponding to a single gene are
spotted onto the array so as to ensure statistically significant
results. Where the nucleic acid member is "spotted" onto the solid
support in a spot comprising an overall circular dimension, the
diameter of the "spot" will generally range from about 10 to 5.000
.mu.m, usually from about 20 to 2.000 .mu.m and more usually from
about 100 to 200 .mu.m.
[0319] Control nucleic acid members may be present on the array
including nucleic acid members comprising oligonucleotides or
nucleic acids corresponding to genomic DNA, housekeeping genes,
vector sequences, plant nucleic acid sequence, negative and
positive control genes, and the like. Control nucleic acid members
are calibrating or control genes whose function is not to tell
whether a particular "key" gene of interest is expressed, but
rather to provide other useful information, such as background or
basal level of expression.
[0320] Other control nucleic acids are spotted on the array and
used as target expression control nucleic acids and mismatch
control nucleotides to monitor non-specific binding or
cross-hybridization to a nucleic acid in the sample other than the
target to which the probe is directed. Mismatch probes thus
indicate whether a hybridization is specific or not. For example,
if the target is present, the perfectly matched probes should be
consistently brighter than the mismatched probes. In addition, if
all control mismatches are present, the mismatch probes are used to
detect a mutation.
Solid Substrate
[0321] An array according to the invention comprises either a
flexible or rigid substrate. A flexible substrate is capable of
being bent, folded or similarly manipulated without breakage.
Examples of solid materials which are flexible solid supports with
respect to the present invention include membranes, e.g., nylon,
flexible plastic films, and the like. By "rigid" is meant that the
support is solid and does not readily bend, i.e., the support is
not flexible. As such, the rigid substrates of the subject arrays
are sufficient to provide physical support and structure to the
associated nucleic acids present thereon under the assay conditions
in which the array is employed, particularly under high throughput
handling conditions.
[0322] The substrate may be biological, non-biological, organic,
inorganic, or a combination of any of these, existing as particles,
strands, precipitates, gels, sheets, tubing, spheres, beads,
containers, capillaries, pads, slices, films, plates, slides,
chips, etc. The substrate may have any convenient shape, such as a
disc, square, sphere, circle, etc. The substrate is preferably flat
or planar but may take on a variety of alternative surface
configurations. The substrate may be a polymerized Langmuir
Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO.sub.2,
SIN.sub.4, modified silicon, or any one of a wide variety of gels
or polymers such as (poly)tetrafluoroethylene,
(poly)vinylidenedifluoride, polystyrene, polycarbonate, or
combinations thereof. Other substrate materials will be readily
apparent to those of skill in the art upon review of this
disclosure.
[0323] In a preferred embodiment the substrate is flat glass or
single-crystal silicon. According to some embodiments, the surface
of the substrate is etched using well-known techniques to provide
for desired surface features. For example, by way of formation of
trenches, v-grooves, mesa structures, or the like, the synthesis
regions may be more closely placed within the focus point of
impinging light, be provided with reflective "mirror" structures
for maximization of light collection from fluorescent sources,
etc.
[0324] Surfaces on the solid substrate will usually, though not
always, be composed of the same material as the substrate.
Alternatively, the surface may be composed of any of a wide variety
of materials, for example, polymers, plastics, resins,
polysaccharides, silica or silica-based materials, carbon, metals,
inorganic glasses, membranes, or any of the above-listed substrate
materials. In some embodiments the surface may provide for the use
of caged binding members which are attached firmly to the surface
of the substrate. Preferably, the surface will contain reactive
groups, which are carboxyl, amino, hydroxyl, or the like. Most
preferably, the surface will be optically transparent and will have
surface Si--OH functionalities, such as are found on silica
surfaces.
[0325] The surface of the substrate is preferably provided with a
layer of linker molecules, although it will be understood that the
linker molecules are not required elements of the invention. The
linker molecules are preferably of sufficient length to permit
nucleic acids of the invention and on a substrate to hybridize to
other nucleic acid molecules and to interact freely with molecules
exposed to the substrate.
[0326] Often, the substrate is a silicon or glass surface,
(poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,
polycarbonate, a charged membrane, such as nylon 66 or
nitrocellulose, or combinations thereof. In a preferred embodiment,
the solid support is glass. Preferably, at least one surface of the
substrate will be substantially flat. Preferably, the surface of
the solid support will contain reactive groups, including, but not
limited to, carboxyl, amino, hydroxyl, thiol, or the like. In one
embodiment, the surface is optically transparent. In a preferred
embodiment, the substrate is a poly-lysine coated slide or Gamma
amino propyl silane-coated Corning Microarray Technology-GAPS or
CMT-GAP2 coated slides.
[0327] Any solid support to which a nucleic acid member may be
attached may be used in the invention. Examples of suitable solid
support materials include, but are not limited to, silicates such
as glass and silica gel, cellulose and nitrocellulose papers,
nylon, polystyrene, polymethacrylate, latex, rubber, and
fluorocarbon resins such as TEFLON.TM..
[0328] The solid support material may be used in a wide variety of
shapes including, but not limited to slides and beads. Slides
provide several functional advantages and thus are a preferred form
of solid support. Due to their flat surface, probe and
hybridization reagents are minimized using glass slides. Slides
also enable the targeted application of reagents, are easy to keep
at a constant temperature, are easy to wash and facilitate the
direct visualization of RNA and/or DNA immobilized on the solid
support. Removal of RNA and/or DNA immobilized on the solid support
is also facilitated using slides.
[0329] The particular material selected as the solid support is not
essential to the invention, as long as it provides the described
function. Normally, those who make or use the invention will select
the best commercially available material based upon the economics
of cost and availability, the expected application requirements of
the final product, and the demands of the overall manufacturing
process.
Spotting Method
[0330] In one aspect, the invention provides for arrays where each
nucleic acid member comprising the array is spotted onto a solid
support.
[0331] Preferably, spotting is carried out as follows. PCR products
(.about.40 ul) of cDNA clones from osteoarthritis, fetal or normal
cartilage cDNA libraries, in the same 96-well tubes used for
amplification, are precipitated with 4 ul (1/10 volume) of 3M
sodium acetate (pH 5.2) and 100 ul (2.5 volumes) of ethanol and
stored overnight at -20.degree. C. They are then centrifuged at
3,300 rpm at 4.degree. C. for 1 hour. The obtained pellets are
washed with 50 ul ice-cold 70% ethanol and centrifuged again for 30
minutes. The pellets are then air-dried and resuspended well in 20
ul 3.times.SSC or in 50% dimethylsulfoxide (DMSO) overnight. The
samples are then spotted, either singly or in duplicate, onto
slides using a robotic GMS 417 or 427 arrayer (Affymetrix, Ca).
[0332] The boundaries of the spots on the microarray may be marked
with a diamond scriber (as the spots become invisible after
post-processing). The arrays are rehydrated by suspending the
slides over a dish of warm particle free ddH.sub.20 for
approximately one minute (the spots will swell slightly but will
not run into each other) and snap-dried on a 70-80.degree. C.
inverted heating block for 3 seconds. Nucleic acid is then UV
crosslinked to the slide (Stratagene, Stratalinker, 65 mJ--set
display to "650" which is 650.times.100 uJ) or the array is baked
at 80 C for two to four hours prior to hybridization. The arrays
are placed in a slide rack. An empty slide chamber is prepared and
tilled with the following solution: 3.0 grams of succinic anhydride
(Aldrich) was dissolved in 189 ml of 1-methyl-2-pyrrolidinone
(rapid addition of reagent is crucial); immediately after the last
flake of succinic anhydride is dissolved, --21.0 ml of 0.2 M sodium
borate is mixed in and the solution is poured into the slide
chamber. The slide rack is plunged rapidly and evenly in the slide
chamber and vigorously shaken up and down for a few seconds, making
sure the slides never leave the solution, and then mixed on an
orbital shaker for 15-20 minutes. The slide rack is then gently
plunged in 95.degree. C. ddH.sub.20 for 2 minutes, followed by
plunging five times in 95% ethanol. The slides are then air dried
by allowing excess ethanol to drip onto paper towels. The arrays
are stored in the slide box at room temperature until use.
[0333] Numerous methods may be used for attachment of the nucleic
acid members of the invention to the substrate (a process referred
to as "spotting"). For example, nucleic acids are attached using
the techniques of, for example U.S. Pat. No. 5,807,522, which is
incorporated herein by reference, for teaching methods of polymer
attachment.
[0334] Alternatively, spotting may be carried out using contact
printing technology as is known in the art.
Use of a Microarray
[0335] Nucleic acid arrays according to the invention can be used
in high throughput techniques that can assay a large number of
nucleic acids in a sample comprising one or more target nucleic
acid sequences. The arrays of the subject invention find use in a
variety of applications, including gene expression analysis,
diagnosis of osteoarthritis and prognosis of osteoarthritis,
monitoring a patient's response to therapy, drug screening, and the
like.
[0336] The arrays are also useful in broad scale expression
screening for drug discovery and research, such as the effect of a
particular active agent on the expression pattern of genes of the
invention, where such information is used to reveal drug efficacy
and toxicity, environmental monitoring, disease research and the
like.
[0337] Arrays can be made using at least one, more preferably a
combination of these sequences, as a means of diagnosing
osteoarthritis, or for purposes of monitoring efficacy of treatment
and of identifying stage specific osteoarthritis.
[0338] The choice of a standard sample would be well understood by
a person skilled in the art, and would include a sample
complementary to RNA isolated from one or more normal individuals,
wherein a normal individual is an individual not suffering from
osteoarthritis. In the case of monitoring efficacy of treatment or
identifying stage specific osteoarthritis, it would be understood
by a person skilled in the art that a control would include samples
from persons suffering various degrees of osteoarthritis and/or
persons responding to treatment. Standard samples would also
include a sample complementary to RNA isolated from chondrocytes,
or from blood, or from synovial fluid.
Target Preparation
[0339] The targets for the arrays according to the invention are
preferably derived from human cartilage, blood or synovial
fluid.
[0340] A target nucleic acid is capable of binding to a nucleic
acid probe or nucleic acid member of complementary sequence through
one or more types of chemical bonds, usually through complementary
base pairing, usually through hydrogen bond formation.
[0341] As used herein, a nucleic acid derived from an mRNA
transcript: or a "nucleic acid corresponding to an mRNA" refers to
a nucleic acid for which synthesis of the mRNA transcript or a
sub-sequence thereof has ultimately served as a template. Thus, a
cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA, a DNA amplified from the cDNA, an RNA transcribed from the
amplified DNA, etc., are all derived from or correspond to the mRNA
transcript and detection of such derived or corresponding products
is indicative of or proportional to the presence and/or abundance
of the original transcript in a sample. Thus, suitable target
nucleic acid samples include, but are not limited to, mRNA
transcripts of a gene or genes, cDNA reverse transcribed from the
mRNA, cRNA transcribed from the cDNA, DNA amplified from a gene or
genes, RNA transcribed from amplified DNA, and the like. The
nucleic acid targets used herein are preferably derived from human
cartilage, blood or synovial fluid. Preferably, the targets are
nucleic acids derived from human cartilage, blood or synovial fluid
extracts. Nucleic acids can be single- or double-stranded DNA, RNA,
or DNA-RNA hybrids synthesized from human cartilage, blood or
synovial fluid mRNA extracts using methods known in the art, for
example, reverse transcription or PCR.
[0342] In the simplest embodiment, such a nucleic acid target
comprises total mRNA or a nucleic acid sample corresponding to mRNA
(e.g., cDNA) isolated from cartilage, blood, or synovial fluid
samples. In another embodiment, total mRNA is isolated from a given
sample using, for example, an acid guanidinium-phenol-chloroform
extraction method and polyA+ mRNA is isolated by oligo dT column
chromatography or by using (dT)n magnetic beads (see, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.),
Vols. 1-3, Cold Spring Harbor Laboratory, (1989), or Current
Protocols in Molecular Biology, F. Ausubel et al., ed. Greene
Publishing and Wiley-Interscience, New York (1987). In a preferred
embodiment, total RNA is extracted using TRIzol.RTM. reagent
(GIBCO/BRL, Invitrogen Life Technologies, Cat. No. 15596). Purity
and integrity of RNA is assessed by absorbance at 260/280 nm and
agarose gel electrophoresis followed by inspection under
ultraviolet light.
[0343] In some embodiments, it is desirable to amplify the target
nucleic acid sample prior to hybridization, for example, when
synovial fluid is used. One of skill in the art will appreciate
that whatever amplification method is used, if a quantitative
result is desired, care must be taken to use a method that
maintains or controls for the relative frequencies of the amplified
nucleic acids. Methods of "quantitative" amplification are well
known to those of skill in the art. For example, quantitative PCR
involves simultaneously co-amplifying a known quantity of a control
sequence using the same primers. This provides an internal standard
that may be used to calibrate the PCR reaction. The high density
array may then include probes specific to the internal standard for
quantification of the amplified nucleic acid. Detailed protocols
for quantitative PCR are provided in PCR Protocols, A Guide to
Methods and Applications, Innis et al., Academic Press, Inc. N.Y.,
(1990).
[0344] Other suitable amplification methods include, but are not
limited to polymerase chain reaction (PCR) (Innis, et al., PCR
Protocols. A Guide to Methods and Application. Academic Press, Inc.
San Diego, (1990)), ligase chain reaction (LCR) (see Wu and
Wallace, 1989. Genomics, 4:560; Landegren, et al. 1988, Science,
241:1077 and Barringer, et al., 1990, Gene, 89:117, transcription
amplification (Kwoh, et al. 1989, Proc. Natl. Acad. Sci. USA, 86:
1173), and self-sustained sequence replication (Guatelli, et al.,
1990, Proc. Nat. Acad. Sci. USA, 87: 1874).
[0345] In a particularly preferred embodiment, the target nucleic
acid sample mRNA is reverse transcribed with a reverse
transcriptase and a primer consisting of oligo dT and a sequence
encoding the phage T7 promoter to provide single-stranded DNA
template. The second DNA strand is polymerized using a DNA
polymerase. After synthesis of double-stranded cDNA, T7 RNA
polymerase is added and RNA is transcribed from the cDNA template.
Successive rounds of transcription from each single cDNA template
results in amplified RNA. Methods of in vitro transcription are
well known to those of skill in the art (see, e.g., Sambrook,
supra.) and this particular method is described in detail by Van
Gelder, et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 1663-1667 who
demonstrate that in vitro amplification according to this method
preserves the relative frequencies of the various RNA transcripts.
Moreover, Eberwine et al. Proc. Natl. Acad. Sci. USA, 89: 3010-3014
provide a protocol that uses two rounds of amplification via in
vitro transcription to achieve greater than 10.sup.6 fold
amplification of the original starting material thereby permitting
expression monitoring even where biological samples are
limited.
Labeling of Target or Nucleic Acid Probe
[0346] Either the target or the probe can be labeled.
[0347] Any analytically detectable marker that is attached to or
incorporated into a molecule may be used in the invention. An
analytically detectable marker refers to any molecule, moiety or
atom which is analytically detected and quantified.
[0348] Detectable labels suitable for use in the present invention
include any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include biotin for staining
with labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.TM.), fluorescent dyes (e.g. fluorescein, texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., .sup.3H, .sup.125I, 35S, .sup.14C, or .sup.32P), enzymes
(e.g., horse radish peroxidase, alkaline phosphatase and others
commonly used in an ELISA), and colorimetric labels such as
colloidal gold or colored glass or plastic (e.g., polystyrene,
polypropylene, latex, etc.) beads. Patents teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241, the entireties of
which are incorporated by reference herein.
[0349] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and detecting the reaction product produced
by the action of the enzyme on the substrate, and colorimetric
labels are detected by simply visualizing the colored label.
[0350] The labels may be incorporated by any of a number of means
well known to those of skill in the art. However, in a preferred
embodiment, the label is simultaneously incorporated during the
amplification step in the preparation of the sample nucleic acids.
Thus, for example, polymerase chain reaction (PCR) with labeled
primers or labeled nucleotides will provide a labeled amplification
product. In a preferred embodiment, transcription amplification, as
described above, using a labeled nucleotide (e.g.
fluorescein-labeled UTP and/or CTP) incorporates a label into the
transcribed nucleic acids.
[0351] Alternatively, a label may be added directly to the original
nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the
amplification product after the amplification is completed. Means
of attaching labels to nucleic acids are well known to those of
skill in the art and include, for example, nick translation or
end-labeling (e.g. with a labeled RNA) by kinasing of the nucleic
acid and subsequent attachment (ligation) of a nucleic acid linker
joining the sample nucleic acid to a label (e.g., a
fluorophore).
[0352] In a preferred embodiment, the fluorescent modifications are
by cyanine dyes e.g. Cy-3/Cy-5 dUTP, Cy-3/Cy-5 dCTP (Amersham
Pharmacia) or alexa dyes (Khan, et al. 1998, Cancer Res.
58:5009-5013).
[0353] In a preferred embodiment, the two target samples used for
comparison are labeled with different fluorescent dyes which
produce distinguishable detection signals, for example, targets
made from normal cartilage are labeled with Cy5 and targets made
from mild osteoarthritis cartilage are labeled with Cy3. The
differently labeled target samples are hybridized to the same
microarray simultaneously. In a preferred embodiment, the labeled
targets are purified using methods known in the art, e.g., by
ethanol purification or column purification.
[0354] In a preferred embodiment, the target will include one or
more control molecules which hybridize to control probes on the
microarray to normalize signals generated from the microarray.
Preferably, labeled normalization targets are nucleic acid
sequences that are perfectly complementary to control
oligonucleotides that are spotted onto the microarray as described
above. The signals obtained from the normalization controls after
hybridization provide a control for variations in hybridization
conditions, label intensity, "reading" efficiency and other factors
that may cause the signal of a perfect hybridization to vary
between arrays. In a preferred embodiment, signals (e.g.,
fluorescence intensity) read from all other probes in the array are
divided by the signal (e.g., fluorescence intensity) from the
control probes, thereby normalizing the measurements.
[0355] Preferred normalization targets are selected to reflect the
average length of the other targets present in the sample, however,
they are selected to cover a range of lengths. The normalization
control(s) also can be selected to reflect the (average) base
composition of the other probes in the array, however, in a
preferred embodiment, only one or a few normalization probes are
used and they are selected such that they hybridize well (i.e.,
have no secondary structure and do not self hybridize) and do not
match any target molecules.
[0356] Normalization probes are localized at any position in the
array or at multiple positions throughout the array to control for
spatial variation in hybridization efficiency. In a preferred
embodiment, normalization controls are located at the corners or
edges of the array as well as in the middle.
Hybridization Conditions
[0357] Nucleic acid hybridization involves providing a denatured
probe or target nucleic acid member and target nucleic acid under
conditions where the probe or target nucleic acid member and its
complementary target can form stable hybrid duplexes through
complementary base pairing. The nucleic acids that do not form
hybrid duplexes are then washed away leaving the hybridized nucleic
acids to be detected, typically through detection of an attached
detectable label. It is generally recognized that nucleic acids are
denatured by increasing the temperature or decreasing the salt
concentration of the buffer containing the nucleic acids. Under low
stringency conditions (e.g., low temperature and/or high salt)
hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even
where the annealed sequences are not perfectly complementary. Thus
specificity of hybridization is reduced at lower stringency.
Conversely, at higher stringency (e.g., higher temperature or lower
salt) successful hybridization requires fewer mismatches.
[0358] The invention provides for hybridization conditions
comprising the Dig hybridization mix (Boehringer); or
formamide-based hybridization solutions, for example as described
in Ausubel et al. supra and Sambrook et al. supra.
[0359] Methods of optimizing hybridization conditions are well
known to those of skill in the art (see, e.g., Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 24:
Hybridization With Nucleic acid Probes, P. Tijssen, ed. Elsevier,
N.Y., (1993)).
[0360] Following hybridization, non-hybridized labeled or unlabeled
nucleic acid is removed from the support surface, conveniently by
washing, thereby generating a pattern of hybridized target nucleic
acid on the substrate surface. A variety of wash solutions are
known to those of skill in the art and may be used. The resultant
hybridization patterns of labeled, hybridized oligonucleotides
and/or nucleic acids may be visualized or detected in a variety of
ways, with the particular manner of detection being chosen based on
the particular label of the test nucleic acid, where representative
detection means include scintillation counting, autoradiography,
fluorescence measurement, calorimetric measurement, light emission
measurement and the like.
Image Acquisition and Data Analysis
[0361] Following hybridization and any washing step(s) and/or
subsequent treatments, as described above, the resultant
hybridization pattern is detected. In detecting or visualizing the
hybridization pattern, the intensity or signal value of the label
will be not only be detected but quantified, by which is meant that
the signal from each spot of the hybridization will be measured and
compared to a unit value corresponding to the signal emitted by a
known number of end labeled target nucleic acids to obtain a count
or absolute value of the copy number of each end-labeled target
that is hybridized to a particular spot on the array in the
hybridization pattern.
[0362] Methods for analyzing the data collected from hybridization
to arrays are well known in the art. For example, where detection
of hybridization involves a fluorescent label, data analysis can
include the steps of determining fluorescent intensity as a
function of substrate position from the data collected, removing
outliers. i.e., data deviating from a predetermined statistical
distribution, and calculating the relative binding affinity of the
test nucleic acids from the remaining data. The resulting data is
displayed as an image with the intensity in each region varying
according to the binding affinity between associated
oligonucleotides and/or nucleic acids and the test nucleic
acids.
[0363] The following detection protocol is used for the
simultaneous analysis of two cartilage samples to be compared,
where each sample is labeled with a different fluorescent dye.
[0364] Each element of the microarray is scanned for the first
fluorescent color. The intensity of the fluorescence at each array
element is proportional to the expression level of that gene in the
sample.
[0365] The scanning operation is repeated for the second
fluorescent label. The ratio of the two fluorescent intensities
provides a highly accurate and quantitative measurement of the
relative gene expression level in the two tissue samples.
[0366] In a preferred embodiment, fluorescence intensities of
immobilized target nucleic acid sequences were determined from
images taken with a custom confocal microscope equipped with laser
excitation sources and interference filters appropriate for the Cy3
and Cy5 fluors. Separate scans were taken for each fluor at a
resolution of 225 .mu.m.sup.2 per pixel and 65.536 gray levels.
Image segmentation to identify areas of hybridization,
normalization of the intensities between the two fluor images, and
calculation of the normalized mean fluorescent values at each
target are as described (Khan, et al., 1998, Cancer Res.
58:5009-5013. Chen, et al., 1997, Biomed. Optics 2:364-374).
Normalization between the images is used to adjust for the
different efficiencies in labeling and detection with the two
different fluors. This is achieved by equilibrating to a value of
one the signal intensity ratio of a set of internal control genes
spotted on the array.
[0367] In another preferred embodiment, the array is scanned in the
Cy 3 and Cy5 channels and stored as separate 16-bit TIFF images.
The images are incorporated and analysed using software which
includes a gridding process to capture the hybridization intensity
data from each spot on the array. The fluorescence intensity and
background-subtracted hybridization intensity of each spot is
collected and a ratio of measured mean intensities of Cy5 to Cy3 is
calculated. A linear regression approach is used for normalization
and assumes that a scatter plot of the measured Cy5 versus Cy3
intensities should have a slope of one. The average of the ratios
is calculated and used to rescale the data and adjust the slope to
one. A ratio of expression not equal to 1 is used as an indication
of differential gene expression.
[0368] In a particularly preferred embodiment, where it is desired
to quantify the transcription level (and thereby expression) of one
or more nucleic acid sequences in a sample, the target nucleic acid
sample is one in which the concentration of the mRNA transcript(s)
of the gene or genes, or the concentration of the nucleic acids
derived from the mRNA transcript(s), is proportional to the
transcription level (and therefore expression level) of that gene.
Similarly, it is preferred that the hybridization signal intensity
be proportional to the amount of hybridized nucleic acid. While it
is preferred that the proportionality be relatively strict (e.g., a
doubling in transcription rate results in a doubling in mRNA
transcript in the sample nucleic acid pool and a doubling in
hybridization signal), one of skill will appreciate that the
proportionality can be more relaxed and even non-linear and still
provide meaningful results. Thus, for example, an assay where a 5
fold difference in concentration of the target mRNA results in a 3-
to 6-fold difference in hybridization intensity is sufficient for
most purposes. Where more precise quantification is required,
appropriate controls are run to correct for variations introduced
in sample preparation and hybridization as described herein. In
addition, serial dilutions of "standard" target mRNAs are used to
prepare calibration curves according to methods well known to those
of skill in the art. Of course, where simple detection of the
presence or absence of a transcript is desired, no elaborate
control or calibration is required.
[0369] For example, if an nucleic acid member on an array is not
labeled after hybridization, this indicates that the gene
comprising that nucleic acid member is not expressed in either
sample. If a nucleic acid member is labeled with a single color, it
indicates that a labeled gene was expressed only in one sample. The
labeling of a nucleic acid member comprising an array with both
colors indicates that the gene was expressed in both samples. Even
genes expressed once per cell are detected (1 part in 100.000
sensitivity). A difference in expression intensity in the two
samples being compared is indicative of differential expression,
the ratio of the intensity in the two samples being not equal to
1.0, preferably less than 0.7 or greater than 1.2, more preferably
less than 0.5 or greater than 1.5.
RT-PCR
[0370] In aspect of the invention, the level of the expression of
the RNA products of the biomarkers of the invention can be measured
by amplifying the RNA products of the biomarkers from a sample
using reverse transcription (RT) in combination with the polymerase
chain reaction (PCR). In accordance with one embodiment of the
invention, the RT can be quantitative as would be understood to a
person skilled in the art.
[0371] Total RNA, or mRNA from a sample is used as a template and a
primer specific to the transcribed portion of a biomarker of the
invention is used to initiate reverse transcription. Methods of
reverse transcribing RNA into cDNA are well known and described in
Sambrook et al., 1989, supra. Primer design can be accomplished
utilizing commercially available software (e.g., Primer Designer
1.0, Scientific Sofware etc.). The product of the reverse
transcription is subsequently used as a template for PCR.
[0372] PCR provides a method for rapidly amplifying a particular
nucleic acid sequence by using multiple cycles of DNA replication
catalyzed by a thermostable. DNA-dependent DNA polymerase to
amplify the target sequence of interest. PCR requires the presence
of a nucleic acid to be amplified, two single-stranded
oligonucleotide primers flanking the sequence to be amplified, a
DNA polymerase, deoxyribonucleoside triphosphates, a buffer and
salts.
[0373] The method of PCR is well known in the art. PCR, is
performed as described in Mullis and Faloona, 1987. Methods
Enzymol., 155: 335, which is incorporated herein by reference. PCR
is performed using template DNA (at least 1 fg; more usefully,
1-1000 ng) and at least 25 .mu.mol of oligonucleotide primers. A
typical reaction mixture includes: 2 .mu.l of DNA, 25 .mu.mol of
oligonucleotide primer, 2.5 .mu.l of 10H PCR buffer 1
(Perkin-Elmer, Foster City, Calif.), 0.4 .mu.l of 1.25 .mu.M dNTP,
0.15 .mu.l (or 2.5 units) of Taq DNA polymerase (Perkin Elmer,
Foster City, Calif.) and deionized water to a total volume of 25
.mu.l. Mineral oil is overlaid and the PCR is performed using a
programmable thermal cycler.
[0374] The length and temperature of each step of a PCR cycle, as
well as the number of cycles, are adjusted according to the
stringency requirements in effect. Annealing temperature and timing
are determined both by the efficiency with which a primer is
expected to anneal to a template and the degree of mismatch that is
to be tolerated. The ability to optimize the stringency of primer
annealing conditions is well within the knowledge of one of
moderate skill in the art. An annealing temperature of between
30.degree. C. and 72.degree. C. is used. Initial denaturation of
the template molecules normally occurs at between 92.degree. C. and
99.degree. C. for 4 minutes, followed by 20-40 cycles consisting of
denaturation (94-99.degree. C. for 15 seconds to 1 minute),
annealing (temperature determined as discussed above; 1-2 minutes),
and extension (72.degree. C. for 1 minute). The final extension
step is generally carried out for 4 minutes at 72.degree. C., and
may be followed by an indefinite (0-24 hour) step at 4.degree.
C.
[0375] QRT-PCR, which is quantitative in nature, can also be
performed to provide a quantitative measure of gene expression
levels. In QRT-PCR reverse transcription and PCR can be performed
in two steps, or reverse transcription combined with PCR can be
performed concurrently. One of these techniques, for which there
are commercially available kits such as Taqman (Perkin Elmer.
Foster City. CA), is performed with a transcript-specific antisense
probe. This probe is specific for the PCR product (e.g. a nucleic
acid fragment derived from a gene) and is prepared with a quencher
and fluorescent reporter probe complexed to the 5' end of the
oligonucleotide. Different fluorescent markers are attached to
different reporters, allowing for measurement of two products in
one reaction. When Taq DNA polymerase is activated, it cleaves off
the fluorescent reporters of the probe bound to the template by
virtue of its 5'-to-3' exonuclease activity. In the absence of the
quenchers, the reporters now fluoresce. The color change in the
reporters is proportional to the amount of each specific product
and is measured by a fluorometer; therefore, the amount of each
color is measured and the PCR product is quantified. The PCR
reactions are performed in 96 well plates so that samples derived
from many individuals are processed and measured simultaneously.
The Taqman system has the additional advantage of not requiring gel
electrophoresis and allows for quantification when used with a
standard curve.
[0376] A second technique useful for detecting PCR products
quantitatively without is to use an intercolating dye such as the
commercially available QuantiTect SYBR Green PCR (Qiagen, Valencia
Calif.). RT-PCR is performed using SYBR green as a fluorescent
label which is incorporated into the PCR product during the PCR
stage and produces a flourescense proportional to the amount of PCR
product.
[0377] Both Taqman and QuantiTect SYBR systems can be used
subsequent to reverse transcription of RNA. Reverse transcription
can either be performed in the same reaction mixture as the PCR
step (one-step protocol) or reverse transcription can be performed
first prior to amplification utilizing PCR (two-step protocol).
[0378] Additionally, other systems to quantitatively measure mRNA
expression products are known including Molecular Beacons.RTM.
which uses a probe having a fluorescent molecule and a quencher
molecule, the probe capable of forming a hairpin structure such
that when in the hairpin form, the fluorescence molecule is
quenched, and when hybridized the flourescense increases giving a
quantitative measurement of gene expression.
[0379] Additional techniques to quantitatively measure RNA
expression include, but are not limited to, polymerase chain
reaction, ligase chain reaction, Qbeta replicase (see, e.g.,
International Application No. PCT/US87/00880), isothermal
amplification method (see, e.g., Walker et al. (1992) PNAS
89:382-396), strand displacement amplification (SDA), repair chain
reaction, Asymmetric Quantitative PCR (see, e.g., U.S. Publication
No. US200330134307A1) and the multiplex microsphere bead assay
described in Fuja et al., 2004, Journal of Biotechnology
108:193-205.
[0380] The level of gene expression can be measured by amplifying
RNA from a sample using transcription based amplification systems
(TAS), including nucleic acid sequence amplification (NASBA) and
3SR. See. e.g., Kwoh et al (1989) PNAS USA 6:1173; International
Publication No. WO 88/10315; and U.S. Pat. No. 6,329,179. In NASBA,
the nucleic acids may be prepared for amplification using
conventional phenol/chloroform extraction, heat denaturation,
treatment with lysis buffer and minispin columns for isolation of
DNA and RNA or guanidinium chloride extraction of RNA. These
amplification techniques involve annealing a primer that has target
specific sequences. Following polymerization, DNA/RNA hybrids are
digested with RNase H while double stranded DNA molecules are heat
denatured again. In either case the single stranded DNA is made
fully double stranded by addition of second target specific primer,
followed by polymerization. The double-stranded DNA molecules are
then multiply transcribed by a polymerase such as T7 or SP6. In an
isothermal cyclic reaction, the RNA's are reverse transcribed into
double stranded DNA, and transcribed once with a polymerase such as
T7 or SP6. The resulting products, whether truncated or complete,
indicate target specific sequences.
[0381] Several techniques may be used to separate amplification
products. For example, amplification products may be separated by
agarose, agarose-acrylamide or polyacrylamide gel electrophoresis
using conventional methods. See Sambrook et al., 1989. Several
techniques for detecting PCR products quantitatively without
electrophoresis may also be used according to the invention (see
for example PCR Protocols, A Guide to Methods and Applications,
Innis et al., Academic Press, Inc. N.Y., (1990)). For example,
chromatographic techniques may be employed to effect separation.
There are many kinds of chromatography which may be used in the
present invention: adsorption, partition, ion-exchange and
molecular sieve, HPLC, and many specialized techniques for using
them including column, paper, thin-layer and gas chromatography
(Freifelder, Physical Biochemistry Applications to Biochemistry and
Molecular Biology, 2nd ed. Wm. Freeman and Co., New York, N.Y.,
1982).
[0382] Another example of a separation methodology is done by
covalently labeling the oligonucleotide primers used in a PCR
reaction with various types of small molecule ligands. In one such
separation, a different ligand is present on each oligonucleotide.
A molecule, perhaps an antibody or avidin if the ligand is biotin,
that specifically binds to one of the ligands is used to coat the
surface of a plate such as a 96 well ELISA plate. Upon application
of the PCR reactions to the surface of such a prepared plate, the
PCR products are bound with specificity to the surface. After
washing the plate to remove unbound reagents, a solution containing
a second molecule that binds to the first ligand is added. This
second molecule is linked to some kind of reporter system. The
second molecule only binds to the plate if a PCR product has been
produced whereby both oligonucleotide primers are incorporated into
the final PCR products. The amount of the PCR product is then
detected and quantified in a commercial plate reader much as ELISA
reactions are detected and quantified. An ELISA-like system such as
the one described here has been developed by the Raggio Italgene
company under the C-Track trade name.
[0383] Amplification products must be visualized in order to
confirm amplification of the nucleic acid sequences of interest.
One typical visualization method involves staining of a gel with
ethidium bromide and visualization under UV light. Alternatively,
if the amplification products are integrally labeled with radio- or
fluorometrically-labeled nucleotides, the amplification products
may then be exposed to x-ray film or visualized under the
appropriate stimulating spectra, following separation.
[0384] In one embodiment, visualization is achieved indirectly.
Following separation of amplification products, a labeled, nucleic
acid probe is brought into contact with the amplified nucleic acid
sequence of interest. The probe preferably is conjugated to a
chromophore but may be radiolabeled. In another embodiment, the
probe is conjugated to a binding partner, such as an antibody or
biotin, where the other member of the binding pair carries a
detectable moiety.
[0385] In another embodiment, detection is by Southern blotting and
hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art and
may be found in many standard books on molecular protocols. See
Sambrook et al., 1989, supra. Briefly, amplification products are
separated by gel electrophoresis. The gel is then contacted with a
membrane, such as nitrocellulose, permitting transfer of the
nucleic acid and non-covalent binding. Subsequently, the membrane
is incubated with a chromophore-conjugated probe that is capable of
hybridizing with a target amplification product. Detection is by
exposure of the membrane to x-ray film or ion-emitting detection
devices.
[0386] One example of the foregoing is described in U.S. Pat. No.
5,279,721, incorporated by reference herein, which discloses an
apparatus and method for the automated electrophoresis and transfer
of nucleic acids. The apparatus permits electrophoresis and
blotting without external manipulation of the gel and is ideally
suited to carrying out methods according to the present
invention.
Nuclease Protection Assays
[0387] In another embodiment of the invention, Nuclease protection
assays (including both ribonuclease protection assays and S1
nuclease assays) can be used to detect and quantitate the RNA
products of the biomarkers of the invention. In nuclease protection
assays, an antisense probe (labeled with, e.g., radiolabeled or
nonisotopic) hybridizes in solution to an RNA sample. Following
hybridization, single-stranded, unhybridized probe and RNA are
degraded by nucleases. An acrylamide gel is used to separate the
remaining protected fragments. Typically, solution hybridization is
more efficient than membrane-based hybridization, and it can
accommodate up to 100 .mu.g of sample RNA, compared with the 20-30
.mu.g maximum of blot hybridizations.
[0388] The ribonuclease protection assay, which is the most common
type of nuclease protection assay, requires the use of RNA probes.
