Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF DIAGNOSIS AND TREATMENT FOR ASTHMA AND OTHER
RESPIRATORY DISEASES BASED ON HAPLOTYPE ASSOCIATION
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/487,072, filed on July 14, 2003 and U.S. Provisional Application No.
60/559,611, filed on April 5, 2004. The entire teachings of the above
applications
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Bronchial asthma [Morbidity Number (MIM) 600807], the most common
chronic disease affecting children and young adults, is a complex genetic
disorder
is with several overlapping phenotypes (Cookson and Moffatt 2000; Weiss 2001).
There is strong evidence for a genetic component in asthma (Bleecker et al.,
1997;
Kauffmann et al., 2002). Multiple environmental factors are also known to
modulate the clinical expression of asthma as well as the asthma-associated
phenotypes: bronchial hyperresponsiveness, atopy and elevated IgE (Koppelman
et
2o al., 1999; Cookson 1999; Holloway et al., 1999). It is a commonly held view
that
asthma is caused by multiple interacting genes some having a protective effect
and
others contributing to the disease pathogenesis, with each gene having its own
tendency to be influenced by the environment (Koppelman et al, and Postma,
1999;
Cookson, 1999; Holloway et al., 1999). Thus, the complex nature of the asthma
2s phenotype, together with substantial locus heterogeneity and environmental
influence, has made it difficult to uncover genetic factors that underlie
asthma.
Numerous loci and candidate genes have been reported to show linkage and
association to asthma and atopy. While some studies reporting these
observations are
compelling, no asthma gene conferring high risk has been mapped such that it
meets
3o stringent criteria for genome-wide significance.
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SUMMARY OF THE INVENTION
As described herein, a gene on chromosome I4q24 has been identified that
plays a major role is asthma. The gene MAP3K9 (the asthma gene) encodes a
kinase that is part of the Mixed Lineage Kinase (MLK) family. The protein
encoded
by the MAP3K9 gene is designated as MAP3K9 (herein) or more commonly as
MLK-1. This locus has been designated asthma locus one (hereinafter referred
to as
"AS 1 "). The present invention relates to methods of treatment using
inhibitors to
the asthma gene products.
The invention pertains to methods of treatment (prophylactic and/or
therapeutic)
to for certain diseases and conditions (e.g., asthma and other respiratory
diseases)
associated with MAP3K9 or with other members of the JNK pathway, for example,
members ofthe MLK family kinases (e.g., MLKl, MLK2, MLK3(SPRK, PTK1),
MLK4, LZK, DLK (ZPK, MUK) and MLK6), in particular, MLK1; and/or with other
members of the JNK pathway (as shown in FIG. 1), receptors and/or binding
agents of .
1s the enzymes; the transcription factor AP-I and its individual components, c
jun and v-
fos, and receptors fox the MLK family kinases. The methods include the
following:
methods of treatment for asthma or susceptibility to asthma; and methods of
treatment
for respiratory diseases associated with MAP3K9 or with other members of the
MLK
family.
20 In the methods of the invention, a MLK kinase family inhibitor is
administered
to an individual in a therapeutically effective amount. The MLK kinase family
inhibitor
can be an agent that inhibits or antagonizes a member of the JKN pathway, in
particular
the MLK family kinase pathway (e.g., MLKl, MLK2, MLK3) that are members of a
subset of the JNK pathway, and the transcription factor AP-1 and its
individual
2s components, c-jun and v-fos. For example, the MLK kinase family inhibitor
synthesis
inhibitor can be an agent that inhibits or antagonizes MAP3K9 polypeptide
(MLK1)
activity (e.g., a MAP3K9 inhibitor, for example, a compound (1), CEP-1347, or
a
compound of Formula IV) and/or MAP3K9 nucleic acid expression, as described
herein
(e.g., a MAP3K9 nucleic acid antagonist). In one aspect, the agent alters
activity and/or
3o nucleic acid expression of MAP3K9. In another aspect, the agents used in
the methods
are represented by formula I and further described in Tables A and B, and
their optically
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pure stereoisomers, mixtures of stereoisomers, salts, chemical derivatives,
and
analogues. In other aspects, the agent used in the methods is CEP-1347 as
shown in
Formula III, its, optically pure stereoisomers, mixtures of stereoisomers,
salts, chemical
derivatives, and analogues or a compound of Structural Formula IV, its
optically pure
stereoisomers, mixtures of stereoisomers, salts, chemical derivatives, and
analogues. In
another aspect, the agent alters metabolism or inhibits activity of an MLKl
protein
(e.g., MLKl kinase), or an MLK kinase family member.
In certain aspects of the invention, the individual is an individual who has
at
least one risk factor, such as an at-risk haplotype for asthma; an at-risk
haplotype in the
1o MAP3K9 gene; a polymorphism in a MAP3K9 nucleic acid; dysregulation of
MAP3K9
mRNA expression; dysregulation of a MAP3K9 mRNA isoform; increased MLKl
protein expression; increased MLKI biochemical activity; and increased MKL1
protein
isoform expression.
The invention further pertains to methods of assessing response to treatment
~5 with a MLK kinase family protein, for example MLK1, by assessing a level of
a MLK
kinase family protein in the individual before treatment, and comparing the
level to a
level of the MLK kinase family protein assessed during or after treatment. A
level that
is significantly lower during or after treatment, than before treatment, is
indicative of
efficacy of the treatment with the MLK kinase family protein. The level of the
MLK
2o kinase family protein can be measured using a biochemical assay of enzyme
activity, or
using methods that allow direct quantitation of the amount of MLK kinase
protein, e.g.,
by enzyme-linked immunosorbent assay (EIA). The invention additionally
pertains to
methods of assessing response to treatment with a MLK kinase family protein,
by
stimulating production of a MLK kinase family protein or a MLK kinase family
protein
25 in a first test sample from the individual (e.g., a sample comprising
leukocytes) before
treatment, and comparing the level of the MLK kinase family protein with a
level of
production of the MLK kinase family protein in a second test sample from the
individual, during or after treatment. A level of production of the MLK kinase
family
protein or MLK kinase family protein in the second test sample that is
significantly
30 lower than the level in the first test sample is indicative of efficacy of
the treatment.
Similarly, the invention encompasses methods of assessing response to
treatment with a
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MLK kinase family inhibitor, by assessing a level of an inflammatory marker
(e.g., IL-2
and TNFa) in the individual before treatment, and during or after treatment. A
level of
the inflammatory protein marker during or after treatment, that is
significantly lower
than the level of inflammatory marker before treatment, is indicative of
e~cacy of the
treatment. The first sample can also be a "control level" of the MLK kinase
family
protein that has been determined by large sampling of individual without any
incidence
of asthma.
The present invention also relates to isolated nucleic acid molecules
comprising
the asthma gene located within AS 1 locus. It has also been discovered that
particular
1o combinations of genetic markers ("haplotypes"), are present at a higher
than expected
frequency in patients with phenotypes associated with asthma and a
susceptibility to
asthma. The markers that are included in the haplotypes described herein are
associated
with the genomic region that directs expression of MAP3K9 kinase.
In one embodiment, the invention is directed to a method of diagnosing astlnna
1s or a susceptibility to asthma in an individual, comprising detecting the
presence or
absence of an at-risk haplotype, comprising a haplotype selected from the
group
.consisting of: haplotype 1, haplotype 2, haplotype 3, haplotype 4, haplotype
5,
haplotype 6, haplotype 7 and combinations thereof; wherein the presence of the
haplotype is indicative of asthma or a susceptibility to asthma. In a
particular
2o embodiment, the invention is directed to assaying for the presence of a
first nucleic acid
molecule in a sample, comprising contacting said sample with a second nucleic
acid
molecule comprising the one or more haplotypes described herein. In one
embodiment,
determining the presence or absence of the haplotype comprises enzymatic
amplification of nucleic acid from the individual. In a particular embodiment,
2s determining the presence or absence of the haplotype further comprises
electrophoretic
analysis. For example, in one embodiment, determining the presence or absence
of the
haplotype comprises restriction fragment length polymorphism analysis. In
another
embodiment, determining the presence or absence of the haplotype comprises
sequence
analysis.
3o In another embodiment, the invention is directed to a method of diagnosing
asthma or a.susceptibility to asthma in an individual, comprising detecting
the presence
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or absence of an at-risk haplotype comprising haplotype 6 (shown in Table 1),
wherein
the presence of the haplotype is indicative of asthma or a susceptibility to
asthma. In a
particular embodiment, determining the presence or absence of the haplotype
comprises
enzymatic amplification of nucleic acid from the individual. In a particular
embodiment, determining the presence or absence of the haplotype further
comprises
electrophoretic analysis. For example, in one embodiment, determining the
presence or
absence of the haplotype comprises restriction fragment length polymorphism
analysis.
In another embodiment, determining the presence or absence of the haplotype
comprises sequence analysis.
to In another embodiment, the invention is directed to a method of~,diagnosing
asthma or a susceptibility to asthma in an individual, comprising detecting
the presence
or absence of an at-risk haplotype comprising haplotype 7 (shown in Table 1),
wherein
the presence of the haplotype is indicative of asthma or a susceptibility to
asthma. In a
particular embodiment, determining the presence or absence of the haplotype
comprises
15 enzymatic amplification of nucleic acid from the individual. In a
particular
embodiment, determining the presence or absence of the haplotype further
comprises
electrophoretic analysis. For example, in one embodiment, determining the
presence or
absence of the haplotype comprises restriction fragment length polymorphism
analysis.
In another embodiment, determining the presence or absence of the haplotype
2o comprises sequence analysis.
In another embodiment, the invention is directed to a kit for assaying a
sample
for the presence of a haplotype associated with asthma, wherein the haplotype
comprises two or more specific alleles, and wherein the kit comprises one or
more
nucleic acids capable of detecting the presence or absence of two or more of
the specific
2s alleles, thereby indicating the presence or absence of the haplotype in the
sample. In a
particular embodiment, the nucleic acid comprises a contiguous nucleotide
sequence
that is completely complementary to a region comprising specific allele of the
haplotype.
In another embodiment, the invention is directed to a reagent kit for assaying
a
3o sample for the presence of a haplotype associated with asthma, wherein the
haplotype
comprises two or more specific alleles, comprising in separate containers: a)
one or
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more labeled nucleic acids capable of detecting one or more specific alleles
of the
haplotype; and b) reagents for detection of said label. In a particular
embodiment, the
labeled nucleic acid comprises a contiguous nucleotide sequence that is
completely
complementary to a region comprising specific allele of the haplotype.
s In yet another embodiment, the invention is directed to a reagent kit for
assaying
a sample for the presence of a haplotype associated with asthma, wherein the
haplotype
comprises two or more specific alleles, wherein the kit comprises one or more
nucleic
acids comprising a nucleotide sequence that is at least partially
complementary to a part
of the nucleotide sequence of the MAP3K9 gene, and wherein the nucleic acid is
1o capable of acting as a primer for a primer extension reaction capable of
detecting one or
more of the specific alleles of the haplotype.
In another embodiment, the invention is directed to a method for the diagnosis
and identification of susceptibility to asthma in an individual, comprising:
screening in
a sample from the individual to be diagnosed for an at=risk haplotype
associated with
~s MAP3K9 that is more frequently present in an individual susceptible to
asthma
compared to an individual who is not susceptible to asthma wherein the at-risk
haplotype increases the risk significantly. In a particular embodiment, the
significant
increase is at least about 20°l°. In another embodiment, the
significant increase is
identified as an odds ratio of at least about 1.2.
2o The invention further pertains to a method of diagnosing asthma or a
susceptibility to asthma in an individual, comprising detecting in a sample
from the
individual to be diagnosed the presence or absence of at least one marker of
an at-risk
haplotype associated with the Mf1P3K9 gene selected from the group consisting
of:
DG14S205, DG14S428, D14S1002, DG14S4399, DG14S404, D14S251, DG14S1300,
2s DG14S266, DG14S462, DG14S448 and DG145406, wherein the presence of one or
more markers is indicative of asthma or a susceptibility to asthma.
In another embodiment, the invention is directed to a method for diagnosing a
susceptibility to asthma in an individual, comprising determining in a sample
from the
individual to be diagnosed the presence or absence in the individual of a
haplotype,
3o comprising two or more alleles selected from the group consisting of one or
a
combination of the markers that comprise the haplotypes set forth in Table l,
wherein
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the presence of the haplotype is indicative of susceptibility to asthma. In a
particular
embodiment, determining the presence or absence of the haplotype further
comprises
electrophoretic analysis. For example, in one embodiment, determining the
presence or
absence of the haplotype comprises restriction fragment length polymorphism
analysis.
In another embodiment, determining the presence or absence of the haplotype
comprises sequence analysis.
In yet another embodiment, the invention is directed to a method for
diagnosing
a susceptibility to asthma in an individual, comprising obtaining a nucleic
acid sample
from the individual; and analyzing the nucleic acid sample for the presence or
absence
of a haplotype comprising two or more alleles selected from the group
consisting of one
or a combination of the markers that comprise the haplotypes set forth in
Table l,
wherein the presence of the haplotype is indicative of susceptibility to
asthma.
The present invention relates to isolated nucleic acid molecules comprising
the
asthma gene located within AS1 locus. In one embodiment, the isolated nucleic
acid
molecule comprises a nucleotide sequence of SEQ ID NO: 1 or the complement
thereof;
wherein the nucleic acid molecule can optionally comprise one ore more of the
SNPs
set forth in the Examples. The invention further relates to a nucleic acid
molecule that
hybridizes under high stringency conditions to a nucleotide sequence of SEQ ID
NO: 1
and the complement thereof. The invention additionally relates to isolated
nucleic acid
2o molecules (e.g., cDNA molecules) encoding a MAP3K9 polypeptide (e.g.,
encoding a
polypeptide of SEQ ID NO: 2).
Also contemplated by the invention is a method of assaying for the presence of
a
first nucleic acid molecule in a sample, comprising contacting said sample
with a
second nucleic acid molecule, where the second nucleic acid molecule comprises
at
least one (or more) nucleic acid sequences) selected from the sequences
described
herein, wherein the nucleic acid sequence hybridizes to the first nucleic acid
under high
stringency conditions. In certain embodiments, the second nucleic acid
molecule
contains one or more polymorphism(s), described herein.
The invention also relates to a vector comprising an isolated nucleic acid
3o molecule of the invention, optionally including one or more of the
polymorphisms
described herein) operably linked to a regulatory sequence, as well as to a
recombinant
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_g_
host cell comprising the vector. The invention also provides a method for
producing a
polypeptide encoded by an isolated nucleic acid molecule having a
polymorphism,
comprising culturing the recombinant host cell under conditions suitable for
expression
of the nucleic acid molecule.
Also contemplated by the invention is a method of assaying for the presence of
a
polypeptide encoded by an isolated nucleic acid molecule of the invention in a
sample,
the method comprising contacting the sample with an antibody that specifically
binds to
the encoded polypeptide.
The invention further pertains to a method of identifying an agent that alters
1o expression of a MAP3K9 nucleic acid, comprising: contacting a solution
containing a
nucleic acid comprising the promoter region of the MAP3K9 gene operably linked
to a
reporter gene, with an agent to be tested; assessing the level of expression
of the
reporter gene in the presence of the agent; and comparing the level of
expression of the
reporter gene in the presence of the agent with a level of expression of the
reporter gene
is in the absence of the agent; wherein if the level of expression of the
reporter gene in the
presence of the agent differs, by an amount that is statistically significant,
from the level
of expression in the absence of the agent, then the agent is an agent that
alters
expression of the MAP3K9 gene or nucleic acid. An agent identified by this
method is
also contemplated.
2o The invention additionally comprises a method of identifying an agent that
alters
expression of a MAP3K9 nucleic acid, comprising contacting a solution
containing a
nucleic acid of the invention or a derivative or fragment thereof, with an
agent to be
tested; comparing expression of the nucleic acid, derivative or fragment in
the presence
of the agent with expression of the nucleic acid, derivative or fragment in
the absence of
2s the agent; wherein if expression of the nucleic acid, derivative or
fragment in the
presence of the agent differs, by an amount that is statistically significant,
from the
expression in the absence of the agent, then the agent is an agent that alters
expression
of the MAP3K9 nucleic acid. In certain embodiments, the expression of the
nucleic
acid, derivative or fragment in the presence of the agent comprises expression
of one or
3o more splicing variants(s) that differ in kind or in quantity from the
expression of one or
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more splicing variants) the absence of the agent. Agents identified by this
method are
also contemplated.