Oligonucleotides and other single-stranded DNA probes can only be
used in assays containing S1 nuclease. The single-stranded,
antisense probe must typically be completely homologous to target
RNA to prevent cleavage of the probe:target hybrid by nuclease.
Northern Blots
[0389] A standard Northern blot assay can also be used to ascertain
an RNA transcript size, identify alternatively spliced RNA
transcripts, and the relative amounts of RNA products of the
biomarker of the invention, in accordance with conventional
Northern hybridization techniques known to those persons of
ordinary skill in the art. In Northern blots, RNA samples are first
separated by size via electrophoresis in an agarose gel under
denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Nonisotopic or
high specific activity radiolabeled probes can be used including
random-primed, nick-translated, or PCR-generated DNA probes, in
vitro transcribed RNA probes, and oligonucleotides. Additionally,
sequences with only partial homology (e.g. cDNA from a different
species or genomic DNA fragments that might contain an exon) may be
used as probes. The labeled probe, e.g., a radiolabelled cDNA,
either containing the full-length, single stranded DNA or a
fragment of that DNA sequence may be at least 20, at least 30, at
least 50, or at least 100 consecutive nucleotides in length. The
probe can be labeled by any of the many different methods known to
those skilled in this art. The labels most commonly employed for
these studies are radioactive elements, enzymes, chemicals that
fluoresce when exposed to ultraviolet light, and others. A number
of fluorescent materials are known and can be utilized as labels.
These include, but are not limited to, fluorescein, rhodamine,
auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular
detecting material is anti-rabbit antibody prepared in goats and
conjugated with fluorescein through an isothiocyanate. Proteins can
also be labeled with a radioactive element or with an enzyme. The
radioactive label can be detected by any of the currently available
counting procedures. Non-t limiting examples of isotopes include
.sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36Cl, .sup.51Cr,
.sup.57Co, .sup.58Co, .sup.59Fe .sup.90Y, .sup.125I, .sup.131I, and
.sup.186Re. Enzyme labels are likewise useful, and can be detected
by any of the presently utilized colorimetric, spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques.
The enzyme is conjugated to the selected particle by reaction with
bridging molecules such as carbodiimides, diisocyanates,
glutaraldehyde and the like. Any enzymes known to one of skill in
the art can be utilized. Examples of such enzymes include, but are
not limited to, peroxidase, beta-D-galactosidase, urease, glucose
oxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos.
3,654,090, 3,850,752, and 4,016,043 are referred to by way of
example for their disclosure of alternate labeling material and
methods.
[0390] 5.18 Techniques to Measure the Protein Products of the
Biomarkers of the Invention
Protein Products
[0391] Standard techniques can also be utilized for determining the
amount of the protein or proteins of interest present in a sample.
For example, standard techniques can be employed using. e.g.,
immunoassays such as, for example. Western blot,
immunoprecipitation followed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), immunocytochemistry,
and the like to determine the amount of the protein or proteins of
interest present in a sample. A preferred agent for detecting a
protein of interest is an antibody capable of binding to a protein
of interest, preferably an antibody with a detectable label.
[0392] For such detection methods, protein from the sample to be
analyzed can easily be isolated using techniques which are well
known to those of skill in the art. Protein isolation methods can,
for example, be such as those described in Harlow and Lane (Harlow,
E. and Lane. D. Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1988)).
[0393] Preferred methods for the detection of the protein or
proteins of interest involve their detection via interaction with a
protein-specific antibody. For example, antibodies directed a
protein of interest can be utilized as described herein. Antibodies
can be generated utilizing standard techniques well known to those
of skill in the art. See, e.g., Section 5.5.1 of this application
and Section 5.2 of U.S. Publication No. 20040018200 for a more
detailed discussion of such antibody generation techniques, which
is incorporated herein by reference. Briefly, such antibodies can
be polyclonal, or more preferably, monoclonal. An intact antibody,
or an antibody fragment (e.g., Fab or F(ab').sub.2) can, for
example, be used. Preferably, the antibody is a human or humanized
antibody.
[0394] For example, antibodies, or fragments of antibodies,
specific for a protein of interest can be used to quantitatively or
qualitatively detect the presence of the protein. This can be
accomplished, for example, by immunofluorescence techniques.
Antibodies (or fragments thereof) can, additionally, be employed
histologically, as in immunofluorescence or immunoelectron
microscopy, for in situ detection of a protein of interest. In situ
detection can be accomplished by removing a histological specimen
(e.g., a biopsy specimen) from a patient, and applying thereto a
labeled antibody thereto that is directed to a protein. The
antibody (or fragment) is preferably applied by overlaying the
labeled antibody (or fragment) onto a biological sample. Through
the use of such a procedure, it is possible to determine not only
the presence of the protein of interest, but also its distribution,
its presence in cells (e.g. chondrocytes and lymphocytes) within
the sample. A wide variety of well-known histological methods (such
as staining procedures) can be utilized in order to achieve such in
situ detection.
[0395] Immunoassays for a protein of interest typically comprise
incubating a biological sample of a detectably labeled antibody
capable of identifying a protein of interest, and detecting the
bound antibody by any of a number of techniques w ell-known in the
art. As discussed in more detail, below, the term "labeled" can
refer to direct labeling of the antibody via, e.g., coupling (i.e.
physically linking) a detectable substance to the antibody, and can
also refer to indirect labeling of the antibody by reactivity with
another reagent that is directly labeled. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody.
[0396] For example, the biological sample can be brought in contact
with and immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
can then be washed with suitable buffers followed by treatment with
the detectably labeled fingerprint gene-specific antibody. The
solid phase support can then be washed with the buffer a second
time to remove unbound antibody. The amount of bound label on solid
support can then be detected by conventional means.
[0397] By "solid phase support or carrier" in the context of
proteinaceous agents is intended any support capable of binding an
antigen or an antibody. Well-known supports or carriers include
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite. The nature of the carrier can be either
soluble to some extent or insoluble for the purposes of the present
invention. The support material can have virtually any possible
structural configuration so long as the coupled molecule is capable
of binding to an antigen or antibody. Thus, the support
configuration can be spherical, as in a bead, or cylindrical, as in
the inside surface of a test tube, or the external surface of a
rod. Alternatively, the surface can be flat such as a sheet, test
strip, etc. Preferred supports include polystyrene beads. Those
skilled in the art will know many other suitable carriers for
binding antibody or antigen, or will be able to ascertain the same
by use of routine experimentation.
[0398] One of the ways in which a specific antibody can be
detectably labeled is by linking the same to an enzyme and use in
an enzyme immunoassay (EIA) (Voller, A. "The Enzyme Linked
Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7,
Microbiological Associates Quarterly Publication. Walkersville,
Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler,
J. E., 1981, Meth. Enzymol. 73:482-523; Maggio. E. (ed.), 1980,
Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et
al. (eds.), 1981, Enzyme Immunoassay. Kgaku Shoin, Tokyo). The
enzyme which is bound to the antibody will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual means.
Enzymes which can be used to detectably label the antibody include,
but are not limited to, malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection can also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0399] Detection can also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect a
protein of interest through the use of a radioimmunoassay (RIA)
(see, for example. Weintraub, B., Principles of Radioimmunoassays,
Seventh Training Course on Radioligand Assay Techniques. The
Endocrine Society, March, 1986, which is incorporated by reference
herein). The radioactive isotope (e.g., .sup.125I, .sup.131I,
.sup.35S or .sup.3H) can be detected by such means as the use of a
gamma counter or a scintillation counter or by autoradiography.
[0400] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0401] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylene riaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0402] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemi
luminescent labeling compounds are luminol, isoluminol, theromatic
acridinium ester, imidazole, acridinium salt and oxalate ester.
[0403] Likewise, a bioluminescent compound can be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
Protein Arrays
[0404] Polypeptides which specifically and/or selectively bind to
the protein products of the biomarkers of the invention can be
immobilized on a protein array. The protein array can be used as a
diagnostic tool, e.g., to screen medical samples (such as isolated
cells, blood, synovial fluid, sera, biopsies, and the like) for the
presence of the polypeptides protein products of the biomarkers of
the invention. The protein array can also include antibodies as
well as other ligands, e.g., that bind to the polypeptides encoded
by the biomarkers of the invention.
[0405] Methods of producing polypeptide arrays are described, e.g.,
in De Wildt et al., 2000, Nature Biotech. 18:989-994; Lueking et
al., 1999, Anal. Biochem. 270:103-111: Ge, 2000, Nuc. Acids Res.
28:e3; MacBeath and Schreiber, 2000, Science 289:1760-1763;
International Publication Nos. WO 01/40803 and WO 99/51773A1: and
U.S. Pat. No. 6,406,921. Polypeptides for the array can be spotted
at high speed, e.g., using commercially available robotic
apparatus. e.g., from Genetic MicroSystems and Affymetrix (Santa
Clara, Calif., USA) or BioRobotics (Cambridge, UK). The array
substrate can be, for example, nitrocellulose, plastic, glass,
e.g., surface-modified glass. The array can also include a porous
matrix, e.g., acrylamide, agarose, or another polymer.
[0406] For example, the array can be an array of antibodies, e.g.,
as described in De Wildt, supra. Cells that produce the polypeptide
ligands can be grown on a filter in an arrayed format. Polypeptide
production is induced, and the expressed antibodies are immobilized
to the filter at the location of the cell. Information about the
extent of binding at each address of the array can be stored as a
profile, e.g., in a computer database.
[0407] In one embodiment the array is an array of protein products
of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50, all or any combination of the biomarkers of the invention. In
one aspect, the invention provides for antibodies that are bound to
an array which selectively bind to the protein products of the
biomarkers of the invention.
[0408] 5.19 Protein Production
[0409] Standard recombinant nucleic acid methods can be used to
express a polypeptide or antibody of the invention (e.g., a protein
product of a biomarker of the invention). Generally, a nucleic acid
sequence encoding the polypeptide is cloned into a nucleic acid
expression vector. Of course, if the protein includes multiple
polypeptide chains, each chain must be cloned into an expression
vector, e.g., the same or different vectors, that are expressed in
the same or different cells. If the protein is sufficiently small,
i.e., the protein is a peptide of less than 50 amino acids, the
protein can be synthesized using automated organic synthetic
methods. Polypeptides comprising the 5' region, 3' region or
internal coding region of a biomarker of the invention, are
expressed from nucleic acid expression vectors containing only
those nucleotide sequences corresponding to the 5' region, 3'
region or internal coding region of a biomarker of the invention.
Methods for producing antibodies directed to protein products of a
biomarker of the invention, or polypeptides encoded by the 5'
region, 3' region or internal coding regions of a biomarker of the
invention.
[0410] The expression vector for expressing the polypeptide can
include, in addition to the segment encoding the polypeptide or
fragment thereof, regulatory sequences, including for example, a
promoter, operably linked to the nucleic acid(s) of interest. Large
numbers of suitable vectors and promoters are known to those of
skill in the art and are commercially available for generating the
recombinant constructs of the present invention. The following
vectors are provided by way of example. Bacterial: pBs,
phagescript. PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene. La Jolla, Calif., USA); pTrc99A,
pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3,
pBPV, pMSG, and pSVL (Pharmacia). One preferred class of preferred
libraries is the display library, which is described below.
[0411] Methods well known to those skilled in the art can be used
to construct vectors containing a polynucleotide of the invention
and appropriate transcriptional/translational control signals.
These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Sambrook & Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and
Ausubel et al., Current Protocols in Molecular Biology (Greene
Publishing Associates and Wiley Interscience, N.Y. (1989). Promoter
regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ. T3, T7,
gpt, lambda P, and trc. Eukaryotic promoters include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, mouse metallothionein-I, and various art-known tissue
specific promoters. In specific embodiments, the promoter is an
inducible promoter. In other embodiments, the promoter is a
constitutive promoter. In yet other embodiments, the promoter is a
tissue-specific promoter.
[0412] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae auxotrophic markers (such as
URA3, LEU2, HIS3, and TRPl genes), and a promoter derived from a
highly expressed gene to direct transcription of a downstream
structural sequence. Such promoters can be derived from operons
encoding glycolytic enzymes such as 3-phosphoglycerate kinase
(PGK), a-factor, acid phosphatase, or heat shock proteins, among
others. The polynucleotide of the invention is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, a nucleic acid of the invention
can encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product. Useful
expression-vectors for bacteria are constructed by inserting a
polynucleotide of the invention together with suitable translation
initiation and termination signals, optionally in operable reading
phase with a functional promoter. The vector will comprise one or
more phenotypic selectable markers and an origin of replication to
ensure maintenance of the vector and to, if desirable, provide
amplification within the host. Suitable prokaryotic hosts for
transformation include E. coli, Bacillus subtilis, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus, although others may also be
employed as a matter of choice.
[0413] As a representative but nonlimiting example, useful
expression vectors for bacteria can comprise a selectable marker
and bacterial origin of replication derived from commercially
available plasmids comprising genetic elements of the well known
cloning vector pBR322 (ATCC 37017). Such commercial vectors
include, for example, pKK223-3 (Pharmacia Fine Chemicals. Uppsala.
Sweden) and pGEM1 (Promega. Madison, Wis., USA).
[0414] The present invention provides host cells genetically
engineered to contain the polynucleotides of the invention. For
example, such host cells may contain nucleic acids of the invention
introduced into the host cell using known transformation,
transfection or infection methods. The present invention also
provides host cells genetically engineered to express the
polynucleotides of the invention, wherein such polynucleotides are
in operative association with a regulatory sequence heterologous to
the host cell which drives expression of the polynucleotides in the
cell.
[0415] The present invention further provides host cells containing
the vectors of the present invention, wherein the nucleic acid has
been introduced into the host cell using known transformation,
transfection or infection methods. The host cell can be a
eukaryotic host cell, such as a mammalian cell, a lower eukaryotic
host cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a bacterial cell. Introduction of the
recombinant construct into the host cell can be effected, for
example, by calcium phosphate transfection, DEAE, dextran mediated
transfection, or electroporation (Davis. L. et al., Basic Methods
in Molecular Biology (1986)). Cell-free translation systems can
also be employed to produce such proteins using RNAs derived from
the DNA constructs of the present invention.
[0416] Any host/vector system can be used to express one or more of
the genes listed in Table 2 or splice variants. Appropriate cloning
and expression vectors for use with prokaryotic and eukaryotic
hosts are described by Sambrook et al., in Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989),
the disclosure of which is incorporated herein by reference in its
entirety. The most preferred host cells are those which do not
normally express the particular polypeptide or which expresses the
polypeptide at low natural level.
[0417] In a specific embodiment, the host cells are engineered to
express an endogenous gene comprising the polynucleotides of the
invention under the control of inducible regulatory elements, in
which case the regulatory sequences of the endogenous gene may be
replaced by homologous recombination. As described herein, gene
targeting can be used to replace a gene's existing regulatory
region with a regulatory sequence isolated from a different gene or
a novel regulatory sequence synthesized by genetic engineering
methods. Such regulatory sequences may be comprised of promoters,
enhancers, scaffold-attachment regions, negative regulatory
elements, transcriptional initiation sites, regulatory protein
binding sites or combinations of said sequences. Alternatively,
sequences which affect the structure or stability of the RNA or
protein produced may be replaced, removed, added, or otherwise
modified by targeting, including polyadenylation signals. mRNA
stability elements, splice sites, leader sequences for enhancing or
modifying transport or secretion properties of the protein, or
other sequences which alter or improve the function or stability of
protein or RNA molecules.
[0418] The host of the present invention may also be a yeast or
other fungi. In yeast, a number of vectors containing constitutive
or inducible promoters may be used. For a review see, Ausubel et
al. (eds), Current Protocols in Molecular Biology, Vol. 2, Greene
Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Grant et
al., 1987, "Expression and Secretion Vectors for Yeast", Methods
Enzymol. 153:516-544; Glover. DNA Cloning, Vol. II, IRL Press,
Wash. D.C., Ch. 3 (1986); Bitter, 1987. "Heterologous Gene
Expression in Yeast", Methods Enzymol. 152:673-684; and Strathern
et al. (eds), The Molecular Biology of the Yeast Saccharomyces,
Cold Spring Harbor Press, Vols. I and II (1982).
[0419] Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins. Potentially suitable bacterial strains include
Escherichia coli, enterobacteriaceae such as Serratia marescans,
bacilli such as Bacillus subtilis, Salmonella typhimurium,
pseudomonads or any bacterial strain capable of expressing
heterologous proteins. If the protein is made in yeast or bacteria,
it may be necessary to modify the protein produced therein, for
example by phosphorylation or glycosylation of the appropriate
sites, in order to obtain the functional protein. Such covalent
attachments may be accomplished using known chemical or enzymatic
methods.
[0420] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the monkey COS cells such as COS-7 lines of monkey
kidney fibroblasts, described by Gluzman, 1981. Cell 23:175 (1981),
Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human
epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells,
normal diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants. HeLa cells, mouse L cells, BHK,
HL-60, U937. HaK, C127, 3T3, or Jurkat cells, and other cell lines
capable of expressing a compatible vector. Mammalian expression
vectors will comprise an origin of replication, a suitable promoter
and also any necessary ribosome-binding sites, polyadenylation
site, splice donor and acceptor sites, transcriptional termination
sequences, and 5' flanking nontranscribed sequences.
[0421] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents.
Recombinant polypeptides produced in bacterial culture are usually
isolated by initial extraction from cell pellets, followed by one
or more salting-out, aqueous ion exchange or size exclusion
chromatography steps. In some embodiments, the template nucleic
acid also encodes a polypeptide tag. e.g., penta- or
hexa-histidine.
[0422] Recombinant proteins can be isolated using an techniqe
well-known in the art. Scopes (Protein Purification. Principles and
Practice, Springer-Verlag, New York (1994)), for example, provides
a number of general methods for purifying recombinant (and
non-recombinant) proteins. The methods include, e.g., ion-exchange
chromatography, size-exclusion chromatography, affinity
chromatography, selective precipitation, dialysis, and hydrophobic
interaction chromatography.
[0423] Variations, modifications, and other implementations of what
is described herein will occur to those of ordinary skill in the
art without departing from the spirit and scope of the
invention.
[0424] In order that the invention described herein may be more
fully understood, the following example is set forth. It should be
understood that this example is for illustrative purposes only and
are not to be construed as limiting this invention in any
manner.
[0425] 5.20 Methods for Identifying Compounds for Use in the
Prevention, Treatment, Management or Amelioration Osteoarthritis or
a Symptom Thereof
[0426] 5.20.1 Methods for Identifying Compounds that Modulate the
Expression or Activity of a Biomarker
[0427] The present invention provides methods of identifying
compounds that bind to the products of the biomarkers of the
invention. The present invention also provides methods for
identifying compounds that modulate the expression and/or activity
of the products of the biomarkers of the invention. The compounds
identified via such methods are useful for the development of one
or more animal models to study osteoarthritis. Further, the
compounds identified via such methods are useful as lead compounds
in the development of prophylactic and therapeutic compositions for
prevention, treatment, management and/or amelioration of
osteoarthritis or a symptom thereof. Such methods are particularly
useful in that the effort and great expense involved in testing
potential prophylactics and therapeutics in vivo is efficiently
focused on those compounds identified via the in vitro and ex vivo
methods described herein.
[0428] The present invention provides a method for identifying a
compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof, said method
comprising: (a) contacting a cell expressing a protein product of
one or more biomarkers of the invention or a fragment thereof, or a
RNA product of one or more biomarkers of the invention or a
fragment thereof with a test compound; and (b) determining the
ability of the test compound to bind to the protein product,
protein fragment, RNA product, or RNA portion so that if a compound
binds to the protein product, protein fragment, RNA product, RNA
portion, a compound to be tested for an ability to prevent, treat,
manage or ameliorate osteoarthritis or a symptom thereof is
identified. The cell, for example, can be a yeast cell or a cell of
mammalian origin. Determining the ability of the test compound to
bind to the protein product, protein fragment, RNA product, or RNA
portion can be accomplished, for example, by coupling the test
compound with a radioisotope or enzymatic label such that binding
of the test compound to the protein product, protein fragment, RNA
product, or RNA portion can be determined by detecting the labeled
compound in a complex. For example, test compounds can be labeled
with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively, test
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product. In a specific embodiment, the
assay comprises contacting a cell which expresses a protein product
of one or more biomarkers of the invention or a fragment thereof,
or a RNA product of one or more biomarkers of the invention or a
fragment thereof, with a known compound which binds the protein
product, protein fragment, RNA product, or RNA portion to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
the protein product, protein fragment, RNA product, or RNA portion,
wherein determining the ability of the test compound to interact
with the protein product, protein fragment, RNA product, or RNA
portion comprises determining the ability of the test compound to
preferentially bind to the protein product, protein fragment, RNA
product, or RNA portion as compared to the known compound.
[0429] The present invention provides a method for identifying a
compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof, said method
comprising: (a) contacting a protein product of one or more
biomarkers of the invention or a fragment thereof, or a RNA product
of one or more biomarkers of the invention or a portion thereof
with a test compound; and (b) determining the ability of the test
compound to bind to the protein product, protein fragment, RNA
product, or RNA portion so that if a compound binds to the protein
product, protein fragment, RNA product, or RNA portion, a compound
to be tested for an ability to prevent, treat, manage or ameliorate
osteoarthritis or a symptom thereof is identified. Binding of the
test compound to the protein product or protein fragment can be
determined either directly or indirectly. In a specific embodiment,
the assay includes contacting a protein product of one or more
biomarkers of the invention or a fragment thereof, or a RNA product
of one or more biomarkers of the invention or a portion thereof
with a known compound which binds the protein product, protein
fragment, RNA product, or RNA portion to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with the protein
product, protein fragment, RNA product, or RNA portion, wherein
determining the ability of the test compound to interact with the
protein product, protein fragment, RNA product, or RNA portion
comprises determining the ability of the test compound to
preferentially bind to the protein product, protein fragment, RNA
product, or RNA portion as compared to the known compound.
Techniques well known in the art can be used to determine the
binding between a test compound and a protein product of a
biomarker of the invention or a fragment thereof, or a RNA product
of a biomarker of the invention or a portion thereof.
[0430] In some embodiments of the above assay methods of the
present invention, it may be desirable to immobilize a RNA product
of a biomarker of the invention or a portion thereof, or its target
molecule to facilitate separation of complexed from uncomplexed
forms of the RNA product or RNA portion, the target molecule or
both, as well as to accommodate automation of the assay. In more
than one embodiment of the above assay methods of the present
invention, it may be desirable to immobilize either a protein
product of a biomarker of the invention or a fragment thereof, or
its target molecule to facilitate separation of complexed from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of a test compound to
a protein product of a biomarker of the invention or a fragment
thereof can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase (GST) fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.)
or glutathione derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or a protein product of a biomarker of
the invention or a fragment thereof, and the mixture incubated
under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components and complex formation is measured either
directly or indirectly, for example, as described above.
Alternatively, the complexes can be dissociated from the matrix,
and the level of binding of a protein product of a biomarker of the
invention or a fragment thereof can be determined using standard
techniques.
[0431] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a protein product of a biomarker of the invention or a
fragment thereof, or a target molecule can be immobilized utilizing
conjugation of biotin and streptavidin. A biotinylated protein
product of a biomarker of the invention or a target molecule can be
prepared from biotin-NHS (N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals;
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with a protein product of a
biomarker of the invention or a fragment thereof can be derivatized
to the wells of the plate, and protein trapped in the wells by
antibody conjugation. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with a protein product of a biomarker of the invention, as
well as enzyme-linked assays which rely on detecting an enzymatic
activity associated with a protein product of a biomarker of the
invention or a fragment thereof, or target molecule.
[0432] The interaction or binding of a protein product of a
biomarker of the invention or a fragment thereof to a test compound
can also be determined using such proteins or protein fragments as
"bait proteins" in a two-hybrid assay or three hybrid assay (see,
e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell
72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;
Bartel et al. (1993) Bio/Techniques 14:920-924; lwabuchi et al.
(1993) Oncogene 8:1693-1696; and International Publication No. WO
94/10300).
[0433] The present invention provides a method for identifying a
compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof, said method
comprising: (a) contacting a cell expressing a protein or RNA
product of one or more biomarkers of the invention with a test
compound; (b) determining the amount of the protein or RNA product
present in (a); and (c) comparing the amount in (a) to that present
in a corresponding control cell that has not been contacted with
the test compound, so that if the amount of the protein or RNA
product is altered relative to the amount in the control, a
compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof is identified. In a
specific embodiment, the expression level(s) is altered by 5%, 10%,
15%, 25%, 30%, 40%, 50%, 5 to 25%, 10 to 30%, at least 1 fold, at
least 1.5 fold, at least 2 fold, 4 fold, 5 fold, 10 fold, 25 fold,
1 to 10 fold, or 5 to 25 fold relative to the expression level in
the control as determined by utilizing an assay described herein
(e.g., a microarray or RT-PCR) or an assay well known to one of
skill in the art. In alternate embodiments, such a method comprises
determining the amount of the protein or RNA product of at least 2,
at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 12, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, 1 to 5, 1-10, 5-10, 5-25, or 10-40, all or
any combination of the biomarkers of the invention present in the
cell and comparing the amounts to those present in the control.
[0434] The cells utilized in the cell-based assays described herein
can be engineered to express a biomarker of the invention utilizing
techniques known in the art. See, e.g., Section III entitled
"Recombinant Expression Vectors and Host Cells" of U.S. Pat. No.
6,245,527, which is incorporated herein by reference.
Alternatively, cells that endogenously express a biomarker of the
invention can be used. For example, chondrocytes may be used.
[0435] In a specific embodiment, chondrocytes are isolated from a
"normal" individual, or an individual with mild, moderate, marked
or severe osteoarthritis and are incubated in the presence and
absence of a test compound for varying amounts of time (i.e., 30
min, 1 hr, 5 hr, 24 hr, 48 hr and 96 hrs). When screening for
prophylactic or therapeutic agents, a clone of the full sequence of
a biomarker of the invention or functional portion thereof is used
to transfect chondrocytes. The transfected chondrocytes are
cultured for varying amounts of time (i.e., 1, 2, 3, 5, 7, 10, or
14 days) in the presence or absence of test compound. Following
incubation, target nucleic acid samples are prepared from the
chondrocytes and hybridized to a nucleic acid probe corresponding
to a nucleic acid sequence which is differentially expressed in a
chondrocyte derived from at least any two of the following of:
normal, mild osteoarthritic, moderate osteoarthritic and severe
osteoarthritic. The nucleic acid probe is labeled, for example,
with a radioactive label, according to methods well-known in the
art and described herein. Hybridization is carried out by northern
blot, for example as described in Ausubel et al., supra or Sambrook
et al. supra). The differential hybridization, as defined herein,
of the target to the samples on the array from normal relative to
RNA from any one of mild osteoarthritic, moderate osteoarthritic,
marked osteoarthritic and severe osteoarthritic is indicative of
the level of expression of RNA corresponding to a differentially
expressed chondrocyte specific nucleic acid sequence. A change in
the level of expression of the target sequence as a result of the
incubation step in the presence of the test compound, is indicative
of a compound that increases or decreases the expression of the
corresponding chondrocyte specific nucleic acid sequence.
[0436] The present invention also provides a method for identifying
a compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof, said method
comprises: (a) contacting a cell-free extract (e.g., a chondrocyte
extract) with a nucleic acid sequence encoding a protein or RNA
product of one or more biomarkers of the invention and a test
compound; (b) determining the amount of the protein or RNA product
present in (a); and (c) comparing the amount(s) in (a) to that
present to a corresponding control that has not been contacted with
the test compound, so that if the amount of the protein or RNA
product is altered relative to the amount in the control, a
compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof is identified. In a
specific embodiment, the expression level(s) is altered by 5%, 10%,
15%, 25%, 30%, 40%, 50%, 5 to 25%, 10 to 30%, at least 1 fold, at
least 1.5 fold, at least 2 fold, 4 fold, 5 fold, 10 fold, 25 fold,
1 to 10 fold, or 5 to 25 fold relative to the expression level in
the control sample determined by utilizing an assay described
herein (e.g., a microarray or RT-PCR) or an assay well known to one
of skill in the art. In alternate embodiments, such a method
comprises determining the amount of a protein or RNA product of at
least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 12, at least 15,
at least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, 1 to 5, 1-10, 5-10, 5-25, or 10-40, all or
any combination of the biomarkers of the invention present in the
extract and comparing the amounts to those present in the
control.
[0437] In certain embodiments, the amount of RNA product of a
biomarker of the invention is determined, in other embodiments, the
amount of protein product of a biomarker of the invention is
determined, while in still other embodiments, the amount of RNA and
protein product of a biomarker of the invention is determined.
Standard methods and compositions for determining the amount of RNA
or protein product of a biomarker of the invention can be utilized.
Such methods and compositions are described in detail above.
[0438] In specific embodiments, in a screening assay described
herein, the amount of protein or RNA product of a biomarker of the
invention is determined utilizing kits. Such kits comprise
materials and reagents required for measuring the expression of at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, or more protein or RNA products of at least
1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 15, at least
20, at least 25, at least 30, at least 35, at least 40, at least
45, at least 50, all or any combination of the biomarkers of the
invention. In specific embodiments, the kits may further comprise
one or more additional reagents employed in the various methods,
such as: (1) reagents for purifying RNA from blood, chondrocytes or
synovial fluid; (2) primers for generating test nucleic acids; (3)
dNTPs and/or rNTPs (either premixed or separate), optionally with
one or more uniquely labeled dNTPs and/or rNTPs (e.g., biotinylated
or Cy3 or Cy5 tagged dNTPs); (4) post synthesis labeling reagents,
such as chemically active derivatives of fluorescent dyes; (5)
enzymes, such as reverse transcriptases, DNA polymerases, and the
like; (6) various buffer mediums. e.g., hybridization and washing
buffers; (7) labeled probe purification reagents and components,
like spin columns, etc.; and (8) protein purification reagents; (9)
signal generation and detection reagents. e.g.,
streptavidin-alkaline phosphatase conjugate, chemifluorescent or
chemiluminescent substrate, and the like. In particular
embodiments, the kits comprise prelabeled quality controlled
protein and or RNA transcript (preferably, mRNA) for use as a
control.
[0439] In some embodiments, the kits are RT-PCR kits. In other
embodiments, the kits are nucleic acid arrays and protein arrays.
Such kits according to the subject invention will at least comprise
an array having associated protein or nucleic acid members of the
invention and packaging means therefore. Alternatively the protein
or nucleic acid members of the invention may be prepackaged onto an
array.
[0440] In a specific embodiment, kits for measuring a RNA product
of a biomarker of the invention comprise materials and reagents
that are necessary for measuring the expression of the RNA product.
For example, a microarray or RT-PCR kit may be used and contain
only those reagents and materials necessary for measuring the
levels of RNA products of at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 15, at least 20, at least 25, at least 30,
at least 35, at least 40, at least 45, at least 50, all or any
combination of the biomarkers of the invention. Alternatively, in
some embodiments, the kits can comprise materials and reagents that
are not limited to those required to measure the levels of RNA
products of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, all or any combination of the biomarkers of the invention.
For example, a microarray kit may contain reagents and materials
necessary for measuring the levels of RNA products 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, all or any
combination of the biomarkers of the invention, in addition to
reagents and materials necessary for measuring the levels of the
RNA products of at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 15, at least 20, at least 25, at least 30, at least
35, at least 40, at least 45, at least 50 or more genes other than
the biomarkers of the invention. In a specific embodiment, a
microarray or RT-PCR kit contains reagents and materials necessary
for measuring the levels of RNA products of at least 1, at least 2,
at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 15, at least 20, at
least 25, at least 30, at least 35, at least 40, at least 45, at
least 50, all or any combination of the biomarkers of the
invention, and 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225,
250, 300, 350, 400, 450, or more genes that are not biomarkers of
the invention, or 1-10, 1-100, 1-150, 1-200, 1-300, 1-400, 1-500,
1-1000, 25-100, 25-200, 25-300, 25-400, 25-500, 25-1000, 100-150,
100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 genes that
are not biomarkers of the invention.
[0441] For nucleic acid micoarray kits, the kits generally comprise
probes attached to a solid support surface. The probes may be
labeled with a detectable label. In a specific embodiment, the
probes are specific for the 5' region, the 3' region, the internal
coding region, an exon(s), an intron(s), an exon junction(s), or an
exon-intron junction(s), of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50, all or any combination of the biomarkers of
the invention. The microarray kits may comprise instructions for
performing the assay and methods for interpreting and analyzing the
data resulting from the performance of the assay. The kits may also
comprise hybridization reagents and/or reagents necessary for
detecting a signal produced when a probe hybridizes to a target
nucleic acid sequence. Generally, the materials and reagents for
the microarray kits are in one or more containers. Each component
of the kit is generally in its own a suitable container.
[0442] For RT-PCR kits, the kits generally comprise pre-selected
primers specific for particular RNA products (e.g., an exon(s), an
intron(s), an exon junction(s), and an exon-intron junction(s)) of
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, all
or any combination of the biomarkers of the invention. The RT-PCR
kits may also comprise enzymes suitable for reverse transcribing
and/or amplifying nucleic acids (e.g., polymerases such as Taq),
and deoxynucleotides and buffers needed for the reaction mixture
for reverse transcription and amplification. The RT-PCR kits may
also comprise probes specific for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25, 30, 35, 40, 45, 50, all or any combination of the
biomarkers of the invention. The probes may or may not be labeled
with a detectable label (e.g., a fluorescent label). Each component
of the RT-PCR kit is generally in its own suitable container. Thus,
these kits generally comprise distinct containers suitable for each
individual reagent, enzyme, primer and probe. Further, the RT-PCR
kits may comprise instructions for performing the assay and methods
for interpreting and analyzing the data resulting from the
performance of the assay.
[0443] For antibody based kits, the kit can comprise, for example:
(1) a first antibody (which may or may not be attached to a solid
support) which binds to protein of interest (e.g., a protein
product of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 15, 30, 35, 40,
45, 50, all or any combination of the biomarkers of the invention);
and, optionally, (2) a second, different antibody which binds to
either the protein, or the first antibody and is conjugated to a
detectable label (e.g. a fluorescent label, radioactive isotope or
enzyme). The antibody-based kits may also comprise beads for
conducting an immunoprecipitation. Each component of the
antibody-based kits is generally in its own suitable container.
Thus, these kits generally comprise distinct containers suitable
for each antibody. Further, the antibody-based kits may comprise
instructions for performing the assay and methods for interpreting
and analyzing the data resulting from the performance of the
assay.
[0444] Reporter gene-based assays may also be conducted to identify
a compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof. In a specific
embodiment, the present invention provides a method for identifying
a compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof, said method
comprising: (a) contacting a compound with a cell expressing a
reporter gene construct comprising a reporter gene operably linked
to a regulatory element of a biomarker of the invention (e.g. a
promoter/enhancer element); (b) measuring the expression of said
reporter gene; and (c) comparing the amount in (a) to that present
in a corresponding control cell that has not been contacted with
the test compound, so that if the amount of expressed reporter gene
is altered relative to the amount in the control cell, a compound
to be tested for an ability to prevent, treat, manage or ameliorate
osteoarthritis or a symptom thereof is identified. In accordance
with this embodiment, the cell may naturally express the biomarker
or be engineered to express the biomarker. In another embodiment,
the present invention provides a method for identifying a compound
to be tested for an ability to prevent, treat, manage or ameliorate
osteoarthritis or a symptom thereof, said method comprising: (a)
contacting a compound with a cell-free extract and a reporter gene
construct comprising a reporter gene operably linked to a
regulatory element of a biomarker of the invention (e.g., a
promoter/enhancer element); (b) measuring the expression of said
reporter gene; and (c) comparing the amount in (a) to that present
in a corresponding control that has not been contacted with the
test compound, so that if the amount of expressed reporter gene is
altered relative to the amount in the control, a compound to be
tested for an ability to prevent, treat, manage or ameliorate
osteoarthritis or a symptom thereof is identified.
[0445] Any reporter gene well-known to one of skill in the art may
be used in reporter gene constructs used in accordance with the
methods of the invention. Reporter genes refer to a nucleotide
sequence encoding a RNA transcript or protein that is readily
detectable either by its presence (by, e.g. RT-PCR, Northern blot,
Western Blot, ELISA, etc.) or activity. Non-limiting examples of
reporter genes are listed in Table 4, infra. Reporter genes may be
obtained and the nucleotide sequence of the elements determined by
any method well-known to one of skill in the art. The nucleotide
sequence of a reporter gene can be obtained, e.g., from the
literature or a database such as GenBank. Alternatively, a
polynucleotide encoding a reporter gene may be generated from
nucleic acid from a suitable source. If a clone containing a
nucleic acid encoding a particular reporter gene is not available,
but the sequence of the reporter gene is known, a nucleic acid
encoding the reporter gene may be chemically synthesized or
obtained from a suitable source (e.g., a cDNA library, or a cDNA
library generated from, or nucleic acid, preferably poly A+ RNA,
isolated from, any tissue or cells expressing the reporter gene) by
PCR amplification. Once the nucleotide sequence of a reporter gene
is determined, the nucleotide sequence of the reporter gene may be
manipulated using methods well-known in the art for the
manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology. John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate reporter genes having a different amino
acid sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
TABLE-US-00002 TABLE 4 Reporter Genes and the Properties of the
Reporter Gene Products Reporter Gene Protein Activity &
Measurement CAT (chloramphenicol Transfers radioactive acetyl
groups to acetyltransferase) chloramphenicol or detection by thin
layer chromatography and autoradiography GAL (beta-galactosidase)
Hydrolyzes colorless galactosides to yield colored products. GUS
(beta-glucuronidase) Hydrolyzes colorless glucuronides to yield
colored products. LUC (luciferase) Oxidizes luciferin, emitting
photons GFP (green fluorescent Fluorescent protein without
substrate protein) SEAP (secreted alkaline Luminescence reaction
with suitable phosphatase) substrates or with substrates that
generate chromophores HRP (horseradish In the presence of hydrogen
oxide, oxidation peroxidase) of 3,3',5,5'-tetramethylbenzidine to
form a colored complex AP (alkaline phosphatase) Luminescence
reaction with suitable substrates or with substrates that generate
chromophores
[0446] In accordance with the invention, cells that naturally or
normally express one or more, all or any combination of the
biomarkers of the invention can be used in the methods described
herein. Alternatively, cells can be engineered to express one or
more, all or any combination of the biomarkers of the invention, or
a reporter gene using techniques well-known in the art and used in
the methods described herein. Examples of such techniques include,
but are not to, calcium phosphate precipitation (see, e.g., Graham
& Van der Eb, 1978, Virol. 52:546), dextran-mediated
transfection, calcium phosphate mediated transfection, polybrene
mediated transfection, protoplast fusion, electroporation,
encapsulation of the nucleic acid in liposomes, and direct
microinjection of the nucleic acid into nuclei.
[0447] In a specific embodiment, the cells used in the methods
described herein are chondrocytes, lymphocytes (T or B
lymphocytes), monocytes, neutrophils, macrophages, eosinophils,
basophils, erythrocytes or platelets. In a preferred embodiment,
the cells used in the methods described herein are chondrocytes. In
another preferred embodiment, the cells used in the methods
described herein are lymphocytes. In another embodiment, the cells
used in the methods described herein are immortalized cell lines
derived from a source, e.g., a tissue.
[0448] Any cell-free extract that permits the translation, and
optionally but preferably, the transcription, of a nucleic acid can
be used in accordance with the methods described herein. The
cell-free extract may be isolated from cells of any species origin.
For example, the cell-free translation extract may be isolated from
human cells, cultured mouse cells, cultured rat cells, Chinese
hamster ovary (CHO) cells, Xenopus oocytes, rabbit reticulocytes,
wheat germ, or rye embryo (see. e.g., Krieg & Melton, 1984,
Nature 308:203 and Dignam et al. 1990 Methods Enzymol.
182:194-203). Alternatively, the cell-free translation extract,
e.g., rabbit reticulocyte lysates and wheat germ extract, can be
purchased from, e.g., Promega, (Madison, Wis.). In a preferred
embodiment, the cell-free extract is an extract isolated from human
cells. In a specific embodiment, the human cells are HeLa cells,
lymphocytes, or chondrocytes.
[0449] In addition to the ability to modulate the expression levels
of RNA and/or protein products a biomarker of the invention, it may
be desirable, at least in certain instances, that compounds
modulate the activity of a protein product of a biomarker of the
invention. Thus, the present invention provides methods of
identifying compounds to be tested for an ability to prevent,
treat, manage or ameliorate osteoarthritis or a symptom thereof,
comprising methods for identifying compounds that modulate the
activity of a protein product of one or more biomarkers of the
invention. Such methods can comprise: (a) contacting a cell
expressing a protein product of one or more biomarkers of the
invention with a test compound; (b) determining the activity level
of the protein product; and (c) comparing the activity level to
that in a corresponding control cell that has not been contacted
with the test compound, so that if the level of activity in (a) is
altered relative to the level of activity in the control cell, a
compound to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof is identified. In a
specific embodiment, the activity level(s) is altered by 5%, 10%,
15%, 25%, 30%, 40%, 50%, 5 to 25%, 10 to 30%, at least 1 fold, at
least 1.5 fold, at least 2 fold, 4 fold, 5 fold, 10 fold, 25 fold,
1 to 10 fold, or 5 to 25 fold relative to the activity level in the
control as determined by utilizing an assay described herein (e.g.,
a microarray or RT-PCR) or an assay well known to one of skill in
the art. In alternate embodiments, such a method comprises
determining the activity level of a protein product of at least 2,
at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 12, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, 1 to 5, 1-10, 5-10, 5-25, or 10-40, all or
any combination of the biomarkers of the invention present in the
cell and comparing the activity levels to those present in the
control.
[0450] The present invention provides methods of identifying
compounds to be tested for an ability to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof, comprising: (a)
contacting a cell-free extract with a nucleic acid encoding a
protein product of one or more biomarkers of the invention and a
test compound; (b) determining the activity level of the protein
product; and (c) comparing the activity level to that in a
corresponding control that has not been contacted with the test
compound, so that if the level of activity in (a) is altered
relative to the level of activity in the control, a compound to be
tested for an ability to prevent, treat, manage or ameliorate
osteoarthritis or a symptom thereof is identified. In a specific
embodiment, the activity level(s) is altered by 5%, 10%, 15%, 25%,
30%, 40%, 50%, 5 to 25%, 10 to 30%, at least 1 fold, at least 1.5
fold, at least 2 fold 4 fold 5 fold, 10 fold, 25 fold 1 to 10 fold,
or 5 to 25 fold relative to the activity level in the control as
determined by utilizing an assay described herein (e.g. a
microarray or RT-PCR) or an assay well known to one of skill in the
art. In alternate embodiments, such a method comprises determining
the activity level of a protein product of at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 12, at least 15, at least 20, at
least 25, at least 30, at least 35, at least 40, at least 45, at
least 50, 1 to 5, 1-10, 5-10, 5-25, or 10-40, all or any
combination of the biomarkers of the invention present in the
sample and comparing the activity levels to those present in the
control.
[0451] Standard techniques can be utilized to determine the level
of activity of a protein product of a biomarker of the invention.
See, e.g., Table 5, infra, for examples of activities of protein
products of biomarkers of the invention that can be determined
using techniques well known in the art.
TABLE-US-00003 TABLE 5 Gene Symbol Activity ABCA1 Regulation of
cholesterol and phospholipid efflux ABCG1 Regulation of cholesterol
and phospholipid efflux BCL6 Regulation of T cell and B cell
interactions; the Expression of B7-1/CD80 ACP1 Regulation of cell
motility; Interaction with and dephosphorylation of FAK ADPRT
Interaction with proliferating cell nuclear antigen (PCNA);
Transfer of ADP-ribose moieties to itself and to nuclear acceptor
proteins ANGPTL2 Sprouting in vascular endothelial cells B2M
Interaction with MHC1 alpha 3 domain BMPR2 Phosphorylation of Smads
1, 5, and 8 CLIC4 Interaction with ERK7 Egr1 LDLR transcription;
Activation of human PPARgamma1 gene expression IKBKAP Interaction
with NF-kappaB-inducing kinase (NlK) and IKB kinases (IKKs) IL13RA1
Phosphorylation of IL13RA1; Binding to Tyk2: Activation of Jak1,
Tyk2, the insulin receptor substrate-1 and STAT6 ILF1 Binding to
the interleukin-2 (IL-2) promoter; Regulation of IL-2 gene
expression NCOA1 Association with STAT3 PAIP2 Interaction with PABP
PDCD5 Translocation to the nucleus PDK4 Phosphorylation of pyruvate
dehydrogenase complex (PDC) PF4 Binding to Heparin; Anti-heparin
activities SETBP1 Interaction with SET TSG-6 Binding to hyaluronan
and glycosaminoglycans TNFA1P6 Role in extracellular remodeling and
cellular proliferation Yes1 Tyrosine kinase activity
[0452] 5.20.2 Biological Activity of the Compounds
[0453] Upon identification of compounds to be tested for an ability
to prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof (for convenience referred to herein as a "lead" compound),
the compounds can be further investigated. For example, the
compounds identified via the present methods can be further tested
in vivo in accepted animal models of inflammation, preferably,
arthritis and more preferably, osteoarthritis. Further, the
compounds identified via the methods can be analyzed with respect
to their specificity. In particular, the compounds can be tested
for an effect on manufacture of type II collagen and proteoglycans
by chondrocytes. Techniques for such additional compound
investigation are well known to one of skill in the art.
[0454] In one embodiment, the effect of a lead compound can be
assayed by measuring the cell growth or viability of the target
cell. Such assays can be carried out with representative cells of
cell types involved in osteoarthritis (e.g., chondrocytes).
Alternatively, instead of culturing cells from a patient, a lead
compound may be screened using cells of a cell line.
[0455] Many assays well-known in the art can be used to assess the
survival and/or growth of a patient cell or cell line following
exposure to a lead compound: for example, cell proliferation can be
assayed by measuring Bromodeoxyuridine (BrdU) incorporation (see.
e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al.,
1988, J. Immunol. Meth. 107:79) or (.sup.3H)-thymidine
incorporation (see, e.g., Chen, J., 1996, Oncogene 13:1395-403;
Jeoung, J., 1995, J. Biol. Chem. 270:18367-73), by direct cell
count, by detecting changes in transcription, translation or
activity of known genes such as proto-oncogenes (e.g., jos, myc) or
cell cycle markers (Rb, cdc2, cyclin A, D1, D2, D3, E, etc). The
levels of such protein and RNA (e.g., mRNA) and activity can be
determined by any method well known in the art. For example,
protein can be quantitated by known immunodiagnostic methods such
as Western blotting or immunoprecipitation using commercially
available antibodies. mRNA can be quantitated using methods that
are well known and routine in the art, for example, using northern
analysis, RNase protection, the polymerase chain reaction in
connection with the reverse transcription. Cell viability can be
assessed by using trypan-blue staining or other cell death or
viability markers known in the art. In a specific embodiment, the
level of cellular ATP is measured to determined cell viability.
Differentiation can be assessed, for example, visually based on
changes in morphology.
[0456] One example of a chondrocyte proliferation assay is as
follows: Chondrocytes are retrieved from human severe OA cartilage
slices as previously described. (Doherty P J, Zhang H, Trembley L.
Manolopoulos V and Marshall K W., 1998, Osteoarthritis and
Cartilage 6:153-160). Cells are then washed, counted and seeded at
1.times.10.sup.4 cells/well in a flat-bottomed 96-well plate
(Corning) in DMEM++. After cells attach to the plate, they are
washed with DMEM only, and then incubated in DMEM with or without
10% FCS along with different concentrations of lead compound for 48
hours. The cell number in each well is then determined by adding 10
.mu.l of WST-1 (a tetrazolium salt that can be cleaved to formazan
by mitochondrial dehydrogenases in live cells. Roche) to each well,
mixing thoroughly for 1 min. and incubating at 37.degree. for 1.5
hours. Then the plate is scanned by a microplate autoreader
(BIO-TEK Instruments) at an absorbance of 450 nm. The number of
viable cells is reflected by the amount of formazan formed which is
quantified by measuring absorbance at 450 nm. (Lang I, Hoffmann C.
Olip H, Pabst M A, Hahn T. Dohr G, Desoye G., 2001, Differential
mitogenic responses of human macrovascular and microvascular
endothelial cells to cytokines underline their phenotypic
heterogeneity. Cell Prolif 34:143-55).
[0457] The effect on manufacture of type II collagen and
proteoglycans by chondrocytes exposed to a lead compound can be
determined using techniques well known in the art. Further, any
assay well known in the art for assessing the efficacy of a therapy
for prevention, treatment, management or amelioration of a
condition, in particular osteoarthritis, can be performed using the
lead compounds.
Animal Models
[0458] Compounds can be tested in suitable animal model systems
prior to use in humans. Such animal model systems include but are
not limited to rats, mice, chicken, cows, monkeys, pigs, dogs,
rabbits, etc. Any animal system well-known in the art may be used.
In certain embodiments, compounds are tested in a mouse model.
Compounds can be administered repeatedly.
[0459] Accepted animal models can be utilized to determine the
efficacy of the compounds identified via the methods described
above for the prevention, treatment, management and/or amelioration
of osteoarthritis or a symptom thereof. Such models can include the
various experimental animal models of inflammatory arthritis known
in the art and described in Crofford L. J. and Wilder R. L.
"Arthritis and Autoimmunity in Animals", in Arthritis and Allied
Conditions: A Textbook of Rheumatology, McCarty et al. (eds.),
Chapter 30 (Lee and Febiger, 1993). The principle animal models for
arthritis or inflammatory disease known in the art and widely used
include: adjuvant-induced arthritis rat models, collagen-induced
arthritis rat and mouse models and antigen-induced arthritis rat,
rabbit and hamster models, all described in Crofford L. J. and
Wilder R. L. "Arthritis and Autoimmunity in Animals", in Arthritis
and Allied Conditions: A Textbook of Rheumatology, McCarty et al.
(eds.), Chapter 30 (Lee and Febiger, 1993), incorporated herein by
reference in its entirety.
[0460] In one embodiment, the efficacy of a compound for the
prevention, treatment, management and/or amelioration of
osteoarthritis or a symptom thereof is determined using a
carrageenan-induced arthritis rat model. Carrageenan-induced
arthritis has also been used in rabbit, dog and pig in studies of
chronic arthritis or inflammation. Quantitative histomorphometric
assessment is used to determine therapeutic efficacy. The methods
for using such a carrageenan-induced arthritis model is described
in Hansra P. et al., "Carrageenan-Induced Arthritis in the Rat,"
Inflammation, 24(2): 141-155, (2000). Also commonly used are
zymosan-induced inflammation animal models as known and described
in the art.
[0461] The anti-inflammatory activity of the compounds can be
assessed by measuring the inhibition of carrageenan-induced paw
edema in the rat, using a modification of the method described in
Winter C. A. e t al., "Carrageenan-Induced Edema in Hind Paw of the
Rat as an Assay for Anti-inflammatory Drugs" Proc. Soc. Exp. Biol
Med. 111, 544-547, (1962). This assay has been used as a primary in
vivo screen for the anti-inflammatory activity of most NSAIDs, and
is considered predictive of human efficacy. The anti-inflammatory
activity of the test compound is expressed as the percent
inhibition of the increase in hind paw weight of the test group
relative to the vehicle dosed control group.
[0462] In another embodiment, the efficacy of a compound for the
prevention, treatment, management and/or amelioration of
osteoarthritis or a symptom thereof is determined using a
collagen-induced arthritis (CIA) model. CIA is an animal model for
the human autoimmune disease rheumatoid arthritis (RA) (Trenthorn
et al., 1977, J. Exp. Med., 146:857). This disease can be induced
in many species by the administration of heterologous type II
collagen (Courtenay et al. 1980. Nature 283:665: Cathcart et at,
1986, Lab. Invest., 54:26). With respect to animal models of
arthritis see, in addition. e.g., Holmdahl, R., 1999, Curr. Biol.
15:R528-530.
[0463] In another embodiment, the efficacy of a compound for the
prevention, treatment, management and/or amelioration of
osteoarthritis or a symptom thereof is determined using assays that
determine bone formation and/or bone loss. Animal models such as
ovariectomy-induced bone resorption mice, rat and rabbit models are
known in the art for obtaining dynamic parameters for bone
formation. Using methods such as those described by Yositake et al.
or Yamamoto et al., bone volume is measured in vivo by
microcomputed tomography analysis and bone histomorphometry
analysis. Yoshitake et al. "Osteopontin-Deficient Mice Are Resist
ant to Ovariectomy-Induced Bone Resorption." Proc. Natl. Acad. Sci.
96:8156-8160, (1999); Yamamoto et al., "The Integrin Ligand
Echistatin Prevents Bone Loss in Ovariectomized Mice and Rats,"
Endocrinology 139(3):1411-1419, (1998), both incorporated herein by
reference in their entirety.
Toxicity
[0464] The toxicity and/or efficacy of a compound identified in
accordance with the invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). Cells and cell lines that can be used to
assess the cytotoxicity of a compound identified in accordance with
the invention include, but are not limited to, peripheral blood
mononuclear cells (PBMCs), Caco-2 cells, and Huh7 cells. The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD.sub.50/ED.sub.50. A
compound identified in accordance with the invention that exhibits
large therapeutic indices is preferred. While a compound identified
in accordance with the invention that exhibits toxic side effects
may be used, care should be taken to design a delivery system that
targets such agents to the site of affected tissue in order to
minimize potential damage to uninfected cells and, thereby, reduce
side effects.
[0465] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of a compound
identified in accordance with the invention for use in humans. The
dosage of such agents lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
any agent used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 (i.e. the
concentration of the compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
Design of Congeners or Analogs
[0466] The compounds which display the desired biological activity
can be used as lead compounds for the development or design of
congeners or analogs having useful pharmacological activity. For
example, once a lead compound is identified, molecular modeling
techniques can be used to design variants of the compound that can
be more effective. Examples of molecular modeling systems are the
CHARM and QUANTA programs (Polygen Corporation. Waltham, Mass.).
CHARM performs the energy minimization and molecular dynamics
functions. QUANTA performs the construction, graphic modelling and
analysis of molecular structure. QUANTA allows interactive
construction, modification, visualization, and analysis of the
behavior of molecules with each other.
[0467] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen et al., 1988,
Acta Pharmaceutical Fennica 97:159-166; Ripka, 1998, New Scientist
54-57; McKinaly & Rossmann, 1989, Annu. Rev. Pharmacol.
Toxiciol. 29:111-122; Perry & Davies. OSAR: Quantitative
Structure-Activity Relationships in Drug Design pp. 189-193 (Alan
R. Liss, Inc. 1989); Lewis & Dean, 1989, Proc. R. Soc. Lond.
236:125-140 and 141-162; Askew et al., 1989, J. Am. Chem. Soc.
111:1082-1090. Other computer programs that screen and graphically
depict chemicals are available from companies such as BioDesign,
Inc. (Pasadena, Calif.). Allelix, Inc. (Mississauga. Ontario,
Canada), and Hypercube, Inc. (Cambridge. Ontario). Although these
are primarily designed for application to drugs specific to
particular proteins, they can be adapted to design of drugs
specific to any identified region. The analogs and congeners can be
tested for binding to the proteins of interest (i.e., the protein
products of a biomarker of the invention) using the above-described
screens for biologic activity. Alternatively, lead compounds with
little or no biologic activity, as ascertained in the screen, can
also be used to design analogs and congeners of the compound that
have biologic activity.
[0468] 5.20.3 Compounds
[0469] Compounds that can be tested and identified methods
described herein can include, but are not limited to, compounds
obtained from any commercial source, including Aldrich (1001 West
St. Paul Ave., Milwaukee, Wis. 53233), Sigma Chemical (P.O. Box
14508, St. Louis, Mo. 63178). Fluka Chemie AG (Industriestrasse 25,
CH-9471 Buchs, Switzerland (Fluka Chemical Corp. 980 South 2nd
Street, Ronkonkoma, N.Y. 11779)). Eastman Chemical Company, Fine
Chemicals (P.O Box 431, Kingsport, Tenn. 37662), Boehringer
Mannheim GmbH (Sandhofer Strasse 116, D-68298 Mannheim), Takasago
(4 Volvo Drive, Rockleigh, N.J. 07647), SST Corporation (635
Brighton Road. Clifton, N.J. 07012). Ferro (111 West Irene Road,
Zachary, La. 70791), Riedel-deHaen Aktiengesellschaft (P.O. Box
D-30918, Seelze, Germany). PPG Industries Inc., Fine Chemicals (One
PPG Place, 34th Floor, Pittsburgh, Pa. 15272). Further any kind of
natural products may be screened using the methods of the
invention, including microbial, fungal, plant or animal
extracts.
[0470] Compounds from large libraries of synthetic or natural
compounds can be screened. Numerous means are currently used for
random and directed synthesis of saccharide, peptide, and nucleic
acid-based compounds. Synthetic compound libraries are commercially
available from a number of companies including Maybridge Chemical
Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon
Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.).
A rare chemical library is available from Aldrich (Milwaukee,
Wis.). Combinatorial libraries are available and are prepared.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available from
e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are
readily produceable by methods well known in the art. Additionally,
natural and synthetically produced libraries and compounds are
readily modified through conventional chemical, physical, and
biochemical means.
[0471] Furthermore, diversity libraries of test compounds,
including small molecule test compounds, may be utilized. Libraries
screened using the methods of the present invention can comprise a
variety of types of compounds. Examples of libraries that can be
screened in accordance with the methods of the invention include,
but are not limited to, peptoids; random biooligomers; diversomers
such as hydantoins, benzodiazepines and dipeptides; vinylogous
polypeptides; nonpeptidal peptidomimetics; oligocarbamates;
peptidyl phosphonates; peptide nucleic acid libraries; antibody
libraries; carbohydrate libraries; and small molecule libraries
(preferably, small organic molecule libraries). In some
embodiments, the compounds in the libraries screened are nucleic
acid or peptide molecules. In a non-limiting example, peptide
molecules can exist in a phage display library. In other
embodiments, the types of compounds include, but are not limited
to, peptide analogs including peptides comprising non-naturally
occurring amino acids, e.g. D-amino acids, phosphorous analogs of
amino acids, such as .alpha.-amino phosphoric acids and
.alpha.-amino phosphoric acids, or amino acids having non-peptide
linkages, nucleic acid analogs such as phosphorothioates and PNAs,
hormones, antigens, synthetic or naturally occurring drugs,
opiates, dopamine, serotonin, catecholamines, thrombin,
acetylcholine, prostaglandins, organic molecules, pheromones,
adenosine, sucrose, glucose, lactose and galactose. Libraries of
polypeptides or proteins can also be used in the assays of the
invention.
[0472] In a specific embodiment, the combinatorial libraries are
small organic molecule libraries including, but not limited to
benzodiazepines, isoprenoids, thiazolidinones, metathiazanones,
pyrrolidines, morpholino compounds, and benzodiazepines. In another
embodiment, the combinatorial libraries comprise peptoids: random
bio-oligomers; benzodiazepines; diversomers such as hydantoins,
benzodiazepines and dipeptides; vinylogous polypeptides;
nonpeptidal peptidomimetics; oligocarbamates: peptidyl
phosphonates; peptide nucleic acid libraries; antibody libraries;
or carbohydrate libraries. Combinatorial libraries are themselves
commercially available For example, libraries may be commercially
obtained from, e.g., Specs and BioSpecs B.V. (Rijswijk, The
Netherlands), Chembridge Corporation (San Diego, Calif.), Contract
Service Company (Dolgoprudny, Moscow Region. Russia), Comgenex USA
Inc. (Princeton. NJ), Maybridge Chemicals Ltd. (Cornwall PL34 OHW,
United Kingdom). Asinex (Moscow, Russia), ComGenex (Princeton,
N.J.), Ru, Tripos, Inc. (St. Louis, Mo.), ChemStar. Ltd (Moscow,
Russia), 3D Pharmaceuticals (Exton. Pa.), and Martek Biosciences
(Columbia. Md.).
[0473] In a preferred embodiment, the library is preselected so
that the compounds of the library are more amenable for cellular
uptake. For example, compounds are selected based on specific
parameters such as, but not limited to, size, lipophilicity,
hydrophilicity, and hydrogen bonding, which enhance the likelihood
of compounds getting into the cells. In another embodiment, the
compounds are analyzed by three-dimensional or four-dimensional
computer computation programs.
[0474] The combinatorial compound library for use in accordance
with the methods of the present invention may be synthesized. There
is a great interest in synthetic methods directed toward the
creation of large collections of small organic compounds, or
libraries, which could be screened for pharmacological, biological
or other activity. The synthetic methods applied to create vast
combinatorial libraries are performed in solution or in the solid
phase, i.e., on a solid support. Solid-phase synthesis makes it
easier to conduct multi-step reactions and to drive reactions to
completion with high yields because excess reagents can be easily
added and washed away after each reaction step. Solid-phase
combinatorial synthesis also tends to improve isolation,
purification and screening. However, the more traditional solution
phase chemistry supports a wider variety of organic reactions than
solid-phase chemistry.
[0475] Combinatorial compound libraries of the present invention
may be synthesized using the apparatus described in U.S. Pat. No.
6,190,619 to Kilcoin et al., which is hereby incorporated by
reference in its entirety. U.S. Pat. No. 6,190,619 discloses a
synthesis apparatus capable of holding a plurality of reaction
vessels for parallel synthesis of multiple discrete compounds or
for combinatorial libraries of compounds.
[0476] In one embodiment, the combinatorial compound library can be
synthesized in solution. The method disclosed in U.S. Pat. No.
6,194,612 to Boger et al., which is hereby incorporated by
reference in its entirety, features compounds useful as templates
for solution phase synthesis of combinatorial libraries. The
template is designed to permit reaction products to be easily
purified from unreacted reactants using liquid/liquid or
solid/liquid extractions. The compounds produced by combinatorial
synthesis using the template will preferably be small organic
molecules. Some compounds in the library may mimic the effects of
non-peptides or peptides. In contrast to solid phase synthesize of
combinatorial compound libraries, liquid phase synthesis does not
require the use of specialized protocols for monitoring the
individual steps of a multistep solid phase synthesis (Egner et
al., 1995. J. Org. Chem. 60:2652; Anderson et al., 1995, J. Org.
Chem. 60:2650; Fitch et al. 1994, J. Org. Chem. 59:7955; Look et
al. 1994, J. Org. Chem. 49:7588; Metzger et al., 1993, Angew.
Chem., Int. Ed. Engl. 32:894: Youngquist et al. 1994, Rapid Commun.
Mass Spect. 8:77: Chu et al. 1995, J. Am. Chem. Soc. 117:5419;
Brummel et al., 1994. Science 264:399; and Stevanovic et al., 1993.
Bioorg. Med. Chem. Lett. 3:431).
[0477] Combinatorial compound libraries useful for the methods of
the present invention can be synthesized on solid supports. In one
embodiment, a split synthesis method, a protocol of separating and
mixing solid supports during the synthesis, is used to synthesize a
library of compounds on solid supports (see e.g., Lam et al., 1997,
Chem. Rev. 97:41-448; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci.
USA 90:10922-10926 and references cited therein). Each solid
support in the final library has substantially one type of compound
attached to its surface. Other methods for synthesizing
combinatorial libraries on solid supports, wherein one product is
attached to each support, will be known to those of skill in the
art (see, e.g., Nefzi et al. 1997, Chem. Rev. 97:449-472).
[0478] In some embodiments of the present invention, compounds can
be attached to solid supports via linkers. Linkers can be integral
and part of the solid support, or they may be nonintegral that are
either synthesized on the solid support or attached thereto after
synthesis. Linkers are useful not only for providing points of
compound attachment to the solid support, but also for allowing
different groups of molecules to be cleaved from the solid support
under different conditions, depending on the nature of the linker.
For example, linkers can be, inter alia, electrophilically cleaved,
nucleophilically cleaved, photocleavable, enzymatically cleaved,
cleaved by metals, cleaved under reductive conditions or cleaved
under oxidative conditions. In a preferred embodiment, the
compounds are cleaved from the solid support prior to high
throughput screening of the compounds.
[0479] If the library comprises arrays or microarrays of compounds,
wherein each compound has an address or identifier, the compound
can be deconvoluted, e.g., by cross-referencing the positive sample
to original compound list that was applied to the individual test
assays.
[0480] If the library is a peptide or nucleic acid library, the
sequence of the compound can be determined by direct sequencing of
the peptide or nucleic acid. Such methods are well known to one of
skill in the art.
[0481] A number of physico-chemical techniques can be used for the
de novo characterization of compounds. Examples of such techniques
include, but are not limited to, mass spectrometry. NMR
spectroscopy. X-ray crytallography and vibrational
spectroscopy.
[0482] 5.21 Use of Identified Compounds to Prevent, Treat, Manage
or Ameliorate Osteoarthritis or a Symptom Thereof
[0483] The present invention provides methods of preventing,
treating, managing or ameliorating osteoarthritis or a symptom
thereof, said methods comprising administering to a subject in need
thereof one or more compounds identified in accordance with the
methods of the invention. In certain embodiments, the subject has
mild, moderate, marked or severe osteoarthritis. In a preferred
embodiment, the subject is human.
[0484] In one embodiment, the invention provides a method of
preventing, treating, managing or ameliorating osteoarthritis or a
symptom thereof, said method comprising administering to a subject
in need thereof a dose of a prophylactically or therapeutically
effective amount of one or more compounds identified in accordance
with the methods of the invention. In a specific embodiment, a
compound identified in accordance with the methods of the invention
is not administered to prevent, treat, or ameliorate osteoarthritis
or a symptom thereof, if such compound has been used previously to
prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof. In another embodiment, a compound identified in accordance
with the methods of the invention is not administered to prevent,
treat, or ameliorate osteoarthritis or a symptom thereof, if such
compound has suggested to be used to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof. In another
embodiment, a compound identified in accordance with the methods of
the invention specifically binds to and/or alters the expression
and/or activity level of a protein or RNA product of only one
biomarker of the invention. In another embodiment, a compound
identified in accordance with the methods of the invention is not
administered to prevent, treat, or ameliorate osteoarthritis or a
symptom thereof, if such compound binds to and/or alters the
expression and/or activity of a protein or RNA product of one, two,
three, all or any combination of the following biomarkers: B2M,
TNFAIP6, PDCD5, and EGR 1. In yet another embodiment, a compound
identified in accordance with the methods of the invention binds to
and/or alters the expression and/or activity level of a protein or
RNA product of at least 2, at least 3, at least 4, at least 5, at
least 10, at least 15, at least 20, at least 25, at least 30, at
least 35, at least 40 or more biomarkers of the invention.
[0485] In a specific embodiment, a compound identified in
accordance with the methods of the invention increases or decreases
the anabolic and/or the catabolic activity of a chondrocyte.
Preferably, such a compound increases or decreases the anabolic
and/or catabolic activity of a chondrocyte by greater than
1.0-fold, more preferably, 1.5-5-fold, and most preferably,
5-100-fold, as compared to an untreated chondrocyte. In another
embodiment, a compound identified in accordance with the methods of
the invention ameliorates at least one of the symptoms and/or
changes associated with osteoarthritis including cartilage
degeneration, or pain, swelling, weakness and/or loss of functional
ability in the afflicted joints, associated with cartilage
degeneration. In a particular embodiment, the prophylactic or
therapeutic agent administered to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof is a synthetic
compound or a natural product (e.g. a plant extract or culture
supernatant), or a mixture of compounds.
[0486] The invention also provides methods of preventing, treating,
managing or ameliorating osteoarthritis or a symptom thereof, said
methods comprising administering to a subject in need thereof one
or more of the compounds identified utilizing the screening methods
described herein, and one or more other therapies (e.g.,
prophylactic or therapeutic agents and surgery). In a specific
embodiment, such therapies are currently being used, have been used
or are known to be useful in the prevention, treatment, management
or amelioration of osteoarthritis or a symptom thereof (including,
but not limited to the prophylactic or therapeutic agents listed in
Section 5.21.2 hereinbelow). The therapies (e.g., prophylactic or
therapeutic agents) of the combination therapies of the invention
can be administered sequentially or concurrently. In a specific
embodiment, the combination therapies of the invention comprise a
compound identified in accordance with the invention and at least
one other therapy that has the same mechanism of action as said
compound. In another specific embodiment, the combination therapies
of the invention comprise a compound identified in accordance with
the methods of the invention and at least one other therapy (e.g.,
prophylactic or therapeutic agent) which has a different mechanism
of action than said compound. The combination therapies of the
present invention improve the prophylactic or therapeutic effect of
a compound of the invention by functioning together with the
compound to have an additive or synergistic effect. The combination
therapies of the present invention reduce the side effects
associated with the therapies (e.g., prophylactic or therapeutic
agents).
[0487] The prophylactic or therapeutic agents of the combination
therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, the prophylactic or
therapeutic agents of the combination therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
The prophylactic or therapeutic agents may be administered to a
subject by the same or different routes of administration.
[0488] In specific embodiment, a pharmaceutical composition
comprising one or more compounds identified in an assay described
herein is administered to a subject, preferably a human, to
prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof. In accordance with the invention, the pharmaceutical
composition may also comprise one or more prophylactic or
therapeutic agents. Preferably, such agents are currently being
used, have been used or are known to be useful in the prevention,
treatment, management or amelioration of osteoarthritis or a
symptom thereof.
[0489] A compound identified in accordance with the methods of the
invention may be used as a first, second, third, fourth or fifth
line of therapy for osteoarthritis. The invention provides methods
for treating, managing or ameliorating osteoarthritis or a symptom
thereof in a subject refractory to conventional therapies for
osteoarthritis, said methods comprising administering to said
subject a dose of a prophylactically or therapeutically effective
amount of a compound identified in accordance with the methods of
the invention.
[0490] The invention provides methods for treating, managing or
ameliorating osteoarthritis or a symptom thereof in a subject
refractory to existing single agent therapies for osteoarthritis,
said methods comprising administering to said subject a dose of a
prophylactically or therapeutically effective amount of a compound
identified in accordance with the methods of the invention and a
dose of a prophylactically or therapeutically effective amount of
one or more other therapies (e.g., prophylactic or therapeutic
agents). The invention also provides methods for treating or
managing a osteoarthritis by administering a compound identified in
accordance with the methods of the invention in combination with
any other therapy (e.g. surgery) to patients who have proven
refractory to other therapies but are no longer on these therapies.
The invention also provides methods for the treatment or management
of a patient having osteoarthritis and immunosuppressed by reason
of having previously undergone other therapies. The invention also
provides alternative methods for the treatment or management of
osteoarthritis where hormonal therapy and/or biological
therapy/immunotherapy has proven or may prove too toxic, i.e.,
results in unacceptable or unbearable side effects, for the subject
being treated or managed.
[0491] 5.21.1 Compounds for Use in Preventing, Treating, Managing
or Ameliorating Osteoarthritis or a Symptom Thereof
[0492] Representative, non-limiting examples of compounds that can
used in accordance with the methods of the invention to prevent,
treat, manage and/or ameliorate osteoarthritis or a symptom thereof
are described in detail below.
[0493] First, such compounds can include, for example, antisense,
ribozyme, or triple helix compounds that can downregulate the
expression or activity of a protein or RNA product of a biomarker
of the invention. Such compounds are described in detail in the
subsection below.
[0494] Second, such compounds can include, for example, antibody
compositions that can modulate the expression of a protein or RNA
product of a biomarker of the invention, or the activity of a
protein product of a biomarker of the invention. In a specific
embodiment, the antibody compositions downregulate the expression a
protein or RNA product of a biomarker of the invention, or the
activity of a protein product of a biomarker of the invention. Such
compounds are described in detail in the subsection below.
[0495] Third, such compounds can include, for example, protein
products of a biomarker of the invention. The invention encompasses
the use of peptides or peptide mimetics selected to mimic a protein
product of a biomarker of the invention to prevent, treat, manage
or ameliorate osteoarthritis or a symptom thereof. Further, such
compounds can include, for example, dominant-negative polypeptides
that can modulate the expression a protein or RNA product of a
biomarker of the invention, or the activity of a protein product of
a biomarker of the invention.