Representative agents that alter expression of a MAP3K9 nucleic acid
contemplated by the invention include, for example, antisense nucleic acids to
a
MAP3K9 gene or nucleic acid; a MAP3K9 gene or nucleic acid; a MAP3K9
polypeptide; a MAP3K9 gene or nucleic acid receptor, or other receptor; a
MAP3K9
binding agent; a peptidomimetic; a fusion protein; a prodrug thereof; an
antibody; and a
ribozyme. A method of altering expression of a MAP3K9 nucleic acid, comprising
contacting a cell containing a nucleic acid with such an agent is also
contemplated.
1o The invention further pertains to a method of identifying a polypeptide
which
interacts with a MAP3K9 polypeptide (e.g., a MAP3K9 polypeptide encoded-by a
nucleic acid of the invention, such as a nucleic acid comprising one or more
polymorphism(s) described herein), comprising employing a yeast two-hybrid
system
using a first vector which comprises a nucleic acid encoding a DNA binding
domain
and a MAP3K9 polypeptide, splicing variant, or a fragment or derivative
thereof, and a
second vector which comprises a nucleic acid encoding a transcription
activation
domain and a nucleic acid encoding a test polypeptide. If transcriptional
activation
occurs in the yeast two-hybrid system, the test polypeptide is a polypeptide,
which
interacts with a MAP3K9 polypeptide.
2o In certain methods of the invention, an asthma therapeutic agent is used.
The
asthma therapeutic agent can be ari agent that alters (e.g., enhances or
inhibits)
MAP3K9 polypeptide activity and/or MAP3K9 nucleic acid expression, as
described
herein (e.g., a nucleic acid agonist or antagonist).
Asthma therapeutic agents can alter polypeptide activity or nucleic acid
expression of a MAP3K9 nucleic acid by a variety of means, such as, for
example, by
providing additional polypeptide or upregulating the transcription or
translation of the
nucleic acid encoding the MAP3K9 polypeptide; by altering posttranslational
processing of the MAP3K9 polypeptide; by altering transcription of splicing
variants; or
by interfering with polypeptide activity (e.g., by binding to the MAP3K9
polypeptide,
or by binding to another polypeptide that interacts with MAP3K9, such as a
MAP3K9
binding agent as described herein), by altering (e.g., downregulating) the
expression,
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transcription or translation of a nucleic acid encoding MAP3K9; or by altering
interaction among MAP3K9 and a MAP3K9 binding agent.
In a further embodiment, the invention relates to asthma therapeutic agent,
such
as an agent selected from the group consisting of a MAP3K9 nucleic acid or
fragment
s or derivative thereof; a polypeptide encoded by a MAP3K9 nucleic acid (e.g.,
encoded
by a MAP3K9 nucleic acid having one or more polymorphism(s) such as those
described herein); a MAP3K9 receptor; a MA1'3K9 binding agent; a
peptidomimetic; a
fusion protein; a prodrug; an antibody; an agent that alters MAP3K9 gene or
nucleic
acid expression; an agent that alters activity of a polypeptide encoded by a
MA.P3K9
1o gene or nucleic acid; an agent that alters posttranscriptional processing
of a polypeptide
encoded by a MAP3K9 gene or nucleic acid; an agent that alters interaction of
a
MAP3K9 polypeptide with a MAP3K9 binding agent or receptor; an agent that
alters
transcription of splicing variants encoded by a MAP3K9 gene. or nucleic acid;
and
ribozymes. The invention also relates to pharmaceutical compositions
comprising at
1s least one asthma therapeutic agent as described herein.
The present invention pertains to methods of diagnosing a susceptibility to
asthma in an individual, comprising detecting a polymorphism in a MAP3K9
nucleic
acid, wherein the presence of the polymorphism in the nucleic acid is
indicative of a
susceptibility to asthma. The invention additionally pertains to methods of
diagnosing
2o asthma in an individual, comprising detecting a polymorphism in a MAP3K9
nucleic
acid, wherein the presence of the polymorphism in the nucleic acid is
indicative of
asthma. In one embodiment, in diagnosing asthma or susceptibility to asthma by
detecting the presence of a polymorphism in a MAP3K9 nucleic acid, the
presence of
the polymorphism in the MA.P3K9 nucleic acid can be indicated, for example, by
the
2s presence of one or more of the polymorphisms indicated in the Example
Section.
In other embodiments, the invention relates to methods of diagnosing a
susceptibility to asthma in an individual, comprising detecting an alteration
in the
expression or composition of a polypeptide encoded by a MAP3K9.nucleic acid in
a test
sample, in comparison with the expression or composition of a polypeptide
encoded by
3o a MAP3K9 nucleic acid in a control sample, wherein the presence of an
alteration in
expression or composition of the polypeptide in the test sample is indicative
of a
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susceptibility to asthma. The invention additionally relates to a method of
diagnosing
asthma in an individual, comprising detecting an alteration in the expression
or
composition of a polypeptide encoded by a MAP3K9 nucleic acid in;a test
sample, in
comparison with the expression or composition of a polypeptide encoded by
MAP3K9
nucleic acid in a control sample, wherein the presence of an alteration in
expression or
composition of the polypeptide in the test sample is indicative of asthma.
The invention also pertains to a method of treating a disease or condition
associated with a MAP3K9 polypeptide (e.g., asthma) in an individual,
comprising
administering a asthma therapeutic agent to the individual, in a
therapeutically effective
to amount. In certain embodiments, the asthma therapeutic agent is a MAP3K9
agonist; in
other embodiments, the asthma therapeutic agent is a MAP3K9 antagonist.
A transgenic animal comprising a nucleic acid selected from the group
consisting of an exogenous MAP3K9 gene or nucleic acid and a nucleic acid
encoding
a MAP3K9 polypeptide, is further contemplated by the invention.
15 In yet another embodiment, the invention relates to a method for assaying a
sample for the presence of a MAP3K9 nucleic acid, comprising contacting the
sample
with a nucleic acid comprising a contiguous nucleotide sequence which is at
least
partially complementary to a part of the sequence of said MAP3K9 nucleic acid
under
conditions appropriate for hybridization, and assessing whether hybridization
has
20 occurred between a MAP3K9 nucleic acid and said nucleic acid comprising a
contiguous nucleotide sequence which is at least partially complementary to a
part of
the sequence of said MAP3K9 nucleic acid; wherein if hybridization has
occurred, a
MAP3K9 nucleic acid is present in sample. In certain embodiments, the
contiguous
nucleotide sequence is completely complementary to a part of the sequence of
said
25 MAP3K9 nucleic acid. If desired, amplification of at least part of said
MAP3K9
nucleic acid can be performed.
In certain other embodiments, the contiguous nucleotide sequence is 100 or
fewer nucleotides in length and is either at least 80% identical to a
contiguous sequence
of nucleotides, at least 80% identical to the complement of a contiguous
sequence of
3o nucleotides; or capable of selectively hybridizing to said MAP3K9 nucleic
acid.
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In other embodiments, the invention relates to a reagent for assaying a sample
for the presence of a MAP3K9 gene or nucleic acid, the reagent comprising a
contiguous nucleotide sequence which is at least partially complementary to a
part of
the nucleic acid sequence of said MAP3K9 gene or nucleic acid; or comprising a
s contiguous nucleotide sequence which is completely complementary to a part
of the
nucleic acid sequence of said IVIAP3K9 gene or nucleic acid. Also contemplated
by the
invention is a reagent kit, e.g., for assaying a sample for the presence of a
MAP3K9
nucleic acid, comprising (e.g., in separate containers) one or more labeled
nucleic acids
comprising a contiguous nucleotide sequence which is at least partially
complementary
1o to a part of the nucleic acid sequence of the MAP3K9 nucleic acid, and
reagents for
detection of said label. In certain embodiments,-the labeled nucleic acid
comprises a
contiguous nucleotide sequence that is completely complementary to a part of
the
nucleotide sequence of said MAP3K9 gene or nucleic acid. In other embodiments,
the
labeled nucleic acid can comprise a contiguous nucleotide sequence which is at
least
15 partially complementary to a part of the nucleotide sequence of said MAP3K9
gene or
nucleic acid, and which is capable of acting as a primer for said MAP3K9
nucleic acid
when maintained under conditions for primer extension.
The invention also provides for the use of a nucleic acid which is 100 or
fewer
nucleotides in length and which is either: a) at least 80% identical to a
contiguous
2o sequence of nucleotides; b) at least 80% identical to the complement of a
contiguous
sequence of nucleotides; or c) capable of selectively hybridizing to said
MAP3K9
nucleic acid, for assaying a sample for the presence of a IVIAP3K9 nucleic
acid.
In yet another embodiment, the use of a first nucleic acid which is 100 or
fewer
nucleotides in length and which is either: a) at least 80% identical to a
contiguous
25 sequence of nucleotides; b) at least 80% identical to the complement of a
contib ous
sequence of nucleotides; or c) capable of selectively hybridizing to said
MAP3K9
nucleic acid; for assaying a sample for the presence of a MAP3K9 gene or
nucleic acid
that has at least one nucleotide difference from the first nucleic acid (e.g.,
a SNP as set
forth herein), such as for diagnosing a susceptibility to a disease or
condition associated
3o with a MAP3K9.
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The invention also pertains to the use of a MLK family kinase inhibitor for
the
manufactor of a medicament for treatment for asthma in an individual, wherein
the
individual has at least one risk factor selected from the group consisting of
an at-risk
haplotype forasthma; an at-risk haplotype in the MAP3K9 gene; a polymorphism
in a
MAP3K9 nucleic acid; dysregulation of MAP3K9 mRNA expression, dysregulation of
a MAP3K9 mRNA isoform; increased MLK1 protein expression; increased MLKl
biochemical activity; and increased MKL1 protein isoform expression. In
certain
embodiements, wherein the MLK family kinase inhibitor is a MLK1 inhibitor, for
example CEP-1347 (Formula III) and its optically pure stereoisomers, mixtures
of
1o stereoisomers and salts or an indolocarbazole derivative and its optically
pure
stereoisomers, mixtures of stereoisomers and salts.
Also contemplated by the present invention is use of a first nucleic acid
molecule for diagnosing asthma or a susceptibility to asthma in a sample from
an
individual to be diagnosed, comprising detecting in the sample the presence or
absence
15 of a second nucleic acid molecule of at least one marker of an at-risk
haplotype
associated with the MAP3K9 gene selected from the group consisting of
haplotype 1,
2, 3, 4, 5, 6, 7 of Table 1 and combinations thereof by contact with the first
nucleic acid,
wherein the presence of one or more markers is indicative of asthma or a
susceptibility
to asthma. The presence or absence of the marker can be accomplished by
enzymatic
2o amplification of nucleic acids, electrophoretic analysis, restriction
fragment length
polymorphism analysis or sequence analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the invention
25 will be apparent from the following more particular description of
preferred aspects
of the invention, as illustrated in the accompanying drawings.
The patent or application file contains at least one drawing executed in
color.
Copies of this patent or patent application publication with color drawings
will be
provided by the Office upon request and payment of the necessary fee.
3o FIG. 1 is an illustration of the JNK signaling cascade.
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FIG. 2 shows examples of asthma pedigrees used in the linkage analysis.
Unaffected siblings of patients are not shown and sex indicators have been
shuffled
for some individuals in the top two generations to protect privacy. The
darkened
squares and circles represent affected men and women, respectively. The
slashed
symbols represent deceased individuals.
FIG. 3 shows multipoint allele-sharing Iod score of chromosome I4. A
framework genome scan is shown by a dotted line. A fine mapping lod score of
4.00 was detected within the peak region after adding 34 microsatellite
markers to
obtain a marker density of less than 0.2 cM, using deCODE's high-density
genetic
to map to determine genetic distances. The multipoint lod score is on the y-
axis and
centimorgan distance from the p-terminus of the chromosome is on the x-axis.
FIG. 4 shows mean age, gender, smoking history, % patients with positive
skin tests, mean total IgE Level, asthma severity level and lung function
values
expressed as % predicted forced expiratory volume in one second (% FEVl),
forced
1s expiratory volume in one second/forced vital capacity ratio (FEV 1/FVC) and
% of
patients requiring < 2.0 mg/ml and <8 mg/ml of methacholine, respectively, to
produce 20% drop in FEV 1 (PCZO) for the asthma study population.
FIG. 5 shows a map of the MAP3K9 gene with SNPs and microsatellites.
FIGS. 6:1 and 6.2 show the linkage disequilibrium (LD) plot for chr 14q24.2-
20 3 region with all markers present. Mapping of the linkage disequilibrium
blocks
using multiple microsatellite and SNP markers (x- and y-axis), covering the
one lod
drop on chromosome 14q24.2-3 (FIG. 6.1) and the 3 LD blocks (FIG. 6.2) that
show
the strongest association to asthma and include the MAP3I~9gene. The d-dimer
plot
is shown above the transverse line (i.e., LD of any two markers next to each
other)
25 and the corresponding p-values are shown below the Line. The significance
of the d-
dimer and the p values is reflected in the difference in black and white
intensities
(vertical axis to the right). FIG. 6.2 shows a narrowly focused section of the
linkage
disequilibrium plot of FIG. 6.1 in the region where the markers comprising the
haplotypes reside.
3o FIGS. 7:1 to 7.19 show the nucleic acid sequence for MAP3K9 with coding
regions (SEQ ID NO: 1). The upper case letters indicate the coding regions
(exons).
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FIG. 8 is the amino acid sequence of MAP3K9 (SEQ ID NO: 2).
FIG. 9 is a graph showing the MAP3K9 expression in asthma airway tissue
using RT-PCR. OLD: Surgical resection of non-cancer tissue from individuals;
OLD-1 and OLD-2, with obstructive lung disease. CO: Control Lung (surgical
resection of non-cancer tissue from 2 individuals; CO-1 and CO-2). The results
show a markedly enhance expression of Map3K9 expression in asthma compared to
control airway tissue.
FIG. 10 is a graph showing MAP3K9 expression in PBM cells from asthma
patients vs. control subjects. The results indicate a significant enhanced
expression
Zo in PBM cells from asthma patients for variant b comparable to lung data.
FIGS. 1 l.l-11.17 shows the mRNA and amino acid sequences for the splice
variants a-e.
FIGS. 12.1 to 12.430 are a table of microsatellite and SNP markers with the
forward and reverse primer sequences. Also provided are the amplimer sequences
1s and the nucleic acid start and end positions.
DETAILED DESCRIPTION OF THE INVENTION
Extensive genealogical information for a population with population-based
lists of patients has been combined with powerful genome sharing methods to
map
2o the first major locus in asthma with genome-wide significance. A genome-
wide
scan on patients, related within 6 meiotic events, diagnosed with asthma and
their
unaffected relatives has been completed. Locus AS 1 on chromosome 14q24 has
been identified through linkage studies to be associated with asthma. This
locus
does not correspond to known susceptibility loci for asthma, and represents
the first
2s mapping of a gene for asthma on chromosome 14q24. Until now there have been
no
known linkage studies of asthma in humans showing connection to this region of
the
chromosome. Based on the linkage studies conducted, Applicants have discovered
a
direct relationship between the genome wide significant (GWS) AS 1 locus and
asthma.
3o Linkage and association studies have identified a gene MAP3K9, that is
located on 14q24.2-3 and is in the middle of a region where there is a
significant 3-
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marker haplotype that overlays the MAP3K9 gene and is present in up to 14% of
patients and 5% in controls (Relative Risk 2.784). This haplotype is the most
common of seven overlapping haplotypes that extend over a 286 kb region
covering
one whole and a part of two other LD blocks. See Tables 1 and 2 in the
Examples
Section. All the patients studied have physician-diagnosed asthma (2/3 of who
also
have atopy as determined by positive skin tests and/or elevated IgE). All
seven
genetic marker haplotypes are shown in Table 1 and their p-values (p-val) and
relative risk ratios (r) are shown in Table 2. Initially the MAP3K9 gene was
isolated
from a human epithelial tumor cell line. Expression of MAP3K9 has been found
in
to lung tumor cell and different cell lines from the immune system as well as
in smooth
muscle cells.
MAP3K9 is a part of Mitogen-Activated Protein Kinase (MAPK) signal
transduction pathways, which are among the most widespread mechanisms of
eukaryotic cell regulation. In all eukaryotic cells there are multiple MAPK
15 pathways, each reacting to different stimuli. The regulation of MAP3Ks
represents
an entry point into the MAPK pathways and is therefore complex.