[0496] The methods also encompasses the use derivatives, analogs
and fragments of a protein product of a biomarker of the invention
to prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof. In particular, the invention encompasses the use of
fragments of a protein product of a biomarker of the invention
comprising one or more domains of such a protein(s) to prevent,
treat, manage or ameliorate osteoarthritis or a symptom thereof. In
another specific embodiment, the invention encompasses the use of a
protein product of a biomarker of the invention, or an analog,
derivative or fragment of such a protein which is expressed as a
fusion, or chimeric protein product (comprising the protein,
fragment, analog, or derivative joined via a peptide bond to a
heterologous protein sequence).
[0497] In specific embodiments, an antisense oligonucleotide of at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, or more of biomarkers of the invention are
administered to prevent, treat, manage or ameliorate osteoarthritis
or a symptom thereof. In other embodiments, one or more of protein
products of a biomarker of the invention or a fragment, analog, or
derivative thereof are administered to prevent, treat, manage or
ameliorate osteoarthritis or a symptom thereof. In other
embodiment, one or more antibodies that specifically bind to a
protein product of the invention are administered to prevent,
treat, manage or ameliorate osteoarthritis or a symptom thereof. In
other embodiments, one or more dominant-negative polypeptides are
administered to prevent, treat, manage or ameliorate osteoarthritis
or a symptom thereof.
[0498] Antisense, Ribozyme, Triple-Helix Compositions
[0499] Representative, non-limiting examples of antisense molecules
include those listed in Table 6.
TABLE-US-00004 TABLE 6 Gene Examples of Antisense Symbol Sequences
SEQ ID NO: B2M 5'-TTAAAGGACTTAACGATACA-3' 1 BCL6
5'-TGTAGAGCCGAGTTAAACGC-3' 2 C1QR1 5'-GCTCTGAGTCTCAGTAATAA-3' 3
CCNC 5'-TAAGATACGGTCCATAAGAG-3' 4 EBNA1BP2
5'-CTTCGTTACAAACCGTCGGA-3' 5 FLJ32234 5'-CTTTTACACTTCTATGGAAG-3 6
G2AN 5'-TCAGAGTGACCCTGGGTCCG-3' 7 HSPCA 5'-CGGAAAGTCCGTCTTTAACG-3'
8 IKBKAP 5'-ACCTAGTCCTCAGACACACA-3' 9 IL13RA1
5'-CTCACTCTTCGGATCGTAAA-3' 10 LAMC1 5'-TCGTCCGGAACACATGACTA-3' 11
MAFB 5'-GTCCCGTCCCGTCCCTTCGA-3' 12 PAIP2 5'-GTTCGTAGTAGTTACTTCTA-3'
13 PER1 5'-GGTCCTTATGATGGTCGTCA-3' 14 PF4
5'-CAACGACGAGGACGGTGAAC-3' 15 TNFAIP6 5'-TTTTTAACCTAAAGTACAGA-3' 16
WDR9 5'-ACTATCCTGTCCTGTATCTT-3' 17 IRF1 5'-TCTAGGGTACCTTCGTACGA-3'
18 ABCA1 5'-CTTCGGTTAGGACTCTTGTG-3' 19 ABCG1
5'-TCTGACGCACAGGACGTTTT-3' 20 NCOA1 5'-GGTCGATAATGCCCACATCT-3' 21
CLIC4 5'-GTGCATTTAAAGACCTACCG-3' 22 SFRS6
5'-CCACCTATGTCGTCAGCCTC-3' 23 HSPCB 5'-CTTCTGGTGAACCGTCAGTT-3' 24
ADPRT 5'-CACTTCCGGTACTAACTCTT-3' 25 ACP1 5'-ACAGCTAGTGGGTAACGTCT-3'
26 PMSCL2 5'-GAAGGGCCTGCGGCTGTCGA-3' 27 FLJ13612
5'-GTCGGCGGCGGAGTAGTTCC-3' 28 SLC5A6 5'-TCTCTAGATGGCTAAACCCT-3' 29
YES1 5'-ACCTTGGTGCTTTCATCGTT-3' 30 CLN3 5'-AGAGATGCCGACGACACGAG-3'
31 LCMT2 5'-GTTGTAAAAGCCGTCGATTT-3' 32 NXN
5'-AGTCCCTTCAGTAACGTCCC-3' 33 ANGPTL2 5'-TCTGCATGTTCGTTCCCAAA-3' 34
BMPR2 5'-ATCAACCTCTACTCTCTCAG-3' 35 CLECSF6
5'-ATTATGGCCTAAGGGGTTCG-3' 36 DNAPTP6 5'-GTGTCTTCCGTTGTCTGATG-3' 37
F2RL1 5'-GAACTTCTAACGGATAGTGT-3' 38 FLJ11000
5'-GTTGTCACTACTCTCTCGCA-3' 39 FLJ11142 5'-TTAACCGCAGTAACCCCAAG-3'
40 ILF1 5'-TGCGGGGGCCCGCCGCCGCC-3' 41 PDCD5
5'-CTCTTTGTCATAGAATCGGG-3' 42 PDK4 5'-AGTTGCCGCGGCCGGACCAC-3' 43
PPIF 5'-CCCGAGGCCGCTGGGCAGGA-3' 44 SETBP1
5'-TGGAGTTTCCTAAAGTCGGT-3' 45 ZFR 5'-GTCGTTGACTGATACCAATA-3' 46
EGR1 5'-TGCGGCTTGTGACTGTAAAA-3' 47 TSPAN2
5'-CTCATAAAGATACACCCCGA-3' 48
[0500] In addition, standard techniques can be utilized to produce
antisense, triple helix, or ribozyme molecules for use as part of
the methods described herein. First, standard techniques can be
utilized for the production of antisense nucleic acid molecules,
i.e., molecules which are complementary to a sense nucleic acid
encoding a polypeptide of interest, e.g., complementary to the
coding strand of a double-stranded cDNA molecule or complementary
to an mRNA sequence. Accordingly, an antisense nucleic acid can
hydrogen bond to a sense nucleic acid. The antisense nucleic acid
can be complementary to an entire coding strand, or to only a
portion thereof, e.g., all or part of the protein coding region (or
open reading frame). An antisense nucleic acid molecule can be
antisense to all or part of a non-coding region of the coding
strand of a nucleotide sequence encoding a polypeptide of interest.
The non-coding regions ("5' and 3' untranslated regions") are the
5' and 3' sequences that flank the coding region and are not
translated into amino acids.
[0501] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length.
An antisense nucleic acid of the invention can be constructed using
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid
e.g., an antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxy
methylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N.sup.6-adenine,
7-methylguanine, 5-methaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),
wybutoxosine, 4-thiouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil. 4thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil,
(acp3)w, and 2,6-diaminopurine. Alternatively, the antisense
nucleic acid can be produced biologically using an expression
vector into which a nucleic acid has been subcloned in an antisense
orientation (i.e., RNA transcribed from the inserted nucleic acid
will be of an antisense orientation to a target nucleic acid of
interest).
[0502] Antisense nucleic acid molecules administered to a subject
or generated in situ such that they hybridize with or bind to
cellular mRNA encoding the polypeptide of interest to thereby
inhibit expression, e.g., by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the
case of an antisense nucleic acid molecule which binds to DNA
duplexes, through specific interactions in the major groove of the
double helix. An example of a route of administration of antisense
nucleic acid molecules of the invention includes direct injection
at a tissue. e.g., a joint (e.g., a knee, hip, elbow, and knuckle),
site. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically, bind to
receptors or antigens expressed on a selected cell. e.g., a T cell
or chondrocyte, surface. e.g., by linking the antisense nucleic
acid molecules to peptides or antibodies which bind to cell surface
receptors or antigens. The antisense nucleic acid molecules can
also be delivered to cells using vectors, e.g., gene therapy
vectors, described below. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[0503] An antisense nucleic acid molecule of interest can be an
.alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic
acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .alpha.-units,
the strands run parallel to each other (Gaultier et al., 1987,
Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid
molecule can also comprise a 2''-o-methylribonucleotide (Inoue et
al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA
analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
[0504] Ribozymes are catalytic RNA molecules with ribonuclease
activity that are capable of cleaving a single-stranded nucleic
acid, such as an mRNA, to which they have a complementary region,
and can also be generated using standard techniques. Thus,
ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and
Gerlach, 1988. Nature 334:585-591)) can be used to catalytically
cleave mRNA transcripts to thereby inhibit translation of the
protein encoded by the mRNA. A ribozyme having specificity for a
nucleic acid molecule encoding a polypeptide of interest can be
designed based upon the nucleotide sequence of a cDNA disclosed
herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can
be constructed in which the nucleotide sequence of the active site
is complementary to the nucleotide sequence to be cleaved in a Cech
et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.
5,116,742. Alternatively, an mRNA encoding a polypeptide of
interest can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel and Szostak, 1993. Science 261:1411-1418.
[0505] Triple helical structures can also be generated using well
known techniques. For example, expression of a polypeptide of
interest can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the gene encoding the
polypeptide (e.g., the promoter and/or enhancer) to form triple
helical structures that prevent transcription of the gene in target
cells. See generally Helene, 1991, Anticancer Drug Des.
6(6):569-84; Helene, 1992, Ann. N.Y. Acad. Sci. 660:27-36; and
Maher, 1992, Bioassays 14(12):807-15.
[0506] In various embodiments, nucleic acid compositions can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve. e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids
(see Hyrup et al., 1996. Bioorganic & Medicinal Chemistry 4(1):
5-23). As used herein, the terms "peptide nucleic acids" or "NAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. 1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl.
Acad. Sci. USA 93: 14670-675.
[0507] PNAs can, for example, be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other
helper groups to PNA, by the formation of PNA-DNA chimeras, or by
the use of liposomes or other techniques of drug delivery known in
the art. For example, PNA-DNA chimeras can be generated which may
combine the advantageous properties of PNA and DNA. Such chimeras
allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases,
to interact with the DNA portion while the PNA portion would
provide high binding affinity and specificity. PNA-DNA chimeras can
be linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras
can be performed as described in Hyrup, 1996, supra, and Finn et
al., 1996. Nucleic Acids Res. 24(17):3357-63. For example, a DNA
chain can be synthesized on a solid support using standard
phosphoramidite coupling chemistry and modified nucleoside analogs.
Compounds such as 5'(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite can be used as a link between the PNA and the 5'
end of DNA (Mag et al., 1989 Nucleic Acids Res. 17:5973-88). PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
et al., 1996, Nucleic Acids Res. 24(17):3357-63). Alternatively,
chimeric molecules can be synthesized with a 5' DNA segment and a
3' PNA segment (Peterser et al., 1975, Bioorganic Med. Chem. Lett.
5:1119-11124).
[0508] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al., 1987. Proc. Natl. Acad.
Sci. USA 84:648-652; International Publication No. WO 88/09810) or
the blood-brain barrier (see, e.g., International Publication No.
WO 89/10134). In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.,
1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g.,
Zon, 1988. Pharm. Res. 5:539-549). To this end, the oligonucleotide
may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent,
hybridization-triggered cleavage agent, etc.
Antibody Compositions
[0509] In one embodiment, antibodies that specifically bind to one
or more protein products of one or more biomarkers of the invention
are administered to a subject, preferably a human, to prevent,
treat, manage or ameliorate osteoarthritis or a symptom thereof. In
another embodiment, any combination of antibodies that specifically
bind to one or more protein products of one or more biomarkers of
the invention are administered to a subject, preferably a human, to
prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof. In a specific embodiment, one or more antibodies that
specifically bind to one or more protein products of one or more
biomarkers of the invention are administered to a subject,
preferably a human, in combination with other types of therapies
(e.g., NSAIDS) to prevent, treat, manage or ameliorate
osteoarthritis or a symptom thereof. In certain embodiments,
antibodies known in the art that specifically bind to one or more
protein products of one or more biomarkers of the invention are
administered to a subject, preferably a human, alone or in
combination with other types of therapies (e.g., NSAIDS) to
prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof. In other embodiments, antibodies known in the art that
specifically bind to one or more protein products of one or more
biomarkers of the invention are not administered to a subject,
preferably a human, alone or in combination with other types of
therapies (e.g., NSAIDS) to prevent, treat, manage or ameliorate
osteoarthritis or a symptom thereof.
[0510] One or more antibodies that specifically bind to one or more
protein products of one or more biomarkers of the invention can be
administered to a subject, preferably a human, using various
delivery systems are known to those of skill in the art. For
example, such antibodies can be administered by encapsulation in
liposomes, microparticles or microcapsules. See, e.g., U.S. Pat.
No. 5,762,904, U.S. Pat. No. 6,004,534, and International
Publication No. WO 99/52563. In addition, such antibodies can be
administered using recombinant cells capable of expressing the
antibodies, or retroviral, other viral vectors or non-viral vectors
capable of expressing the antibodies.
[0511] Antibodies that specifically bind one or more protein
products of one or more biomarkers of the invention can be obtained
from any known source. Alternatively, antibodies that specifically
bind to one or more protein products of one or more biomarkers of
the invention can be produced by any method known in the art for
the synthesis of antibodies, in particular, by chemical synthesis
or preferably, by recombinant expression techniques.
[0512] Antibodies include, but are not limited to, polyclonal
antibodies, monoclonal antibodies, bispecific antibodies,
multispecific antibodies, human antibodies, humanized antibodies,
camelised antibodies, chimeric antibodies, single-chain Fvs (scFv)
(see e.g., Bird et al. (1988) Science 242:423-426; and Huston et
al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883), single chain
antibodies, single domain antibodies, Fab fragments, F(ab')
fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above. The term "antibody", as used herein, refers to
immunoglobulin molecules and immunologically active fragments of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site. Immunoglobulin molecules can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2) or
subclass. Examples of immunologically active fragments of
immunoglobulin molecules include F(ab) fragments (a monovalent
fragment consisting of the VL, VH, CL and CH1 domains) and
F(ab').sub.2 fragments (a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region) which
can be generated by treating the antibody with an enzyme such as
pepsin or papain. Immunologically active fragments also include,
but are not limited to, Fd fragments (consisting of the VH and CH1
domains), Fv fragments (consisting of the VL and VII domains of a
single arm of an antibody), dAb fragments (consisting of a VH
domain; Ward et al., (1989) Nature 341:544-546), and isolated
complementarity determining regions (CDRs). Antibodies that
specifically bind to an antigen can be produced by any method known
in the art for the synthesis of antibodies, in particular, by
chemical synthesis or preferably, by recombinant expression
techniques.
[0513] Polyclonal antibodies that specifically bind to an antigen
can be produced by various procedures well-known in the art. For
example, a human antigen can be administered to various host
animals including, but not limited to, rabbits, mice, rats. etc. to
induce the production of sera containing polyclonal antibodies
specific for the human antigen. Various adjuvants may be used to
increase the immunological response, depending on the host species,
and include but are not limited to, Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and corynebacterium parvum. Such
adjuvants are also well known in the art.
[0514] The term "monospecific antibody" refers to an antibody that
displays a single binding specificity and affinity for a particular
target, e.g., epitope. This term includes monoclonal antibodies.
Monoclonal antibodies can be prepared using a wide variety of
techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. See, e.g., U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439,
4,411,993 and 4,196,265; Kennett et al (eds.), Monoclonal
Antibodies, Hybridomas: A New Dimension in Biological Analyses,
Plenum Press (1980); and Harlow and Lane (eds.). Antibodies. A
Laboratory Manual, Cold Spring Harbor Laboratory Press (1988),
which are incorporated herein by reference. For example, monoclonal
antibodies can be produced using hybridoma techniques including
those known in the art and taught, for example, in Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies
and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said
references incorporated by reference in their entireties). Other
techniques that enable the production of antibodies through
recombinant techniques (e.g. techniques described by William D.
Huse et al., 1989, Science, 246: 1275-1281; L. Sastry et al., 1989,
Proc. Natl. Acad. Sci. USA, 86: 5728-5732; and Michelle Alting-Mees
et al., Strategies in Molecular Biology, 3: 1-9 (1990) involving a
commercial system available from Stratacyte, La Jolla. Calif.) may
also be utilized to construct monoclonal antibodies. The term
"monoclonal antibody" as used herein is not limited to antibodies
produced through hybridoma technology. The term "monoclonal
antibody" refers to an antibody that is derived from a single
clone, including any eukaryotic, prokaryotic, or phage clone, and
not the method by which it is produced.
[0515] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with a protein product of a
biomarker of the invention, and once an immune response is
detected, e.g., antibodies specific for the protein are detected in
the mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC. Hybridomas are selected and cloned by
limited dilution. Additionally, a RIMMS (repetitive immunization
multiple sites) technique can be used to immunize an animal
(Kilptrack et al., 1997, Hybridoma 16:381-9, incorporated by
reference in its entirety). The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0516] Accordingly, the present invention provides methods of
generating antibodies by culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with a protein product of a biomarker of the invention, with
myeloma cells and then screening the hybridomas resulting from the
fusion for hybridoma clones that secrete an antibody able to bind
to the protein or protein fragment.
[0517] Antibody fragments which recognize specific epitopes of a
protein product of a biomarker of the invention may be generated by
any technique known to those of skill in the art. For example, Fab
and F(ab')2 fragments of the invention may be produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes
such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2 fragments). F(ab')2 fragments contain the variable region,
the light chain constant region and the CH1 domain of the heavy
chain. Further, the antibodies of the present invention can also be
generated using various phage display methods known in the art.
[0518] In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In particular. DNA
sequences encoding VH and VL domains are amplified from animal cDNA
libraries (e.g. human or murine cDNA libraries of affected
tissues). The DNA encoding the VH and VL domains are recombined
together with an scFv linker by PCR and cloned into a phagemid
vector. The vector is electroporated in E. coli and the E. coli is
infected with helper phage. Phage used in these methods are
typically filamentous phage including fd and M13 and the VH and VL
domains are usually recombinantly fused to either the phage gene
III or gene VIII. Phage expressing an antigen binding domain that
binds to a particular antigen can be selected or identified with
antigen, e.g., using labeled antigen or antigen bound or captured
to a solid surface or bead. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., 1995, J. Immunol. Methods
182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;
Kettleborough et al. 1994. Eur. J. Immunol. 24:952-958; Persic et
al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in
Immunology 57:191-280: PCT Application No. PCT/GB91/O1 134;
International Publication Nos. WO 90/02809, WO 91/10737, WO
92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and
WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,
5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,
5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0519] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce Fab. Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in
International Publication No. WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6):864-869; Sawai et al., 1995. AJRI 34:26-34; and
Better et al., 1988, Science 240:1041-1043 (said references
incorporated by reference in their entireties).
[0520] To generate whole antibodies. PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques know n to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a VH constant region, e.g., the
human gamma 4 constant region, and the PCR amplified VL domains can
be cloned into vectors expressing a VL constant region, e.g., human
kappa or lamba constant regions. Preferably, the vectors for
expressing the VH or VL domains comprise an EF-1a promoter, a
secretion signal, a cloning site for the variable domain, constant
domains, and a selection marker such as neomycin. The VH and VL
domains may also cloned into one vector expressing the necessary
constant regions. The heavy chain conversion vectors and light
chain conversion vectors are then co-transfected into cell lines to
generate stable or transient cell lines that express full-length
antibodies, e.g. IgG, using techniques known to those of skill in
the art.
[0521] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use human or
chimeric antibodies. Completely human antibodies are particularly
desirable for therapeutic treatment of human subjects. Human
antibodies can be made by a variety of methods known in the art
including phage display methods described above using antibody
libraries derived from human immunoglobulin sequences. See also
U.S. Pat. Nos. 4,444,887 and 4,716,111; and International
Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654,
WO 96/34096, WO 96/33735, and WO 91/10741: each of which is
incorporated herein by reference in its entirety.
[0522] Antibodies can also be produced by a transgenic animal. In
particular, human antibodies can be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the J.sub.H
region prevents endogenous antibody production. The modified
embryonic stem cells are expanded and microinjected into
blastocysts to produce chimeric mice. The chimeric mice are then be
bred to produce homozygous offspring which express human
antibodies. The transgenic mice are immunized in the normal fashion
with a selected antigen, e.g., all or a portion of a polypeptide of
the invention. Monoclonal antibodies directed against the antigen
can be obtained from the immunized, transgenic mice using
conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., International Publication
Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos.
5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806,
5,814,318, and 5,939,598, which are incorporated by reference
herein in their entirety. In addition, companies such as Abgenix.
Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0523] U.S. Pat. No. 5,849,992, for example, describes a method of
expressing an antibody in the mammary gland of a transgenic mammal.
A transgene is constructed that includes a milk-specific promoter
and nucleic acids encoding the antibody of interest and a signal
sequence for secretion. The milk produced by females of such
transgenic mammals includes, secreted-therein, the antibody of
interest. The antibody can be purified from the milk, or for some
applications, used directly.
[0524] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different immunoglobulin
molecules. Methods for producing chimeric antibodies are known in
the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al.,
1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.
Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,
4,816,397, and 6,331,415, which are incorporated herein by
reference in their entirety.
[0525] A humanized antibody is an antibody or its variant or
fragment thereof which is capable of binding to a predetermined
antigen and which comprises a framework region having substantially
the amino acid sequence of a human immunoglobulin and a CDR having
substantially the amino acid sequence of a non-human immuoglobulin.
A humanized antibody comprises substantially all of at least one,
and typically two, variable domains (Fab, Fab', F(ab').sub.2, Fabc,
Fv) in which all or substantially all of the CDR regions correspond
to those of a non-human immunoglobulin (i.e., donor antibody) and
all or substantially all of the framework regions are those of a
human immunoglobulin consensus sequence. Preferably, a humanized
antibody also comprises at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin.
Ordinarily, the antibody will contain both the light chain as well
as at least the variable domain of a heavy chain. The antibody also
may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy
chain. The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4.
Usually the constant domain is a complement fixing constant domain
where it is desired that the humanized antibody exhibit cytotoxic
activity, and the class is typically IgG.sub.1. Where such
cytotoxic activity is not desirable, the constant domain may be of
the IgG.sub.2 class. The humanized antibody may comprise sequences
from more than one class or isotype, and selecting particular
constant domains to optimize desired effector functions is within
the ordinary skill in the art. The framework and CDR regions of a
humanized antibody need not correspond precisely to the parental
sequences, e.g., the donor CDR or the consensus framework may be
mutagenized by substitution, insertion or deletion of at least one
residue so that the CDR or framework residue at that site does not
correspond to either the consensus or the import antibody. Such
mutations, however, will not be extensive. Usually, at least 75% of
the humanized antibody residues will correspond to those of the
parental FR and CDR sequences, more often 90%, and most preferably
greater than 95%. Humanized antibody can be produced using variety
of techniques known in the art, including but not limited to,
CDR-grafting (European Patent No. EP 239,400; International
Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,
5,530,101, and 5,585,089), veneering or resurfacing (European
Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular
Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein
Engineering 7(6):805-814; and Roguska et al., 1994, PNAS
91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and
techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat.
No. 5,766,886, WO 9317105, Tan et al., 2002, J. Immunol.
169:1119-25, Caldas et al., 2000, Protein Eng. 13(5):353-60, Morea
et al., 2000, Methods 20(3):267-79, Baca et al., 1997, J. Biol.
Chem. 272(16):10678-84, Roguska et al., 1996, Protein Eng.
9(10):895-904, Couto et al., 1995, Cancer Res. 55 (23
Supp):5973s-5977s, Couto et al., 1995, Cancer Res. 55(8):1717-22,
Sandhu J S, 1994, Gene 150(2):409-10, and Pedersen et al. 1994, J.
Mol. Biol. 235(3):959-73. Often, framework residues in the
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and
Riechmann et al., 1988, Nature 332:323, which are incorporated
herein by reference in their entireties.)
[0526] Single domain antibodies, for example, antibodies lacking
the light chains, can be produced by methods well-known in the art.
See Riechmann et al., 1999, J. Immuno. 231:25-38; Nuttall et al.,
2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J.
Biotechnol. 74(4):277302; U.S. Pat. No. 6,005,079; and
International Publication Nos. WO 94/04678, WO 94/25591, and WO
01/44301, each of which is incorporated herein by reference in its
entirety.
[0527] Further, the antibodies that specifically bind to an antigen
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" an antigen using techniques well known to those skilled in
the art. (See. e.g. Greenspan & Bona, 1989, FASEB J.
7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).
Such antibodies can be used, alone or in combination with other
therapies, in the prevention, treatment, management or amelioration
of osteoarthritis or a symptom thereof.
[0528] The invention encompasses polynucleotides comprising a
nucleotide sequence encoding an antibody or fragment thereof that
specifically binds to an antigen. The invention also encompasses
polynucleotides that hybridize under high stringency, intermediate
or lower stringency hybridization conditions to polynucleotides
that encode an antibody of the invention.
[0529] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. The nucleotide sequences encoding known antibodies can be
determined using methods well known in the art, i.e., nucleotide
codons known to encode particular amino acids are assembled in such
a way to generate a nucleic acid that encodes the antibody. Such a
polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., 1994, BioTechniques 17:242), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, fragments, or
variants thereof, annealing and ligating of those oligonucleotides,
and then amplification of the ligated oligonucleotides by PCR.
[0530] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0531] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described
in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual,
2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and
Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY, which are both incorporated by reference
herein in their entireties), to generate antibodies having a
different amino acid sequence, for example to create amino acid
substitutions, deletions, and/or insertions.
[0532] Once a polynucleotide encoding an antibody molecule, heavy
or light chain of an antibody, or fragment thereof (preferably, but
not necessarily, containing the heavy or light chain variable
domain) of the invention has been obtained, the vector for the
production of the antibody molecule may be produced by recombinant
DNA technology using techniques well-known in the art.
[0533] In one preferred embodiment, monoclonal antibodies are
produced in mammalian cells. Preferred mammalian host cells for
expressing the clone antibodies or antigen-binding fragments
thereof include Chinese Hamster Ovary (CHO cells) (including dhfr-
CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad.
Sci. USA 77:4216-4220), used with a DHFR selectable marker. e.g.,
as described in Kaufman and Sharp (1982. Mol. Biol. 159:601-621),
lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS
cells, and a cell from a transgenic animal. e.g., a transgenic
mammal. For example, the cell is a mammary epithelial cell.
[0534] In addition to the nucleic acid sequence encoding the
diversified immunoglobulin domain, the recombinant expression
vectors may carry additional sequences, such as sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0535] In an exemplary system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr.sup.-
CHO cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to enhancer/promoter regulatory
elements (e.g., derived from SV40, CMV, adenovirus and the like,
such as a CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the antibody heavy and light
chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. For example, some antibodies can be isolated by
affinity chromatography with a Protein A or Protein G.
[0536] For antibodies that include an Fc domain, the antibody
production system preferably synthesizes antibodies in which the Fc
region is glycosylated. For example, the Fc domain of IgG molecules
is glycosylated at asparagine 297 in the CH2 domain. This
asparagine is the site for modification with biantennary-type
oligosaccharides. It has been demonstrated that this glycosylation
is required for effector functions mediated by Fc.gamma. receptors
and complement C1q (Burton and Woof, 1992, Adv. Immunol. 51:1-84;
Jefferis et al., 1998, Immunol. Rev. 163:59-76). In a preferred
embodiment, the Fc domain is produced in a mammalian expression
system that appropriately glycosylates the residue corresponding to
asparagine 297. The Fe domain can also include other eukaryotic
post-translational modifications.
[0537] Once an antibody molecule has been produced by recombinant
expression, it may be purified by any method known in the art for
purification of an immunoglobulin molecule, for example, by
chromatography (e.g. ion exchange, affinity, particularly by
affinity for the specific antigen after Protein A, and sizing
column chromatography), centrifugation, differential solubility, or
by any other standard technique for the purification of proteins.
Further, the antibodies or fragments thereof may be fused to
heterologous polypeptide sequences known in the art to facilitate
purification.
Gene Therapy Techniques
[0538] Gene therapy refers to therapy performed by the
administration to a subject of an expressed or expressible nucleic
acid. Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0539] In specific embodiments, one or more antisense
oligonucleotides for one or more biomarkers of the invention are
administered to prevent, treat, manage or ameliorate osteoarthritis
or a symptom thereof, by way of gene therapy. In other embodiments,
one or more nucleic acid molecules comprising nucleotides encoding
one or more antibodies that specifically bind to one or more
protein products of one or more biomarkers of the invention are
administered to prevent, treat, manage or ameliorate osteoarthritis
or a symptom thereof, by way of gene therapy. In other embodiments,
one or more nucleic acid molecules comprising nucleotides encoding
protein products of one or more biomarkers of the invention or
analogs, derivatives or fragments thereof, are administered to
prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof, by way of gene therapy. In yet other embodiments, one or
more nucleic acid molecules comprising nucleotides encoding one or
more dominant-negative polypeptides of one or more protein products
of one or more biomarker of the invention are administered to
prevent, treat, manage or ameliorate osteoarthritis or a symptom
thereof, by way of gene therapy.
[0540] For general reviews of the methods of gene therapy, see
Goldspiel et al. 1993. Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993. Ann. Rev. Pharmacol.
Toxicol. 32:573-596: Mulligan, 1993. Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993. TIBTECH 11(5):155-215). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology. John Wiley & Sons, NY; and Kriegler, 1990, Gene
Transfer and Expression. A Laboratory Manual, Stockton Press,
NY.
[0541] In one aspect, a composition of the invention comprises
nucleic acid sequences encoding one or more antibodies that
specifically bind to one or more protein products of one or more
biomarkers of the invention, said nucleic acid sequences being part
of expression vectors that express one or more antibodies in a
suitable host. In particular, such nucleic acid sequences have
promoters operably linked to the antibodies, said promoter being
inducible or constitutive, and, optionally, tissue-specific.
[0542] In another aspect, a composition of the invention comprises
nucleic acid sequences encoding dominant-negative polypeptides of
one or protein products of one or more biomarkers of the invention,
said nucleic acid sequences being part of expression vectors that
express dominant-negative polypeptides in a suitable host. In
particular, such nucleic acid sequences have promoters operably
linked to the dominant-negative polypeptides, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the dominant-negative coding sequences and any other desired
sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the dominant-negative nucleic acids
(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989. Nature 342:435-438).
[0543] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0544] In a specific embodiment, the nucleic acid sequence is
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing it as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see. e.g., Wu
and Wu, 1987. J. Biol. Chem. 262:4429-4432) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., International Publication
Nos. WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated
Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992
(Findeis et al.); WO 93/14188 dated Jul. 22, 1993 (Clarke et al.),
WO 93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the
nucleic acid can be introduced intracellularly and incorporated
within host cell DNA for expression, by homologous recombination
(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
[0545] For example, a retroviral vector can be used. These
retroviral vectors have been modified to delete retroviral
sequences that are not necessary for packaging of the viral genome
and integration into host cell DNA. The nucleic acid sequences
encoding the antibodies of interest, or proteins of interest or
fragments thereof to be used in gene therapy are cloned into one or
more vectors, which facilitates delivery of the gene into a
patient. More detail about retroviral vectors can be found in
Boesen et al., 1994. Biotherapy 6:291-302, which describes the use
of a retroviral vector to deliver the mdr1 gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are: Clowes et al. 1994, J. Clin. Invest.
93:644-651; Kiem et al. 1994, Blood 83:1467-1473: Salmons and
Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and
Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
[0546] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al. 1992. Cell 68:143-155;
Mastrangeli et al., 1993. J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang, et al., 1995, Gene Therapy
2:775-783. In a preferred embodiment, adenovirus vectors are
used.
[0547] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146).
[0548] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0549] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et
al., 1993. Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0550] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) and/or
chondrocytes are preferably administered intravenously. The amount
of cells envisioned for use depends on the desired effect, patient
state, etc., and can be determined by one skilled in the art.
[0551] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, chondrocytes, fibroblasts, muscle
cells, hepatocytes; blood cells such as T lymphocytes. B
lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
megakaryocytes, granulocytes; various stem or progenitor cells, in
particular hematopoietic stem or progenitor cells, e.g., as
obtained from bone marrow, umbilical cord blood, peripheral blood,
fetal liver, etc.
[0552] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0553] In one embodiment in which recombinant cells are used in
gene therapy, nucleic acid sequences encoding antibodies of
interest, or proteins of interest or fragments thereof are
introduced into the cells such that they are expressible by the
cells or their progeny, and the recombinant cells are then
administered in vivo for therapeutic effect. In a specific
embodiment, stem or progenitor cells are used. Any stem and/or
progenitor cells which can be isolated and maintained in vitro can
potentially be used in accordance with this embodiment of the
present invention (see, e.g., International Publication No. WO
94/08598, dated Apr. 28, 1994; Stemple and Anderson, 1992, Cell
71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow
and Scott, 1986, Mayo Clinic Proc. 61:771).
[0554] Promoters that may be used to control the expression of
nucleic acid sequences encoding antibodies of interest, proteins of
interest or fragments thereof may be constitutive, inducible or
tissue-specific. Non-limiting examples include the SV40 early
promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),
the promoter contained in the 3' long terminal repeat of Rous
sarcoma virus (Yamamoto, et al. 1980, Cell 22:787-797), the herpes
thymidine kinase promoter (Wagner et al. 1981, Proc. Natl. Acad.
Sci. USA 78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:39-42);
prokaryotic expression vectors such as the .beta.-lactamase
promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA
75:3727-3731), or the lac promoter (DeBoer et al., 1983. Proc.
Natl. Acad. Sci. USA 80:21-25): see also "Useful proteins from
recombinant bacteria" in Scientific American, 1980, 242:74-94;
plant expression vectors comprising the nopaline synthetase
promoter region (Herrera-Estrella et al., Nature 303:209-213) or
the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,
1981, Nucl. Acids Res. 9:2871), and the promoter of the
photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter
elements from yeast or other fungi such as the Gal 4 promoter, the
ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following animal
transcriptional control regions, which exhibit tissue specificity
and have been utilized in transgenic animals: elastase I gene
control region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor
Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology
7:425-515); insulin gene control region which is active in
pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region which is active in lymphoid
cells (Grosschedl et al., 1984. Cell 38:647-658; Adames et al.,
1985, Nature 318:533-538; Alexander et al., 1987, Mol.
[0555] Cell. Biol. 7:1436-1444), mouse mammary tumor virus control
region which is active in testicular, breast, lymphoid and mast
cells (Leder et al., 1986, Cell 45:485-495), albumin gene control
region which is active in liver (Pinkert et al., 1987, Genes and
Devel. 1:268-276), alpha-fetoprotein gene control region which is
active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al. 1987, Science 235:53-58; alpha
1-antitrypsin gene control region which is active in the liver
(Kelsey et al. 1987, Genes and Devel. 1:161-171), beta-globin gene
control region which is active in myeloid cells (Mogram et al.,
1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94;
myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead et al., 1987, Cell
48:703-712): myosin light chain-2 gene control region which is
active in skeletal muscle (Sani, 1985, Nature 314:283-286), and
gonadotropic releasing hormone gene control region which is active
in the hypothalamus (Mason et al., 1986. Science
234:1372-1378).
[0556] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0557] 5.21.2 Anti-Inflammatory Therapies
[0558] Anti-inflammatory agents have exhibited success in the
treatment, management and amelioration of osteoarthritis and are
now a common and a standard therapy for such disorder. Any
anti-inflammatory agent well-known to one of skill in the art can
be used in the compositions and methods of the invention.
Non-limiting examples of anti-inflammatory agents include
non-steroidal anti-inflammatory drugs (NSAIDs), steroidal
anti-inflammatory drugs, beta-agonists, anticholingeric agents, and
methyl xanthines. Examples of NSAIDs include, but are not limited
to, aspirin, ibuprofen, celecoxib (CELEBREX.TM.), diclofenac
(VOLTAREN.TM.), etodolac (LODINE.TM.), fenoprofen (NALFONT.TM.),
indomethacin (INDOCIN.TM.), ketoralac (TORADOL.TM.), oxaprozin
(DAYPROT.TM.), nabumentone (RELAFENT.TM.), sulindac (CLINORIL.TM.),
tolmentin (TOLECTIN.TM.), rofecoxib (VIOXX.TM.), naproxen
(ALEVE.TM., NAPROSYN.TM.), ketoprofen (ACTRON.TM.) and nabumetone
(RELAFEN.TM.). Such NSAIDs function by inhibiting a cyclooxygenase
enzyme (e.g., COX-1 and/or COX-2). Examples of steroidal
anti-inflammatory drugs include, but are not limited to,
glucocorticoids, dexamethasone (DECADRON.TM.), cortisone,
hydrocortisone, prednisone (DELTASONET.TM.), prednisolone,
triamcinolone, azulfidine, and eicosanoids such as prostaglandins,
thromboxanes, and leukotrienes.
[0559] 5.22 Pharmaceutical Compositions
[0560] Biologically active compounds identified using the methods
of the invention or a pharmaceutically acceptable salt thereof can
be administered to a patient, preferably a mammal, more preferably
a human, suffering from osteoarthritis. In a specific embodiment, a
compound or pharmaceutically acceptable salt thereof is
administered to a patient, preferably a mammal, more preferably a
human, suffering from the following stage of osteoarthritis: mild,
moderate, marked or severe. In another embodiment, a compound or a
pharmaceutically acceptable salt thereof is administered to a
patient, preferably a mammal, more preferably a human, as a
preventative measure against osteoarthritis. In accordance with
these embodiments, the patient may be a child, an adult or elderly,
wherein a "child" is a subject between the ages of 24 months of age
and 18 years of age, an "adult" is a subject 18 years of age or
older, and "elderly" is a subject 65 years of age or older.
[0561] When administered to a patient, the compound or a
pharmaceutically acceptable salt thereof is preferably administered
as component of a composition that optionally comprises a
pharmaceutically acceptable vehicle. The composition can be
administered orally, or by any other convenient route, for example,
by infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal
mucosa, etc.) and may be administered together with another
biologically active agent. Administration can be systemic or local.
Various delivery systems are known, e.g., encapsulation in
liposomes, microparticles, microcapsules, capsules, etc., and can
be used to administer the compound and pharmaceutically acceptable
salts thereof.
[0562] Methods of administration include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, oral, sublingual, intranasal,
intracerebral, intravaginal, transdermal, rectally, by inhalation,
or topically, particularly to the ears, nose, eyes, or skin. The
mode of administration is left to the discretion of the
practitioner. In most instances, administration will result in the
release of the compound or a pharmaceutically acceptable salt
thereof into the bloodstream.
[0563] In specific embodiments, it may be desirable to administer
the compound or a pharmaceutically acceptable salt thereof locally.
This may be achieved, for example, and not by way of limitation, by
local infusion during surgery, topical application, e.g., in
conjunction with a wound dressing after surgery, by injection, by
means of a catheter, by means of a suppository, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as sialastic membranes, or
fibers. In a specific embodiment, a compound is administered
locally to a joint affected by osteoarthritis.
[0564] In certain embodiments, it may be desirable to introduce the
compound or a pharmaceutically acceptable salt thereof into the
central nervous system by any suitable route, including
intraventricular, intrathecal and epidural injection.
Intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0565] Pulmonary administration can also be employed, e.g. by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent, or via perfusion in a fluorocarbon or synthetic pulmonary
surfactant. In certain embodiments, the compound and
pharmaceutically acceptable salts thereof can be formulated as a
suppository, with traditional binders and vehicles such as
triglycerides.
[0566] In another embodiment, the compound and pharmaceutically
acceptable salts thereof can be delivered in a vesicle, in
particular a liposome (see Langer, 1990, Science 249:1527-1533;
Treat et al., in Liposomes in the Therapy of Infectious Disease and
Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.
353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally
ibid.).
[0567] In yet another embodiment, the compound and pharmaceutically
acceptable salts thereof can be delivered in a controlled release
system (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra, vol. 2, pp. 115-138 (1984)). Other
controlled-release systems discussed in the review by Langer, 1990,
Science 249:1527-1533 may be used. In one embodiment, a pump may be
used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989,
N. Engl. J. Med. 321:574). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see
also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.
Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet
another embodiment, a controlled-release system can be placed in
proximity of a target RNA of the compound or a pharmaceutically
acceptable salt thereof, thus requiring only a fraction of the
systemic dose.
[0568] The compounds described herein can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the active compound and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0569] The invention includes methods for preparing pharmaceutical
compositions for modulating the expression or activity of a
polypeptide or nucleic acid of interest. Such methods comprise
formulating a pharmaceutically acceptable carrier with an agent
that modulates expression or activity of a polypeptide or nucleic
acid of interest. Such compositions can further include additional
active agents. Thus, the invention further includes methods for
preparing a pharmaceutical composition by formulating a
pharmaceutically acceptable carrier with an agent that modulates
expression or activity of a polypeptide or nucleic acid of interest
and one or more additional active compounds.
[0570] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Intravenous administration is preferred. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0571] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water. Cremophor EL.TM. (BASE; Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0572] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide or antibody)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0573] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
[0574] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0575] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[0576] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0577] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0578] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0579] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0580] For antibodies, the preferred dosage is 0.1 mg/kg to 100
mg/kg of body weight (more preferably, 0.1 to 20 mg/kg, 0.1-10
mg/kg, or 0.1 to to 1.0 mg/kg). If the antibody is to act in the
brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.
Generally, partially human antibodies and fully human antibodies
have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al. (1997, J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14:193).
[0581] In a specific embodiment, an effective amount of protein or
polypeptide (i.e., an effective dosage) ranges from about 0.001 to
30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body
weight, more preferably about 0.1 to 20 mg/kg body weight, and even
more preferably about 0.1 to 1.0 mg/kg, 1 to 10 mg/kg, 2 to 9
mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
[0582] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a protein,
polypeptide, or antibody can include a single treatment or,
preferably, can include a series of treatments.
[0583] In addition to those compounds described above, the present
invention encompasses the use of small molecules that modulate
expression or activity of a nucleic acid or polypeptide of
interest. Non-limiting examples of small molecules include
peptides, peptidomimetics, amino acids, amino acid analogs,
polynucleotides, polynucleotide analogs, nucleotides, nucleotide
analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0584] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the ken of the
ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention. Exemplary doses
include milligram or microgram amounts of the small molecule per
kilogram of subject or sample weight (e.g., about 1 microgram per
kilogram to about 500 milligrams per kilogram, about 100 micrograms
per kilogram to about 5 milligrams per kilogram, or about 1
microgram per kilogram to about 50 micrograms per kilogram). It is
furthermore understood that appropriate doses of a small molecule
depend upon the potency of the small molecule with respect to the
expression or activity to be modulated. Such appropriate doses may
be determined using the assays described herein. When one or more
of these small molecules is to be administered to a subject (e.g.,
a human) in order to modulate expression or activity of a
polypeptide or nucleic acid of the invention, a physician,
veterinarian, or researcher may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. In addition, it is
understood that the specific dose level for any particular animal
subject will depend upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degre of expression or activity to be
modulated.
[0585] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0586] 5.23 Kits
[0587] The present invention provides kits for measuring the
expression of the protein and RNA products of at least 1, at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 15, at least 20, at
least 25, at least 30, at least 35, at least 40, at least 45, at
least 50, all or any combination of the biomarkers of the
invention. Such kits comprise materials and reagents required for
measuring the expression of such protein and RNA products. In
specific embodiments, the kits may further comprise one or more
additional reagents employed in the various methods, such as: (1)
reagents for purifying RNA from blood, chondrocytes or synovial
fluid; (2) primers for generating test nucleic acids; (3) dNTPs
and/or rNTPs (either premixed or separate), optionally with one or
more uniquely labeled dNTPs and/or rNTPs (e.g., biotinylated or Cy3
or Cy5 tagged dNTPs); (4) post synthesis labeling reagents, such as
chemically active derivatives of fluorescent dyes; (5) enzymes,
such as reverse transcriptases, DNA polymerases, and the like; (6)
various buffer mediums, e.g., hybridization and washing buffers;
(7) labeled probe purification reagents and components, like spin
columns, etc.; and (8) protein purification reagents; (9) signal
generation and detection reagents. e.g., streptavidin-alkaline
phosphatase conjugate, chemifluorescent or chemiluminescent
substrate, and the like. In particular embodiments, the kits
comprise prelabeled quality controlled protein and or RNA isolated
from a sample (e.g., blood or chondrocytes or synovial fluid) for
use as a control.
[0588] In some embodiments, the kits are RT-PCR kits. In other
embodiments, the kits are nucleic acid arrays and protein arrays.
Such kits according to the subject invention will at least comprise
an array having associated protein or nucleic acid members of the
invention and packaging means therefore. Alternatively the protein
or nucleic acid members of the invention may be prepackaged onto an
array.
[0589] In some embodiments, the kits are Quantitative RT-PCR kits.
In one embodiment, the quantitative RT-PCR kit includes the
following: (a) primers used to amplify each of a combination of
biomarkers of the invention; (b) buffers and enzymes including an
reverse transcripate; (c) one or more thermos table polymerases;
and (d) Sybr.RTM. Green. In a preferred embodiment, the kit of the
invention also includes (a) a reference control RNA and (b) a
spiked control RNA.
[0590] The invention provides kits that are useful for (a)
diagnosing individuals as having arthritis, (b) differentiating
between two stages of osteoarthritis (OA) and (c) diagnosing
individuals as having a particular stage of osteoarthritis (OA).
For example, in a particular embodiment of the invention a kit is
comprised a forward and reverse primer wherein the forward and
reverse primer are designed to quantitate expression of all of the
species of mRNA corresponding to each of the biomarkers as
identified in accordance with the invention useful in determining
whether an individual has mild OA or does not have OA. In certain
embodiments, at least one of the primers is designed to span an
exon junction.
[0591] The invention provides kits that are useful for detecting,
diagnosing, monitoring and prognosing osteoarthritis based upon the
expression of protein or RNA products of at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 15, at least 20, at least 25,
at least 30, at least 35, at least 40, at least 45, at least 50,
all or any combination of the biomarkers of the invention in a
sample. In certain embodiments, such kits do not include the
materials and reagents for measuring the expression of a protein or
RNA product of a biomarker of the invention that has been suggested
by the prior art to be associated with osteoarthritis. In other
embodiments, such kits include the materials and reagents for
measuring the expression of a protein or RNA product of a biomarker
of the invention that has been suggested by the prior art to be
associated with osteoarthritis and at least 1, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 15, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45 or more genes other
than the biomarkers of the invention.
[0592] The invention provides kits useful for monitoring the
efficacy of one or more therapies that a subject is undergoing
based upon the expression of a protein or RNA product of at least
1, at least 1 at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 15, at least
20, at least 25, at least 30, at least 35, at least 40, at least
45, at least 50, all or any combination of the biomarkers of the
invention in a sample. In certain embodiments, such kits do not
include the materials and reagents for measuring the expression of
a protein or RNA product of a biomarker of the invention that has
been suggested by the prior art to be associated with
osteoarthritis. In other embodiments, such kits include the
materials and reagents for measuring the expression of a protein or
RNA product of a biomarker of the invention that has been suggested
by the prior art to be associated with osteoarthritis and at least
1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 15, at least
20, at least 25, at least 30, at least 35, at least 40, at least 45
or more genes other than the biomarkers of the invention.
[0593] The invention provides kits using for determining whether a
subject will be responsive to a therapy based upon the expression
of a protein or RNA product of at least 1, at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 15, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least 50, all
or any combination of the biomarkers of the invention in a sample.
In certain embodiments, such kits do not include the materials and
reagents for measuring the expression of a protein or RNA product
of a biomarker of the invention that has been suggested by the
prior art to be associated with osteoarthritis. In other
embodiments, such kits include the materials and reagents for
measuring the expression of a protein or RNA product of a biomarker
of the invention that has been suggested by the prior art to be
associated with osteoarthritis and at least 1, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 15, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45 or more genes other
than the biomarkers of the invention.
[0594] The invention provides kits for measuring the expression of
a RNA product of at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 15, at least 20, at least 25, at least 30, at least
35, at least 40, at least 45, at least 50, all or any combination
of the biomarkers of the invention in a sample. In a specific
embodiment, such kits comprise materials and reagents that are
necessary for measuring the expression of a RNA product of a
biomarker of the invention. For example, a microarray or RT-PCR kit
may be produced for osteoarthritis and contain only those reagents
and materials necessary for measuring the levels of RNA products of
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
15, at least 20, at least 25, at least 30, at least 35, at least
40, at least 45, at least 50, all or any combination of the
biomarkers of the invention. Alternatively, in some embodiments,
the kits can comprise materials and reagents that are not limited
to those required to measure the levels of RNA products of 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, all or any
combination of the biomarkers of the invention. For example, a
microarray kit may contain reagents and materials necessary for
measuring the levels of RNA products of not necessarily associated
with or indicative of osteoarthritis, in addition to reagents and
materials necessary for measuring the levels of the RNA products of
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
15, at least 20, at least 25, at least 30, at least 35, at least
40, at least 45, at least 50, all or any combination of the
biomarkers of the invention. In a specific embodiment, a microarray
or RT-PCR kit contains reagents and materials necessary for
measuring the levels of RNA products of at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 15, at least 20, at least 25,
at least 30, at least 35, at least 40, at least 45, at least 50,
all or any combination of the biomarkers of the invention, and 1,
2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400,
450, or more genes other than the biomarkers of the invention, or
1-10, 1-100, 1-150, 1-200, 1-300, 1-400, 1-500, 1-1000, 25-100,
25-200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300,
100-400, 100-500, 100-1000, 500-1000 other genes than the
biomarkers of the invention.
[0595] For nucleic acid micoarray kits, the kits generally comprise
probes attached to a solid support surface. The probes may be
labeled with a detectable label. In a specific embodiment, the
probes are specific for an exon(s), an intron(s), an exon
junction(s), or an exon-intron junction(s)), of RNA products of 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, all or
any combination of the biomarkers of the invention. The microarray
kits may comprise instructions for performing the assay and methods
for interpreting and analyzing the data resulting from the
performance of the assay. In a specific embodiment, the kits
comprise instructions for diagnosing osteoarthritis. The kits may
also comprise hybridization reagents and/or reagents necessary for
detecting a signal produced when a probe hybridizes to a target
nucleic acid sequence. Generally, the materials and reagents for
the microarray kits are in one or more containers. Each component
of the kit is generally in its own a suitable container.
[0596] For RT-PCR kits, the kits generally comprise pre-selected
primers specific for particular RNA products (e.g., an exon(s), an
intron(s), an exon junction(s), and an exon-intron junction(s)) of
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, all
or any combination of the biomarkers of the invention. The RT-PCR
kits may also comprise enzymes suitable for reverse transcribing
and/or amplifying nucleic acids (e.g., polymerases such as Taq),
and deoxynucleotides and buffers needed for the reaction mixture
for reverse transcription and amplification. The RT-PCR kits may
also comprise probes specific for RNA products of 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, all or any combination
of the biomarkers of the invention. The probes may or may not be
labeled with a detectable label (e.g., a fluorescent label). Each
component of the RT-PCR kit is generally in its own suitable
container. Thus, these kits generally comprise distinct containers
suitable for each individual reagent, enzyme, primer and probe.
Further, the RT-PCR kits may comprise instructions for performing
the assay and methods for interpreting and analyzing the data
resulting from the performance of the assay. In a specific
embodiment, the kits contain instructions for diagnosing
osteoarthritis.
[0597] In a specific embodiment, the kit is a real-time RT-PCR kit.
Such a kit may comprise a 96 well plate and reagents and materials
necessary for SYBR Green detection. The kit may comprise reagents
and materials so that beta-actin can be used to normalize the
results. The kit may also comprise controls such as water, phospate
buffered saline, and phage MS2 RNA. Further, the kit may comprise
instructions for performing the assay and methods for interpreting
and analyzing the date resulting from the performance of the assay.
In a specific embodiment, the instructions state that the level of
a RNA product of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,
40, 45, 50, all or any combination of the biomarkers of the
invention should be examined at two concentrations that differ by,
e.g., 5 fold to 10-fold.
[0598] For antibody based kits, the kit can comprise, for example:
(1) a first antibody (which may or may not be attached to a solid
support) which binds to protein of interest (e.g., a protein
product of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, all or any combination of the biomarkers of the invention);
and, optionally, (2) a second, different antibody which binds to
either the protein, or the first antibody and is conjugated to a
detectable label (e.g., a fluorescent label, radioactive isotope or
enzyme). The antibody-based kits may also comprise beads for
conducting an immunoprecipitation. Each component of the
antibody-based kits is generally in its own suitable container.
Thus, these kits generally comprise distinct containers suitable
for each antibody. Further, the antibody-based kits may comprise
instructions for performing the assay and methods for interpreting
and analyzing the data resulting from the performance of the assay.
In a specific embodiment, the kits contain instructions for
diagnosing osteoarthritis.
6. EXAMPLES
[0599] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention
Example 1
Microarray Construction
[0600] An array according to one aspect of the invention was
constructed as follows.
[0601] PCR products (.about.40 ul) of cDNA clones from OA cartilage
cDNA libraries, in the same 96-well tubes used for amplification,
are precipitated with 4 ul (1/10 volume) of 3M sodium acetate (pH
5.2) and 100 ul (2.5 volumes) of ethanol and stored overnight at
-20.degree. C. They are then centrifuged at 3,300 rpm at 4.degree.
C. for 1 hour. The obtained pellets were washed with 50 ul ice-cold
70% ethanol and centrifuged again for 30 minutes. The pellets are
then air-dried and resuspended well in 50% dimethylsulfoxide (DMSO)
or 20 ul 3.times.SSC overnight. The samples are then deposited
either singly or in duplicate onto Gamma Amino Propyl Silane
(Corning CMT-GAPS or CMT-GAP2, Catalog No. 40003, 40004) or poly
lysine-coated slides (Sigma Cat. No. P0425) using a robotic GMS 417
or 427 arrayer (Affymetrix. CA). The boundaries of the DNA spots on
the microarray are marked with a diamond scriber. The invention
provides for arrays where 10-20.000 PCR products are spotted onto a
solid support to prepare an array.
[0602] The arrays are rehydrated by suspending the slides over a
dish of warm particle free ddH.sub.2O for approximately one minute
(the spots will swell slightly but not run into each other) and
snap-dried on a 70-80.degree. C. inverted heating block for 3
seconds. DNA is then UV crosslinked to the slide (Stratagene,
Stratalinker, 65 mJ--set display to "650" which is 650.times.100
uJ) or baked at 80 C for two to four hours. The arrays are placed
in a slide rack. An empty slide chamber is prepared and filled with
the following solution: 3.0 grams of succinic anhydride (Aldrich)
is dissolved in 189 ml of 1-methylrrolidinone (rapid addition of
reagent is crucial); immediately after the last flake of succinic
anhydride dissolved, 21.0 ml of 0.2 M sodium borate is mixed in and
the solution is poured into the slide chamber. The slide rack is
plunged rapidly and evenly in the slide chamber and vigorously
shaken up and down for a few seconds, making sure the slides never
leave the solution, and then mixed on an orbital shaker for 15-20
minutes. The slide rack is then gently plunged in 95.degree. C.
ddH.sub.20 for 2 minutes, followed by plunging five times in 95%
ethanol. The slides are then air dried by allowing excess ethanol
to drip onto paper towels. The arrays are then stored in the slide
box at room temperature until use.
Example 2
RNA Isolation
From Whole Blood
[0603] 100 ul whole blood is obtained in a microcentrifuge tube and
spun at 2.000 rpm (800 g) for 5 min at 4.degree. C. and the
supernatant removed. Pelleted cells are homogenized using
TRIzol.RTM. (GIBCO/BRL) in a ratio of approximately 6 .mu.l of
TRIzol.RTM. for every 10 .mu.l of the original blood sample and
vortexed well. Samples are left for 5 minutes at room temperature.
RNA is extracted using 12 .mu.l of chloroform per 10 .mu.l of
TRIzol.RTM.. Sample is centrifuged at 12,000.times.g for 5 minutes
at 4.degree. C. and upper layer is collected. To upper layer,
isopropanol is added in ratio of 5 .mu.l per 10 .mu.l of
TRIzol.RTM.. Sample is left overnight at -20.degree. C. or for one
hour at -20.degree. C. RNA is pelleted in accordance with known
methods. RNA pellet air dried, and pellet resuspended in DEPC
treated ddH.sub.2O. RNA samples can also be stored in 75% ethanol
where the samples are stable at room temperature for
transportation.
From Centrifuged Lysed Blood
[0604] 10 ml whole blood is obtained in a Vacutainer and spun at
2.000 rpm (800 g) for 5 min at 4.degree. C. and the plasma layer
removed. Lysis Buffer is added to blood sample in a ratio of 3
parts Lysis Buffer to 1 part blood (Lysis Buffer (1 L) 0.6 g EDTA;
1.0 g KHCO.sub.2, 8.2 g NH.sub.4Cl adjusted to pH 7.4 (using
NaOH)). Sample is mixed and placed on ice for 5-10 minutes until
transparent. Lysed sample is centrifuged at 1000 rpm for 10 minutes
at 4.degree. C., and supernatant is aspirated. Pellet is
resuspended in 5 ml Lysis Buffer, and centrifuged again at 1000 rpm
for 10 minutes at 4.degree. C. Pelleted cells are homogenized using
TRIzol.RTM. (GIBCO/BRL) in a ratio of approximately 6 ml of
TRIzol.RTM. for every 10 ml of the original blood sample and
vortexed well. Samples are left for 5 minutes at room temperature.
RNA is extracted using 1.2 ml of chloroform per 1 ml of
TRIzol.RTM.. Sample is centrifuged at 12,000.times.g for 5 minutes
at 4.degree. C. and upper layer is collected. To upper layer,
isopropanol is added in ratio of 0.5 ml per 1 ml of TRIzol.RTM..
Sample is left overnight at -20.degree. C. or for one hour at
-20.degree. C. RNA is pelleted in accordance with known methods.
RNA pellet air dried, and pellet resuspended in DEPC treated
ddH.sub.2O. RNA samples can also be stored in 75% ethanol where the
samples are stable at room temperature for transportation.
From Serum Free Whole Blood
[0605] 10 ml whole blood is obtained in a Vacutainer and spun at
2,000 rpm (800 g) for 5 min at 4.degree. C. and the plasma layer
removed. Pelleted cells are homogenized using TRIzol.RTM.
(GIBCO/BRL) in a ratio of approximately 6 ml of TRIzol.RTM. for
every 10 ml of the original blood sample and vortexed well. Samples
are left for 5 minutes at room temperature. RNA is extracted using
1.2 ml of chloroform per 1 ml of TRIzol.RTM.. Sample is centrifuged
at 12,000.times.g for 5 minutes at 4.degree. C. and upper layer is
collected. To upper layer, isopropanol is added in ratio of 0.5 ml
per 1 ml of TRIzol.RTM.. Sample is left overnight at -20.degree. C.
or for one hour at -20.degree. C. RNA is pelleted in accordance
with known methods, RNA pellet air dried, and pellet resuspended in
DEPC treated ddH.sub.2O. RNA samples can also be stored in 75%
ethanol where the samples are stable at room temperature for
transportation.
Example 3
Target Nucleic Acid Preparation and Hybridization
[0606] Preparation of Fluorescent DNA Probe from mRNA
[0607] Fluorescently labeled target nucleic acid samples of RNA are
prepared for analysis with an array of the invention.
[0608] 1 .mu.g Oligo-dT primers are annealed to 10 ug of total RNA
isolated from blood from patient diagnosed with osteoarthritis or
suspected of having osteoarthritis in a total volume of 10 ul, by
heating to 70.degree. C. for 10 min, and cooled on ice. The mRNA is
reverse transcribed by incubating the sample at 42.degree. C. for
40 min in a 25 .mu.l volume containing a final concentration of 50
mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 25 mM DTT, 25 mM
unlabeled dNTPs, 400 units of Superscript II (200 U/uL, Gibco BRL),
and 15 mM of Cy3 or Cy5 (Amersham). The reaction is stopped by the
addition of 2.5 .mu.l of 55500 mM EDTA and 5 .mu.l of 1M NaOH, and
incubation at 65.degree. C. for 10 min. The reaction mixture is
neutralized by addition of 12.5 .mu.l of 1M TrisHCl (pH7.6).
[0609] The labeled target nucleic acid sample is purified by
centrifugation in a Centricon-30 micro-concentrator (Amicon). If
two different target nucleic acid samples (e.g., two samples
derived from different patients) are being analyzed and compared by
hybridization to the same array, each target nucleic acid sample is
labeled with a different fluorescent label (e.g., Cy3 and Cy5) and
separately concentrated. The separately concentrated target nucleic
acid samples (Cy3 and Cy5 labeled) are combined into a fresh
centricon, washed with 500 .mu.l TE, and concentrated again to a
volume of less than 7 .mu.l, 14 of 10 .mu.g/.mu.l polyA RNA (Sigma.
#P9403) and 1 .mu.l of 10 .mu.g/ultRNA (Gibco-BRL, #15401-011) is
added and the volume is adjusted to 9.5 .mu.l with distilled water.
For final target nucleic acid preparation 2.1 .mu.l 20.times.SSC
(1.5M NaCl, 150 mM NaCltrate (pH8.0)) and 0.35 .mu.l 10% SDS is
added.
Hybridization
[0610] Labeled nucleic acid is denatured by heating for 2 min at
100.degree. C., and incubated at 37.degree. C. for 20-30 min before
being placed on a nucleic acid array under a 22 mm.times.22 mm
glass cover slip. Hybridization is carried out at 65.degree. C. for
14 to 18 hours in a custom slide chamber with humidity maintained
by a small reservoir of 3.times.SSC. The array is washed by
submersion and agitation for 2-5 min in 2.times.SSC with 0.1% SDS,
followed by 1.times.SSC, and 0.1.times.SSC. Finally, the array is
dried by centrifugation for 2 min in a slide rack in a Beckman GS-6
tabletop centrifuge in Microplus carriers at 650 RPM for 2 min.
Example 4
Real Time RT PCR
[0611] Real time RT PCR was performed on the genes as disclosed in
Table 1 and FIGS. 1 to 4 using the SYBR.RTM. Green Kit from Qiagen
(Product Number 204143). An example of the experimental results for
one of the genes identified in FIGS. 1 to 4 is shown in FIG. 5.
[0612] Either a one step (reverse transcription and PCR combined)
or a two step (reverse transcription first and then subsequent PCR)
can be used. In the case of the two step protocol, reverse
transcription was first performed using the High-Capacity cDNA
Archive Kit from Applied Biosystems (Product number 432217) and
following the protocol utilized therein.
[0613] More specifically purified RNA as described previously
herein was incubated with Reverse Transcriptase buffer, dNTPs,
Random primers and Reverse transcriptase and incubated for
25.degree. C. for 10 minutes and subsequently for 37.degree. C. for
two hours and the resulting mixture utilized as the starting
product for quantitative PCR.
[0614] cDNA resulting from reverse transcription was incubated with
the QuantiTect SYBR.RTM. Green PCR Master Mix as provided and no
adjustments were made for magnesium concentration.
Uracil-N-Glycosylase was not added. 5 .mu.M of both forward primer
and reverse primer specific to the genes of the invention were
added and the reaction was incubated and monitored in accordance
with the standard protocol utilizing the ABI PRISM 7700/ABI GeneAmp
5700/iCycler/DNA Engine Opticon.
TABLE-US-00005 TABLE 7 Gene Forward Primer Reverse Primer Size B2M
GAATTCACCCCCACTGAAAA CCTCCATGATGCTGCTTACA 111 (SEQ ID NO: 49) (SEQ
ID NO: 50) G2AN TTCCTGCTGCGTCGATTCTCA TTATCACCACCCGCTCAATCCA 106
(SEQ ID NO: 51) (SEQ ID NO: 52) IL13RA1 CATTGTTCCAGTCATCGTCGCA
TCTTGCCAGGATCAGGAATTGG 101 (SEQ ID NO: 53) (SEQ ID NO: 54) TNFAIP6
AAGGATGGGGATTCAAGGAT AATTCACACACCGCCTTAGC 133 (SEQ ID NO: 55) (SEQ
ID NO: 56) WDR9 TTGCAGGCCCTGTTGATTTGTG CCAAACTAACGCAGACAGCCTC 128
(SEQ ID NO: 57) (SEQ ID NO: 58) WWP2 GGCACTACACCAAGAACAGCAA
TGCTACCGATGAGTTCGGCA 147 (SEQ ID NO: 59) (SEQ ID NO: 60) BCL6
CAAGGCATTGGTGAAGACAA CGGCTCACAACAATGACAAC 140 (SEQ ID NO: 61) (SEQ
ID NO: 62) C1QR1 GCCATGGAGAACCAGTACAGTC GAGTTCAAAGCTCTGAGGATGGTG
105 (SEQ ID NO: 63) (SEQ ID NO: 64) CCNC CCCTTGCATGGAGGATAGTG
CATTGCCTGGCATCTTTCTG 127 (SEQ ID NO: 65) (SEQ ID NO: 66) EBNA1BP2
GCCTCCATCAGCTCAAAGTC TTGCACCTTCTTCCCGTATT 179 (SEQ ID NO: 67) (SEQ
ID NO: 68) FLJ32234 GCGGAGGATGAAGTTGTGA GAGTCCTTATTCAAAGTAATCGAAGG
191 (SEQ ID NO: 69) (SEQ ID NO: 70) HSPCA ATGATTGGCCAGTTCGGTGTTG
TTCACCTGTGTCTGTCCTCACT 147 (SEQ ID NO: 71) (SEQ ID NO: 72) LAMC1
CAGGCTCCATGAAGCAACAGA GCACTTCTCTCACTGTATGTCCCAC 120 (SEQ ID NO: 73)
(SEQ ID NO: 74) PAIP2 AAGATCCAAGTCGCAGCAGT
TCCCATAACTCCTCTTTAACTTGTC 154 (SEQ ID NO: 75) (SEQ ID NO: 76) ZFR
CGAGAAGAGAACATGAGGGAAGGA TAGAGCAGCCAGAGCGTCAA 102 (SEQ ID NO: 77)
(SEQ ID NO: 78) IKBKAP GCAGCCCAGCTTAACTTTACCA
TAATCCTGGGCACACTCTTCCA 125 (SEQ ID NO: 79) (SEQ ID NO: 80) ABCA1
AGTTGGCAAGGTTGGTGAGT ATGGCTGTAGAGAGCTTGCGTT 111 (SEQ ID NO: 81)
(SEQ ID NO 82) ABCG1 TGCAGGTGGCCACTTTCGTG CCTTCGAACCCATACCTGACA 139
(SEQ ID NO: 83) (SEQ ID NO: 84) IRF1 ACTCCAGCACTGTCGCCAT
TGGGTGACACCTGGAAGTTGTA 112 (SEQ ID NO: 85) (SEQ ID NO: 86) NCOA1
TCCAGTATCCAGGAGCAGGAA AGAACTCTGAGGAGGAGGGATT 110 (SEQ ID NO: 87)
(SEQ ID NO 88) CLIC4 TGCCCTCCCAAGTACTTAAAGC CAGTGCTTCATTAGCCTCTGG
123 (SEQ ID NO: 89) (SEQ ID NO: 90) ACP1 TCAGAGAATTGGAGGGTAGACAGC
TTTGGTAATCTGCCGGGCAAC 132 (SEQ ID NO: 91) (SEQ ID NO: 92) ADPRT
CCCGTGACAGGCTACATGTTT AAGGGCAACTTCTCCCAACA 126 (SEQ ID NO: 93) (SEQ
ID NO: 94) ANGPTL2 AGACGTACAAGCAAGGGTTTGG CACCAGGAGTTTGTAGTTGCCT
(SEQ ID NO: 95) (SEQ ID NO: 96) BMPR2 AAATAGCCTGGCAGTGAGGT
TTCAGCCATCCTTTCCTCAGCA 106 (SEQ ID NO: 97) (SEQ ID NO: 98) C19orf13
CAATAGAGAACGGAGACCAACCTG ACCTCCTCTGCCTCTGTAT 112 (SEQ ID NO: 99)
(SEQ ID NO: 100) CLECSF6 ATATGCCCGTGGAAGAGACA
TGAGCCTCCATTCTAGCACAGT 136 (SEQ ID NO: 101) (SEQ ID NO: 102) CLN3
TGCCAAGCATCTACCTCGTCTT ATCACTGGTCTCCAGGGCGAT 104 (SEQ ID NO: 103)
(SEQ ID NO: 104) DNAPTP6 GGTCACAGAAGGCAACAGACTACT
TGCCTTAGGCTTGCTTGGGTTA 139 (SEQ ID NO: 105) (SEQ ID NO: 106) EFHD1
TTCCGGGAGTTCCTGCTCATTT AGTTCTTGGCACCTTTGACACC 130 (SEQ ID NO: 107)
(SEQ ID NO: 108) EGR1 GACCGCAGAGTTCTTTTCCTG CACAAGGTGTTGCCACTGTT
150 (SEQ ID NO: 109) (SEQ ID NO: 110) EXOSC10
AGTTGACTTGGAGCACCACTCT TTCGAAGCTCGAGGGTGTCAAT 107 (SEQ ID NO: 111)
(SEQ ID NO: 112) F2RL1 TGGCACCATCCAAGGAACCAAT
TTCCAGTGAGGACAGATGCAGA 146 (SEQ ID NO: 113) (SEQ ID NO: 114)
FLJ11000 TGTGGTCTCTGCACCCCTTT TGACCACAATCACGAGGACT 120 (SEQ ID NO:
115) (SEQ ID NO: 116) FLJ11142 TCGTTTCTTGGTGACTGCTGGA
TCCAAACCTGGGAGATGGAACT 112 (SEQ ID NO: 117) (SEQ ID NO: 118) FOXK2
AGACAGCCCGAAGGATGATT TTGTCCGCAGTCCTGTAGTA 150 (SEQ ID NO: 119) (SEQ
ID NO: 120) HSPCB CCACTTGGCAGTCAAGCACTTT TGATGAACACACGGCGGACATA 146
(SEQ ID NO: 121) (SEQ ID NO: 122) LCMT2 TGGCCAGTTCATGCTGCAA
ATTCATTCATGTCCACGGCACC 141 (SEQ ID NO: 123) (SEQ ID NO: 124) MAFB
CCCTTGTTTCTTTGGGTGAG ACGTTCTCTATGCGGTTTG 119 (SEQ ID NO: 125) (SEQ
ID NO: 126) NXN TGTAGATTCTGAGGATGACGGAG CCTCCTCCTCTTTGGCTTTGTACT
101 (SEQ ID NO: 127) (SEQ ID NO: 128) PDCD5 ATGGCGGACGAGGAGCTTGA
CTCATTTCTGCTTCCCTGTGCT 119 (SEQ ID NO: 129) (SEQ ID NO: 130) PDK4
ACTCGGATGCTGATGAACCA AAGGCATCTTGGACCACTGCTA 109 (SEQ ID NO: 131)
(SEQ ID NO: 132) PER1 TAAGCGTAAATGTGCCTCCTCC TGACGGCGGATCTTTCTTGGT
109 (SEQ ID NO: 133) (SEQ ID NO: 134) PF4 CCAACTGATAGCCACGCTGAAGAA
AATGCACACACGTAGGCAGCTA 123 (SEQ ID NO: 135) (SEQ ID NO: 136) PF4V1
GTTGCTGCTCCTGCCACTT GTGGCTATCAGTTGGGCAGT 171 (SEQ ID NO: 137) (SEQ
ID NO: 138) PPIF TGCTGGAGCTGAAGGCAGAT TGGCACATGAAGGAAGGGAT 127 (SEQ
ID NO: 139) (SEQ ID NO: 140) SETBP1 GAAGGCTTTGGAACGTACAGG
GGGACTTGGCATCCCTGGAG 106 (SEQ ID NO: 141) (SEQ ID NO: 142) SFRS6
TGGACAAACTGGATGGCACAGA ATCGAGACCTGGATCTGCTT 111 (SEQ ID NO: 143)
(SEQ ID NO: 144) SLC5A6 CCAGACCAGTTCGTCCTGTACTTT
TATAGTGCTGAGAGAGCCGCTGAA 105 (SEQ ID NO: 145) (SEQ ID NO: 146)
TSPAN-2 AGGTCTGACGATCTTTGGCA GACAGCTCCTGTGACATTTGGT (SEQ ID NO:
147) (SEQ ID NO: 148) YES1 ATCCAGGTATGGTGAACCGTGA
TCAGGGTCCTTCTTCCAACAC 124 (SEQ ID NO: 149) (SEQ ID NO: 150) ZNF397
GCAGCAGGTCCCAGCTAGT TGGAGGTGGATGTCTGTTGACT 96 (SEQ ID NO: 151) (SEQ
ID NO: 152) HSPCAL3 ATGATTGGCCAGTTCGGTGTTG TTCACCTGTGTCTGTCCTCACT
147 (SEQ ID NO: 153) (SEQ ID NO: 154)
Example 5
Taqman.RTM.