Several kinases have been targeted therapeutically in various diseases and
their function shown to be effectively modulated using small molecules. An
example is the development of new small molecule inhibitors ~of p38 kinase
that are
2o being considered a potential new therapy for asthma. The MAP3K9 kinase is a
gene
that overlays the center of a haplotype that is almost three times more common
in
patients than in controls (i.e., RR 2.8); no asthma gene has been isolated
today that
carries a higher risk than this gene. The gene is in the pathway of cell
signaling that
involves mitogenic and second messenger activities (including IP3 regulation).
2s Thus, this gene is a strong therapeutic target candidate for the
development of new
small molecule therapy for patients with asthma.
MAP3K9 is a member of the Mixed Lineage Kinase (MLK) family. The
kinase domain of MLK family kinases has amino acid sequence similarity to both
the tyrosine-specific and the serine/threonine-specific kinase classes
although
3o MAP3K9 is a serine/threonine kinase. Known serine/threonine phosphorylation
substrates of MLK family members are the kinases MKK7 or MKK4. MLK family
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kinases, MKK7 and MKK4 are all known members of the JNK signalling cascade
(see FIG. 1). Within the JNK signalling cascade there are three tiers of
kinases
linking stimuli such as cellular stress, injury or cytokines through the JNKs
to
transcriptional regulation via phosphorylation of c- Jun and related
transcription
factors (Jung & JunD).
The JNKs and a substrate of the JNKs, c-Jun have been implicated in the
positive regulatory control of cell death or apoptosis. This could relate to
asthma
through two fundamental processes: 1) dysregulation of immune function through
inappropriate cell death or lack thereof; and 2) inappropriate vascular and
airway
io smooth muscle cell hyperplasia and consequent airway thickening increasing
the
risk or severity of asthma. While the MAP3K9 gene itself has never been
associated
with asthma, the JNK pathway has been implicated in the following processes
that
relate to various aspects of asthma:
15 1. Nitric oxide-induced AP-1 activation in human bronchial epithelial cells
T-helper type 2 cell differentiation and production of the pro-
inflammatory cytokines IFN-gamma and TGF-beta;
2. Allergen-induced airway inflammation in mice (this is reduced in Jun-B
20 deficient mice); and
3. LPS induced IL-10 and IL-13 production by mast cells relating to airway
inflammation in asthma.
25 The MLK-1 & the JNK pathway have been shown to regulate TNFa & IL-lb
secretion, both of which are important cytokines in asthma. Moreover, the TH2-
type
cytokines, IL10, IL13 and ILS, all of which are effective modulators of airway
smooth muscle (ASM) contractility and relaxation, exert their effects on
airway
hyperresponsiveness, at least in part, through the induced expression and
autocrine
so action of ILlbeta (Hakonarson and Grunstein, Respir Physiol Neuf-obiol. Sep
16;137(2-3):263-76 (2003); Nakae S et al., Intlfnmunol. Apr;lS(4):483-90
(2003)).
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Apart from regulation of IL-lb and TNFa secretion, the MLK-1 signaling
pathway,
through JNK and c jun, is involved in the regulation of various other pro-
inflammatory cytokines, including IL6, IL8 as well as various chemokines and
TH1-
type cytokines (1L2, IL12, lFNg) through its regulation of AP-1 transcription
factor
activity. IL-lb and TNFa have been shown to be critically involved in the
local
regulation of airway inflammation (Wuyts et al., Respir Med. Ju1;97(7):811-7
(2003)). In addition, ILIb and to a lesser extend, TNFa are key regulators of
ASM
contractility and relaxation, two of the cardinal phenotypic features of
asthma
(Hakonarson et al. Mechanism of cytokine-induced modulation of beta-
adrenoceptor
1o responsiveness in airway smooth muscle. (Hakonarson et al., ,l Clih Invest.
Jun
1;97(11):2593-600 (1996); Autocrine role of interleukin lbeta in altered
responsiveness of atopic asthmatic sensitized airway smooth muscle. (J Clin
Invest.
Jan 1;99(1):117-24 (1997)). Both ILIb and TNFa are potent mitogens in ASM and
mucus glands (Page et al., Ff~ont Biosci. 5: 258-267, (2000); Stylianou et
al.,Int. J.
Biochena Cell Biol. Oct;30(10):1075-9 (1998)) and thereby account for, at
least in
part, the increased ASM muscle mass and mucus hypersecretion, the other
principal
features of the asthma phenotype. Therefore, an inhibitor of MLK-1 such as the
compound, CEP-1347, that has been shown to potently inhibit the release of TNF
IL-1 secretion in various cellular systems, would be anticipated to block
asthma.
2o Chemical inhibitors of MLK family kinases have been described (Maroney
et al., JBC, 276(27): 25302-25308 (2001)). For example, CEP-1347 (formula III)
directly inhibits MLK family kinases including MLK1, MLK2 and MLK3.
Inhibitory potency defined as the concentration of CEP-1347 needed to inhibit
MLK
kinase activity in a standardized biochemical assay is 38nM ~l7nM, 51 nM~9nM
and 23 nM ~O.lnM for MLK1, MLK2 and MLK3 respectively. CEP-1347 also
effectively inhibits the activity of MLK kinases within intact cells with
inhibitory
potencies of 61 nM ~ 11 nM, 82 nM ~ lOnM and 39 nM ~3nM for MLK1, MLK2
and MLK3, respectively. The kinetics of CEP-1347 inhibition of MLK kinases is
consistent with a mode of action competitive with the binding of adenosine
3o triphosphate in the MLK active site.
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In addition, CEP-1347 has been shown to inhibit the production of TNFa and
IL-1(3 by cultured cells under standard in vit~'o pharmacological testing
procedures
(see WO 97/49406). Also in animals, CEP-1347 reduces production of TNFa and
IL-1 /3 by mice after challenge with lipopolysaccharide (LPS) and provides
protection from LPS-induced death. Since ILlb and TNFa are key regulators of
ASM contractility (i.e., bronchial hyperresponsiveness) and relaxation, two of
the
cardinal phenotypic features of asthma and are the principal cytokines
responsible
for ASM and epithelial gland hypertrophy and hyperplacia as well as having
profound autocrine effects that promote local airway inflammation, inhibition
of IL-
lb and TNFa would be anticipated to benefit asthma. Thus, CEP-1347 possesses
pharmaceutic properties indicative of its potential benefit for the treatment
of human
diseases including asthma resulting from MAP3K9 gene dysregulation and
consequent dysregulated production of MLKI.
TARGET AT-RISK POPULATIONS
Target populations for the methods described herein, include individuals
having an at-risk factor in a MAP3K9 gene haplotype or a polymorphism in the
MAP3K9 gene. These at-risk individuals with the MAP3K9 DNA risk haplotype
are a subset of all patients with asthma. Risk populations also include
individuals
2o with dysregulation of MAP3K9 gene transcription and dysregulation of a
MAP3K9
mRNA isoform for example, an increase in RNA transcripts of MLK1 protein or an
isoform of the protein. At-risk populations can have increased MLK1
biochemical
activity, increased levels of MLK1 protein or a particular MLK1 protein
isoform
Level can be increased. Thus, at-risk populations with MAP3K9 gene associated
asthma can have differences with DNA sequences, RNA regulation and protein
expression. These underlying genetic and protein differences can be
manipulated in
the type and extent of treatment provided.
Isolation and identification of target populations for treatment of
individuals
are advantageous for many reasons. For example, it can be possible to identify
3o individuals in a specific at-risk population that respond to a specific
treatment, such
as treatment with one of the compounds disclosed herein. Samples from
individuals
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with asthma can be tested in a diagnostic assay such as those described herein
to
assist in identifying the underlying~genetic cause of the disease.
Direct efficient treatment of the subset of the population with an underlying
genetic cause of asthma is possible utilizing the diagnositic and treatments
described
s in the instant application. Knowledge of the underlying cause is useful in
identifying an individual likely to be a responder to a particular treatment
designed
for the underlying cause from an individual that is a non-responder to this
treatment.
For example, if an asthmatic individual is diagnosed as a carrier of a DNA
based
haplotype or variant of the isofonn of MAP3K9 protein, this individual would
be
to more likely to repond to one of the compounds described herein that
interfere with
the perturbed 3NK pathway. Thus, an individual identified by diagnosis as a
target
at-risk population can have a treatment tailored for the specific diagnosis,
thereby
reducing possible side effects or other deleterious reactions an individual
can have
with conventional treatment.
15 Generally, conventional treatments typically correct only the symptoms
associated with the disease and do not prevent, delay or arrest the
progression of the
disease. Therefore, specific diagnosis of the target at-risk population and
subsequent
treatments as described herein, allows the patient to he treated to not only
reduce the
symptoms associated with the disease but also hold the progression of the
disease by
2o remodling the underlying genetic problem.
NUCLEIC ACID THERAPEUTIC AGENTS
In another aspect, a nucleic acid of the invention; a nucleic acid
complementary to a nucleic acid of the invention; or a portion of such a
nucleic acid
25 (e.g., an oligonucleotide as described below); or a nucleic acid encoding a
member
of the MLK pathway (e.g., MAP3K9), can be used in "antisense" therapy, in
which
a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the
mRNA
and/or genomic DNA of a nucleic acid is administered or generated in situ. The
antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA
inhibits
3o expression of the polypeptide encoded by that mRNA and/or DNA, e.g., by
inhibiting translation andlor transcription. Binding of the antisense nucleic
acid can
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be by conventional base pair complementarity, or, for example, in the case of
binding to DNA duplexes, through specific interaction in the major groove of
the
double helix.
An antisense construct can be delivered, for example, as an expression
plasmid as described above. When the plasmid is transcribed in the cell, it
produces
RNA that is complementary to a portion of the mRNA and/or DNA that encodes the
polypeptide for the member of the IVIZ,K pathway (e.g., MAP3K9).
Alternatively,
the antisense construct can be an oligonucleotide probe that is generated ex
vivo and
introduced into cells; it then inhibits expression by hybridizing with the
mRNA
Io and/or genomic DNA of the polypeptide. In one aspect, the oligonucleotide
probes
are modified oligonucleotides that are resistant to endogenous nucleases,
e.g.,
exonucleases and/or endonucleases, thereby rendering them stable i~r vivo.
Exemplary nucleic acid molecules for use as antisense oligonucleotides are
phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also
U.S. Pat. Nos. 5,176,996, 5,264,564 and 5,256,775). Additionally, general
approaches to constructing oligomers useful in antisense therapy are also
described,
for example, by Van der Krol et al. (Biotech~iques 6:958-976 (1988)); and
Stein et
al. (Cancer Res. 48:2659-2668 (1988)). With respect to antisense DNA,
oligodeoxyriboriucleotides derived from the translation initiation site are
preferred.
2o To perform antisense therapy, oligonucleotides (mRNA, cDNA or DNA) are
designed that are complementary to mRNA encoding the polypeptide. The
antisense
oligonucleotides bind to mIRNA transcripts and prevent translation. Absolute
complementarity, although preferred, is not required. A sequence
"complementary"
to a portion of an RNA, as referred to herein, indicates that a sequence has
sufficient
complementarity to be able to hybridize with the RNA, forming a stable duplex;
in
the case of double-stranded antisense nucleic acids, a single strand of the
duplex
DNA may thus be tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the length of
the
antisense nucleic acid, as described in detail above. Generally, the longer
the
3o hybridizing nucleic acid, the more base mismatches with an RNA it may
contain and
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still form a stable duplex (or triplex, as the case may be). One skilled in
the art can
ascertain a tolerable degree of mismatch by use of standard procedures.
The oligonucleotides used in antisense therapy can be DNA, RNA, or
chimeric mixtures or derivatives or modified versions thereof, single-stranded
or
s double-stranded. The oligonucleotides can be modified at the base moiety,
sugar
moiety, or phosphate backbone, for example, to improve stability of the
molecule,
hybridization, etc. The oligonucleotides can include other appended groups
such as
peptides (e.g. for targeting host cell receptors i~ vivo), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci.
USA
86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-652
(1987); PCT International Publication No. WO 88/09810) or the blood-brain
barrier
(see, e.g., PCT International Publication No. WO 89/10134), or hybridization-
triggered cleavage agents (see, e.g., Krol et al., BioTeclaniques 6:958-976
(1988)) or
intercalating agents. (See, e.g., Zon, Pharm.Res. 5: 539-549 (1988)). To this
end,
1s the oligonucleotide may be conjugated to another molecule (e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, hybridization-
triggered
cleavage agent).
The antisense molecules are delivered to cells that express the member of the
MLK pathway i~ vivo. A number of methods can be used for delivering antisense
DNA or RNA to cells; e.g., antisense molecules can be injected directly into
the
tissue site, or modified antisense molecules, designed to target the desired
cells (e.g.,
antisense linked to peptides or antibodies that specifically bind receptors or
antigens
expressed on the target cell surface) can be administered systematically.
Alternatively, in a preferred aspect, a recombinant DNA construct is utilized
in
2s which the antisense oligonucleotide is placed under the control of a strong
promoter
(e.g., pol III or pol II). The use of such a construct to transfect target
cells in the
patient results in the transcription of sufficient amounts of single stranded
RNAs that
will form complementary base pairs with the endogenous transcripts and thereby
prevent translation of the mRNA. For example, a vector can be introduced in
vivo
such that it is taken up by a cell and directs the transcription of an
antisense RNA.
Such a vector can remain episomal or become chromosomally integrated, as long
as
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it can be transcribed to produce the desired antisense RNA. Such vectors can
be
constructed by recombinant DNA technology methods standard in the art and
described above. For example, a plasmid, cosmid, YAC or viral vector can be
used
to prepare the recombinant DNA construct that can be introduced directly into
the
tissue site. Alternatively, viral vectors can be used which selectively infect
the
desired tissue, in which case administration may be accomplished by another
route
(e.g., systemically).
In another aspect of the invention, small double-stranded interfering RNA
(RNA interference (RNAi)) can be used. RNAi is a post-transcription process,
in
1o which double-stranded RNA is introduced, and sequence-specific gene
silencing
results, though catalytic degradation of the targeted mRNA. See, e.g.,
Elbashir,
S.M, et al., Natuf°e 411:494-498 (2001); Lee, N.S., Nature Biotech.
19:500-505
(2002); Lee, S-K. et al., Nature Medicine 8(7):681-686 (2002) the entire
teachings
of these references are incorporated herein by reference.
1s RNAi is used routinely to investigate gene function in a high throughput
fashion or to modulate gene expression in human diseases (Chi et al., PNAS,100
(11):6343-6346 (2003)).
Introduction of long double standed RNA leads to sequence-specific
degradation of homologous gene transcripts. The long double stranded RNA is
2o metabolized to small 21-23 nucleotide siRNA (small interfering RNA). The
siRNA
then binds to protein complex RISC (RNA-induced silencing complex) with dual
function helicase. The helicase has RNAas activity and is able to unwind the
RNA.
The unwound si RNA allows an antisense strand to bind to a target. This
results in
sequence dependent degradation of cognate mRNA. Aside from endogenous RNAi,
25 exogenous RNAi, chemically synthesized or recombinantly produced can also
be
used.
Using non-intronic portions of the MAP3K9 gene such as corresponding
mRNA portions of SEQ ID NO: 1 , target regions of the MAP3K9 gene that are
accessible for RNAi are targeted and silenced. With this technique it is
possible to
3o conduct a RNAi gene walk of the nucleic acids of MAP3K9 and determine the
amount of inhibition of the protein product. Thus, it is possible to design
gene-
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specific therapeutics by directly targeting the mRNAs of Asthma-related MAP3K9
gene.
Endogenous expression of a member of the MLK pathway (e.g., MAP3K9)
can also be reduced by inactivating or "knocking out" the gene or its promoter
using
targeted homologous recombination (e.g., see Smithies et al., Nature 317:230-
234
(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-
321 (1989)). For example, an altered, non-functional gene of a member of the
MLK
pathway (or a completely unrelated DNA sequence) flanked by DNA homologous to
the endogenous gene (either the coding regions or regulatory regions of the
gene)
1o can be used, with or without a selectable marker and/or a negative
selectable marker,
to transfect cells that express the gene in vivo. Insertion of the DNA
construct, via
targeted homologous recombination, results in inactivation of the gene. The
recombinant DNA constructs can be directly administered or targeted to the
required
site in vivo using appropriate vectors, as described above. Alternatively,
expression
of non-altered genes can be increased using a similar method: targeted
homologous
recombination can be used to insert a DNA construct comprising a non-altered
functional gene, or the complement thereof, or a portion thereof, in place of
a gene
in the cell, as described above. In another aspect, targeted homologous
recombination can be used to insert a DNA construct comprising a nucleic acid
that
2o encodes a polypeptide variant that differs from that present in the cell.