[0615] Quantitative real time RT PCR was also performed using the
QuantiTect.TM. Probe RT-PCR system from Qiagen (Product Number
204343) in conjunction with a TaqMan.RTM. dual labelled probe and
primers corresponding to the gene of interest. The TaqMan.RTM.
probe and primers can be ordered from Applied Biosystems
Assays-On-Demand.TM..
[0616] The dual labelled probe contains both a fluorophore and a
quencher molecule. The proximity of the fluorescent reporter with
the quencher prevents the reporter from fluorescing, but during the
PCR extension step, the 5'-3' exonuclease activity of the Taq DNA
polymerase releases the fluorophore which allows it to fluoresce.
As such, the amount of fluorescence correlates with the amount of
PCR product generated.
[0617] TaqMan.RTM. quantitative PCR was performed on the TNFAIP6
gene in order to confirm the quantitative real time data obtained
using SYBR.RTM. green. An example of the experimental results is
showin in FIG. 6 and FIG. 8. More specifically the TaqMan.RTM.
probe was incubated with the QuantiTect PCR Master Mix, primers
specific for the TNFAIP6 gene and cDNA resulting from RT PCR as
described previously to a final volume of 45 ul and the PCR
reaction performed using standard conditions. Results from this
experiment can be seen in FIG. 6. It would be understood by a
person skilled in the art that similar results can be obtained for
the other genes of the invention.
Example 6
Statistical Analysis of Real Time PCR Results
[0618] Real Time PCR analysis on blood samples isolated from
individuals categorized as normal or having mild OA, moderate OA,
marked OA or severe OA were statistically analyzed using known
methods in order to confirm the usefulness of the genes disclosed
as biomarkers.
[0619] Preferably individuals having similar age and body mass
index (BMI) were selected for further analysis. Selection of
samples for which comparisons could be made on the basis of age and
BMI were determined using KW One Way Analysis of Variance on Ranks
as would be understood by a person skilled in the art.
[0620] Delta CT value and MW Rank Sum tests were utilized on age
and BMI matched sample sets of approximately 20 to 50 in size. Box
plots were done on the sample sets so as to determine fold change
differences as between said sets. FIGS. 5, 7 and 8 show a
representative analysis for G2AN, PER1 and ZFR respectively. As
would be clear to a person skilled in the art, similar analysis can
be performed for any of the sequences identified herein.
Example 7
Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Mild Osteoarthritis as Compared with Gene
Expression Profiles from Normal Individuals Using the Isolated
Biomarker Described in FIG. 1
[0621] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0622] Blood samples are taken from patients who are clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0623] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzol.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing full length cDNA
sequences for each of the 19 genes as described in FIG. 1.
Detection of specific hybridization to the array is then measured
by scanning with a GMS Scanner 418 and processing of the
experimental data with Scanalyzer software (Michael Eisen, Stanford
University), followed by GeneSpring software (Silicon Genetics, CA)
analysis. Differential expression of the 19 genes in blood samples
from patients with osteoarthritis as compared to healthy patients
is determined by statistical analysis using the Wilcox Mann Whitney
rank sum test (Glantz S A. Primer of Biostatistics. 5th ed. New
York, USA: McGraw-Hill Medical Publishing Division, 2002).
Differential expression of each of the 19 genes described in FIG. 1
is diagnostic for osteoarthritis.
Example 8
Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Moderate Osteoarthritis as Compared with Gene
Expression Profiles from Normal Individuals Using the Isolated
Biomarker Described in FIG. 2
[0624] This example demonstrates the use of the claimed invention
to diagnose moderate osteoarthritis by detecting differential gene
expression in blood samples taken from patients with moderate OA as
compared to blood samples taken from healthy patients.
[0625] Blood samples are taken from patients who were clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0626] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzoI.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing full length cDNA
sequences for each of the 4 genes as described in FIG. 2. Detection
of specific hybridization to the array is then measured by scanning
with a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen, Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the 4 genes in blood samples from
patients with moderate osteoarthritis as compared to healthy
patients is determined by statistical analysis using the Wilcox
Mann Whitney rank sum test (Glantz S A. Primer of Biostatistics.
5th ed. New York, USA: McGraw-Hill Medical Publishing Division,
2002). Differential expression of each of the 4 genes described in
FIG. 2 is diagnostic for moderate osteoarthritis.
Example 9
Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Marked Osteoarthritis as Compared with Gene
Expression Profiles from Individuals Having Moderate Osteoarthritis
Using the Isolated Biomarker Described in FIG. 3
[0627] This example demonstrates the use of the claimed invention
to diagnose marked osteoarthritis by detecting differential gene
expression in blood samples taken from patients with marked OA as
compared to blood samples taken from patients with moderate OA.
[0628] Blood samples are taken from patients who are clinically
diagnosed with marked osteoarthritis as defined herein. Gene
expression profiles are then analyzed and compared to profiles from
patients who were clinically diagnosed with moderate OA. In each
case, the diagnosis and staging of osteoarthritis is corroborated
by a skilled Board certified physician.
[0629] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzol.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing full length cDNA
sequences for each of the 2 genes as described in FIG. 3. Detection
of specific hybridization to the array is then measured by scanning
with a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen, Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the 2 genes in blood samples from
patients with marked osteoarthritis as compared to patients with
moderate osteoarthritis is determined by statistical analysis using
the Wilcox Mann Whitney rank sum test (Glantz S A. Primer of
Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
2 genes described in FIG. 3 is diagnostic for marked
osteoarthritis.
Example 10
Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Severe Osteoarthritis as Compared with Gene
Expression Profiles from Individuals Having Marked Osteoarthritis
Using the Isolated Biomarker Described in FIG. 4
[0630] This example demonstrates the use of the claimed invention
to diagnose severe osteoarthritis by detecting differential gene
expression in blood samples taken from patients with severe OA as
compared to blood samples taken from patients with marked OA.
[0631] Blood samples are taken from patients who are clinically
diagnosed with severe osteoarthritis as defined herein. Gene
expression profiles are then analyzed and compared to profiles from
patients who are clinically diagnosed with marked OA. In each case,
the diagnosis and staging of osteoarthritis is corroborated by a
skilled Board certified physician.
[0632] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzol.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing full length cDNA
sequences for each of the 4 genes as described in FIG. 4. Detection
of specific hybridization to the array is then measured by scanning
with a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen, Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the 4 genes in blood samples from
patients with severe osteoarthritis as compared to patients with
marked osteoarthritis is determined by statistical analysis using
the Wilcox Mann Whitney rank sum test (Glantz S A. Primer of
Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
4 genes described in FIG. 4 is diagnostic for severe
osteoarthritis.
Example 11
Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Osteoarthritis as Compared with Gene Expression
Profiles from Healthy Individuals Using the 5' Regions of the 19
Genes Described in FIG. 1
[0633] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0634] Blood samples are taken from patients who are clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0635] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzol.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing DNA sequences of 25
nucleotides in length corresponding to the 5' region of each of the
19 genes as described in FIG. 1. Detection of specific
hybridization to the array is then measured by scanning with a GMS
Scanner 418 and processing of the experimental data with Scanalyzer
software (Michael Eisen, Stanford University), followed by
GeneSpring software (Silicon Genetics, CA) analysis. Differential
expression of the 19 genes in blood samples from patients with
osteoarthritis as compared to healthy patients is determined by
statistical analysis using the Wilcox Mann Whitney rank sum test
(Glantz S A. Primer of Biostatistics. 5th ed. New York, USA:
McGraw-Hill Medical Publishing Division, 2002). Differential
expression of each of the 19 genes described in FIG. 1 is
diagnostic for osteoarthritis.
Example 12
Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Osteoarthritis as Compared with Gene Expression
Profiles from Healthy Individuals Using the 3' Regions of the 19
Genes Described in FIG. 1
[0636] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0637] Blood samples are taken from patients who were clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0638] Total mRNA from a drop of blood taken from each patient is
first isolated using TRIzol.RTM. reagent (GIBCO) and fluorescently
labeled probes for each blood sample are then generated, denatured
and hybridized to a microarray containing DNA sequences of 50
nucleotides in length corresponding to the 3' region of each of the
19 genes as described in FIG. 1. Detection of specific
hybridization to the array is then measured by scanning with a GMS
Scanner 418 and processing of the experimental data with Scanalyzer
software (Michael Eisen, Stanford University), followed by
GeneSpring software (Silicon Genetics, CA) analysis. Differential
expression of the 19 genes in blood samples from patients with
osteoarthritis as compared to healthy patients is determined by
statistical analysis using the Wilcox Mann Whitney rank sum test
(Glantz S A. Primer of Biostatistics. 5th ed. New York, USA:
McGraw-Hill Medical Publishing Division, 2002). Differential
expression of each of the 19 genes described in FIG. 1 is
diagnostic for osteoarthritis.
Example 13
Analysis of Gene Expression Profiles of Blood Samples from
Individuals Having Osteoarthritis as Compared with Gene Expression
Profiles from Healthy Individuals Using the Internal Coding Regions
of the 19 Genes Described in FIG. 1
[0639] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0640] Blood samples are taken from patients who are clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0641] Total mRNA from a drop of blood taken from each patient
wisas first isolated using TRIzol.RTM. reagent (GIBCO) and
fluorescently labeled probes for each blood sample are then
generated, denatured and hybridized to a microarray containing DNA
sequences of 70 nucleotides in length corresponding to the internal
coding region of each of the 19 genes as described in FIG. 1.
Detection of specific hybridization to the array is then measured
by scanning with a GMS Scanner 418 and processing of the
experimental data with Scanalyzer software (Michael Eisen, Stanford
University), followed by GeneSpring software (Silicon Genetics, CA)
analysis. Differential expression of the 19 genes in blood samples
from patients with osteoarthritis as compared to healthy patients
is then determined by statistical analysis using the Wilcox Mann
Whitney rank sum test (Glantz S A. Primer of Biostatistics. 5th ed.
New York. USA: McGraw-Hill Medical Publishing Division, 2002).
Differential expression of each of the 19 genes described in FIG. 1
is diagnostic for osteoarthritis.
Example 14
Analysis of Blood Samples from Individuals Having Osteoarthritis as
Compared with Blood Samples from Healthy Individuals Using
Monoclonal Antibodies Directed to the Polypeptides Encoded by the
19 Genes Described in FIG. 1
[0642] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0643] Blood samples are taken from patients who are clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0644] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cyt Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to full length
polypeptides encoded by the 19 genes described in FIG. 1. Detection
of specific binding to the array is then measured by scanning with
a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen, Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the 19 genes in blood samples from
patients with osteoarthritis as compared to healthy patients is
determined by statistical analysis using the Wilcox Mann Whitney
rank sum test (Glantz S A. Primer of Biostatistics. 5th ed. New
York, USA: McGraw-Hill Medical Publishing Division, 2002).
Differential expression of each of the 19 genes described in FIG. 1
is diagnostic for osteoarthritis.
Example 15
Analysis of Blood Samples from Individuals Having Moderate
Osteoarthritis as Compared with Blood Samples from Healthy
Individuals Using Monoclonal Antibodies Directed to the
Polypeptides Encoded by the 4 Genes Described in FIG. 2
[0645] This example demonstrates the use of the claimed invention
to diagnose moderate osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0646] Blood samples are taken from patients who are clinically
diagnosed with moderate osteoarthritis as defined herein. Gene
expression profiles are then analyzed and compared to profiles from
patients unaffected by OA. In each case, the diagnosis of moderate
osteoarthritis is corroborated by a skilled Board certified
physician.
[0647] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to full length
polypeptides encoded by the 4 genes described in FIG. 2. Detection
of specific binding to the array is then measured by scanning with
a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen, Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the 4 genes in blood samples from
patients with moderate osteoarthritis as compared to healthy
patients is determined by statistical analysis using the Wilcox
Mann Whitney rank sum test (Glantz S A. Primer of Biostatistics.
5th ed. New York, USA: McGraw-Hill Medical Publishing Division,
2002). Differential expression of each of the 4 genes described in
FIG. 2 is diagnostic for moderate osteoarthritis.
Example 16
Analysis of Blood Samples from Individuals Having Marked
Osteoarthritis as Compared with Blood Samples from Individuals
Having Moderate Arthritis Using Monoclonal Antibodies Directed to
the Polypeptides Encoded by the 2 Genes Described in FIG. 3
[0648] This example demonstrates the use of the claimed invention
to diagnose marked osteoarthritis by detecting differential gene
expression in blood samples taken from patients with marked OA as
compared to blood samples taken from patients with moderate OA.
[0649] Blood samples are taken from patients who are clinically
diagnosed with marked osteoarthritis as defined herein. Gene
expression profiles are then analyzed and compared to profiles from
patients with moderate OA. In each case, the diagnosis of the stage
of osteoarthritis is corroborated by a skilled Board certified
physician.
[0650] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to full length
polypeptides encoded by the 2 genes described in FIG. 3. Detection
of specific binding to the array is then measured by scanning with
a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen. Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the 2 genes in blood samples from
patients with marked osteoarthritis as compared to patients with
moderate OA is determined by statistical analysis using the Wilcox
Mann Whitney rank sum test (Glantz S A. Primer of Biostatistics.
5th ed. New York, USA: McGraw-Hill Medical Publishing Division,
2002). Differential expression of each of the 2 genes described in
FIG. 3 is diagnostic for marked osteoarthritis.
Example 17
Analysis of Blood Samples from Individuals Having Severe
Osteoarthritis as Compared with Blood Samples from Individuals
Having Marked Osteoarthritis Using Monoclonal Antibodies Directed
to the Polypeptides Encoded by the 4 Genes Described in FIG. 4
[0651] This example demonstrates the use of the claimed invention
to diagnose severe osteoarthritis by detecting differential gene
expression in blood samples taken from patients with severe OA as
compared to blood samples taken from patients with marked.
[0652] Blood samples are taken from patients who are clinically
diagnosed with severe osteoarthritis as defined herein. Gene
expression profiles are then analyzed and compared to profiles from
patients with marked OA. In each case, the diagnosis of the stage
of osteoarthritis is corroborated by a skilled Board certified
physician.
[0653] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to full length
polypeptides encoded by the 4 genes described in FIG. 4. Detection
of specific binding to the array is then measured by scanning with
a GMS Scanner 418 and processing of the experimental data with
Scanalyzer software (Michael Eisen, Stanford University), followed
by GeneSpring software (Silicon Genetics, CA) analysis.
Differential expression of the 4 genes in blood samples from
patients with severe osteoarthritis as compared to patients with
marked OA is determined by statistical analysis using the Wilcox
Mann Whitney rank sum test (Glantz S A. Primer of Biostatistics.
5th ed. New York, USA: McGraw-Hill Medical Publishing Division,
2002). Differential expression of each of the 4 genes described in
FIG. 4 is diagnostic for severe osteoarthritis.
Example 18
Analysis of Blood Samples from Individuals Having Osteoarthritis as
Compared with Blood Samples from Healthy Individuals Using
Monoclonal Antibodies Directed to the Amino Terminal Region of
Polypeptides Encoded by the 5' Regions of the 19 Genes Described in
FIG. 1
[0654] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0655] Blood samples are taken from patients who are clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0656] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to amino terminal
regions of polypeptides encoded by the 5' regions of the 19 genes
described in FIG. 1. Detection of specific binding to the array is
then measured by scanning with a GMS Scanner 418 and processing of
the experimental data with Scanalyzer software (Michael Eisen,
Stanford University), followed by GeneSpring software (Silicon
Genetics, CA) analysis. Differential expression of the 19 genes in
blood samples from patients with osteoarthritis as compared to
healthy patients is determined by statistical analysis using the
Wilcox Mann Whitney rank sum test (Glantz S A. Primer of
Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
19 genes described in FIG. 1 is diagnostic for osteoarthritis.
Example 19
Analysis of Blood Samples from Individuals Having Osteoarthritis as
Compared with Blood Samples from Healthy Individuals Using
Monoclonal Antibodies Directed to the Carboxy Terminal Region of
Polypeptides Encoded By the 3' Regions of the 19 Genes Described in
FIG. 1
[0657] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0658] Blood samples are taken from patients who were clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0659] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to the carboxy terminal
regions of polypeptides encoded by the 3' regions of the 19 genes
described in FIG. 1. Detection of specific binding to the array is
then measured by scanning with a GMS Scanner 418 and processing of
the experimental data with Scanalyzer software (Michael Eisen,
Stanford University), followed by GeneSpring software (Silicon
Genetics. CA) analysis. Differential expression of the 19 genes in
blood samples from patients with osteoarthritis as compared to
healthy patients is determined by statistical analysis using the
Wilcox Mann Whitney rank sum test (Glantz S A. Primer of
Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
19 genes described in FIG. 1 is diagnostic for osteoarthritis.
Example 20
Analysis of Blood Samples from Individuals Having Osteoarthritis as
Compared with Blood Samples from Healthy Individuals Using
Antibodies Directed to the Internal Polypeptide Region of
Polypeptides Encoded by the Internal Coding Region of the 19 Genes
Described in FIG. 1
[0660] This example demonstrates the use of the claimed invention
to diagnose osteoarthritis by detecting differential gene
expression in blood samples taken from patients with OA as compared
to blood samples taken from healthy patients.
[0661] Blood samples are taken from patients who were clinically
diagnosed with osteoarthritis as defined herein. Gene expression
profiles are then analyzed and compared to profiles from patients
unaffected by OA. In each case, the diagnosis of osteoarthritis is
corroborated by a skilled Board certified physician.
[0662] Total cellular protein from blood taken from each patient is
first isolated and labelled using the BD Clontech Protein
Extraction and labelling kit (Catalogue #K1848-1 or #631786).
Briefly, the Extraction Protocol consists of three main steps:
mechanically disrupting the cells, solubilizing the cells, and
centrifuging the extract The process may start with a cell pellet
or frozen tissue and may use any method of mechanical
disruption--French press, sonication, mincing, or grinding. Once
disrupted, the sample is solubilized by adding the
Extraction/Labeling Buffer (1:20 w/v). Because the Buffer is
formulated for labeling with N-hydroxysuccinimide (NHS)-ester dyes
(e.g. Cy3 and CyS dyes), it does not contain any protease
inhibitors or reducing agents that would compete for reaction with
the dye. After extraction, the sample is centrifuged to pellet
insoluble material such as chromosomal DNA. The soluble extract is
then labelled with Cy3 and Cy5 Fluorescent Dyes (monofunctional
NHS-esters). The labelled proteins are then incubated with an array
of monoclonal antibodies which are directed to internal polypeptide
regions of polypeptides encoded by the internal coding regions of
the 19 genes described in FIG. 1. Detection of specific binding to
the array is then measured by scanning with a GMS Scanner 418 and
processing of the experimental data with Scanalyzer software
(Michael Eisen, Stanford University), followed by GeneSpring
software (Silicon Genetics, CA) analysis. Differential expression
of the 19 genes in blood samples from patients with osteoarthritis
as compared to healthy patients is determined by statistical
analysis using the Wilcox Mann Whitney rank sum test (Glantz S A.
Primer of Biostatistics. 5th ed. New York, USA: McGraw-Hill Medical
Publishing Division, 2002). Differential expression of each of the
19 genes described in FIG. 1 is diagnostic for osteoarthritis.
Example 21
Application of Logistic Regression to a Subset of Nine of the
Biomarkers of the Invention to Identify Combinations Useful in
Differentiating a Stage of OA from Non OA
[0663] RNA was isolated from blood samples of 259 patients with
mild osteoarthritis as classified using the system of Marshall
(supra) and 82 normal subjects. Primers as disclosed in Table 4 in
Example 4 were used to provide data corresponding to the level of
expression of the population of RNA products disclosed in Table 2.
A Reference dataset consisting of .DELTA.Ct values arising from the
QRT-PCR for nine of the biomarkers (EGR1, G2AN, HSPCA, IKBKAP,
ILI3RA1, LAMC1, MAFB, PF4, TNFAIP6) was utilized for input into
logistic regression to determine the diagnostic capabilities of
different combinations of .DELTA.Ct values from these 9 candidate
biomarkers. Of the 2.sup.9-1=511 possible biomarker combinations,
254 combinations were well-behaved in maximum-likelihood logistic
regression and gave significant discrimination (ROC Area>0.5) of
"mild osteoarthritis" vs. "control". It is noteworthy that
combinations of as few as 3 biomarkers produced ROC Areas>0.85.
Table 9 below presents the logistic regression parameters for those
combinations of 1, 2, 3, 4, 5, 6, 7 or 8 biomarkers giving the
greatest ROC Areas for a fixed number of genes.
TABLE-US-00006 TABLE 9 Noise ROC Eqn.sup.1 # (genes) T statistic
Factor.sup.2 Area Constant EGR1 G2AN HSPCA 128 1 5.8757 0.37838
0.80893 -7.7261 0 0 0 MC.sup.3 384 2 6.3994 0.39773 0.82311 -11.183
0 0 0 MC.sup.3 192 2 6.3621 0.48444 0.82436 -4.0057 0 0 0 MC.sup.3
208 3 7.8386 0.76278 0.87151 -12.789 0 0 0 MC.sup.3 464 4 8.1562
0.79025 0.87818 -14.324 0 0 0 214 5 8.3676 1.0311 0.89111 -10.806 0
-1.1191 1.8456 246 6 8.7395 1.0134 0.89487 -12.662 0 -1.1052 1.6446
222 6 8.5553 1.1738 0.8957 -14.995 0 -2.2536 1.2969 223 7 8.6763
1.215 0.89779 -14.654 0.14331 -2.4461 1.2095 503 8 9.2433 1.0239
0.9028 -14.777 0.0506 -0.94879 1.6983 Eqn.sup.1 IKBKAP IL13RA1
LAMC1 MAFB PF4 TNFAIP6 128 0 0 0 0 1.2613 0 MC.sup.3 1.2349 384 0 0
0 0 1.2648 0.3976 MC.sup.3 1.2223 0.3901 192 0 0 0 -0.6799 1.4647 0
MC.sup.3 -0.6664 1.4298 208 0 1.7416 0 -1.0808 1.5045 0 MC.sup.3
1.6667 -1.0387 1.4276 464 0 1.5216 0 -1.3204 1.6344 0.45691 214 0
1.7009 0 -1.3922 1.5245 0 246 0 1.6012 0.47022 -1.6293 1.4304 0 222
1.4864 1.7789 0 -1.139 1.3704 0 223 1.6415 1.731 0 -1.2236 1.3221 0
503 0 1.2055 0.45795 -1.9036 1.5243 0.50045
[0664] The six biomarker combinations with greatest overall ROC
Areas (0.89-0.90) are presented in FIG. 2, and their logistic
regression parameter values are given in Table 10.
TABLE-US-00007 TABLE 10 Noise ROC Eqn.sup.1 # (genes) T statistic
Factor.sup.2 Area Constant EGR1 G2AN HSPCA 471 7 8.8868 1.0428
0.89487 -13.048 0.1155 -0.96585 1.8829 246 6 8.7395 1.0134 0.89487
-12.662 0 -1.1052 1.6446 247 7 8.7478 1.0145 0.89529 -12.503
2.20E-02 -1.1186 1.6405 222 6 8.5553 1.1738 0.8957 -14.995 0
-2.2536 1.2969 223 7 8.6763 1.215 0.89779 -14.654 0.14331 -2.4461
1.2095 503 8 9.2433 1.0239 0.9028 -14.777 5.06E-02 -0.94879 1.6983
Eqn.sup.1 IKBKAP IL13RA1 LAMC1 MAFB PF4 TNFAIP6 471 0 1.3037 0
-1.7394 1.6159 0.50523 246 0 1.6012 0.47022 -1.6293 1.4304 0 247 0
1.5997 0.45789 -1.6383 1.4287 0 222 1.4864 1.7789 0 -1.139 1.3704 0
223 1.6415 1.731 0 -1.2236 1.3221 0 503 0 1.2055 0.45795 -1.9036
1.5243 0.50045 .sup.1Equation # (out of 2.sup.9 - 1 = 511 possible
equations) in exhaustive logistic regression analysis using LogReg
v 12 m .sup.2.+-.1 .sigma. in the logit function, propageted from
an input error .sigma. (Ct) .apprxeq. 0.3 cycle .sup.31000
MonteCarlo samplings of exact logistic distribution, using
LogXact-5
TABLE-US-00008 TABLE 11 Combinations of Two Genes Useful as
Biomarkers of OA as Compared with Normal B2M G2AN IL13RA1 IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC B2M n/a G2AN IL13RA1 IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC B2M B2M B2M B2M B2M B2M B2M B2M
B2M G2AN B2M n/a IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC
G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN 1L13RA1 B2M G2AN n/a
IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 IKBKAP B2M G2AN
IL13RA1 n/a TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP TNFAIP6 B2M G2AN IL13RA1
IKPKAP n/a WDR9 WWP2 BCL6 C1QR1 CCNC TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 WDR9 B2M G2AN
IL13RA1 IKPKAP TNFAIP6 n/a WWP2 BCL6 C1QR1 CCNC WDR9 WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 WWP2 B2M G2AN IL13RA1 IKPKAP TNFAIP6 WDR9
n/a BCL6 C1QR1 CCNC WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2
BCL6 B2M G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 n/a C1QR1 CCNC BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 C11QR1 B2M G2AN IL13RA1
IKPKAP TNFAIP6 WDR9 WWP2 BCL6 n/a CCNC C11QR1 C11QR1 C11QR1 C11QR1
C11QR1 C11QR1 C11QR1 C11QR1 C11QR1 CCNC B2M G2AN IL13RA1 IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 n/a CCNC CCNC CCNC CCNC CCNC CCNC CCNC
CCNC CCNC FLJ32234 B2M G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6
C1QR1 CCNC FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 HSPCA B2M G2AN IL13RA1 IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC HSPCA HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA LAMC1 B2M G2AN IL13RA1 IKPKAP TNFAIP6
WDR9 WWP2 BCL6 C1QR1 CCNC LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 PAIP2 B2M G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 IRF1 B2M G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6
C1QR1 CCNC IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 NCOA1
B2M G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC NCOA1
NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 CLIC4 B2M
G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC CLIC4 CLIC4
CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 ABCA1 B2M G2AN
IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 B2M G2AN IL13RA1
IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC ABCG1 ABCG1 ABCG1 ABCG1
ABCG1 ABCG1 ABCG1 ABCG1 ABCG1 ABCG1 FLJ32334 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 B2M FLJ32334 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M B2M B2M G2AN FLJ32334
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN
G2AN G2AN G2AN G2AN G2AN 1L13RA1 FLJ32334 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 IKBKAP FLJ32334 HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP TNFAIP6 FLJ32334 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 WDR9 FLJ32334 HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 WDR9 WWP2 FLJ32334 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 BCL6 FLJ32334
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 C11QR1 FLJ32334 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 C11QR1 C11QR1 C11QR1 C11QR1 C11QR1 C11QR1
C11QR1 C11QR1 C11QR1 CCNC FLJ32334 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC
FLJ32234 n/a HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 HSPCA FLJ32334 n/a LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA LAMC1
FLJ32334 HSPCA n/a PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 PAIP2 FLJ32334 HSPCA LAMC1 n/a
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 IRF1 FLJ32334 HSPCA LAMC1 PAIP2 n/a NCOA1 CLIC4 ABCA1
ABCG1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 NCOA1 FLJ32334 HSPCA
LAMC1 PAIP2 IRF1 n/a CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 NCOA1 CLIC4 FLJ32334 HSPCA LAMC1 PAIP2 IRF1 NCOA1
n/a ABCA1 ABCG1 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4
ABCA1 FLJ32334 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 n/a ABCG1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 FLJ32334 HSPCA
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 ABCG1 ABCG1 ABCG1 ABCG1
ABCG1 ABCG1 ABCG1 ABCG1 ABCG1
TABLE-US-00009 TABLE 12 Combinations of Three Genes Useful as
Biomarkers of OA as Compared with Normal B2M G2AN IL13RA1 IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 (a) B2M B2M N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6
WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN
G2AN G2AN G2AN B2M B2M B2M B2M B2M B2M B2M B2M B2M 1L13RA1 N/A N/A
N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 B2M B2M B2M
B2M B2M B2M B2M B2M IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6
C1QR1 CCNC FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP B2M B2M B2M B2M B2M B2M B2M TNFAIP6 N/A N/A N/A N/A N/A WDR9
WWP2 BCL6 C1QR1 CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 B2M B2M B2M B2M B2M B2M WDR9 N/A N/A N/A N/A N/A
N/A WWP2 BCL6 C1QR1 CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 B2M B2M
B2M B2M B2M WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC
FLJ32334 WWP2 WWP2 WWP2 WWP2 B2M B2M B2M B2M BCL6 N/A N/A N/A N/A
N/A N/A N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 B2M B2M B2M
C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1
B2M B2M CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC
B2M FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (b) G2AN
B2M N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC
FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M G2AN G2AN G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A 1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1
CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN IKBKAP N/A
N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP G2AN G2AN G2AN G2AN G2AN
G2AN G2AN TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6 C1QR1 CCNC
FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 G2AN G2AN
G2AN G2AN G2AN G2AN WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1
CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 G2AN G2AN G2AN G2AN G2AN
WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC FLJ32334 WWP2 WWP2
WWP2 WWP2 G2AN G2AN G2AN G2AN BCL6 N/A N/A N/A N/A N/A N/A N/A N/A
C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 G2AN G2AN G2AN C1QR1 N/A N/A N/A
N/A N/A N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 G2AN G2AN CCNC
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC G2AN FLJ32234
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IRF1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCG1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (c) IL13RA1 B2M N/A
G2AN N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 B2M B2M
B2M B2M B2M B2M B2M B2M B2M IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 IL13RA1 G2AN N/A N/A N/A IKPKAP TNFAIP6
WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN
G2AN G2AN IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 1L13RA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IKBKAP
N/A N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IL13RA1 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 IL13RA1 TNFAIP6 N/A N/A N/A N/A N/A WDR9
WWP2 BCL6 C1QR1 CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 CCNC FLJ32334 WDR9
WDR9 WDR9 WDR9 WDR9 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 WWP2
N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC FLJ32334 WWP2 WWP2 WWP2
WWP2 IL13RA1 IL13RA1 IL13RA1 IL13RA1 BCL6 N/A N/A N/A N/A N/A N/A
N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 IL13RA1 IL13RA1 IL13RA1
C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1
IL13RA1 IL13RA1 CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
FLJ32334 CCNC IL13RA1 FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A HSPCA N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A NCOA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A (d) IKPKAP B2M N/A G2AN IL13RA1 N/A TNFAIP6 WDR9 WWP2 BCL6
C1QR1 CCNC FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M IKPKAP
IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP G2AN N/A
N/A IL13RA1 N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN
G2AN G2AN G2AN G2AN G2AN G2AN G2AN IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP IKPKAP IKPKAP IKPKAP 1L13RA1 N/A N/A N/A N/A TNFAIP6 WDR9
WWP2 BCL6 C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP IKBKAP N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A TNFAIP6
N/A N/A N/A N/A N/A WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP IKPKAP WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 CCNC
FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC FLJ32334
WWP2 WWP2 WWP2 WWP2 IKPKAP IKPKAP IKPKAP IKPKAP BCL6 N/A N/A N/A
N/A N/A N/A N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 IKPKAP
IKPKAP IKPKAP C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC
FLJ32334 C1QR1 C1QR1 IKPKAP IKPKAP CCNC N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A FLJ32334 CCNC IKPKAP FLJ32234 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A HSPCA N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A (e) TNFAIP6 B2M N/A G2AN IL13RA1 IKPKAP N/A
WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M
B2M TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 G2AN N/A N/A IL13RA1 IKPKAP N/A WDR9 WWP2 BCL6 C1QR1 CCNC
FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 1L13RA1 N/A N/A N/A
IKPKAP N/A WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 IKBKAP N/A N/A N/A N/A N/A WDR9
WWP2 BCL6 C1QR1 CCNC FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A WDR9 N/A N/A N/A N/A N/A
N/A WWP2 BCL6 C1QR1 CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 WWP2 N/A N/A N/A N/A N/A N/A N/A
BCL6 C1QR1 CCNC FLJ32334 WWP2 WWP2 WWP2 WWP2 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 CCNC
FLJ32334 BCL6 BCL6 BCL6 TNFAIP6 TNFAIP6 TNFAIP6 C1QR1 N/A N/A N/A
N/A N/A N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 TNFAIP6 TNFAIP6
CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC TNFAIP6
FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (f) WDR9 B2M
N/A G2AN IL13RA1 IKPKAP TNFAIP6 N/A WWP2 BCL6 C1QR1 CCNC FLJ32334
B2M B2M B2M B2M B2M B2M B2M B2M B2M WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6 N/A WWP2 BCL6
C1QR1 CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 1L13RA1 N/A N/A N/A IKPKAP
TNFAIP6 N/A WWP2 BCL6 C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
IKBKAP N/A N/A N/A N/A TNFAIP6 N/A WWP2 BCL6 C1QR1 CCNC FLJ32334
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 TNFAIP6 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 CCNC FLJ32334
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A WWP2 N/A N/A N/A
N/A N/A N/A N/A BCL6 C1QR1 CCNC FLJ32334 WWP2 WWP2 WWP2 WWP2 WDR9
WDR9 WDR9 WDR9 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 CCNC
FLJ32334 BCL6 BCL6 BCL6 WDR9 WDR9 WDR9 C1QR1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 WDR9 WDR9 CCNC N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC WDR9 FLJ32234 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IRF1 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A (g) WWP2 B2M N/A G2AN IL13RA1
IKPKAP TNFAIP6 WDR9 N/A BCL6 C1QR1 CCNC FLJ32334 B2M B2M B2M B2M
B2M B2M B2M B2M B2M WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2
G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9 N/A BCL6 C1QR1 CCNC
FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN WWP2 WWP2 WWP2
WWP2 WWP2 WWP2 WWP2 WWP2 1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9
N/A BCL6 C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 IKBKAP
N/A N/A N/A N/A TNFAIP6 WDR9 N/A BCL6 C1QR1 CCNC FLJ32334 IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 TNFAIP6 N/A N/A N/A N/A N/A WDR9 N/A
BCL6 C1QR1 CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
WWP2 WWP2 WWP2 WWP2 WWP2 WDR9 N/A N/A N/A N/A N/A N/A N/A BCL6
C1QR1 CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WWP2 WWP2 WWP2 WWP2 WWP2
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A BCL6 N/A N/A N/A N/A
N/A N/A N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 WWP2 WWP2 WWP2
C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1
WWP2 WWP2 CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334
CCNC WWP2 FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
HSPCA N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
NCOA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
(h) BCL6 B2M N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 N/A C1QR1
CCNC FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6
WDR9 WWP2 N/A C1QR1 CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN
G2AN G2AN BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 1L13RA1 N/A N/A
N/A IKPKAP TNFAIP6 WDR9 WWP2 N/A C1QR1 CCNC FLJ32334 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2 N/A C1QR1
CCNC FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 N/A C1QR1
CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 BCL6 BCL6
BCL6 BCL6 BCL6 WDR9 N/A N/A N/A N/A N/A N/A WWP2 N/A C1QR1 CCNC
FLJ32334 WDR9 WDR9 WDR9 WDR9 BCL6 BCL6 BCL6 BCL6 WWP2 N/A N/A N/A
N/A N/A N/A N/A N/A C1QR1 CCNC FLJ32334 WWP2 WWP2 WWP2 BCL6 BCL6
BCL6 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 N/A N/A
N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 BCL6 BCL6
CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC BCL6
FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (i) C1QR1 B2M
N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 N/A CCNC FLJ32334
B2M B2M B2M B2M B2M B2M B2M B2M B2M C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9
WWP2 BCL6 N/A CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 1L13RA1 N/A N/A N/A
IKPKAP TNFAIP6 WDR9 WWP2 BCL6 N/A CCNC FLJ32334 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 N/A
CCNC FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6
N/A CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 N/A
CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 C1QR1 C1QR1 C1QR1 C1QR1 WWP2 N/A
N/A N/A N/A N/A N/A N/A BCL6 N/A CCNC FLJ32334 WWP2 WWP2 WWP2 C1QR1
C1QR1 C1QR1 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334
BCL6 BCL6 C1QR1 C1QR1 C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC
C1QR1 FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (j)
CCNC B2M N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 N/A
FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M CCNC CCNC CCNC CCNC
CCNC CCNC CCNC CCNC CCNC G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9
WWP2 BCL6 C1QR1 N/A FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN G2AN
G2AN CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC 1L13RA1 N/A N/A N/A
IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 N/A FLJ32334 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 CCNC CCNC CCNC CCNC CCNC
CCNC CCNC IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 N/A
FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP CCNC CCNC CCNC
CCNC CCNC CCNC TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6 C1QR1 N/A
FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 CCNC CCNC CCNC
CCNC CCNC WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 N/A FLJ32334
WDR9 WDR9 WDR9 WDR9 CCNC CCNC CCNC CCNC WWP2 N/A N/A N/A N/A N/A
N/A N/A BCL6 C1QR1 N/A FLJ32334 WWP2 WWP2 WWP2 CCNC CCNC CCNC BCL6
N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 N/A FLJ32334 BCL6 BCL6 CCNC
CCNC C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 C1QR1
CCNC CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32234 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IRF1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (k) FLJ32334 B2M N/A G2AN
IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC N/A B2M B2M B2M
B2M B2M B2M B2M B2M B2M FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 G2AN N/A N/A IL13RA1
IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC N/A G2AN G2AN G2AN G2AN
G2AN G2AN G2AN G2AN FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9
WWP2 BCL6 C1QR1 CCNC N/A 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6
C1QR1 CCNC N/A IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 TNFAIP6 N/A N/A N/A
N/A N/A WDR9 WWP2 BCL6 C1QR1 CCNC N/A TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 WDR9
N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 CCNC N/A WDR9 WDR9 WDR9
WDR9 FLJ32334 FLJ32334 FLJ32334 FLJ32334 WWP2 N/A N/A N/A N/A N/A
N/A N/A BCL6 C1QR1 CCNC N/A WWP2 WWP2 WWP2 FLJ32334 FLJ32334
FLJ32334 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 CCNC N/A BCL6
BCL6 FLJ32334 FLJ32334 C11QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A
CCNC N/A C11QR1 FLJ32334 CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (l)
HSPCA B2M N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC
FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M B2M HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA G2AN N/A N/A IL13RA1
IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA 1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6
C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA
IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6
C1QR1 CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA WDR9 N/A N/A N/A N/A N/A N/A
WWP2 BCL6 C1QR1 CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 HSPCA HSPCA
HSPCA HSPCA HSPCA WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC
FLJ32334 WWP2 WWP2 WWP2 WWP2 HSPCA HSPCA HSPCA HSPCA BCL6 N/A N/A
N/A N/A N/A N/A N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 HSPCA
HSPCA HSPCA C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334
C1QR1 C1QR1 HSPCA HSPCA CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A FLJ32334 CCNC HSPCA FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A HSPCA N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A (m) LAMC1 B2M N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M B2M
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 G2AN
N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334
G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN LAMC1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 1L13RA1 N/A N/A N/A IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 TNFAIP6 N/A N/A
N/A N/A N/A WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 CCNC FLJ32334 WDR9
WDR9 WDR9 WDR9 WDR9 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 WWP2 N/A N/A N/A
N/A N/A N/A N/A BCL6 CIQR1 CCNC FLJ32334 WWP2 WWP2 WWP2 WWP2 LAMC1
LAMC1 LAMC1 LAMC1 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 CCNC
FLJ32334 BCL6 BCL6 BCL6 LAMC1 LAMC1 LAMC1 CIQR1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 LAMC1 LAMC1 CCNC N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC LAMC1
FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (n) PAIP2 B2M
N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334
B2M B2M B2M B2M B2M B2M B2M B2M B2M B2M PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 G2AN N/A N/A IL13RA1 IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN
G2AN G2AN G2AN G2AN PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6 CIQR1 CCNC
FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 IKBKAP N/A
N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 CIQR1 CCNC FLJ32334 IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 PAIP2 TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6 C1QR1
CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 PAIP2
PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 WDR9 N/A N/A N/A N/A N/A N/A WWP2
BCL6 C1QR1 CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC
FLJ32334 WWP2 WWP2 WWP2 WWP2 PAIP2 PAIP2 PAIP2 PAIP2 BCL6 N/A N/A
N/A N/A N/A N/A N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 PAIP2
PAIP2 PAIP2 C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334
C1QR1 C1QR1 PAIP2 PAIP2 CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A FLJ32334 CCNC PAIP2 FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A HSPCA N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A (o) IRF1 B2M N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M B2M
IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 G2AN N/A N/A
IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN G2AN IRF1 IRF1 IRF1 IRF1 IRF1 IRF1
IRF1 IRF1 IRF1 1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6
C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IKBKAP N/A
N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IRF1 IRF1 IRF1 IRF1 IRF1
IRF1 IRF1 TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6 C1QR1 CCNC
FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 IRF1 IRF1
IRF1 IRF1 IRF1 IRF1 WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1
CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 IRF1 IRF1 IRF1 IRF1 IRF1
WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC FLJ32334 WWP2 WWP2
WWP2 WWP2 IRF1 IRF1 IRF1 IRF1 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A
C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 IRF1 IRF1 IRF1 C1QR1 N/A N/A N/A
N/A N/A N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 IRF1 IRF1 CCNC
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC IRF1 FLJ32234
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IRF1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCG1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (p) NCOA1 B2M N/A G2AN
IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 B2M B2M
B2M B2M B2M B2M B2M B2M B2M B2M NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 NCOA1 G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9
WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN G2AN
G2AN G2AN NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1
1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC
FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 IKBKAP N/A
N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP NCOA1 NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6 C1QR1
CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 NCOA1
NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 WDR9 N/A N/A N/A N/A N/A N/A WWP2
BCL6 C1QR1 CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC
FLJ32334 WWP2 WWP2 WWP2 WWP2 NCOA1 NCOA1 NCOA1 NCOA1 BCL6 N/A N/A
N/A N/A N/A N/A N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 NCOA1
NCOA1 NCOA1 C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334
C1QR1 C1QR1 NCOA1 NCOA1 CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A FLJ32334 CCNC NCOA1 FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A HSPCA N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A (q) CLIC4 B2M N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M B2M
CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 G2AN
N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334
G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN CLIC4 CLIC4 CLIC4
CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 1L13RA1 N/A N/A N/A IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 CLIC4 CLIC4 CLIC4 CLIC4
CLIC4 CLIC4 CLIC4 CLIC4 IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 TNFAIP6 N/A N/A
N/A N/A N/A WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4
WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 CCNC FLJ32334 WDR9
WDR9 WDR9 WDR9 WDR9 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 WWP2 N/A N/A N/A
N/A N/A N/A N/A BCL6 C1QR1 CCNC FLJ32334 WWP2 WWP2 WWP2 WWP2 CLIC4
CLIC4 CLIC4 CLIC4 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 CCNC
FLJ32334 BCL6 BCL6 BCL6 CLIC4 CLIC4 CLIC4 C1QR1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 CLIC4 CLIC4 CCNC N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC CLIC4 FLJ32234 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IRF1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A (r) ABCA1 B2M N/A G2AN
IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 B2M B2M
B2M B2M B2M B2M B2M B2M B2M B2M ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 G2AN N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9
WWP2 BCL6 C1QR1 CCNC FLJ32334 G2AN G2AN G2AN G2AN G2AN G2AN G2AN
G2AN G2AN ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
1L13RA1 N/A N/A N/A IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC
FLJ32334 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 IKBKAP N/A
N/A N/A N/A TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 TNFAIP6 N/A N/A N/A N/A N/A WDR9 WWP2 BCL6 C1QR1
CCNC FLJ32334 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 WDR9 N/A N/A N/A N/A N/A N/A WWP2
BCL6 C1QR1 CCNC FLJ32334 WDR9 WDR9 WDR9 WDR9 WDR9 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 WWP2 N/A N/A N/A N/A N/A N/A N/A BCL6 C1QR1 CCNC
FLJ32334 WWP2 WWP2 WWP2 WWP2 ABCA1 ABCA1 ABCA1 ABCA1 BCL6 N/A N/A
N/A N/A N/A N/A N/A N/A C1QR1 CCNC FLJ32334 BCL6 BCL6 BCL6 ABCA1
ABCA1 ABCA1 C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A N/A CCNC FLJ32334
C1QR1 C1QR1 ABCA1 ABCA1 CCNC N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A FLJ32334 CCNC ABCA1 FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A HSPCA N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LAMC1
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A PAIP2 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A IRF1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CLIC4
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCA1 N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A (s) ABCG1 B2M N/A G2AN IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC FLJ32334 B2M B2M B2M B2M B2M B2M B2M B2M B2M B2M
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 G2AN
N/A N/A IL13RA1 IKPKAP TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334
G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 1L13RA1 N/A N/A N/A IKPKAP
TNFAIP6 WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 IKBKAP N/A N/A N/A N/A TNFAIP6 WDR9 WWP2
BCL6 C1QR1 CCNC FLJ32334 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 TNFAIP6 N/A N/A
N/A N/A N/A WDR9 WWP2 BCL6 C1QR1 CCNC FLJ32334 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
WDR9 N/A N/A N/A N/A N/A N/A WWP2 BCL6 C1QR1 CCNC FLJ32334 WDR9
WDR9 WDR9 WDR9 WDR9 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 WWP2 N/A N/A N/A
N/A N/A N/A N/A BCL6 C1QR1 CCNC FLJ32334 WWP2 WWP2 WWP2 WWP2 ABCA1
ABCA1 ABCA1 ABCA1 BCL6 N/A N/A N/A N/A N/A N/A N/A N/A C1QR1 CCNC
FLJ32334 BCL6 BCL6 BCL6 ABCA1 ABCA1 ABCA1 C1QR1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A CCNC FLJ32334 C1QR1 C1QR1 ABCA1 ABCA1 CCNC N/A N/A
N/A N/A N/A N/A N/A N/A N/A N/A FLJ32334 CCNC ABCA1 FLJ32234 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A IRF1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NCOA1 N/A N/A N/A N/A N/A
N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A ABCG1 N/A
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1
(a) B2M B2M N/A N/A N/A N/A N/A N/A N/A N/A G2AN HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN
G2AN B2M B2M B2M B2M B2M B2M B2M B2M 1L13RA1 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 B2M B2M B2M B2M B2M B2M B2M B2M IKBKAP
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP B2M B2M B2M B2M B2M B2M B2M B2M
TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 B2M B2M B2M
B2M B2M B2M B2M B2M WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 B2M B2M B2M B2M B2M
B2M B2M B2M WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 B2M B2M B2M B2M B2M B2M B2M
B2M BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 B2M B2M B2M B2M B2M B2M B2M B2M C1QR1
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 B2M B2M B2M B2M B2M B2M B2M B2M CCNC
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC
CCNC CCNC CCNC CCNC B2M B2M B2M B2M B2M B2M B2M B2M FLJ32234 HSPCA
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 FLJ32234 FLJ32234 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 B2M B2M B2M B2M B2M
B2M B2M B2M HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA B2M B2M B2M B2M B2M B2M
B2M LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 B2M B2M B2M B2M B2M B2M PAIP2 N/A N/A N/A
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 B2M B2M
B2M B2M B2M IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1
IRF1 IRF1 B2M B2M B2M B2M NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1
ABCG1 NCOA1 NCOA1 NCOA1 B2M B2M B2M CLIC4 N/A N/A N/A N/A N/A N/A
ABCA1 ABCG1 CLIC4 CLIC4 B2M B2M ABCA1 N/A N/A N/A N/A N/A N/A N/A
ABCG1 ABCA1 B2M ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (b) G2AN B2M
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M
B2M B2M B2M G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN N/A N/A
N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN
G2AN G2AN G2AN 1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN IKBKAP HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN
TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN CCNC HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC
G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN FLJ32234 HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 G2AN G2AN G2AN G2AN G2AN G2AN
G2AN G2AN HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA G2AN G2AN G2AN G2AN G2AN G2AN
G2AN LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 G2AN G2AN G2AN G2AN G2AN G2AN PAIP2 N/A N/A
N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 G2AN
G2AN G2AN G2AN G2AN IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1
IRF1 IRF1 IRF1 IRF1 G2AN G2AN G2AN G2AN NCOA1 N/A N/A N/A N/A N/A
CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 G2AN G2AN G2AN CLIC4 N/A N/A
N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4 G2AN G2AN ABCA1 N/A N/A N/A
N/A N/A N/A N/A ABCG1 ABCA1 G2AN ABCG1 N/A N/A N/A N/A N/A N/A N/A
N/A (c) IL13RA1 B2M HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
B2M B2M B2M B2M B2M B2M B2M B2M IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 IL13RA1 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN IL13RA1
IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 1L13RA1 N/A
N/A N/A N/A N/A N/A N/A N/A IKBKAP HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 BCL6 HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 CCNC HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC
IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 IL13RA1
HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 IL13RA1 IL13RA1 PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1
ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 IL13RA1 IL13RA1 IL13RA1 IL13RA1
IL13RA1 IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1
IRF1 IL13RA1 IL13RA1 IL13RA1 IL13RA1 NCOA1 N/A N/A N/A N/A N/A
CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 IL13RA1 IL13RA1 IL13RA1 CLIC4
N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4 IL13RA1 IL13RA1
ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 IL13RA1 ABCG1 N/A N/A
N/A N/A N/A N/A N/A N/A (d) IKPKAP B2M HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M B2M IKPKAP IKPKAP
IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP G2AN HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN
G2AN IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 IKPKAP
IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKBKAP N/A N/A N/A
N/A N/A N/A N/A N/A TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP IKPKAP WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP IKPKAP IKPKAP IKPKAP BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 IKPKAP IKPKAP
IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP C1QR1 HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
CCNC HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC
CCNC CCNC CCNC CCNC CCNC IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP IKPKAP FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 FLJ32234 IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA IKPKAP IKPKAP IKPKAP IKPKAP
IKPKAP IKPKAP IKPKAP LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 IKPKAP IKPKAP IKPKAP
IKPKAP IKPKAP IKPKAP PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1
PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 IKPKAP IKPKAP IKPKAP IKPKAP IKPKAP
IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1
IKPKAP IKPKAP IKPKAP IKPKAP NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1
ABCG1 NCOA1 NCOA1 NCOA1 IKPKAP IKPKAP IKPKAP CLIC4 N/A N/A N/A N/A
N/A N/A ABCA1 ABCG1 CLIC4 CLIC4 IKPKAP IKPKAP ABCA1 N/A N/A N/A N/A
N/A N/A N/A ABCG1 ABCA1 IKPKAP ABCG1 N/A N/A N/A N/A N/A N/A N/A
N/A (e) TNFAIP6 B2M HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
B2M B2M B2M B2M B2M B2M B2M B2M TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 1L13RA1
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 IKBKAP HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 N/A N/A N/A N/A N/A N/A N/A N/A
WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 BCL6 HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 CCNC HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1
ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1
IRF1 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 NCOA1 N/A N/A N/A N/A N/A
CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 TNFAIP6 TNFAIP6 TNFAIP6 CLIC4
N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4 TNFAIP6 TNFAIP6
ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 TNFAIP6 ABCG1 N/A N/A
N/A N/A N/A N/A N/A N/A (f) WDR9 B2M HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M B2M WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 IKBKAP HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 N/A N/A N/A N/A N/A N/A N/A N/A
WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WWP2 WWP2 WWP2
WWP2 WWP2 WWP2 WWP2 WWP2 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 WDR9 CCNC HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC
CCNC CCNC CCNC CCNC CCNC CCNC CCNC WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 WDR9 FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 HSPCA N/A LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 LAMC1 N/A N/A PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4
ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 WDR9 WDR9 WDR9 WDR9 WDR9
IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1
WDR9 WDR9 WDR9 WDR9 NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1
NCOA1 NCOA1 NCOA1 WDR9 WDR9 WDR9 CLIC4 N/A N/A N/A N/A N/A N/A
ABCA1 ABCG1 CLIC4 CLIC4 WDR9 WDR9 ABCA1 N/A N/A N/A N/A N/A N/A N/A
ABCG1 ABCA1 WDR9 ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (g) WWP2 B2M
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M
B2M B2M B2M WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 G2AN HSPCA
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN
G2AN G2AN G2AN WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 1L13RA1
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 WWP2 WWP2 WWP2 WWP2
WWP2 WWP2 WWP2 WWP2 IKBKAP HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP WWP2
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 TNFAIP6 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2
WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2
WWP2 N/A N/A N/A N/A N/A N/A N/A N/A BCL6 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 C1QR1 HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 CCNC HSPCA
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC CCNC
CCNC CCNC CCNC WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 FLJ32234
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 FLJ32234 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 WWP2 WWP2
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA WWP2
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 WWP2 WWP2 WWP2 WWP2
WWP2 WWP2 PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 PAIP2
PAIP2 PAIP2 PAIP2 PAIP2 WWP2 WWP2 WWP2 WWP2 WWP2 IRF1 N/A N/A N/A
N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1 WWP2 WWP2 WWP2 WWP2
NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 WWP2
WWP2 WWP2 CLIC4 N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4
WWP2 WWP2 ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 WWP2 ABCG1
N/A N/A N/A N/A N/A N/A N/A N/A (h) BCL6 B2M HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M B2M BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 IKBKAP
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP BCL6 BCL6 BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 WDR9 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 WWP2 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 N/A N/A N/A N/A N/A
N/A N/A N/A C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 CCNC HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 FLJ32234 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 HSPCA N/A
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 LAMC1 N/A N/A
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 PAIP2 N/A N/A N/A IRF1 NCOA1
CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 BCL6 BCL6 BCL6 BCL6
BCL6 IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1
IRF1 BCL6 BCL6 BCL6 BCL6 NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1
ABCG1 NCOA1 NCOA1 NCOA1 BCL6 BCL6 BCL6 CLIC4 N/A N/A N/A N/A N/A
N/A ABCA1 ABCG1 CLIC4 CLIC4 BCL6 BCL6 ABCA1 N/A N/A N/A N/A N/A N/A
N/A ABCG1 ABCA1 BCL6 ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (i)
C1QR1 B2M HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M
B2M B2M B2M B2M B2M B2M C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN
G2AN G2AN G2AN G2AN G2AN G2AN C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 IKBKAP HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 WDR9 HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 WDR9 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 WWP2
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WWP2 WWP2 WWP2 WWP2
WWP2 WWP2 WWP2 WWP2 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 N/A N/A N/A N/A N/A N/A N/A N/A CCNC HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC CCNC CCNC CCNC
CCNC C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 FLJ32234 HSPCA
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 FLJ32234 FLJ32234 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 LAMC1 N/A N/A PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1
ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1
C1QR1 C1QR1 C1QR1 C1QR1 NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1
NCOA1 NCOA1 NCOA1 C1QR1 C1QR1 C1QR1 CLIC4 N/A N/A N/A N/A N/A N/A
ABCA1 ABCG1 CLIC4 CLIC4 C1QR1 C1QR1 ABCA1 N/A N/A N/A N/A N/A N/A
N/A ABCG1 ABCA1 C1QR1 ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (j)
CCNC B2M HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M
B2M B2M B2M B2M B2M CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC G2AN
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN
G2AN G2AN G2AN G2AN CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC 1L13RA1
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 CCNC CCNC CCNC CCNC
CCNC CCNC CCNC CCNC IKBKAP HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP CCNC
CCNC CCNC CCNC CCNC CCNC CCNC CCNC
TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 CCNC CCNC
CCNC CCNC CCNC CCNC CCNC CCNC WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 CCNC CCNC
CCNC CCNC CCNC CCNC CCNC CCNC WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 CCNC CCNC
CCNC CCNC CCNC CCNC CCNC CCNC BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 CCNC CCNC
CCNC CCNC CCNC CCNC CCNC CCNC C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1
CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC N/A N/A N/A N/A N/A
N/A N/A N/A FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 CCNC CCNC CCNC CCNC CCNC CCNC CCNC CCNC HSPCA N/A LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA CCNC CCNC CCNC CCNC CCNC CCNC CCNC LAMC1 N/A N/A PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
CCNC CCNC CCNC CCNC CCNC CCNC PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4
ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 CCNC CCNC CCNC CCNC CCNC
IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1
CCNC CCNC CCNC CCNC NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1
NCOA1 NCOA1 NCOA1 CCNC CCNC CCNC CLIC4 N/A N/A N/A N/A N/A N/A
ABCA1 ABCG1 CLIC4 CLIC4 CCNC CCNC ABCA1 N/A N/A N/A N/A N/A N/A N/A
ABCG1 ABCG1 CCNC ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (k) FLJ32334
B2M HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M
B2M B2M B2M B2M FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN G2AN FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
IKBKAP HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 TNFAIP6 HSPCA
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 WDR9 HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9
WDR9 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 WWP2
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 BCL6 HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6
BCL6 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 C11QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
C11QR1 C11QR1 C11QR1 C11QR1 C11QR1 C11QR1 C11QR1 C11QR1 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334 CCNC
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC
CCNC CCNC CCNC CCNC FLJ32334 FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32234 N/A N/A N/A N/A N/A N/A N/A N/A
HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA FLJ32334 FLJ32334 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 FLJ32334 PAIP2 N/A N/A N/A IRF1 NCOA1
CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 FLJ32334 FLJ32334
FLJ32334 FLJ32334 FLJ32334 IRF1 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1
ABCG1 IRF1 IRF1 IRF1 IRF1 FLJ32334 FLJ32334 FLJ32334 FLJ32334 NCOA1
N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 FLJ32334
FLJ32334 FLJ32334 CLIC4 N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4
CLIC4 FLJ32334 FLJ32334 ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1
ABCA1 FLJ32334 ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (l) HSPCA B2M
N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M
B2M B2M HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA G2AN N/A LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN
G2AN HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA 1L13RA1 N/A LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 1L13RA1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA
IKBKAP N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP HSPCA HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA TNFAIP6 N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA HSPCA WDR9 N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA HSPCA WWP2 N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA BCL6 N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 HSPCA HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA C1QR1 N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA HSPCA CCNC N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
CCNC CCNC CCNC CCNC CCNC CCNC CCNC HSPCA HSPCA HSPCA HSPCA HSPCA
HSPCA HSPCA FLJ32234 N/A LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA N/A N/A N/A N/A N/A
N/A N/A N/A LAMC1 N/A N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA
PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 HSPCA HSPCA HSPCA HSPCA HSPCA IRF1 N/A N/A N/A N/A
NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1 HSPCA HSPCA HSPCA HSPCA
NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 HSPCA
HSPCA HSPCA CLIC4 N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4
HSPCA HSPCA ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 HSPCA
ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (m) LAMC1 B2M HSPCA N/A PAIP2
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 G2AN HSPCA N/A PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 1L13RA1 HSPCA N/A PAIP2 IRF1 NCOA1
CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 IKBKAP HSPCA N/A
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
TNFAIP6 HSPCA N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 LAMC1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 WDR9 HSPCA N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1
ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 LAMC1 LAMC1 WWP2 HSPCA N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 LAMC1 BCL6 HSPCA N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 CIQR1 HSPCA N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 LAMC1 CCNC HSPCA N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC
CCNC CCNC CCNC CCNC CCNC CCNC LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1
LAMC1 FLJ32234 HSPCA N/A PAIP2 IRF1 NCOA1 CLIC4 ABCA1 ABCG1
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 HSPCA N/A N/A PAIP2 IRF1
NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA LAMC1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 N/A N/A N/A N/A N/A N/A N/A N/A
PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 IRF1 N/A N/A N/A N/A
NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1 LAMC1 LAMC1 LAMC1 LAMC1
NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 LAMC1
LAMC1 LAMC1 CLIC4 N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4
LAMC1 LAMC1 ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 LAMC1
ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (n) PAIP2 B2M HSPCA LAMC1 N/A
IRF1 NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M PAIP2
PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 G2AN HSPCA LAMC1 N/A IRF1 NCOA1
CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN PAIP2 PAIP2
PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 1L13RA1 HSPCA LAMC1 N/A IRF1 NCOA1
CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 IKBKAP HSPCA
LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
TNFAIP6 HSPCA LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 PAIP2 PAIP2 WDR9 HSPCA LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1
ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 PAIP2 WWP2 HSPCA LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 BCL6 HSPCA LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 C1QR1 HSPCA LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 CCNC HSPCA LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1 CCNC
CCNC CCNC CCNC CCNC CCNC CCNC PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 FLJ32234 HSPCA LAMC1 N/A IRF1 NCOA1 CLIC4 ABCA1 ABCG1
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 HSPCA N/A LAMC1 N/A IRF1
NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA PAIP2
PAIP2 PAIP2 PAIP2 PAIP2 PAIP2 LAMC1 N/A N/A N/A IRF1 NCOA1 CLIC4
ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 PAIP2 PAIP2 PAIP2 PAIP2
PAIP2 PAIP2 N/A N/A N/A N/A N/A N/A N/A N/A IRF1 N/A N/A N/A N/A
NCOA1 CLIC4 ABCA1 ABCG1 IRF1 IRF1 IRF1 IRF1 PAIP2 PAIP2 PAIP2 PAIP2
NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 PAIP2
PAIP2 PAIP2 CLIC4 N/A N/A N/A N/A N/A N/A ABCA1 ABCG1
CLIC4 CLIC4 PAIP2 PAIP2 ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1
ABCA1 PAIP2 ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (o) IRF1 B2M