Alternatively, endogenous expression of a member of the MLK pathway can
be reduced by targeting deoxyribonucleotide sequences complementary to the
regulatory region of the member of the MLK pathway (i. e., the promoter and/or
enhancers) to form triple helical structures that prevent transcription of the
gene in
target cells in the body. (See generally, Helene, C., Anticancer Drug Des.,
6(6):569-
84 (1991); Helene, C. et al., Ahn. N. Y. Acad. Sci. 660:27-36 (1992); and
Maher, L.
J., Bioassays 14(12):807-15 (1992)). Likewise, the antisense constructs
described
herein, by antagonizing the normal biological activity of one of the members
of the
MLK pathway, can be used in the manipulation of tissue, e.g., tissue
differentiation,
3o both in vivo and fog ex vivo tissue cultures. Furthermore, the anti-sense
techniques
(e.g., microinjection of antisense molecules, or transfection with plasmids
whose
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transcripts are anti-sense with regard to a nucleic acid RNA or nucleic acid
sequence) can be used to investigate the role of one or more members of the
MLK
pathway in the development of disease-related conditions. Such techniques can
be
utilized in cell culture, but can also be used in the creation of transgenic
animals.
The therapeutic agents as described herein can be delivered in a composition,
as described above, or by themselves. They can be administered systemically,
or
can be targeted to a particular tissue. The therapeutic agents can be produced
by a
variety of means, including chemical family kinase; recombinant production;
ifz vivo
production (e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to
Meade et
al.), for example, and can be isolated using standard means such as those
described
herein. In addition, a combination of any of the above methods of treatment
(e.g.,
administration of non-altered polypeptide in conjunction with antisense
therapy
targeting altered mRNA for a member of the MLK pathway; administration of a
first
splicing variant in conjunction with antisense therapy targeting a second
splicing
variant) can also be used.
The invention additionally pertains to use of such therapeutic agents, as
described herein, for the manufacture of a medicament for the treatment of
asthma
and other MAPK39 gene linked respiratory diseases, e.g., using the methods
described herein.
METHODS OF THERAPY
As a result of these discoveries, methods are now available for the treatment
of asthma, and other respiratory diseases including but not limited to:
chronic
obstructive pulmonary disease, chronic bronchitis and other MAP3K9 gene linked
respiratory diseases and potentially also other inflammatory diseases (such as
rheumatoid arthritis, psoriasis, multiple sclerosis and inflammatory bowel
disease)
with the use of MLKI inhibitors, such as agents that inhibit MLK1 kinase
activity
and thus decrease cellular production of cytokines and other inflammatory
mediators
as a consequence of cell stimulation. The term "treatment" as used herein,
refers not
only to ameliorating symptoms associated with the disease or condition, but
also
preventing or delaying the onset of the disease or condition; preventing or
delaying
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the occurrence of a second episode of the disease or condition; lessening the
severity
or frequency of symptoms of the disease or condition; and/or also lessening
the need
for concomitant therapy with other drugs that ameliorate symptoms associated
with
the disease or condition , e.g., corticosteroids. Methods are additionally
available
for assessing an individual's risk for developing asthma and/or other
respiratory
diseases. In one aspect, the individual to be treated is an individual who is
susceptible (at an increased risk) for asthma, or for whom the severity of the
disease
or condition is associated with DNA at-risk haplotypes in the MAP3K9 gene,
dysregulation of MAP3K9 mRNA expression, or increased amount of MLKI
protein and/or biochemical activity and/or an increased amount of a particular
protein isoform or MLK1.
METHODS OF TREATMENT
The present invention encompasses methods of treatment (prophylactic
and/or therapeutic, as described above) for asthma and other respiratory
diseases in
individuals, such as individuals in the target populations described above, as
well as
for other diseases and conditions associated with MAP3K9 or with other members
of the MLK family kinase. Members of the "JNK pathway," in particular members
of the MLK family kinases as used herein, include other polypeptides (e.g.,
2o enzymes, receptors) and other molecules that are associated with JNK
pathway
signaling, including the transcription factors, c jun, v-fos and AP-1, or
production of
an MLK protein, such as transcription of the MAP3K9 gene and production of
MLK-1 protein and or the stability of MLK-1 protein. In particular, the
invention
relates to methods of treatment for asthma or a susceptibility to asthma,
using an
asthma therapeutic agent. An "asthma therapeutic agent" is an agent that
alters (e.g.,
enhances or inhibits) MAP3K9 polypeptide activity and/or MAP3K9 nucleic acid
expression, as described herein (e.g., an asthma nucleic acid antagonist). In
certain
aspects, the asthma therapeutic agent alters activity and/or nucleic acid
expression of
MAP3K9.
Asthma therapeutic agents can alter MAP3K9 polypeptide activity or nucleic
acid expression by a variety of means, such as, for example, by decreasing
MAP3K9
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polypeptide or by downregulating the transcription or translation of the
MAP3K9
nucleic acid; by altering posttranslational processing of the MAP3K9
polypeptide;
by altering transcription of MAP3K9 splicing variants; or by interfering with
MAP3K9 polypeptide activity (e.g., by binding to a MAP3K9 polypeptide), or by
binding to another polypeptide that interacts with MAP3K9, by altering (e.g.,
downregulating) the expression, transcription or translation of a MAP3K9
nucleic
acid, or by altering (e.g., agonizing or antagonizing) activity.
In particular, the invention relates to methods of treatment for asthma or
susceptibility to asthma, for example: for individuals in an at-risk
population such as
l0 those described; as well as methods of treatment for asthma or other
respiratory
diseases; methods for reducing risk of asthma; and/or for decreasing cellular
cytokines through the use of agents that inhibit MLK kinase activity, for
example
CEP-1347, or compounds as encompassed by formula I and Tables A and B. The
invention additionally pertains to use of one or more MKL inhibitors, as
described
15 herein, for the manufacture of a medicament for the treatment asthma and
other
respiratory diseases, e.g., using the methods described herein.
In the methods of the invention, the "asthma therapeutic agent" is a "MLK
family inhibitor". In one aspect, a "MLK family inhibitor" is an agent that
inhibits
MAP3K9 polypeptide activity and/or MAP3K9 nucleic acid expression, as
2o described herein (e.g., a nucleic acid antagonist). In another aspect, a
MLK family
inhibitor is an agent that inhibits polypeptide activity and/or nucleic acid
expression
of multiple members of the MLK family kinases in the JNK pathway. In still
another aspect, a MLK family inhibitor is an agent that alters activity or
metabolism
of a MLK kinase (e.g., an antagonist of a MLK kinase; an antagonist of a MLK
25 kinase activator). In certain aspects, the MLK inhibitor alters activity
and/or nucleic
acid expression of MAP3K9.
MLK family kinase inhibitors can alter polypeptide activity or nucleic acid
expression of a member of the JNK pathway, in a variety of means, such as, fox
example, by catalytically degrading, downregulating or interfering with the
3o expression, transcription or translation of a nucleic acid encoding the
member of the
JNK pathway; by altering posttranslational processing of the polypeptide; by
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altering transcription of splicing variants; or by interfering with
polypeptide activity
(e.g., by binding to the polypeptide, or by binding to another polypeptide
that
interacts with that member of the JNK pathway, such as a MAP3K9 or MLK1 ,
binding agent as described herein or some other binding agent of a member of
the
pathway); by altering interaction among two or more members of the MLK family
kinases in the JNK pathway; or by antagonizing activity of a member of the JNK
pathway.
Representative MLK family kinase inhibitors include the following:
agents that inhibit activity of a member of the MLK signalling pathway (e.g.,
to MAP3K9 proteins, MLKl) for example, CEF-1347, and compounds represented by
formula I and Tables A and B; agents that inhibit activity of activators of
members
of the MLK pathway, such as MLKI activators, MLK2 activators, and MLK3
activators, or agents that bind to a MLK family kinases or otherwise affect
the
activity of the MLK signaling pathway (for example inhibitors of RAC 1/
Cdc42.,
15 MKK4 and MKK7), other agents that alter (e.g., inhibit or antagonize)
expression of
a member of the JNK pathway, such as MAP3K9 or MLK family kinase nucleic
acid expression or polypeptide activity, or that regulate transcription of
MAP3K9
splicing variants or (e.g., agents that affect which splicing variants are
expressed, or
that affect the amount of each splicing variant that is expressed);
polypeptides described herein and/ or splicing variants encoded by the
MAP3K9 nucleic acid or fragments or derivatives thereof;
other polypeptides (e.g., MAP3K9 activators); MAP3K9 binding agents; or
agents that affect (e.g., increase) activity or decrease MAP3K9 polypeptide
stability;
antibodies to MLKs, such as an antibody to an altered MAP3K9 polypeptide,
or an antibody to a non-altered MAP3K9 polypeptide, or an antibody to a
particular
splicing variant encoded by a MAP3K9 nucleic acid as described above; for
3o example MLK3 (A-20):SC 15068, Santa Cruz Biotechnology, Inc., Santa Cruz,
CA)
that can be delivered intracellularly to decrease activity or amount of MLK-1;
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antisense nucleic acids or small double-stranded interfering RNA to nucleic
acids encoding MAP3K9 or a MLK family kinase or other member of the JNK
pathway, or fragments or derivatives thereof, including antisense nucleic
acids to
nucleic acids encoding the MAP3K9 or other MLK family kinase polypeptides, and
vectors comprising such antisense nucleic acids (e.g., nucleic acid, cDNA,
and/or
mRNA, double-stranded interfering RNA, or a nucleic acid encoding an active
fragment or derivative thereof, or an oligonucleotide; for example, the
complement
of one of SEQ ID Nos: 1 or 3, or a nucleic acid complementary to the nucleic
acid
encoding SEQ ID NO: 2, or fragments or derivatives thereof);
other agents that alter (e.g., inhibit ar antagonize) expression of a member
of
the JNK pathway, such as MAP3K9 or MLK family kinase nucleic acid expression
or polypeptide activity, or that regulate transcription of MAP3K9 splicing
variants
or (e.g., agents that affect which splicing variants are expressed, or that
affect the
amount of each splicing variant that is expressed).
More than one MLK family kinase inhibitor can be used concurrently, if
desired.
The therapy is designed to alter activity of a MAP3K9 polypeptide, a MLK
2o family kinase or another member of the JNK pathway in an individual, such
as by
inhibiting or antagonizing activity. For example, a MLK family kinase
inhibitor can
be administered in order to decrease family kinase of MLKs within the
individual, or
to downregulate or decrease the expression or availability of the MAP3K9
nucleic
acid or specific splicing variants of the MAP3K9 nucleic acid. Downregulation
or
decreasing expression or availability of a native MAP3K9 nucleic acid or of a
particular splicing variant could minimize the expression or activity of a
defective
nucleic acid or the particular splicing variant and thereby minimize~the
impact of the
defective nucleic acid or the particular splicing variant.
The MLK family kinase inhibitors) are administered in a therapeutically
3o effective amount, i.e., an amount that is sufficient to treat the disease
or condition,
such as by ameliorating symptoms associated with the disease or condition,
preventing or delaying the onset of the disease or condition, and/or also
lessening
the severity or frequency of symptoms of the disease or condition. The amount
which will be therapeutically effective in the treatment of a particular
individual's
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disease or condition will depend on the symptoms and severity of the disease,
and
can be determined by standard clinical techniques. In addition, irc vitro or
ih vivo
assays may optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation will also depend on the route
of
administration, and the seriousness of the disease or disorder, and should be
decided
according to the judgment of a practitioner and each patient's circumstances.
Effective Bases may be extrapolated from dose-response curves derived from ifz
vitro or animal model test systems.
In certain aspects of the invention, the MLK family kinase inhibitor agent is
1o an agent that inhibits activity of MAP3K9. In certain methods of the
invention, the
agents set forth in Formula I , Tables A and B and Formula III (GEP-1347) can
be
used for prophylactic and/or therapeutic treatment for diseases and conditions
associated with MAP3K9 or with other members of MLK family kinases or other
members of the JNK pathway, or with increased MLK family kinase activity. In
~s particular, they can be used for treatment for asthma or susceptibility to
astluna, such
as for individuals in an at-risk population as described above, (e.g., based
on
identified risk factors) and individual requirement treatment.
In one aspect of the invention, the MLK family kinase inhibitor is an
inhibitor of MLK1 such as CEP-1347 (also known as KT7515, Cephalon, Inc., W.
2o Chester, PA) its optically pure stereoisomers, mixtures of stereoisomers,
salts,
chemical derivatives, analogues, or other compounds inhibiting MAP3K9 that
effectively decrease MLK family kinase when administered to humans.
The compounds contemplated as MLK family kinase inhibitors in the
methods described herein can be represented by the following formula: Formula
I
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R3
"
R~
R2
or a pharmaceutically acceptable salt thereof, wherein:
one of Rl and R2 is selected from the group consisting of
a) --CO(CH2)~ R4, wherein j is 1 to 6, and R4 is selected from the group
consisting of
1) hydrogen and a halogen;
2) --NRS R6, wherein RS and R6 independently are hydrogen, substituted lower
alkyl,
unsubstituted lower alkyl, substituted aryl, unsubstituted aryl, substituted
heteroaryl,
unsubstituted heteroaryl, substituted aralkyl, unsubstituted aralkyl, lower
1o alkylaminocarbonyl, or lower alkoxycarbonyl; or RS and R6 are combined with
a
nitrogen atom to form a heterocyclic group;
4) --SRZ', wherein R2' is selected from the group consisting of:
i) hydrogen;
ii) substituted lower alkyl;
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iii) unsubstituted lower alkyl;
iv) substituted aryl;
v) unsubstituted aryl;
vi) substituted heteroaryl;
s vii) unsubstituted heteroaryl;
viii) substituted aralkyl;
ix) unsubstituted aralkyl;
x) thiazolinyl;
xi) --(CHZ)a C02 R28, wherein a is 1 or 2, and RZ8 is selected from the group
consisting of: hydrogen and lower alkyl; and
xii) --( CHz) a CONRS R6 ; and
5) OR29 (wherein R29 is hydrogen, substituted lower alkyl, unsubstituted lower
alkyl,
or COR3° (wherein R3° is hydrogen, lower alkyl, substituted
aryl, unsubstituted aryl,
substituted heteroaryl, or unsubstituted heteroaryl));
is b) --CH(OH)(CH2)b R4A, wherein b is 1 to 6 and R4A is hydrogen or the same
as R4 ;
c) --( CHZ)d CHR31 C02 R32 wherein d is 0 to 5, R31 is hydrogen, --CONRS R6,
or -
COz R33 (wherein R33 is hydrogen or lower alkyl), and R32 is hydrogen or lower
alkyl;
d) __(CH2)d CHR31 CONRS R6 ;
2o e) --(CH2)~; R' wherein k is 2 to 6, and R' is halogen, C02R8 (wherein R$
is
hydrogen, lower alkyl, substituted aryl, unsubstituted aryl, substituted
heteroaryl, or
unsubstituted heteroaryl), CONRS R6, substituted aryl, unsubstituted aryl,
substituted
heteroaryl, unsubstituted heteroaryl, OR9 (wherein R~ is hydrogen, substituted
lower
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alkyl, unsubstituted lower alkyl, acyl, substituted aryl, or unsubstituted
aryl), SR2~B
(wherein R2~B is the same as R2~), NRio Rn (wherein RI° and Rll are the
same as RS
and R6) or N3;
f) --CH=CH(CHz)m Riz wherein m is 0 to 4, and Rlz is hydrogen, lower alkyl,
CO2R$A (wherein R$A is the same as R8), --CONRS R6, substituted aryl,
unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, OR9A
(wherein
R9A is the same as R9), or NRIOA Rnn (wherein Rlon and RnA are the same as RS
and
R6).
g) --CH=C(COZR3sA) z, wherein R33A 1S the same as R33 ;
1o h) --C=C(CHz)" Ri3, wherein n is 0 to 4, and R13 is the same as Rlz ;
i) --CHZOR44, wherein R44 is substituted lower alkyl;
and the other of Rl or Rz is selected from the group consisting of
j) hydrogen, lower alkyl, halogen, acyl, nitro, NR14 Rls (wherein R14 or Rls
is
hydrogen or lower alkyl, and the other is hydrogen, lower alkyl,' acyl,
carbamoyl,
~s lower alkylaminocarbonyl, substituted arylaminocarbonyl or unsubstituted
arylaminocarbonyl);
k) --CH(SR34) z, wherein R34 is lower alkyl or allcylene;
1) --CHZR35, wherein R35 is OR36 (wherein R36 is tri-lower alkyl silyl in
which the
three lower alkyl groups are the same or different, or is the same as Rz9), or
SR3~
20 (wherein R3' is the same as Rz~);
m) --CO(CHz)q RI6, wherein q is 1 to 6, and R16 is the same as R4 ;
n) --CH(OH)(CHz)e R38, wherein a is 1 to 6, and R3$ is the same as R4A ;
o) --(CHz) fCHR39 COzR4°, wherein f is 0 to 5, R39 is the same as R31
and R4° is the
same as R3z ;
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p) --(CH2)r Rl', wherein r is 2 to 6, and Rl' is the same as R' ;
q) --CH=CH(CH2)tRlB, wherein t is 0 to 4, and Rl8 is the same as Rla ;
r) --CH=C(COZ R33B)Z, wherein R33B 15 the same as R33 ;
s) --C---C(CHZ)" R19, wherein a is 0 to 4, and R19 is the same as R13);
R3 is hydrogen, acyl, or lower alkyl;
X is selected from the group consisting of:
a) hydrogen;
b) formyl;
c) lower aIkoxycarbonyl;
zo d) --CONR2° RZI, wherein:
RZ° and R21 independently are:
hydrogen;
lower alkyl;
--CH ZRZa, wherein R22 is hydroxy, or
1s --NR23 Rz4 (wherein R23 or R24 is hydrogen or lower alkyl, and the other is
hydrogen,
lower alky, or the residue of an a.-amino acid in which the hydroxy group of
the
carboxyl group is excluded, or R23 and R2ø are combined with a nitrogen atom
to
form a heterocyclic group); and
e) --CH=N-R25, wherein R25 is hydroxy, lower alkoxy, amino, guanidino, or
2o imidazolylamino;
Y is hydroxy, lower alkoxy, aralkyloxy, or acyloxy; or
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X and Y combined represent, --X-Y--, =O, --CH20(C=O)O--, --CH20C(=S)O--, --
CH2NR26 C(=O)--(wherein R26 is hydrogen or lower alkyl), --CH.2 NHC(=S)O--, --
CHZOS(=O)O--, or --CH20C(CH3)a O--; and
Wl and WZ are hydrogen, or Wl and WZ together represent oxygen.