HSPCA LAMC1 PAIP2 N/A NCOA1 CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M
B2M B2M IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 G2AN HSPCA LAMC1 PAIP2
N/A NCOA1 CLIC4 ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN IRF1
IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 1L13RA1 HSPCA LAMC1 PAIP2 N/A NCOA1
CLIC4 ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IKBKAP HSPCA LAMC1 PAIP2
N/A NCOA1 CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 TNFAIP6 HSPCA
LAMC1 PAIP2 N/A NCOA1 CLIC4 ABCA1 ABCG1 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1
WDR9 HSPCA LAMC1 PAIP2 N/A NCOA1 CLIC4 ABCA1 ABCG1 WDR9 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 WWP2 HSPCA
LAMC1 PAIP2 N/A NCOA1 CLIC4 ABCA1 ABCG1 WWP2 WWP2 WWP2 WWP2 WWP2
WWP2 WWP2 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 BCL6 HSPCA LAMC1 PAIP2
N/A NCOA1 CLIC4 ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 IRF1
IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 C1QR1 HSPCA LAMC1 PAIP2 N/A NCOA1
CLIC4 ABCA1 ABCG1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 IRF1
IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 CCNC HSPCA LAMC1 PAIP2 N/A NCOA1
CLIC4 ABCA1 ABCG1 CCNC CCNC CCNC CCNC CCNC CCNC CCNC IRF1 IRF1 IRF1
IRF1 IRF1 IRF1 IRF1 FLJ32234 HSPCA LAMC1 PAIP2 N/A NCOA1 CLIC4
ABCA1 ABCG1 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
FLJ32234 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 HSPCA N/A LAMC1 PAIP2
N/A NCOA1 CLIC4 ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA
IRF1 IRF1 IRF1 IRF1 IRF1 IRF1 LAMC1 N/A N/A PAIP2 N/A NCOA1 CLIC4
ABCA1 ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 IRF1 IRF1 IRF1 IRF1 IRF1
PAIP2 N/A N/A N/A N/A NCOA1 CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2
PAIP2 IRF1 IRF1 IRF1 IRF1 IRF1 N/A N/A N/A N/A N/A N/A N/A N/A
NCOA1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1 NCOA1 NCOA1 NCOA1 IRF1
IRF1 IRF1 CLIC4 N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4
IRF1 IRF1 ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 IRF1 ABCG1
N/A N/A N/A N/A N/A N/A N/A N/A (p) NCOA1 B2M HSPCA LAMC1 PAIP2
IRF1 N/A CLIC4 ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 G2AN HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4
ABCA1 ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 NCOA1 1L13RA1 HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4
ABCA1 ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 IKBKAP HSPCA LAMC1 PAIP2
IRF1 N/A CLIC4 ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 TNFAIP6
HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 NCOA1 NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 WDR9 HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1
WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 WWP2 HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1 WWP2
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1
NCOA1 BCL6 HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1 BCL6 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1
C1QR1 HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1
NCOA1 CCNC HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1 CCNC CCNC
CCNC CCNC CCNC CCNC CCNC NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1
FLJ32234 HSPCA LAMC1 PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 HSPCA N/A LAMC1 PAIP2 IRF1 N/A CLIC4
ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA NCOA1 NCOA1 NCOA1
NCOA1 NCOA1 NCOA1 LAMC1 N/A N/A PAIP2 IRF1 N/A CLIC4 ABCA1 ABCG1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 NCOA1 NCOA1 NCOA1 NCOA1 NCOA1 PAIP2
N/A N/A N/A IRF1 N/A CLIC4 ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2
NCOA1 NCOA1 NCOA1 NCOA1 IRF1 N/A N/A N/A N/A N/A CLIC4 ABCA1 ABCG1
IRF1 IRF1 IRF1 NCOA1 NCOA1 NCOA1 NCOA1 N/A N/A N/A N/A N/A N/A N/A
N/A CLIC4 N/A N/A N/A N/A N/A N/A ABCA1 ABCG1 CLIC4 CLIC4 NCOA1
NCOA1 ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 NCOA1 ABCG1 N/A
N/A N/A N/A N/A N/A N/A N/A (q) CLIC4 B2M HSPCA LAMC1 PAIP2 IRF1
NCOA1 N/A ABCA1 ABCG1 B2M B2M B2M B2M B2M B2M B2M CLIC4 CLIC4 CLIC4
CLIC4 CLIC4 CLIC4 CLIC4 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1
ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN CLIC4 CLIC4 CLIC4 CLIC4
CLIC4 CLIC4 CLIC4 1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1
ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 CLIC4
CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 IKBKAP HSPCA LAMC1 PAIP2 IRF1
NCOA1 N/A ABCA1 ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 TNFAIP6 HSPCA
LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1 TNFAIP6 TNFAIP6 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4
CLIC4 WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1 WDR9 WDR9
WDR9 WDR9 WDR9 WDR9 WDR9 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4
WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1 WWP2 WWP2 WWP2
WWP2 WWP2 WWP2 WWP2 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 BCL6
HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1 BCL6 BCL6 BCL6 BCL6
BCL6 BCL6 BCL6 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 C1QR1
HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1 C1QR1 C1QR1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4
CCNC HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1 CCNC CCNC CCNC
CCNC CCNC CCNC CCNC CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4
FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1 FLJ32234
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 CLIC4 CLIC4
CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 HSPCA N/A LAMC1 PAIP2 IRF1 NCOA1 N/A
ABCA1 ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA CLIC4 CLIC4 CLIC4
CLIC4 CLIC4 CLIC4 LAMC1 N/A N/A PAIP2 IRF1 NCOA1 N/A ABCA1 ABCG1
LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 CLIC4 CLIC4 CLIC4 CLIC4 CLIC4 PAIP2
N/A N/A N/A IRF1 NCOA1 N/A ABCA1 ABCG1 PAIP2 PAIP2 PAIP2 PAIP2
CLIC4 CLIC4 CLIC4 CLIC4 IRF1 N/A N/A N/A N/A NCOA1 N/A ABCA1 ABCG1
IRF1 IRF1 IRF1 CLIC4 CLIC4 CLIC4 NCOA1 N/A N/A N/A N/A N/A N/A
ABCA1 ABCG1 NCOA1 NCOA1 CLIC4 CLIC4 CLIC4 N/A N/A N/A N/A N/A N/A
N/A N/A ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 CLIC4 ABCG1
N/A N/A N/A N/A N/A N/A N/A N/A (r) ABCA1 B2M HSPCA LAMC1 PAIP2
IRF1 NCOA1 CLIC4 N/A ABCG1 B2M B2M B2M B2M B2M B2M B2M ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 G2AN HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 N/A ABCG1 G2AN G2AN G2AN G2AN G2AN G2AN G2AN ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 1L13RA1 HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 N/A ABCG1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 IKBKAP HSPCA
LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A ABCG1 IKBKAP IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A ABCG1 TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A
ABCG1 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A ABCG1
WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A ABCG1 BCL6
BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A ABCG1 C1QR1
C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 CCNC HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A ABCG1 CCNC
CCNC CCNC CCNC CCNC CCNC CCNC ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 N/A ABCG1
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 HSPCA N/A LAMC1 PAIP2
IRF1 NCOA1 CLIC4 N/A ABCG1 HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 LAMC1 N/A N/A PAIP2 IRF1 NCOA1
CLIC4 N/A ABCG1 LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 N/A ABCG1 PAIP2
PAIP2 PAIP2 PAIP2 ABCA1 ABCA1 ABCA1 ABCA1 IRF1 N/A N/A N/A N/A
NCOA1 CLIC4 N/A ABCG1 IRF1 IRF1 IRF1 ABCA1 ABCA1 ABCA1 NCOA1 N/A
N/A N/A N/A N/A N/A N/A N/A CLIC4 N/A N/A N/A N/A N/A N/A N/A ABCG1
CLIC4 ABCA1 ABCA1 N/A N/A N/A N/A N/A N/A N/A ABCG1 ABCA1 ABCA1
ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A (s) ABCG1 B2M HSPCA LAMC1
PAIP2 IRF1 NCOA1 CLIC4 ABCA1 N/A B2M B2M B2M B2M B2M B2M B2M ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 G2AN HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 N/A G2AN G2AN G2AN G2AN G2AN G2AN G2AN ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 1L13RA1 HSPCA LAMC1 PAIP2 IRF1
NCOA1 CLIC4 ABCA1 N/A 1L13RA1 1L13RA1 1L13RA1 1L13RA1 1L13RA1
1L13RA1 1L13RA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 IKBKAP
HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 N/A IKBKAP IKBKAP IKBKAP
IKBKAP IKBKAP IKBKAP IKBKAP ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCG1 TNFAIP6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 N/A TNFAIP6
TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 TNFAIP6 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCG1 WDR9 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 N/A WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 WDR9 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCG1 WWP2 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 N/A WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 WWP2 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCG1 BCL6 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 N/A BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 BCL6 ABCA1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCG1 C1QR1 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4
ABCA1 N/A C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 C1QR1 ABCA1 ABCA1
ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 CCNC HSPCA LAMC1 PAIP2 IRF1 NCOA1
CLIC4 ABCA1 N/A
CCNC CCNC CCNC CCNC CCNC CCNC CCNC ABCA1 ABCA1 ABCA1 ABCA1 ABCA1
ABCA1 ABCG1 FLJ32234 HSPCA LAMC1 PAIP2 IRF1 NCOA1 CLIC4 ABCA1 N/A
FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234 FLJ32234
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 HSPCA N/A LAMC1 PAIP2
IRF1 NCOA1 CLIC4 ABCA1 N/A HSPCA HSPCA HSPCA HSPCA HSPCA HSPCA
ABCA1 ABCA1 ABCA1 ABCA1 ABCA1 ABCG1 LAMC1 N/A N/A PAIP2 IRF1 NCOA1
CLIC4 ABCA1 N/A LAMC1 LAMC1 LAMC1 LAMC1 LAMC1 ABCA1 ABCA1 ABCA1
ABCA1 ABCG1 PAIP2 N/A N/A N/A IRF1 NCOA1 CLIC4 ABCA1 N/A PAIP2
PAIP2 PAIP2 PAIP2 ABCA1 ABCA1 ABCA1 ABCG1 IRF1 N/A N/A N/A N/A
NCOA1 CLIC4 ABCA1 N/A IRF1 IRF1 IRF1 ABCA1 ABCA1 ABCG1 NCOA1 N/A
N/A N/A N/A N/A CLIC4 ABCA1 N/A NCOA1 NCOA1 ABCG1 ABCG1 CLIC4 N/A
N/A N/A N/A N/A N/A ABCA1 N/A CLIC4 ABCG1 ABCA1 N/A N/A N/A N/A N/A
N/A N/A N/A ABCG1 N/A N/A N/A N/A N/A N/A N/A N/A
TABLE-US-00010 TABLE 1 Alternative Gene Symbols HGNC_Symbol Locus
Link ID ABCA1 ABCA1 19 ABCG1 ABCG1 9619 ACP1 ACP1 52 ADPRT ADPRT
142 ANGPTL2 ANGPTL2 23452 B2M B2M 567 BCL6 BCL6 604 BMPR2 BMPR2 659
C19orf13 C19orf13 26065 C1QR1 C1QR1 22918 CCNC CCNC 892 CLECSF6
CLECSF6 50856 CLIC4 CLIC4 25932 CLN3 CLN3 1201 DNAPTP6 DNAPTP6
26010 EBNA1BP2 EBNA1BP2 10969 EGR1 EGR1 1958 F2RL1 F2RL1 2150
FLJ11000 FLJ11000 55281 FLJ11142 FLJ11142 55779 FLJ13612 EFHD1
80303 FLJ32234 C6orf151 154007 G2AN GANAB 23193 HSPCA HSPCAL3 3320
HSPCB HSPCB 3326 IKBKAP IKBKAP 8518 IL13RA1 IL13RA1 3597 ILF1 FOXK2
3607 IRF1 IRF1 3659 LAMC1 LAMC1 3915 LCMT2 LCMT2 9836 MAFB MAFB
9935 NCOA1 NCOA1 8648 NXN NXN 64359 PAIP2 PAIP2 51247 PDCD5 PDCD5
9141 PDK4 PDK4 5166 PER1 PER1 5187 PF4 PF4V1 5196 PF4 PF4 5197
PMSCL2 EXOSC10 5394 PPIF PPIF 10105 SETBP1 SETBP1 26040 SFRS6 SFRS6
6431 SLC5A6 SLC5A6 8884 TNFAIP6 TNFAIP6 7130 TSPAN2 TSPAN-2 64521
WDR9 C21orf107 54014 YES1 YES1 7525 ZFR ZFR 51663 ZNF397 ZNF397
84307 WWP2 WWP2 11060
TABLE-US-00011 TABLE 2 Alternative Locus Gene RefSeq_Protein Link
Symbols HGNC_Symbol RefSeq_Accession Accession ID Rta_Transcript
Anotation ABCA1 ABCA1 NM_005502 NP_005493 19 Homo sapiens
ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1), mRNA
ABCG1 ABCG1 NM_207629 NP_997512 9619 Homo sapiens ATP-binding
cassette, sub-family G (WHITE), member 1 (ABCG1), transcript
variant 7, mRNA ABCG1 ABCG1 NM_207628 NP_997511 9619 Homo sapiens
ATP-binding cassette, sub-family G (WHITE), member 1 (ABCG1),
transcript variant 6, mRNA ABCG1 ABCG1 NM_207630 NP_997513 9619
Homo sapiens ATP-binding cassette, sub-family G (WHITE), member 1
(ABCG1), transcript variant 1, mRNA ABCG1 ABCG1 NM_004915 NP_004906
9619 Homo sapiens ATP-binding cassette, sub-family G (WHITE),
member 1 (ABCG1), transcript variant 4, mRNA ABCG1 ABCG1 NM_207627
NP_997510 9619 Homo sapiens ATP-binding cassette, sub-family G
(WHITE), member 1 (ABCG1), transcript variant 5, mRNA ABCG1 ABCG1
NM_207174 NP_997057 9619 Homo sapiens ATP-binding cassette,
sub-family G (WHITE), member 1 (ABCG1), transcript variant 3, mRNA
ABCG1 ABCG1 NM_016818 NP_058198 9619 Homo sapiens ATP-binding
cassette, sub-family G (WHITE), member 1 (ABCG1), transcript
variant 2, mRNA ACP1 ACP1 NM_004300 NP_004291 52 Homo sapiens acid
phosphatase 1, soluble (ACP1), transcript variant 3, mRNA ADPRT
ADPRT NM_001618 NP_001609 142 Homo sapiens ADP-ribosyltransferase
(NAD+; poly (ADP-ribose) polymerase) (ADPRT), mRNA ANGPTL2 ANGPTL2
NM_012098 NP_036230 23452 Homo sapiens angiopoietin-like 2
(ANGPTL2), mRNA B2M B2M NM_004048 NP_004039 567 Homo sapiens
beta-2-microglobulin (B2M), mRNA. BCL6 BCL6 NM_001706 NP_001697 604
Homo sapiens B-cell CLL/lymphoma 6 (zinc finger protein 51) (BCL6),
transcript variant 1, mRNA BCL6 BCL6 NM_138931 NP_620309 604 Homo
sapiens B-cell CLL/lymphoma 6 (zinc finger protein 51) (BCL6),
transcript variant 2, mRNA BMPR2 BMPR2 NM_001204 NP_001195 659 Homo
sapiens bone morphogenetic protein receptor, type II
(serine/threonine kinase) (BMPR2), transcript variant 1, mRNA BMPR2
BMPR2 NM_033346 NP_203132 659 Homo sapiens bone morphogenetic
protein receptor, type II (serine/threonine kinase) (BMPR2),
transcript variant 2, mRNA C19orf13 C19orf13 NM_015578 NP_056393
26065 Homo sapiens chromosome 19 open reading frame 13 (C19orf13),
mRNA C1QR1 C1QR1 NM_012072 NP_036204 22918 Homo sapiens complement
component 1, q subcomponent, receptor 1 (C1QR1), mRNA CCNC CCNC
NM_005190 NP_005181 892 Homo sapiens cyclin C (CCNC), mRNA CLECSF6
CLECSF6 NM_194447 NP_919429 50856 Homo sapiens C-type (calcium
dependent, carbohydrate-recognition domain) lectin, superfamily
member 6 (CLECSF6), transcript variant 3, mRNA CLECSF6 CLECSF6
NM_016184 NP_057268 50856 Homo sapiens C-type (calcium dependent,
carbohydrate-recognition domain) lectin, superfamily member 6
(CLECSF6), transcript variant 1, mRNA CLIC4 CLIC4 NM_013943
NP_039234 25932 Homo sapiens chloride intracellular channel 4
(CLIC4), mRNA CLN3 CLN3 NM_000086 NP_000077 1201 Homo sapiens
ceroid-lipofuscinosis, neuronal 3, juvenile (Batten,
Spielmeyer-Vogt disease) (CLN3), mRNA DNAPTP6 DNAPTP6 NM_015535
NP_056350 26010 Homo sapiens DNA polymerase-transactivated protein
6 (DNAPTP6), mRNA EBNA1BP2 EBNA1BP2 NM_006824 NP_006815 10969 Homo
sapiens EBNA1 binding protein 2 (EBNA1BP2), mRNA EGR1 EGR1
NM_001964 NP_001955 1958 Homo sapiens early growth response 1
(EGR1), mRNA F2RL1 F2RL1 NM_005242 NP_005233 2150 Homo sapiens
coagulation factor II (thrombin) receptor-like 1 (F2RL1), mRNA
FLJ11000 FLJ11000 NM_018295 NP_060765 55281 Homo sapiens
hypothetical protein FLJ11000 (FLJ11000), mRNA FLJ11142 FLJ11142
NM_018338 NP_060808 55779 Homo sapiens hypothetical protein
FLJ11142 (FLJ11142), mRNA FLJ13612 EFHD1 NM_025202 NP_079478 80303
Homo sapiens EF hand domain containing 1 (EFHD1), mRNA FLJ32234
C6orf151 NM_152551 NP_689764 154007 Homo sapiens chromosome 6 open
reading frame 151 (C6orf151), mRNA G2AN GANAB NM_014610 NP_055425
23193 Homo sapiens glucosidase, alpha; neutral AB (GANAB), mRNA
G2AN GANAB NM_198335 NP_938149 23193 Homo sapiens glucosidase,
alpha; neutral AB (GANAB), mRNA G2AN GANAB NM_198334 NP_938148
23193 Homo sapiens glucosidase, alpha; neutral AB (GANAB), mRNA
HSPCA HSPCAL3 XM_084514 XP_084514 3320 Homo sapiens heat shock 90
kDa protein 1, alpha-like 3 (HSPCAL3), mRNA HSPCA HSPCA NM_005348
NP_005339 3320 Homo sapiens heat shock 90 kDa protein 1, alpha
(HSPCA), mRNA HSPCB HSPCB NM_007355 NP_031381 3326 Homo sapiens
heat shock 90 kDa protein 1, beta (HSPCB), mRNA IKBKAP IKBKAP
NM_003640 NP_003631 8518 Homo sapiens inhibitor of kappa light
polypeptide gene enhancer in B-cells, kinase complex-associated
protein (IKBKAP), mRNA IL13RA1 IL13RA1 NM_001560 NP_001551 3597
Homo sapiens interleukin 13 receptor, alpha 1 (IL13RA1), mRNA ILF1
FOXK2 NM_181430 NP_852095 3607 Homo sapiens forkhead box K2
(FOXK2), transcript variant 2, mRNA ILF1 FOXK2 NM_181431 NP_852096
3607 Homo sapiens forkhead box K2 (FOXK2), transcript variant 3,
mRNA ILF1 FOXK2 NM_004514 NP_004505 3607 Homo sapiens forkhead box
K2 (FOXK2), transcript variant 1, mRNA IRF1 IRF1 NM_002198
NP_002189 3659 Homo sapiens interferon regulatory factor 1 (IRF1),
mRNA LAMC1 LAMC1 NM_002293 NP_002284 3915 Homo sapiens laminin,
gamma 1 (formerly LAMB2) (LAMC1), mRNA LCMT2 LCMT2 NM_014793
NP_055608 9836 Homo sapiens leucine carboxyl methyltransferase 2
(LCMT2), mRNA MAFB MAFB NM_005461 NP_005452 9935 Homo sapiens v-maf
musculoaponeurotic fibrosarcoma oncogene homolog B (avian) (MAFB),
mRNA NCOA1 NCOA1 NM_003743 NP_003734 8648 Homo sapiens nuclear
receptor coactivator 1 (NCOA1), transcript variant 1, mRNA NCOA1
NCOA1 NM_147233 NP_671766 8648 Homo sapiens nuclear receptor
coactivator 1 (NCOA1), transcript variant 3, mRNA NCOA1 NCOA1
NM_147223 NP_671756 8648 Homo sapiens nuclear receptor coactivator
1 (NCOA1), transcript variant 2, mRNA NXN NXN NM_022463 NP_071908
64359 Homo sapiens nucleoredoxin (NXN), mRNA PAIP2 PAIP2 NM_016480
NP_057564 51247 Homo sapiens poly(A) binding protein interacting
protein 2 (PAIP2), mRNA PDCD5 PDCD5 NM_004708 NP_004699 9141 Homo
sapiens programmed cell death 5 (PDCD5), mRNA PDK4 PDK4 NM_002612
NP_002603 5166 Homo sapiens pyruvate dehydrogenase kinase,
isoenzyme 4 (PDK4), mRNA PER1 PER1 NM_002616 NP_002607 5187 Homo
sapiens period homolog 1 (Drosophila) (PER1), mRNA PF4 PF4V1
NM_002620 NP_002611 5196 Homo sapiens platelet factor 4 variant 1
(PF4V1), mRNA PF4 PF4 NM_002619 NP_002610 5197 Homo sapiens
platelet factor 4 (chemokine (C-X-C motif) ligand 4) (PF4), mRNA
PMSCL2 EXOSC10 NM_002685 NP_002676 5394 Homo sapiens exosome
component 10 (EXOSC10), mRNA PPIF PPIF NM_005729 NP_005720 10105
Homo sapiens peptidylprolyl isomerase F (cyclophilin F) (PPIF),
nuclear gene encoding mitochondrial protein, mRNA SETBP1 SETBP1
NM_015559 NP_056374 26040 Homo sapiens SET binding protein 1
(SETBP1), mRNA SFRS6 SFRS6 NM_006275 NP_006266 6431 Homo sapiens
splicing factor, arginine/serine-rich 6 (SFRS6), mRNA SLC5A6 SLC5A6
NM_021095 NP_066918 8884 Homo sapiens solute carrier family 5
(sodium-dependent vitamin transporter), member 6 (SLC5A6), mRNA
TNFA1P6 TNFA1P6 NM_007115 NP_009046 7130 Homo sapiens tumor
necrosis factor, alpha-induced protein 6 (TNFA1P6), mRNA TSPAN2
TSPAN-2 NM_005725 NP_005716 64521 Homo sapiens tetraspan 2
(TSPAN-2), mRNA WDR9 C21orf107 NM_018963 NP_061836 54014 Homo
sapiens chromosome 21 open reading frame 107 (C21orf107),
transcript variant 1, mRNA WDR9 C21orf107 NM_033656 NP_387505 54014
Homo sapiens chromosome 21 open reading frame 107 (C21orf107),
transcript variant 2, mRNA YES1 YES1 NM_005433 NP_005424 7525 Homo
sapiens v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1),
mRNA ZFR ZFR NM_016107 NP_057191 51663 Homo sapiens zinc finger RNA
binding protein (ZFR), mRNA ZNF397 ZNF397 NM_032347 NP_115723 84307
Homo sapiens zinc finger protein 397 (ZNF397), mRNA WWP2 WWP2
NM_007014 NP_008945 11060 Homo sapiens WW domain containing E3
ubiquitin protein ligase 2 isoform 1 WWP2 WWP2 NM_199423 NP_955455
11060 Homo sapiens WW domain containing E3 ubiquitin protein ligase
2 isoform 3 WWP2 WWP2 NM_199424 NP_955456 11060 Homo sapiens WW
domain containing E3 ubiquitin protein ligase 2 isoform 2
REFERENCES
[0665] 1. Zaleske D J. Cartilage and Bone Development. Instr Course
Lect 1998; 47:461- [0666] 2. Buckwalter J A, Mankin H J. Articular
Cartilage: Tissue Design and Chondrocyte-Matrix Interactions. Instr
Course Lect 1998:47:477-86. [0667] 3. Westacott C I, Sharif M.
Cytokines in Osteoarthritis: Mediators or Markers of Joint
Destruction? Semin Arthritis Rheum 1996; 25:254-72 [0668] 4. Adams
M D, Kerlavage A R, Fleischmann R D, Fuldner R A. Bult C J, Lee N
H. et al. Initial assessment of human gene diversity and expression
patterns based upon 83 million nucleotides of cDNA sequence. Nature
1995; 377 Suppl:3-174. [0669] 5. Hwang D M, Dempsey A A, Wang R X,
Rezvani M, Barrans J D, Dai K S, et al. A Genome-Based Resource for
Molecular Cardiovascular Medicine: Toward a Compendium of
Cardiovascular Genes. Circulation 1997; 96:4146-203. [0670] 6. Mao
M, Fu G, Wu J S, Zhang Q H, Zhou J. Kan L X, et al. Identification
of genes expressed in human CD34.sup.+ hematopoietic
stem/progenitor cells by expressed sequence tags and efficient
full-length cDNA cloning. Proc Natl Acad Sci 1998; 95:8175-80.
[0671] 7. Hillier L D, Lennon G, Becker M, Bonaldo M F, Chiapelli
B. Chissoe 5, et al. Generation and analysis of 280,000 human
expressed sequence tags. Genome Res. 1996; 6:807-28. [0672] 8.
Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local
alignment search tool. J Mol Biol 1990; 215:403-10. [0673] 9.
Mundlos S, Zabel B. Developmental Expression of Human Cartilage
Matrix Protein. Dev Dyn 1994:199:241-52. [0674] 10. Nakamura 5,
Kamihagi K. Satakeda H. Katayama M, Pan H, Okamoto H, et al.
Enhancement of SPARC (osteonectin) synthesis in arthritic
cartilage. Increased levels in synovial fluids from patients with
rheumatoid arthritis and regulation by growth factors and cytokines
in chondrocyte cultures. Arthritis Rheum 1996; 39:539-51. [0675]
11. Eyre D R, The Collagens of Articular Cartilage. Semin Arthritis
Rheum 1991; 21 (3 Suppl 2):2-11. [0676] 12. Okihana H, Yamada K.
Preparation of a cDNA Library and Preliminary Assessment of 1400
Genes from Mouse Growth Cartilage. J Bone Miner Res 1999; 14;
304-10. [0677] 13. Morrison E H, Ferguson M W J, Bayliss M T,
Archer C W. The developmental of articular cartilage: I. The
spatial and temporal patterns of collagen types. J Anat 1996;
189:9-22. [0678] 14. Treilleux I, Mallein-Gerin F. le Guellec D,
Herbage D. Localization of the Expression of Type I, II, III
Collagens, and Aggrecan Core Protein Genes in Developing Human
Articular Cartilage. Matrix 1992; 12:221-32. [0679] 15. Eyre D R,
Wu J J, Niyibizi C. The collagens of bone and cartilage: Molecular
diversity and supramolecular assembly. In Cohn D V. Glorieux F H.
Martin T J, editors. Calcium Regulation and Bone Metabolism.
Amsterdam. The Netherlands: Elsevier; 1990. p. 188-94. [0680] 16.
Birnbacher R. Amann G, Breitschopf H, Lassmann H, Suchanek G,
Heinz-Erian P. Cellular localization of insulin-like growth factor
II mRNA in the human fetus and the placenta: detection with a
digoxigenin-labeled cRNA probe and immunocytochemistry. Pediatr Res
1998; 43:614-20. [0681] 17. Wang E, Wang J, Chin E. Zhou J. Bondy C
A. Cellular patterns of insulin-like growth factor system gene
expression in murine chondrogenesis and osteogenesis. Endocrinology
1995; 136:2741-51. [0682] 18. van Kleffens M, Groffen C. Rosato R
R, van den Eijnde S M, van Neck J W, Lindenbergh-Kortleve D J, et
al. mRNA expression patterns of the IGF system during mouse limb
bud development, determined by whole mount in situ hybridization.
MolCell Endocrinol 1998; 138:151-61. [0683] 19. Braulke T, Gotz W,
Claussen M. Immunohistochemical localization of insulin-like growth
factor binding protein-1, -3, and -4 in human fetal tissues and
their analysis in media from fetal tissue explants. Growth Regul
1996; 6:55-65. [0684] 20. Kessler E, Takahara K, Biniaminov L,
Brusel M, Greenspan D S. Bone Morphogenetic Protein-1: The Type I
Procollagen C-Proteinase. Science 1996; 271:360-2. [0685] 21.
Ausubel et al., John Weley & Sons, Inc., 1997, Current
Protocols in Molecular Biology [0686] 22. Marshall, K. et al.,
2000, 46.sup.th Annual Meeting, ORS, paper No. 919. [0687] 23.
Kumar, S. et al., 2000, 46.sup.th Annual Meeting, ORS, paper No.
1031. [0688] 24. Marshall K., et al., 2002, 48.sup.th Annual
meeting, ORS (submitted). [0689] 25. Migita K., et al., Biochem
Biophys Res Commun 1997, 239:621-625. [0690] 26. Migita K et al.,
Kidney Int 1999, 55:572-578.
[0691] The contents of all references, patents and patent
applications (including, published patent applications) cited
throughout this application are hereby incorporated by
reference.
EQUIVALENTS
[0692] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
154120DNAArtificial SequenceAntisense sequence - B2M 1ttaaaggact
taacgataca 20220DNAArtificial SequenceAntisense sequence - BCL6
2tgtagagccg agttaaacgc 20320DNAArtificial SequenceAntisense
sequence - C1QR1 3gctctgagtc tcagtaataa 20420DNAArtificial
SequenceAntisense sequence - CCNC 4taagatacgg tccataagag
20520DNAArtificial SequenceAntisense sequence - EBNA1BP2
5cttcgttaca aaccgtcgga 20620DNAArtificial SequenceAntisense
sequence - FLJ32234 6cttttacact tctatggaag 20720DNAArtificial
SequenceAntisense sequence - G2AN 7tcagagtgac cctgggtccg
20820DNAArtificial SequenceAntisense sequence - HSPCA 8cggaaagtcc
gtctttaacg 20920DNAArtificial SequenceAntisense sequence - IKBKAP
9acctagtcct cagacacaca 201020DNAArtificial SequenceAntisense
sequence - IL13RA1 10ctcactcttc ggatcgtaaa 201120DNAArtificial
SequenceAntisense sequence - LAMC1 11tcgtccggaa cacatgacta
201220DNAArtificial SequenceAntisense sequence - MAFB 12gtcccgtccc
gtcccttcga 201320DNAArtificial SequenceAntisense sequence - PAIP2
13gttcgtagta gttacttcta 201420DNAArtificial SequenceAntisense
sequence - PER1 14ggtccttatg atggtcgtca 201520DNAArtificial
SequenceAntisense sequence - PF4 15caacgacgag gacggtgaac
201620DNAArtificial SequenceAntisense sequence - TNFAIP6
16tttttaacct aaagtacaga 201720DNAArtificial SequenceAntisense
sequence - WDR9 17actatcctgt cctgtatctt 201820DNAArtificial
SequenceAntisense sequence - IRF1 18tctagggtac cttcgtacga
201920DNAArtificial SequenceAntisense sequence - ABCA1 19cttcggttag
gactcttgtg 202020DNAArtificial SequenceAntisense sequence - ABCG1
20tctgacgcac aggacgtttt 202120DNAArtificial SequenceAntisense
sequence - NCOA1 21ggtcgataat gcccacatct 202220DNAArtificial
SequenceAntisense sequence - CLIC4 22gtgcatttaa agacctaccg
202320DNAArtificial SequenceAntisense sequence - SFRS6 23ccacctatgt
cgtcagcctc 202420DNAArtificial SequenceAntisense sequence - HSPCB
24cttctggtga accgtcagtt 202520DNAArtificial SequenceAntisense
sequence - ADPRT 25cacttccggt actaactctt 202620DNAArtificial
SequenceAntisense sequence - ACP1 26acagctagtg ggtaacgtct
202720DNAArtificial SequenceAntisense sequence - PMSCL2
27gaagggcctg cggctgtcga 202820DNAArtificial SequenceAntisense
sequence - FLJ13612 28gtcggcggcg gagtagttcc 202920DNAArtificial
SequenceAntisense sequence - SLC5A6 29tctctagatg gctaaaccct
203020DNAArtificial SequenceAntisense sequence - YES1 30accttggtgc
tttcatcgtt 203120DNAArtificial SequenceAntisense sequence - CLN3
31agagatgccg acgacacgag 203220DNAArtificial SequenceAntisense
sequence - LCMT2 32gttgtaaaag ccgtcgattt 203320DNAArtificial
SequenceAntisense sequence - NXN 33agtcccttca gtaacgtccc
203420DNAArtificial SequenceAntisense sequence - ANGPTL2
34tctgcatgtt cgttcccaaa 203520DNAArtificial SequenceAntisense
sequence - BMPR2 35atcaacctct actctctcag 203620DNAArtificial
SequenceAntisense sequence - CLECSF6 36attatggcct aaggggttcg
203720DNAArtificial SequenceAntisense sequence - DNAPTP6
37gtgtcttccg ttgtctgatg 203820DNAArtificial SequenceAntisense
sequence - F2RL1 38gaacttctaa cggatagtgt 203920DNAArtificial
SequenceAntisense sequence - FLJ11000 39gttgtcacta ctctctcgca
204020DNAArtificial SequenceAntisense sequence - FLJ11142
40ttaaccgcag taaccccaag 204120DNAArtificial SequenceAntisense
sequence - ILF1 41tgcgggggcc cgccgccgcc 204220DNAArtificial
SequenceAntisense sequence - PDCD5 42ctctttgtca tagaatcggg
204320DNAArtificial SequenceAntisense sequence - PDK4 43agttgccgcg
gccggaccac 204420DNAArtificial SequenceAntisense sequence - PPIF
44cccgaggccg ctgggcagga 204520DNAArtificial SequenceAntisense
sequence - SETBP1 45tggagtttcc taaagtcggt 204620DNAArtificial
SequenceAntisense sequence - ZFR 46gtcgttgact gataccaata
204720DNAArtificial SequenceAntisense sequence - EGR1 47tgcggcttgt
gactgtaaaa 204820DNAArtificial SequenceAntisense sequence - TSPAN2
48ctcataaaga tacaccccga 204920DNAArtificial SequencePrimer -
forward primer B2M 49gaattcaccc ccactgaaaa 205020DNAArtificial
SequencePrimer - reverse primer B2M 50cctccatgat gctgcttaca
205121DNAArtificial SequencePrimer - forward primer G2AN
51ttcctgctgc gtcgattctc a 215222DNAArtificial SequencePrimer -
reverse primer G2AN 52ttatcaccac ccgctcaatc ca 225322DNAArtificial
SequencePrimer - forward primer IL13RA1 53cattgttcca gtcatcgtcg ca
225422DNAArtificial SequencePrimer - reverse primer IL13RA1
54tcttgccagg atcaggaatt gg 225520DNAArtificial SequencePrimer -
forward primer TNFAIP6 55aaggatgggg attcaaggat 205620DNAArtificial
SequencePrimer - reverse primer TNFAIP6 56aattcacaca ccgccttagc
205722DNAArtificial SequencePrimer - forward primer WDR9
57ttgcaggccc tgttgatttg tg 225822DNAArtificial SequencePrimer -
reverse primer WDR9 58ccaaactaac gcagacagcc tc 225922DNAArtificial
SequencePrimer - forward primer WWP2 59ggcactacac caagaacagc aa
226020DNAArtificial SequencePrimer - reverse primer WWP2
60tgctaccgat gagttcggca 206120DNAArtificial SequencePrimer -
forward primer BCL6 61caaggcattg gtgaagacaa 206220DNAArtificial
SequencePrimer - reverse primer BCL6 62cggctcacaa caatgacaac
206322DNAArtificial SequencePrimer - forward primer C1QR1
63gccatggaga accagtacag tc 226424DNAArtificial SequencePrimer -
reverse primer C1QR1 64gagttcaaag ctctgaggat ggtg
246520DNAArtificial SequencePrimer - forward primer CCNC
65cccttgcatg gaggatagtg 206620DNAArtificial SequencePrimer -
reverse primer CCNC 66cattgcctgg catctttctg 206720DNAArtificial
SequencePrimer - forward primer EBNA1BP2 67gcctccatca gctcaaagtc
206820DNAArtificial SequencePrimer - reverse primer EBNA1BP2
68ttgcaccttc ttcccgtatt 206919DNAArtificial SequencePrimer -
forward primer FLJ32234 69gcggaggatg aagttgtga 197026DNAArtificial
SequencePrimer - reverse primer FLJ32234 70gagtccttat tcaaagtaat
cgaagg 267122DNAArtificial SequencePrimer - forward primer HSPCA
71atgattggcc agttcggtgt tg 227222DNAArtificial SequencePrimer -
reverse primer HSPCA 72ttcacctgtg tctgtcctca ct 227321DNAArtificial
SequencePrimer - forward primer LAMC1 73caggctccat gaagcaacag a
217425DNAArtificial SequencePrimer - reverse primer LAMC1
74gcacttctct cactgtatgt cccac 257520DNAArtificial SequencePrimer -
forward primer PAIP2 75aagatccaag tcgcagcagt 207625DNAArtificial
SequencePrimer - reverse primer PAIP2 76tcccataact cctctttaac ttgtc
257724DNAArtificial SequencePrimer - forward primer ZFR
77cgagaagaga acatgaggga agga 247820DNAArtificial SequencePrimer -
reverse primer ZFR 78tagagcagcc agagcgtcaa 207922DNAArtificial
SequencePrimer - forward primer IKBKAP 79gcagcccagc ttaactttac ca
228022DNAArtificial SequencePrimer - reverse primer IKBKAP
80taatcctggg cacactcttc ca 228120DNAArtificial SequencePrimer -
forward primer ABCA1 81agttggcaag gttggtgagt 208222DNAArtificial
SequencePrimer - reverse primer ABCA1 82atggctgtag agagcttgcg tt
228320DNAArtificial SequencePrimer - forward primer ABCG1
83tgcaggtggc cactttcgtg 208421DNAArtificial SequencePrimer -
reverse primer ABCG1 84ccttcgaacc catacctgac a 218519DNAArtificial
SequencePrimer - forward primer IRF1 85actccagcac tgtcgccat
198622DNAArtificial SequencePrimer - reverse primer IRF1
86tgggtgacac ctggaagttg ta 228721DNAArtificial SequencePrimer -
forward primer NCOA1 87tccagtatcc aggagcagga a 218822DNAArtificial
SequencePrimer - reverse primer NCOA1 88agaactctga ggaggaggga tt
228922DNAArtificial SequencePrimer - forward primer CLIC4
89tgccctccca agtacttaaa gc 229021DNAArtificial SequencePrimer -
reverse primer CLIC4 90cagtgcttca ttagcctctg g 219124DNAArtificial
SequencePrimer - forward primer ACP1 91tcagagaatt ggagggtaga cagc
249221DNAArtificial SequencePrimer - reverse primer ACP1
92tttggtaatc tgccgggcaa c 219321DNAArtificial SequencePrimer -
forward primer ADPRT 93cccgtgacag gctacatgtt t 219420DNAArtificial
SequencePrimer - reverse primer ADPRT 94aagggcaact tctcccaaca
209522DNAArtificial SequencePrimer - forward primer ANGPTL2
95agacgtacaa gcaagggttt gg 229622DNAArtificial SequencePrimer -
reverse primer ANGPTL2 96caccaggagt ttgtagttgc ct
229720DNAArtificial SequencePrimer - forward primer BMPR2
97aaatagcctg gcagtgaggt 209822DNAArtificial SequencePrimer -
reverse primer BMPR2 98ttcagccatc ctttcctcag ca 229924DNAArtificial
SequencePrimer - forward primer C19orf13 99caatagagaa cggagaccaa
cctg 2410019DNAArtificial SequencePrimer - reverse primer C19orf13
100acctcctctg cctctgtat 1910120DNAArtificial SequencePrimer -
forward primer CLECSF6 101atatgcccgt ggaagagaca
2010222DNAArtificial SequencePrimer - reverse primer CLECSF6
102tgagcctcca ttctagcaca gt 2210322DNAArtificial SequencePrimer -
forward primer CLN3 103tgccaagcat ctacctcgtc tt
2210421DNAArtificial SequencePrimer - reverse primer CLN3
104atcactggtc tccagggcga t 2110524DNAArtificial SequencePrimer -
forward primer DNAPTP6 105ggtcacagaa ggcaacagac tact
2410622DNAArtificial SequencePrimer - reverse primer DNAPTP6
106tgccttaggc ttgcttgggt ta 2210722DNAArtificial SequencePrimer -
forward primer EFHD1 107ttccgggagt tcctgctcat tt
2210822DNAArtificial SequencePrimer - reverse primer EFHD1
108agttcttggc acctttgaca cc 2210920DNAArtificial SequencePrimer -
forward primer EGR1 109gaccgcagag tcttttcctg 2011020DNAArtificial
SequencePrimer - reverse primer EGR1 110cacaaggtgt tgccactgtt
2011122DNAArtificial SequencePrimer - forward primer EXOSC10
111agttgacttg gagcaccact ct 2211222DNAArtificial SequencePrimer -
reverse primer EXOSC10 112ttcgaagctc gagggtgtca at
2211322DNAArtificial SequencePrimer - forward primer F2RL1
113tggcaccatc caaggaacca at 2211422DNAArtificial SequencePrimer -
reverse primer F2RL1 114ttccagtgag gacagatgca ga
2211521DNAArtificial SequencePrimer - forward primer FLJ11000
115tgtggtctct gcacctcctt t 2111620DNAArtificial SequencePrimer -
reverse primer FLJ11000 116tgaccacaat cacgaggact
2011722DNAArtificial SequencePrimer - forward primer FLJ11142
117tcgtttcttg gtgactgctg ga 2211822DNAArtificial SequencePrimer -
reverse primer FLJ11142 118tccaaacctg ggagatggaa ct
2211920DNAArtificial SequencePrimer - forward primer FOXK2
119agacagcccg aaggatgatt 2012020DNAArtificial SequencePrimer -
reverse primer FOXK2 120ttgtccgcag tcctgtagta 2012122DNAArtificial
SequencePrimer - forward primer HSPCB 121ccacttggca gtcaagcact tt
2212222DNAArtificial SequencePrimer - reverse primer HSPCB
122tgatgaacac acggcggaca ta 2212320DNAArtificial SequencePrimer -
forward primer LCMT2 123ttggccagtt catgctgcaa 2012422DNAArtificial
SequencePrimer - reverse primer LCMT2 124attcattcat gtccacggca cc
2212520DNAArtificial SequencePrimer - forward primer MAFB
125cccttgtttc tttgggtgag 2012620DNAArtificial SequencePrimer -
reverse primer MAFB 126acgttctcta
tgcggtttgg 2012723DNAArtificial SequencePrimer - forward primer NXN
127tgtagattct gaggatgacg gag 2312824DNAArtificial SequencePrimer -
reverse primer NXN 128cctcctcctc tttggctttg tact
2412920DNAArtificial SequencePrimer - forward primer PDCD5
129atggcggacg aggagcttga 2013022DNAArtificial SequencePrimer -
reverse primer PDCD5 130ctcatttctg cttccctgtg ct
2213120DNAArtificial SequencePrimer - forward primer PDK4
131actcggatgc tgatgaacca 2013222DNAArtificial SequencePrimer -
reverse primer PDK4 132aaggcatctt ggaccactgc ta
2213322DNAArtificial SequencePrimer - forward primer PER1
133taagcgtaaa tgtgcctcct cc 2213421DNAArtificial SequencePrimer -
reverse primer PER1 134tgacggcgga tctttcttgg t 2113524DNAArtificial
SequencePrimer - forward primer PF4 135ccaactgata gccacgctga agaa
2413622DNAArtificial SequencePrimer - reverse primer PF4
136aatgcacaca cgtaggcagc ta 2213719DNAArtificial SequencePrimer -
forward primer PF4V1 137gttgctgctc ctgccactt 1913820DNAArtificial
SequencePrimer - reverse primer PF4V1 138gtggctatca gttgggcagt
2013920DNAArtificial SequencePrimer - forward primer PPIF
139tgctggagct gaaggcagat 2014020DNAArtificial SequencePrimer -
reverse primer PPIF 140tggcacatga aggaagggat 2014122DNAArtificial
SequencePrimer - forward primer SETBP1 141tgaaggcttt ggaacgtaca gg
2214220DNAArtificial SequencePrimer - reverse primer SETBP1
142gggacttggc atccctggag 2014322DNAArtificial SequencePrimer -
forward primer SFRS6 143tggacaaact ggatggcaca ga
2214420DNAArtificial SequencePrimer - reverse primer SFRS6
144atcgagacct ggatctgctt 2014524DNAArtificial SequencePrimer -
forward primer SLC5A6 145ccagaccagt tcgtcctgta cttt
2414624DNAArtificial SequencePrimer - reverse primer SLC5A6
146tatagtgctg agagagccgc tgaa 2414720DNAArtificial SequencePrimer -
forward primer TSPAN-2 147aggtctgacg atctttggca
2014822DNAArtificial SequencePrimer - reverse primer TSPAN-2
148gacagctcct gtgacatttg gt 2214922DNAArtificial SequencePrimer -
forward primer YES1 149atccaggtat ggtgaaccgt ga
2215022DNAArtificial SequencePrimer - reverse primer YES1
150tcagggtcct tcttccaaca ca 2215119DNAArtificial SequencePrimer -
forward primer ZNF397 151gcagcaggtc ccagctagt 1915222DNAArtificial
SequencePrimer - reverse primer ZNF397 152tggaggtgga tgtctgttga ct
2215322DNAArtificial SequencePrimer - forward primer HSPCAL3
153atgattggcc agttcggtgt tg 2215422DNAArtificial SequencePrimer -
reverse primer HSPCAL3 154ttcacctgtg tctgtcctca ct 22
* * * * *