The compounds represented by formula (n are hereinafter referred to as
Compound (I), and the same applies to the compounds of other formula numbers.
In the definitions of the groups in the formulas, lower alkyl means a straight-
chain or branched alkyl group having 1 to 6 carbon atoms, such as methyl,
ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,
neopentyl, 1-
1o ethylpropyl and hexyl. The lower allyl moiety of lower alkoxy, lower
alkoxycarbonyl, lower alkylaminocarbonyl and tri-lower alkylsilyl has the same
meaning as lower alkyl defined above. The aryl moiety of the acyl and the
acyloxy
groups means a straight-chain or branched alkanoyl group having 1 to 6 carbon
atoms, such as formyl, acetyl, propanoyl, butyryl, valeryl, pivaloyl and
hexanoyl, an
15 arylcarbonyl group described below, or a heteroarylcarbonyl group described
below.
The aryl moiety of the aryl, the arylcarbonyl and the arylaminocarbonyl groups
means a group having 6 to 12 carbon atoms such as phenyl, biphenyl and
naphthyl.
The heteroaryl moiety of the heteroaryl and the heteroarylcarbonyl groups
contain at
least one hetero atom selected from O, S, and N, and include pyridyl,
pyrimidyl,
2o pyrrolyl, furyl thienyl, imidazolyl triazolyl, tetrazolyl, quinolyl,
isoquinolyl
benzoimidazolyl thiazolyl and benzothiazolyl. The aralkyl moiety of the
aralkyl and
the aralkyloxy groups means an aralkyl group having 7 to 15 carbon atoms, such
as
benzyl, phenethyl, benzhydryl and naphthylmethyl. The substituted lower alkyl
group has 1 to 3 independently-selected substituents, such as hydroxy, lower
allcoxy,
25 carboxyl, lower alkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino,
dioxolane, dioxane, dithiolane, and dithione. The lower alkyl moiety of the
substituted lower alkyl, and the lower allyl moiety of the lower alkoxy, the
lower
alkoxycarbonyl, and the mono- or di-lower allcylamino in the substituents of
the
substituted lower alkyl group have the same meaning as lower alkyl defined
above.
3o The substituted aryl, the substituted heteroaryl and the substituted
aralkyl groups
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each has 1 to 3 independently-selected substituents, such as lower alkyl,
hydroxy,
lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono- or di-lower
alkylamino, and halogen. The lower alkyl moiety of the lower alkyl the lower
alkoxy, the lower alkoxycarbonyl, and the mono- or di-lower alkylamino groups
among the substituents has the same meaning as lower alkyl defined above. The
heterocyclic group formed with a nitrogen atom includes pyrrolidinyl,
piperidinyl,
piperidino, morpholinyl, morpholino, thiomorpholino, N-methylpiperazinyl,
indolyl,
and isoindolyl. The alpha.-amino acid groups include glycine, alanine,
proline,
glutamic acid and lysine, which may be in the L-form, the D-form or in the
form of a
1o racemate. Halogen includes fluorine, chlorine, bromine and iodine.
Preferably, one of Rl and R2 is selected from the group consisting of --CH2)k
R~, --CH=CH(CHZ)m R'2, --C---C(CHZ)" R13, --CO(CHZ)~ SRZ~, arid --CH2 OR4a.
wherein R44 is methoxymethyl, ethoxymethyl, or methoxyethyl; and the other of
Rl
and RZ is selected from the group consisting of --(CH2)f Rl', --CH=CH(CHZ)t
Rls, --
1s C---C(CHz)u R'9, NRI~ R15, hydrogen, halogen, nitro, --CH20-- (substituted
or
unsubstituted) lower alkyl, --CO(CHZ)a SR2~, --CHzR35, --CHzOH, and.--CH2SR3~
wherein R3' is selected from the group consisting of lower alkyl, pyridyl, and
benzimidazole.
Preferably, R35 is OR36 wherein R~6, preferably, is selected from the group
2o consisting of methoxymethyl, ethoxymethyl, and methoxyethyl.
Preferably, RZ' is selected from the group consisting of substituted or
unsubstituted lower alkyl, substituted or unsubstituted phenyl, pyridyl,
pyrimidinyl,
thiazole, and tetrazole.
Preferably, k and r, independently, are each 2, 3, or 4.
2s Preferably, j and q, independently, are 1 or 2.
Preferably, R' and RI', independently, are selected from the group consisting
of (1) COZ Rs and COzRsA' where Rs and RsA, independently, are hydrogen,
methyl,
ethyl, or phenyl; (2) phenyl, pyridyl, imidazolyl, thiazolyl, or
tetrazolyl;(3) OR9 and
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OR9A where R9 and R9A, independently, are hydrogen, methyl, ethyl, phenyl, or
aryl;
(4) SR2'B where R27B is selected from the group consisting of unsubstituted
lower
alkyl, 2-thiazoline, and pyridyl; and (5) NRl° RI1 and NR14 Ris, where
Rl°, Ri i, Ria,
and Rls, independently, are selected from the group consisting of hydrogen,
methyl,
ethyl, phenyl, carbamoyl, and lower alkylaminocarbonyl.
Preferably, m, n, t and u, independently, are 0 or 1.
Preferably, R12, R13, RI s, and R19, independently, are selected from the
group
consisting of hydrogen, methyl, ethyl, phenyl, pyridyl, imidazole, thiazole,
tetrazole,
COZRB, OR9, and NRl° R11 where R8, R9, Rl°, and Rll have the
preferred vales
to shown above.-
Preferably, R3 is hydrogen or acetyl, mast preferably hydrogen.
Preferably, X is hydroxymethyl or lower alkoxycarbonyl with
methoxycarbonyl being particularly preferred.
Preferably, Y is hydroxy or acetyloxy, most preferably hydroxy.
Preferably, each Wl and WZ is hydrogen.
Most preferred are the actual substituent values shown on the compounds in
Table 1, with Compounds I-157 being especially preferred.
Examples of Compound (I) are shown in Table A and the intermediates are shown
in
2o Table B.
TABLE A
Com-
pound R1 R2 R3 Y
1 CH=CHCOaMe CH=CHC02Me H OH
2 CH=CHCOZEt H H OH
3 CH=CHCOZEt CH=CHC02Et H OH
4 CH=CHCOZMe H H OH
5 CH=CH-C6Hs H H OH
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6 CH=CH-C6H5 CH=CH-C6H5 H QH
7 CH=CH-2-Pyr H H QH
8 CH=CH-2-Pyr CH=CH-2-Pyr H OH
9 CHZCH2-C6H5 CHZCHZ-C6H5 H OH
10 CH2CH2-C6H5 H H OH
11 CH2CH2-2-Pyr CH2CH2-2-Pyr H OH
12 H CH=CHC02Et H OH
13 H CH=CH-2-Pyr H OH
14 H CH2CH2-2-Pyr H OH
15 NO2 CH=CH-2-Pyr Ac OAc
16 N02 CH=CH-2-Pyr H OH
17 NHZ CH2CH2-2-Pyr Ac QAc
18 NH2 CHZCH2-2-Pyr H OH
19 NHCONHEt CH2CH2-2-Pyr H OH
is 20 C---CCH2NMez C---CCH2NMe2 H OH
21 C---CCH20Me I H OH
22 C---CCHZOMe C=CCH20Me H OH
23 C---CCHzOH C---CCHZOH H OH
_ 24 COCH2CI COCH2CI Ac OAc
2o 25 COCH2-1-Pip COCHZ-1-Pip H OH
26 COCH2CH2CI H Ac OAc
27 COCHZCHZCI COCH2CH2CI Ac OAc
28 COCHzCH2-1-Pip H H OH
29 COCHZCHZ-1-Pip COCH2CH2-1-Pip H - OH
25 30 COCH2CH2-1-Morph COCHZCH2-1-Morph H OH
31 COCHZ-1-Morph COCH2-1-Morph H OH
32 COCH2NMe2 COCHZNMez H OH
33a COCHZCI H Ac OAc
33b H COCH2CI Ac -OAc
so 34 COCH2NMe2 H H OH
35 COCHZ-1-THP H H OH
36a COCHZ-1-Morph H Ac OAc
36b H COCHZ-1-Morph Ac OAc
37a COCHZ-1-Morph H H OH
3s 37b H COCHZ-1-Morph H OH
38 COCHZ-1-THP COCHZ-1-THP H OH
39 COCHZ-1-Pipz(4-Me)COCHZ-1-Pipz(4-Me) Ac OAc
.
40 COCHZ-1-Pipz(4-Me)COCH2-1-Pipz(4-Me) H OH
41 H COCHZSEt H OH
40 42a COCHZS-4-Pyr H H OH
42b H COCHZS-4-Pyr H OH
43 COCHzSMe COCH2SMe H OH
44 COCHzSEt COCHZSEt H OH
45 COCHZSCH2Et COCHZSCHa Et H OH
4s 46 COCH2S(CH2)2 OH COCH2S(CH2)2 OH H OH
47 COCHzS-4-Pyr COCH2S-4-Pyr H OH
48 COCH2S-2-Pyr COCHZS-2-Pyr H , OH
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49 COCHaS-2-Pyrm COCH2S-2-Pyrm H OH
50 COCH2S-CsH~(4- COCHzS-CsHa(4-OH) H OH
OH)
S I COCHZS-2-Thiazl COCH2S-2-Thiazl H OH
s 52 COCHZS-5-Tet(1-Me)COCH2S-5-Tet(I-Me) H OH
53 CO(CH2)zSMe CO(CHa)aSMe H OH
54 CO(CH 2)ZOMe CO(CHZ)ZOMe H OH
55 Br CO(CH2)3H H OH
56 CO(CH2)4H CO(CHa)4H Ac OAc
57 COCH2Br COCH~Br AC OAc
58 CH(OH)Me H Ac OAc
59 CH(OH)(CHz)2Cl CH(OH)(CH2)2C1 H OH
60 CH(OH)CHZ-1-Pipz(4-CH(OH)CHa-1-Pipz(4-Me)H OH
Me)
~s 61 C---CCH2NMe2 H H OH
62 Br C---CCH2NMeBn H OH
63 CH=CHCHzNMe2 CH=CHCH2NMeZ Ac OAc
64 CH=CHCH2NMe2 CH=CHCHZNMe2 H OH
65 GH=CHEt H Ac OAc
66 CH=CHEt I~ H OH
67 CH=CHEt I Ac OAc
68 CH=CHEt CH=CHEt H OH
69 (CH2)2C1 (CHZ)2Cl H OH
. 70a (CHZ)2I _ (CH2)2I H OH
2s 70b (CHz)ZOCOH {CHZ)20COH H OH
70c (CHZ)20H (CHZ)ZOH H OH
71 (CHZ)ZOCO-4-Pyr (CH2)20C0-4-Pyr H OH
72a CH2C02Me H H OH
72b CHzC02Me . CH2COZMe H . OH
73a (CH2)3I (CH2)3I H OH
73b {CH2)~OCOH (CHZ)30COH H OH
73c (CHZ)30H (CHZ)3OH H OH
74 (CHZ)30Me (CH2)3OMe H OH
75 (CH~)2-1-Pip (CH2)2-1-Pip H OH
3s 76 (CHZ)2-1-Morph (GH2)2-1-Morph H OH
77 (CHZ)2NEt2 (CHZ)zNEt2 H OH
78 (CHZ)ZNMe(CHZ)20H(CH2)ZNMe(CHZ)aOH H OH
79 (CH2)2NHMe (CH2)aNHMe H OH
80 (CHZ)~,NHCHzC6Hq(4-(CH2)2NHCH2CsH4(4- H OH
Me0) Me0)
81 (CHZ)2N 3 (CH2)2N3 H OH
82 (CHZ)3-1-Pip (CH2)3-1-Pip H OH
88 (CHZ)3-1-Morph (CH2)3-1-Morph H OH
84 (CHZ)3NEt2 (CHz)3NEtz H OH
4s 85 (CH2)3NHCONHEt (CH2)3NHCONHEt H OH
86 (CHZ)3NHCOat-Bu CH2)3NHC02t-Bu H OH
87 (CHZ)2SMe (CHZ)2SMe H OH
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88 (CHZ)ZSEt (CHZ)2SEt H OH
89 (CH2)2SCHZC02Me (CHa)aSCH2COzMe H OH
90 (CHZ)2S(CH2)aCO2Et(CH2)2 S(CH2)2 C02 H OH
Et
91 (CHZ)2S-C6H4(4- (CHZ)2S-C6H~(4-OH) H OH
s ~ OH)
92 (CH2)2S-2-Thiazl (CHz)2S-2-Thiazl H ~ OH
93 (CHZ)ZS-4-Pyr (CH2)zS-4-Pyr H OH
94 (CH2)2S-2-Pyr (CH2)~S-2-Pyr H OH
95 (CH2)3SMe (CHZ)3SMe H OH
Io 96 (CH2)3S-2- (CHz)3S-2- H OH
(Benz)Thiazole (Benz)Thiazole
97 CH=CH-2-Pyr CHO Ac OAc
98 CH=CH-2-Pyr CH20H Ac .
OAc
99 CH=CH-2-Pyr CH20H H OH
15 100 CH=CH-2-Pyr CH20SiMe2t-Bu Ac OAc
101 CH=CH-2-Pyr CHZOSiMe2t-Bu H OH
102 CH=CH-2-Pyr CH20Me H OH
103 CH=CH-2-Pyr CH20Et H OH
104 CH=CH-2-Pyr CH20(CHZ)ZNMe2 H OH
~20105 CH=CH-2-Pyr CHZSEt H OH
106 CH=CH-2-Pyr CHZS(CH~)ZNMe2 H OH
107 CH=CH-2-Pyr CH2S-2-(Benz)Imid H OH
108 CH=CH-2-Pyr CHZS-2-Pyr H OH
109 CH=CH-2-Pyr CH(SEt)2 Ac
25 OAc
110 CH=CH-2-Pyr CH(SEt)2 H OH
111 CHO CH=CH-2-Pyr Ac OAc
112 CH2OH CH=CH-2-Pyr Ac OAc
113 CH20H CH=CH-2-Pyr H OH
30 114 CHZOSiMe2t-Bu CH=CH-2-Pyr Ac pAc
115 CH20SiMe2t-Bu CH=CH-2-Pyr H OH
I16 CH20Me CH=CH-2-Pyr H OH
117 CH20Et CH=CH-2-Pyr H OH
118 CH2SEt CH=CH-2-Pyr ~ H OH
35 119 CH2S-2-Pyr CH=CH-2-Pyr H OH
120 CH2S-2-(Benz)ImidCH=CH-2-Pyr H OH
121 CH=CHEt CH=CH-2-Pyr Ac OAc
122 CH=CHEt CH=CH-2-Pyr H OH
123 (CHZ)2-2-Pyr CHzOSiMezt-Bu Ac OAc
40 124 (CHz)Z-2-Pyr CH20SiMe2t-Bu H OH
125a (CH2)2-2-Pyr CH20Me Ac OAc
125b (CH2)2-2-Pyr ~ CH20Me H OAc
126 (CHZ)2-2-Pyr CH20Me H OH
127a (CH2)2-2-Pyr CH20Et H OH
4.5127b (CHZ)2-2-Pyr CH20H H OH
128 (CH2)2-2-Pyr CHZS-2-Pyr Ac OAc
129 (CH2)z-2-Pyr CH2S-2-Pyr H OH
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130 CH20SiMeat-Bu (CHa)a-2-Pyr Ac
OAc
131 CHaOSiMeat-Bu (CHa)a-2-Pyr H OH
132 CHZOMe (GHa)a-2-Pyr H OH
133 CH20Et (CHa)a-2-Pyr H OH
134 CHZSEt (CHa)a-2-Pyr H OH
135 CH2S(CHa)aNMea (CHa)a-2-Pyr H OH
136 CH2S-2-Pyr (CHa)a-2-Pyr Ac
OAc
137 CH2S-2-Pyr (CHa)a-2-Pyr H OH
*138 C=CCH20Me C=CCH20Me H . OH
139 CH2CH2C02Me CHaCHaCOaMe H OH
140 CH2CHzC02Et CHaCHaCOa,Et H OH
141 Br CH=CH-2-Pyr Ac OAc
142 Br CH=CH-2-Pyr H OH
I43 Br CHaCHa-2-Pyr H OH
144 CH=CH-4-Pyr CH=CH-4-Pyr Ac OAc
145 CH=CH-4-Pyr CH=CH-4-Pyr H OH
146 CH2CH2-4-Pyr CHaCH2-4-Pyr H OH
147 CH=CH-2-Imid H Ac OAc
148 CH=CH-2-Imid H H OH
149 CH2CH2-2-Imid H H OH~
150 CH=C(COZMe)a CH=C(C02Me)a Ac OAc
151 CH2CH(C02Me)a CHaCH(CO2Me)a Ac OAc
152 CH2CH(C02Me)a CH2CH(COZMe)a H OH
153 n-C4H9 (CHa)a-2-Pyr H OH
I54 CH20CHaOMe H H OH
155 CH20CH20Me CH20CH20Me H OH
156 CHaOCHaOEt CH20CHZOEt H OH
157 CH20(CHa)aOMe CH20(CHa)aOMe H OH
Pyr = Pyridyl
Pip = Piperidine
Morph = Morpholine
THP = Tetrahydropyrrole
Pipz = Piperazine
Pyrm = Pyrimidine
Thiazl = Thiazoline
Tet = Tetrazole
Imid = Imidazole
(Benz)Thiazole = Benzothiazole
(Benz)Imid = Benzimidazole
*The COa CH3 group is
replaced with CHa OH.
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TABLE B
Compound Rl R2 R3 Y
A Br H Ac OAc
s B Br CHO Ac OAc
C H CHO Ac OAc
D N02 H Ac OAc
E NO2 CHO Ac OAc
F I I Ac OAc
1o G I ~I H OH
H I H Ac OAc
I Br I Ac OAc
J CHO I Ac OAc
K CH20H I Ac OAc
is
See U.S. Patent No. 6,306,49 and W097I49406, incorporated herein by reference
in their entirety.
2o The compounds are derivatives of the compound K-252a, represented by the
following structure: Formula II.
V
CH3
HO
25 C02CH3
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K-252a has an indolocarbazole skeleton as described in U.S. Patent No.
4,555,402
s and Japanese Published Unexamined Patent Application No. 41489/85. K-252a is
a
natural product indolocarbazole of the bacterium lVoeardiosis species. The
activity
of these compounds can be demonstrated using the cultured pinal cord choline
acetyltransferase (ChAT) assay.
Formula III represents, an ethylthiomethyl analog of K-252a, CEP-1347,
to that exhibited greater efficacy (250% of control) and potency (EC50 = 50
nlV1) than
K-252a in spinal cord ChAT assays (Kaneko et al., J. Med. Chem. Jun
6;40(12):1863-9 (1997)). In certain aspects, the methods of the invention
utilize
CEP-1347 as the MLKl inhibitor.
CH2SCH2CH3
CHgCH2S
Formula III (CEP-1347) (* denotes chiral center)
Some disclosed compounds contain a chiral center. For example, in Formula
III, the * denotes chiral centers. The presence of chiral centers in a
molecule gives
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rise to stereoisomers. For example, a pair of optical isomers, referred to as
"enantiomers", exist for every chiral center in a molecule; and a pair of
diastereomers exist for every chiral center in a compound having two or more
chiral
centers. Where the structural formulas do not explicitly depict
stereochemistry, it is
to be understood that these formulas encompass enantiomers free from the
corresponding optical isomer, racemic mixtures, mixtures enriched in one
enantiomer relative to its corresponding optical isomer, a diastereomer free
of other
diastereomers, a pair of diastereomers free from other diasteromeric pairs,
mixtures
of diasteromers, mixtures of diasteromeric pairs, mixtures of diasteromers in
which
to one diastereomer is enriched relative to the other diastereomer(s) and
mixtures of
diasteromeric pairs in which one diastereomeric pair is enriched relative to
the
other diastereomeric pair(s).
Also compounds of Structural Formula IV (as described in WO
03/064428AI) are useful in the methods described herein as the MLI~ family
kinase inhibitor. Structural Formula IV:
Rs
/N
W
Ni \ \
rR3
N A
R5
wherein A represents O or S;
2o W represents O, NH, NR1;
R4 and RS are independently selected from the group represented by hydrogen,
halogen, cyano, nitro, CI_6-alk(en/yn)yl, C1_6-alk(en/yn)yloxy, C1_6-
alk(en/yn)yloxy-
Cl_6-alk(en/yn)yl, C1_6-alk(en/yn)ylsulfanyl, hydroxy, hydroxy-Cl_6-
alk(en/yn)yl,
halo-C1_6-alk(en/yn)yl, halo-C1_6-alk(en/yn)yloxy, C3_8-cycloalk(en)yl, C3_8-
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cycloalk(en)yl-CI_6-alk(en/yn)yl, acyl, C1_6-alk(en/yn)yloxycarbonyl, Cl_6-
allc(en/yn)ylsulfonyl, -NR~RB and R~R$N-CI_6-alk(en/yn)yl-;
R3 represents hydrogen, halogen, CI~-alk(en/yn)yl, C3_8-cycloalk(en/yn)yl,
aryl, a
heterocycle, hydroxy, hydroxy-C1_6-alk(en/yn)yl, C1_6-alk(en/yn)yloxy, C1_6-
alk(en/yn)yloxy-C1_6-alk(en/yn)yl, C3_$-cycloalk(en/yn)oxy, C~_6-
alk(en/yn)ylsulfanyl, acyl, R~R$N-Cl_6-alk(en/yn)yl or -NR?R8;
or R3 represents a group of the formula ~
-R9-Ar2
wherein R9 represents O, NH, NRl', S, -CONRI'-, - CO- or C1_6-alkyl, C2_6-
alkenyl,
which may optionally be substituted by OH, halogen, Cl_6-alkoxy or C3_s-
cycloalkyl;
R6 represents CI_6-alk(en/yn)yl, C3_$-cycloalk(en/yn)yl, C3_$-cycloallc(en)yl-
C1_s-
alk(en/yn)yl or Arl;
Arl and Ar2 are independently selected from the group represented by aryl, a
heterocycle or a carbocycle all of which may be substituted one or more times
by
halogen, cyano, nitro, Cl_6-alk(en/yn)yl, CI_6-alk(en/yn)yloxy, C1_6-
alk(en/yn)yloxy-
2o Cl_6-alk(en/yn)yl, C1_6-alk(en/yn)yloxy-CI_6-alk(en/yn)yloxy-C1_6-
alk(en/yn)yl
aryloxy-, aryl-CI_6-alk(en/yn) yloxy, halo-CI_6-alk(eniyn)yloxy, C~_s-
alk(en/yn)yl-
sulfanyl, hydroxy, hydroxy-C1_6-alk(en/yn)yl, halo-C1_6-alk(en/yn)yl, cyano-
C1_6-
alk(en/yn)yl, NR~RB, NR~Rg-Cl_6-alk(en/yn)yl, C3_$-cycloalk(en)yl, C3_8-
cycloalk(en)yl-C1_6-alk(en/yn)yl, CI_6-alk(en/yn)ylsulfonyl, aryl, acyl, Ci-s-
alk(en/yn)yloxycarbonyl, C1_6-allc(en/yn)yl-CONRI'-C1_6-alk(en/yn)yl, C1_s-
alk(en/yn)yl-CONRI'-, -CONR~Rg or R~R8NC0-C1_6-alk(en/yn)yl;
R' and R8 are independently selected from the group represented by hydrogen
and
C1_6-alk(en/yn)yl which may be further substituted by hydroxy, halogen, C1_s-
3o alkoxy, cyano, nitro, C3_g-cycloalk(en)yl, C3_g-cycloalk(en)yl-Cl_6-
alk(enlyn)yl, aryl
or a heterocycle; or R' and R$ together with the nitrogen to which they are
attached
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form a 3-7-membered ring which optionally contains one or more further
heteroatoms and may optionally be substituted by halogen, C1.~-alk(en/yn)yl,
hydroxy, hydroxy-Cl_6-alk(en/yn)yl or acyl;
the aryls may be further substituted by halogen, cyano, nitro, Cl_6-
alk(en/yn)yl, Cl~-
alk(en/yn)yloxy, CI_6-alk(en/yn)ylsulfanyl, hydroxy, hydroxy-C1_6-
alk(en/yn)yl,
halo-C1_6-alk(en/yn)yl, halo-C1_6-alk(en/yn)yloxy, C3_$-cycloalk(en)yl, C3_s-
cycloalk(en)yl-Cl_6-alk(en/yn)yl,.acyl, C1_6-allc(en/yn)yloxycarbonyl, C1_s-
allc(en/yn)ylsulfonyl, or -NR~'R8' wherein -NR~'R8' is as defined for -NR~RB
above
1o provided that any aryl substituent on -NR~'R$' is not further substituted;
and Rl and Rl' are independently selected from the group represented by Cl_s-
alk(enlyn)yl, C3_$-cycloalk(en)yl, aryl, hydroxy-CI_6-alk(en/yn)yl, C3_8-
cycloalk(en)yl-Cl_&-alk(en/yn)yl and acyl;
or a pharmaceutically acceptable salt thereof.
The term "alkyl" refers to a monovalent group derived from a straight or
branched chain saturated hydrocarbon by the removal of a single hydrogen atom.
Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-,
iso- and
2o tent-butyl, and the like. The term "hydroxyalkyl" represents an alkyl
group, as
defined above, substituted by one to three hydroxyl groups with the proviso
that no
more than one hydroxy group may be attached to a single carbon atom of the
alkyl
group. The term "alkylamino" refers to a group having the structure -NHR'
wherein
R' is alkyl, as previously defined, examples of alkylamino include
methylamino,
ethylamino, iso-propylamino and the like. The term "alkanoyl" represents an
alkyl
group, as defined above, attached to the parent molecular moiety through a
carbonyl
group. Alkanoyl groups are exemplified by formyl, acetyl, propionyl, butanoyl
and
the like. The term "alkanoylamino" refers to an alkanoyl group, as previously
defined, attached to the parent molecular moiety through a nitrogen atom.
Examples
of alkanoylamino include formamido, acetamido, and the like. The term "N-
alkanoyl-N-alkylamino" refers to an alkanoyl group, as previously defined,
attached
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to the parent molecular moiety through an aminoalkyl group. Examples of N-
alkanoyl-N-alkylamino include N-methylformamido, N-methyl-acetamido, and the
like. The terms "alkoxy" or "alkoxyl" denote an alkyl group, as defined above,
attached to the parent molecular moiety through an oxygen atom. Representative
s alkoxy groups include methoxyl, ethoxyl, propoxyl, butoxyl, and the like.
The term
"alkoxyalkoxyl" refers to an alkyl group, as defined above, attached through
an
oxygen to an alkyl group, as defined above, attached in turn through an oxygen
to
the parent molecular moiety. Examples of alkoxyalkoxyl include
methoxymethoxyl,
methoxyethyoxyl, ethoxyethoxyl and the like. The term "alkoxyalkyl" refers to
an
~o alkoxy group, as defined above, attached through an alkylene group to the
parent
molecular moiety. The term "alkoxycarbonyl" represents an ester group; i.e.,
an
alkoxy group, attached to the parent molecular moiety through a carbonyl group
such as methoxycarbonyl, ethoxycarbonyl, and the like. The term "alkenyl"
denotes
a monovalent group derived from a hydrocarbon containing at least one carbon-
15 carbon double bond by the removal of a single hydrogen atom. Alkenyl groups
include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and
the like.
The term "alkylene" denotes a divalent group derived from a straight or
branched
chain saturated hydrocarbon by the removal of two hydrogen atoms, for example
methylene, 1,2-ethylene, l,l-ethylene, 1,3-propylene, 2,2-dimethylpropylene,
and
2o the like. The term "alkenylene" denotes a divalent group derived from a
straight or
branched chain hydrocarbon containing at least one carbon-carbon double bond.
Examples of alkenylene include -CH=CH-, -CHZ CH=CH-, -C(CH3)=CH-, -CHI
CH=CHCHZ -, and the like. The term "cycloalkylene" refers to a divalent group
derived from a saturated carbocyclic hydrocarbon by the removal of two
hydrogen
2s atoms, for example cyclopentylene, cyclohexylene, and the like. The term
"cycloalkyl" denotes a monovalent group derived from a monocyclic or bicyclic
saturated carbocyclic ring compound by the removal of a single hydrogen atom.
Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
bicyclo[2.2.1]heptanyl, and bicyclo[2.2.2]octanyl. The term "alkynylene"
refers to a
3o divalent group derived by the removal of two hydrogen atoms from a straight
or
branched chain acyclic hydrocarbon group containing a carbon-carbon triple
bond.
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Examples of alkynylene include -CH= CH-, -CH= CH-CHZ -, -CH= CH-CH(CH3)-,
and the like. The term "carbocyclic aryl" denotes a monovalent carbocyclic
ring
group derived by the removal of a single hydrogen atom from a monocyclic or
bicyclic fused or non-fused ring system obeying the "4n+2 p electron" or
Huckel
aromaticity rule. Examples of carbocyclic aryl groups include phenyl, 1- and 2-
naphthyl, biphenylyl, fluorenyl, and the like. The term "(carbocyclic
aryl)alkyl"
refers to a carbocyclic aryl ring group as defined above, attached to the
parent
molecular moiety through an alkylene group. Representative (carbocyclic
aryl)alkyl
groups include phenylmethyl, phenylethyl, phenylpropyl, 1-naphthylmethyl, and
the
like. The term "halo or halogen" denotes fluorine, chlorine, bromine or
iodine. The
term "haloalkyl" denotes an alkyl group, as defined above, having one, two, or
three
halogen atoms attached thereto and is exemplified by such groups as
chloromethyl,
bromoethyl, trifluoromethyl, and the like. The term "hydroxyalkyl" represents
an
alkyl group, as defined above, substituted by one to three hydroxyl groups
with the
proviso that no more than one hydroxy group may be attached to a single carbon
atom of the alkyl group. The term "phenoxy" refers to a phenyl group attached
to
the parent molecular moiety through an oxygen atom. The term "phenylthio"
refers
to a phenyl group attached to the parent molecular moiety through a sulfur
atom.
The term "pyridyloxy" refers to a pyridyl group attached to the parent
molecular
2o moiety through an oxygen atom. The terms "heteroaryl" or "heterocyclic
aryl" as
used herein refers to substituted or unsubstituted 5- or 6-membered ring
aromatic
groups containing one oxygen atom, one, two, three, or four nitrogen atoms,
one
nitrogen and one sulfur atom, or one nitrogen and one oxygen atom. The term
heteroaryl also includes bi-or tricyclic groups in which the aromatic
heterocyclic
ring is fused to one or two benzene rings. Representative heteroaryl groups
are
pyridyl, thienyl, indolyl, pyrazinyl, isoquinolyl, pyrrolyl, pyrimidyl,
benzothienyl,
furyl, benzo[b]furyl, imidazolyl, thiazolyl, carbazolyl, and the like. The
term
"heteroarylalkyl" denotes a heteroaryl group, as defined above, attached to
the
parent molecular moiety through an alkylene group. The term "heteroaryloxy"
3o denotes a heteroaryl group, as defined above, attached to the parent
molecular
moiety through an oxygen atom. The term "heteroarylalkoxy" denotes a
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heteroarylalkyl group, as defined above, attached to the parent molecular
moiety
through an oxygen atom.
The expression CI_ 6-alk (enlyn)yl means a Cl_ 6-alkyl, Cx_ 6-alkenyl or a CZ
_ 6
-alkynyl group. The expression C3_ $-cycloank (en)yl means a C3_ $--cycloalkyl-
or
cycloalkenyl group.
The term CI_ 6_ alkyl refers to a branched or unbranched alkyl group having
from one to six carbon atoms inclusive, including but not limited to methyl,
ethyl, 1-
propyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-2-propyl and 2-methyl-1-propyl.
Similarly, C2_ 6alkenyl and Cz_ 6alkynyl, respectively, designate such groups
to having from two to six carbon atoms, including one double bond and one
triple bond
respectively, including but not limited to ethenyl, propenyl, butenyl,
ethynyl,
propynyl and butynyl.
The term C3_ g-cycloalkyl designates a monocyclic or bicyclic carbocycle
having three to eight C-atoms, including but not limited tb cyclopropyl,
cyclopentyl,
15 cyclohexyl, etc.
The term C3_ S-cycloalkenyl designates a monocyclic or bicyclic carbocycle
having three to eight C-atoms and including one double bond.
In the term C3_ 8-cycloalkyl (en) yl-Cl-6-alk (en/yn) yl, C3_ $-cycloalk(en)
yl
and C1-6-alk (enlyn) yl are as defined above.
2o The terms CI_ 6-alk (en/yn) yloxy, C1_ 6-alk (en/yn) yloxy- CI_ 6-alk
(enlyn) yl,
Cl_ 6-alk (en/yn) ylsulfanyl, hydroxy- C~_ 6-alk (en/yn) yl, halo- Cl_ 6-alk
(en/yn) yl,
halo- C1_6-alk (en/yn) yloxy, Cl_ 6-alk (en/yn) ylsulfonyl, cyano- CI_ 6-alk
(en/yn) yl,
hydroxy- C1_6- alk (en/yn) yl, NR"Ry- Ci_ 6-alk (en/yn) yl, NRl'CO- Ci_ 6
(en/yn) yl
etc. designate such groups in which the Cl_ 6 (en/yn) yl is as defined above.
The
2s terms halo-, hydroxy-, cyano-etc. are to be understood as the C1_ 6 (en/yn)
yl- part
can be substituted one or more times with such substituent. The term halo-
designates
halogen as defined above.
As used herein, the term CI_ 6 (en/yn) yloxycarbonyl refers to groups of the
formula -COO- C1_ 6 (en/yn) yl, wherein C1_ 6 (en/yn) yl are as defined above.
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As used herein, the term acyl refers to formyl, C1_ 6-alk (en/yn) ylcarbonyl,
arylcarbonyl, aryl- C1_6 alk(en/yn) ylcarbonyl, C~_$-cycloalk (en) ylcarbonyl
or a C3_
$-cycloalk (en) yl-C1_6-alk (en/yn) yl-carbonyl group.
The term heterocycle designates rings such as 5-membered monocyclic rings
such as 3H-1, 2, 3-oxathiazole, 1,3, 2-oxathiazole, 1,3, 2-dioxazole, 3H-1,
2,3-
dithiazole, 1,3, 2-dithiazole, 1,2, 3-oxadiazole, 1,2, 3-thiadiazole, 1H l,]
2,3-
triazole, isoxazole, oxazol, isothiazole, thiazole, 1H-imidazole, 1H pyrazole,
1H-
pyrrole, furan or thiophene and 6-membered monocyclic rings such as 1,2, 3-
oxathiazine, 1,2, 4-oxathiazine, 1,2, 5-oxathiazine, 1,4, 2-oxathiazine, 1,4,
3-
oxathiazine, 1,2, 3-dioxazine, 1,2, 4-dioxazine, 4H-1, 3,2-dioxazine, 1,4, 2-
dioxazine, 2H-1, 5,2-dioxazine, 1,2, 3-dithiazine, 1,2, 4-dithiazine, 4H l,
3,2-
dithiazine, 1,4, 2-dithiazine, 2H l, 5,2-dithiazine, 2H l, 2,3-oxadiazine, 2H
1, 2,4-
oxadiazine, 2H 1, 2,5-o~adiazine, 2H l, 2,6-oxadiazine, 2H-1, 3,4-oxadiazine,
2H
1, 2,3-thia- diazine, 2H-1, 2, 4-thiadiazine, 2H 1, 2, 5-thiadiazine, 2H-l,
2,6-
thiadiazine, 2H-l, 3,4-thiadiazine, 1,2, 3-triazine, 1,2, 4-triazine, 2H-1,2-
oxazine,
2H-1, 3-oxazine, 2H-l, 4-oxazine, 2H-l, 2-thiazine, 2H-1, 3-thiazine, 2H-l, 4-
thiazine, pyrazine, pyridazine~-pyrimidine, 4H-l, 3-oxathiin, 1,4-oxathiin, 4H-
1, 3-
dioxin, 1,4-dioxin, 4H-l, 3-dithiin, 1,4-dithiin, pyridine, 2H-pyran or 2H-
thiin,
bicyclic compounds wherein the above rings are fused to a benzene ring, such
as
2o indole, benzofuran, isobenzofuran, benzothiophen, benzimidazol, quinoline,
isoquinoline, dihydroquinoline, or completely saturated rings such as
morpholin,
piperidin, azepin, piperazin, homopiperazin, and ring systems fused to a
benzene
ring, such as benzodioxan, benzodithiodioxan, benzo [[l, 3] dioxol,
dihydroindol,
dihydrobenzofuran or dihydrobenzothiophen.
z5 The term aryl refers to carbocyclic, aromatic systems such as phenyl,
naphtyl, anthracene and phenantrene.
The terms aryloxy and aryl-C1_6-alk (enlyn) yloxy refer to aryl as defined and
C1_6-alk (en/yn) yloxy as defined above. .
The term carbocyclic refers to partly or completely saturated systems such as
3o cyclohexen, indan or flurene.
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The term heteroatom refers to atoms different from carbon and hydrogen,
such as nitrogen, oxygen and sulphur.
Exemplary of organic acid addition salts according to the invention are thane
with malefic, fumaric, benzoic, ascorbic, succinic; oxalic, bin-
methylenesalicylic,
methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic,
citric,
gluconic, lactic, malic, mandelic, cinnamic, citraconic, aspartic, stearic,
palmitic,
itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic and theophylline
acetic acids, as well as the 8-halotheophyllines, for example 8-
bromotheophylline.
Exemplary of inorganic acid addition salts according to the invention are
those with
to hydrochloric, hydrobromic; sulfuric, sulfamic, phosphoric and nitric acids.
The acid
addition salts of the invention are preferably pharmaceutically acceptable
salts
formed with non-toxic acids.
Furthermore, the compounds used in the methods of this invention may exist
in unsolvated as well as in solvated forms with pharmaceutically acceptable
solvents
such as water, ethanol and the like. In general, the solvated forms are
considered
equivalent to the unsolvated forms for the purposes of this invention.
Some of the compounds of the present invention contain chiral centres and
such compounds exist in the form of isomers (e.g,. enantiomers). The invention
includes all such isomers and any mixtures thereof including racemic mixtures.
2o Racemic forms can be resolved into the optical antipodes by known methods,
for example, by chromatography on an optically active matrix. The compounds of
the present invention may also be resolved by the formation of diastereomeric
derivatives.
Methods standard in the art can be utilized for determining compounds that
modulate the MLK family kinase protein activity, in particular MLKl. For
example,
MLK family kinase protein activity can be determined by measuring the activity
of a
substrate of the MLK family kinase. Sueh substrates are well known to those in
the
art. The substrate is preferably a member of the mitogen activated kinase
family or
substrates further down the pathway (e.g., JNK1, JNK2, JNK3, ERKl, ERK2,
3o p38a, p38~3, p38 y, p388, MEKl, MEK2, MKK3, MKK4(SEK1), MEKS, MI~K6,
MKK7, jun AFT2 and ELK1, or other members of the pathway described in FIG. 1).
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Additionally, general substrates of Ser/Thr protein kinases such a myelin
basic
protein (n~IBP) can also be used. Reagents and methods for measuring the
activity of
the substrates are also known to those skilled in the art. The presence of MLK
can
also be determined by measuring the amount of MLK protein or mRNA encoding
the MLK protein, such as the methods described below.
ASSESSMENT FOR AT-RISK HAPLOTYPES
A "haplotype," as described herein, refers to a combination of genetic markers
("alleles"), such as those set forth in Tables 1 and 2. In a certain aspect,
the haplotype
1o can comprise one or more alleles, two or more alleles, three or more
alleles, four or
more alleles, or five or more alleles. The genetic markers are particular
"alleles" at
"polymorphic sites" associated with MAPK9. A nucleotide position at which more
than
one sequence is possible in a population (either a natural population or a
synthetic
population, e.g., a library of synthetic molecules) is referred to herein as a
"polymorphic
is site". Where a polymorphic site is a single nucleotide in length, the site
is referred to as
a single nucleotide polymorphism ("SNP"). For example, if at a particular
chromosomal location, one member of a population has an adenine and another
member
of the population has a thymine at the same position, then this position is a
polymorphic
site, and, more specifically, the polymorphic site is~a SNP. Polymorphic sites
can allow
20 for differences in sequences based on substitutions, insertions or
deletions. Each
version of the sequence with respect to the polymorphic site is referred to
herein as an
"allele" of the polymorphic site. Thus, in the previous example, the SNP
allows for
both an adenine allele and a thymine allele.
Typically, a reference sequence is referred to for a particular sequence.
.Alleles
25 that differ from the reference are referred to as "variant" alleles. For
example, the
reference MAP3K9 sequence is described herein by SEQ ID NO: 1. The term,
"variant
MAP3K9", as used herein, refers to a sequence that differs from SEQ ID NO: 1,
but is
otherwise substantially similar. The genetic markers that make up the
haplotypes
described herein are MAP3K9 variants.
3o Additional variants can include changes that affect a polypeptide, e.g.,
the
MAP3K9 polypeptide. These sequence differences, when compared to a reference
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nucleotide sequence, can include the insertion or deletion of a single
nucleotide, or of
more than~~one nucleotide, resulting in a frame shift; the change of at least
one
nucleotide, resulting in a change in the encoded amino acid; the change of at
least one
nucleotide, resulting in the generation of a premature stop codon; the
deletion of several
s nucleotides, resulting in a deletion of one or more amino acids encoded by
the
nucleotides; the insertion of one or several nucleotides, such as by unequal
recombination or gene conversion, resulting in an interruption of the coding
sequence of
a reading.frame; duplication of all or a part of a sequence; transposition; or
a
rearrangement of a nucleotide sequence, as described in detail above. Such
sequence
to changes alter the polypeptide encoded by a MAP3K9 nucleic acid. For
example, if the
change in the nucleic acid sequence causes a frame shift, the frame shift can
result in a
change in the encoded amino acids, and/or can result in the generation of a
premature
stop codon, causing generation of a truncated polypeptide. Alternatively, a '
polymorphism associated with a susceptibility to MI, ACS, stroke or PAOD can
be a
1s synonymous change in one or more nucleotides (i.e., a change that does not
result in a
change in the amino acid sequence). Such a polymorphism can, for example,
alter
splice sites, affect the stability or transport of mRNA, or otherwise affect
the
transcription or translation of the polypeptide. The polypeptide encoded by
the
reference nucleotide sequence is the "reference" polypeptide with a particular
reference
2o amino acid sequence, and polypeptides encoded by variant alleles are
referred to as
"variant" polypeptides with variant amino acid sequences.
According to NCBI (National Center for Biotechnology Information), AceView,
MAP3K9 is expressed at high levels. The sequence of this gene is supported by
48
sequences from 40 cDNA clones and produces, by alternative splicing, 5
different
2s transcripts aDec03 (variant a), bDec03(variant b), cDec03 (variant c),
dDec03, (variant
d), and eDec03 (variant e), altogether encoding 5 different protein isoforms.
As
indicated in Table C.
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Table C
mRNA NCBI Ace view site data
variant
aDec03 This complete mRNA is 7028 by long. We annotate
here the sequence derived
7028bp from the genome, although the best path through
the available clones differs
AM_5541bpfrom it in ,1 position. It has 13 exons.
It has a very long 3' UTR. The
premessenger covers 82.46 kb on the NCBI
build 34, August 2003 genome. The
protein (1071 aa, 118.9 kDa, pI 6.1) contains
one SH3 domain motif, one protein
kinase motif. It also contains a coiled coil
stretch [Psort2]. Taxblast (threshold '
10~-3) tracks ancestors down to Viruses and
Eukaryota.
bDec03 This complete CDS mRNA is 5096 by long. Its
sequence exactly matches the
AM 5096bpgenome. It has i l exons. It has a very long
3' UTR. The premessenger covers
5.33 kb on the NCBI build 34, August 2003
genome. The protein (869 aa, 96.5
kDa, pI 6.9) contains one protein kinase
motif. It also contains a coiled coil
stretch [Psort2]. Taxblast (threshold 10~-3)
tracks ancestors down to Viruses and
Eukaryota.
cDec03 This mRNA is 1829 by long. It. It has 3 exons.
It may be incomplete at the 5'
end. The premessenger covers 9.56 kb on the
NCBI build 34, August 2003
genome. The protein contains one SH3 domain
motif, one protein kinase motif.
Taxblast (threshold 10~-3) tracks ancestors
down to Viruses and Bacteria and
Eukaryota.
dDec03 This complete CDS mRNA is 572 by Long. It.
It has a single exon. The
572bp remessenger covers 0.57 kb on the NCBI build
34, August 2003 genome. The
AM 569bpprotein (85 aa, 9.2 kDa, pI 7.7) contains
no Pfam motif. It is predicted to Localise
in the cytoplasm [Psort2].
eDec03 This partial mRNA, 3' incomplete is 411 by
long. It. It has a single axon. It is
411bp partial, truncated at the 3' end. The premessenger
covers 0.41 kb on the NCBI
AM 411bpbuild 34, August 2003 genorne. The partial
protein (80 aa, 9.5 kDa, pI 5.3)
contains no Pfam motif. It contains an ER
membrane domain [Psort2]. Taxblast
(threshold 10~-3) tracks ancestors down to
Bilateria.
Haplotypes are a combination of genetic markers, e.g., particular alleles at
polymorphic sites. The haplotypes described herein, e.g., having markers such
as those
5 shown in Table 1 are found more frequently in individuals with asthma than
in
individuals without asthma. Therefore, these haplotypes have predictive value
for
detecting a susceptibility to asthma in an individual. The haplotypes
described herein
are in some cases a combination of various genetic markers, e.g., SNPs and
microsatellites. Therefore, detecting haplotypes can be accomplished by
methods
to known in the art for detecting sequences at polymorphic sites, such as the
methods
described above.
In certain methods described herein, an individual who is at-risk for asthma
is an
individual in whom an at-risk haplotype is identified. In one aspect, the at-
risk
haplotype is one that confers a significant risk of asthma. In one aspect,
significance
1s associated with a haplotype is measured by an odds ratio. In a further
aspect, the
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significance is measured by a percentage. In one aspect, a significant risk is
measured
as an odds ratio of at least about 1.2, including by not limited to: 1.2, 1.3,
1.4, 1.5, 1.6,
1.7, 1.8, and 1.9. In a further aspect, an odds ratio of at least 1.2 is
significant. In a
further aspect, an odds ratio of at least about 1.5 is significant. In a
further aspect, a
significant increase in risk is at least about 1.7 is significant. In a
further aspect, a
significant increase in risk is at least about 20%, including but not limited
to about 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and
98%. In a further aspect, a significant increase in risk is at least about
SO%. It is
understood however, that identifying whether a risk is medically significant
may also
1o depend on a variety of factors, including the specific disease, the
haplotype, and often,
environmental factors.
An at-risk haplotype in, or comprising portions of, the MAP3K9 gene, in one
where the haplotype is more frequently present in an individual at risk for
asthma
(affected), compared to the frequency of its presence in a healthy individual
(control), and wherein the presence of the haplotype is indicative of
susceptibility to
asthma. As an example of a simple test for correlation would be a Fisher-exact
test
on a two by two table. Given a cohort of chromosomes the two by two table is
constructed out of the number of chromosomes that include both of the
haplotypes,
one of the haplotype but not the other and neither of the haplotypes.
2o In certain aspects of the invention, at-risk haplotype is an at-risk
haplotype
within or near MAP3K9 that significantly correlates with a haplotype such as a
halotype shown in Table 1. In other aspects, an at-risk haplotype comprises an
at-
risk haplotype within or near MAP3K9 that significantly correlates with
susceptibility to asthma. In one aspect, the at-risk haplotype is
characterized by the
following microsatellite markers: DG14S399, DG14S404 and DG14S406, wherein
the presence of a 13, 0, 4 haplotype is diagnostic of asthma or a
susceptibility to
asthma. In another aspect, the at-risk haplotype is characterized by the
following
microsatellite markers: DG14S399 and DG14S404, wherein the presence of a 13, 4
haplotype is diagnostic of asthma or a susceptibility to asthma. Other
haplotype
3o aspects are shown in Table 1.
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Standard techniques for genotyping for the presence of SNPs and/or
microsatellite markers can be used, such as fluorescent based techniques
(Chen, et al.,
Ge~zome Res. 9, 492 (1999)), PCR, LCR, Nested PCR and other techniques for
nucleic
acid amplification. In a preferred aspect, the method comprises assessing in
an
individual the presence or frequency of SNPs and/or microsatellites in,
comprising
portions of, the MAP3K9 gene, wherein an excess or higher frequency of the
SNPs
and/or microsatellites compared to a healthy control individual is indicative
that the
individual is susceptible to MI, ACS, stroke or PAOD. See, for example, Table
3
(below) for SNPs and markers that can form haplotypes that can be used as
screening
to tools. These markers and SNPs can be identified in at-risk haploptypes. For
example,
an at-risk haplotype can include microsatellite markers and/or SNPs such as
those set
forth in Table 1. The presence of the haplotype is indicative of a
susceptibility to
asthma, and therefore is indicative of an individual who falls within a target
population
for the treatment methods described herein.
Is Haplotype analysis involves defining a candidate susceptibility locus using
LOD
scores. The defined regions are then ultra-fine mapped with microsatellite
markers with
an average spacing between markers of less than 100Kb. All usable
microsatellite
markers that found in public databases and mapped within that region can be
used. In
addition, microsatellite markers identified within the deCODE genetics
sequence
2o assembly of the human genome can be used. The frequencies of haplotypes in
the
patient and the control .groups using an expectation-maximization algorithm
can be
estimated (Dempster A. et al., 1977. ~: R. Stat. Soc. B, 39:1-389). An
implementation
of this algorithm that can handle missing genotypes and uncertainty with the
phase can
be used. Under the null hypothesis, the patients and the controls are assumed
to have
25 identical frequencies. Using a likelihood approach, an alternative
hypothesis where a
candidate at-risk-haplotype, which can include the markers described herein,
is allowed
to have a highei frequency in patients than controls, while the ratios of the
frequencies
of other haplotypes are assumed to be the same in both groups is tested.
Likelihoods
are maximized separately under both hypotheses and a corresponding 1-df
likelihood
3o ratio statistic is used to evaluate the statistic significance.
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To look for at-risk-haplotypes in the 1-lod drop, for example, association of
all possible combinations of genotyped markers is studied, provided those
markers
span a practical region. The combined patient and control groups can be
randomly
divided into two sets, equal in size to the original group of patients and
controls.
The haplotype analysis is then repeated and the most significant p-value
registered is
determined. This randomization scheme can be repeated, for example, over 100
times to construct an empirical distribution of p-values. In a preferred
aspect, a p-
value of <0.05 is indicative of an at-risk haplotype.
1o A detailed discussion of haplotype analysis follows.
Haplo ype aNalysis
Our general approach to haplotype analysis involves using likelihood-based
inference applied to NEsted MOdels. The method is implemented in our program
NEMO, which allows for many polymorphic markers, SNPs and microsatellites.
15 The method and software are specifically designed for case-control studies
where
the purpose is to identify haplotype groups that confer different risks. It is
also a
tool for studying LD structures.
When investigating haplotypes constructed from many markers, apart from
looking at each haplotype individually, meaningful summaries often require
putting
20 haplotypes into groups. A particular partition of the haplotype space is a
model that
assumes haplotypes within a group have the same risk, while haplotypes in
different
groups can have different risks. Two models/partitions are nested when one,
the
alternative model, is a finer partition compared to the other, the null model,
i.ethe
alternative model allows some haplotypes assumed to have the same risk in the
null
25 model to have different risks. The models are nested in the classical sense
that the
null model is a special case of the alternative model. Hence traditional
generalized
likelihood ratio tests can be used to test the null model against the
alternative model.
Note that, with a multiplicative model, if haplotypes lal and h; are assumed
to have
the same risk, it corresponds to assuming that flp; =~lp~ where f and p denote
3o haplotype frequencies in the affected population and the control population
respectively.
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One common way to handle uncertainty in phase and missing genotypes is a
two-step method of first estimating haplotype counts and then treating the
estimated
counts as the exact counts, a method that can sometimes be problematic (e.g.,
see the
information measure section below) and may require randomization to properly
evaluate statistical significance. In NEMO, maximum likelihood estimates,
likelihood ratios and p-values are calculated directly, with the aid of the EM
algorithm, for the observed data treating it as a missing-data problem.
NEMO allows complete flexibility for partitions. For example, the first
haplotype problem described in the Methods section on Statistical analysis
considers
1o testing whether hl has the same risk as the other haplotypes hz, ..., 7Zk.
Here the
alternative grouping is [hl], [hz, ..., hk] and the null grouping is [hl, ...,
lak]. The
second haplotype problem in the same section involves three haplotypes lal =
G0, hz
= GX and h3 = AX, and the focus is on comparing hl and lZZ. The alternative
grouping is [hl], [hz], [Iz3] and the null grouping is [hi, h2], [h3]. If
composite alleles
15 exist, one could collapse these alleles into one at the data processing
stage, and
performed the test as described. This is a perfectly valid approach, and
indeed,
whether we collapse or not makes no difference if there were no missing
information
regarding phase. But, with the actual data, if each of the alleles making up a
composite correlates differently with the SNP alleles, this will provide some
partial
2o information on phase. Collapsing at the data processing stage will
unnecessarily
increase the amount of missing information. A nested-models/partition
framework
can be used in this scenario. Let hz be split into lZZa, lzzb, ...., lzze, and
h3 be split into
h3~, h3b, ..., h3e. Then the alternative grouping is [hl), [hza, hzb, .. ..,
hze ], [h3Q, hsb,
..., h3e] and the null grouping is [hl, hza, hab, ...., hze], [h3a, h3b~ ...,
h3e]. The same
2s method can be used to handle composite where collapsing at the data
processing
stage is not even an option since L~ represents multiple haplotypes
constructed from
multiple SNPs. Alternatively, a 3-way test with the alternative grouping of
[lal],
[h2ae h2b, ...., ~Z2e], [~Z3a, ~23b, ..., h3e] versus the null grouping of
[hl, hza, hzb, ....,122e~
j23ae h3b~ . .., lZ3e] could also be performed. Note that the generalized
likelihood ratio
3o test-statistic would have two degrees of freedom instead of one.
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Measuning~ infof-rnation
Even though likelihood ratio tests based on likelihoods computed directly for
the observed data, which have captured the information loss due to uncertainty
in
phase and missing genotypes, can be relied on to give valid p-values, it would
still
be of interest to know how much information had been lost due to the
information
being incomplete. Interestingly, one can measure information loss by
considering a
two-step procedure to evaluating statistical significance that appears natural
but
happens to be systematically anti-conservative. Suppose we calculate the
maximum
likelihood estimates for the population haplotype frequencies calculated under
the
to alternative hypothesis that there are differences between the affected
population and
control population, and use these frequency-estimates as estimates of the
observed
frequencies of haplotype counts in the affected sample and in the control
sample.
Suppose we then perform a likelihood ratio test treating these estimated
haplotype
counts as though they are the actual counts. We could also perform a Fisher's
exact
15 test, but we would then need to round off these estimated counts since they
are in
general non-integers. This test will in general be anti-conservative because
treating
the estimated counts as if they were exact counts ignores the uncertainty with
the
counts, overestimates the effective sample size and underestimates the
sampling
variation. It means that the chi-square likelihood-ratio test statistic
calculated this
2o way, denoted by A*, will in general be bigger than A, the likelihood-ratio
test-
statistic calculated directly from the observed data as described in methods.
But A*
is useful because the ratio tlll~* happens to be a good measure of
information, or 1 -
(t1/A*) is a measure of the fraction of information lost due to missing
information.
This information measure for haplotype analysis is described in Nicolae and
Kong,
25 Technical,Report 537, Department of Statistics, University of Statistics,
University
of Chicago, Revised for Bionaett°ics (2003) as a natural extension of
information
measures defined for linkage analysis, and is implemented in MEMO.
Statistical analysis.
3o For single marker association to the disease, the Fisher exact test can be
used to
calculate two-sided p-values for each individual allele. All p-values are
presented
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unadjusted for multiple comparisons unless specifcally indicated. The
presented
frequencies (for microsatellites, SNPs and haplotypes) are allelic frequencies
as
opposed to carrier frequencies. To minimize any bias due the relatedness of
the
patients who were recruited as families for the linkage analysis, first and
second-
degree relatives can be eliminated from the patient list. Furthermore, the
test can be
repeated for association correcting for any remaining relatedness among the
patients,
by extending a variance adjustment procedure described in Risch, N. & Teng, J.
(Gerroffae Res., 8:1278-1288 (1998)). The relative power of family-based and
case-
control designs for linkage disequilibrium studies of complex human diseases
I.
1o DNA pooling. (ibic~ fox sibships so that it can be applied to general
familial
relationships, and present both adjusted and unadjusted p-values for
comparison.
The diffexences are in general very small as expected. To assess the
significance of
single-marker association corrected for multiple testing we carried out a
randomisation test using the same genotype data. Cohorts of patients and
controls
15 can be randomized and the association analysis redone multiple times (e.g.,
up to
500,000 times) and the p-value is the fraction of replications that produced a
p-value
for some marker allele that is lower than or equal to the p-value we observed
using
the original patient and control cohorts.
For both single-marker and haplotype analyses, relative risk (RR) and the
2o population attributable risk (PAR) can be calculated assuming a
multiplicative
model (haplotype relative risk model), (Terwilliger, J.D. & Ott, J., Huna
Hered, 42,
337-46 (1992) and Falk, C.T. & Rubinstein, P, Arrn Hnm Geraet 51 ( Pt 3), 227-
33
(1987)), i.e., that the risks ofthe two alleles/haplotypes a person carries
multiply.
For example, if RR is the risk of A relative to a, then the risk of a person
25 homozygote AA will be RR times that of a heterozygote Aa and RRz times that
of a
homozygote aa. The multiplicative model has a nice property that simplifies
analysis and computations-haplotypes are independent, i.e., in Hardy-Weinberg
equilibrium, within the affected population as well as within the control
population.
As a consequence, haplotype counts of the affecteds and controls each have
3o multinomial distributions, but with different haplotype frequencies under
the
alternative hypothesis. Specifically, for two haplotypes hi and lZf,
risk(h,)/risk(h~) _
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(flpi)l~lp~), where f and p denote respectively frequencies in the affected
population
and in the control population. While there is some power loss if the true
model is
not multiplicative, the loss tends to be mild except for extreme cases. Most
importantly, p-values are always valid since they are computed with respect to
null
hypothesis.
In general, haplotype frequencies are estimated by maximum likelihood and
tests of differences between cases and controls are performed using a
generalized
likelihood ratio test (Rice, J.A. Mathematical Statistics and Data A~zalysis,
602
(International Thomson Publishing, (1995)). deCODE's haplotype analysis
program
1o called MEMO, which stands for NEsted MOdels, can be used to calculate all
the
haplotype results. To handle uncertainties with phase and missing genotypes,
it is
emphasized that we do not use a common two-step approach to association tests,
where haplotype counts are first estimated, possibly with the use of the EM
algorithm, Dempster, (A.P., Laird, N.M. & Rubin, D.B., Jou~~nal of the Royal
15 Statistical Society B, 39, 1-3$ (1971)) and then tests are performed
treating the
estimated counts as though they are true counts, a method that can sometimes
be
problematic and may require randomisation to properly evaluate statistical
significance. Instead, with NEMO, maximum likelihood estimates, likelihood
ratios
and p-values are computed with the aid of the EM-algorithm directly for the
20 observed data, and hence the loss of information due to uncertainty with
phase and
missing genotypes is automatically captured by the likelihood ratios. Even so,
it is
of interest to know how much information is retained, or lost, due to
incomplete
information. Described herein is such a measure that is natural under the
likelihood
framework. For a fixed set of markers, the simplest tests performed compare
one
2s selected haplotype against all the others. Call the selected haplotype hl
and the
others h2, ..., hk. Let p1, .. ., p~ denote the population frequencies of the
haplotypes in
the controls, and f , . . ., fk denote the population frequencies of the
haplotypes in the
affecteds. Under the null hypothesis, f = p; for all i. The alternative model
we use
for the test assumes h2, ..., hk to have the same risk while hl is allowed to
have a
3o different risk. This implies that while pl can be different from f ,
fl(fa+...+f~) = p~
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/(pz+. ..+pk) _ /~1 for i = 2, . . ., k. Denoting fllpl by r, and noting that
(3z+.. .+/~k = l,
the test statistic based on generalized likelihood ratios is
A = 2 ~~(r,Pi, Via, ..., ~~.-1) - f(l,pn ~2, ..., ~~,-i)
where ~ denotes logelikelihood and " and ~ denote maximum likelihood estimates
s under the null hypothesis and alternative hypothesis respectively. A has
asymptotically a chi-square distribution with 1-df, under the null hypothesis.
Slightly more complicated null and alternative hypotheses can also be used.
For
example, let lay be G0, h2 be GX and h3 be AX. When comparing GO against GX,
i.e., this is the test which gives estimated RR of 1.46 and p-value = 0.0002,
the null
1o assumes GO and GX have the same risk but AX is allowed to have a different
risk.
The alternative hypothesis allows, for example, three haplotype groups to have
different risks. This implies that, under the null hypothesis, there is a
constraint that
f /pi =f lpz, or w = ~yl/[fz/pz] = 1. The test statistic based on generalized
likelihood ratios is
15 A ' 2 ~~~1, .fl, 2~a, ~) '- ~(~i, fi, ~z, I)
that again has asymptotically a chi-square distribution with 1-df under the
null
hypothesis. If there are composite haplotypes (for example, h2 and la3), that
is
handled in a natural manner under the nested models framework.
LD between pairs of SNPs can be calculated using the standard definition of
2o D' and R2 (Lewontin, R., Genetics 49, 49-67 (1964) and Hill, W.G. &
Robertson,
A. Theor. Appl. Genet. 22, 226-231 (1968)).Using MEMO, frequencies of the two
marker allele combinations are estimated by maximum likelihood and deviation
from linkage equilibrium is evaluated by a likelihood ratio test. The
definitions of D'
and Rz are extended to include microsatellites by averaging over the values
for all
2s possible allele combination of the two markers weighted by the marginal
allele
probabilities. When plotting all marker combination to elucidate the LD
structure in
a particular region, we plot D' in the upper left corner and the p-value in
the lower
right corner. In the LD plots the markers can be plotted equidistant rather
than
according to their physical location, if desired.
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Statistical Methods for Linkage Analysis
Multipoint, affected-only allele-sharing methods can be used in the analyses
to assess evidence for linkage. Results, both the LOD-score and the non-
parametric
linkage (NPL) score, can be obtained using the program Allegro (Gudbjartsson
et
al., Nat. Genet. 25:12-3, 2000). Our baseline linkage analysis uses the
Spairs scoring function (Whittemore, A.S., Halpern, J. (1994), Biometrics
50:118-
27; Kruglyak L, et al. (1996), Am JHum Genet 58:1347-63), the exponential
allele-
sharing model (Kong, A. and Cox, N.J. (1997), Arn JHum Genet 61:1179-88) and a
family weighting scheme that is halfway, on the log-scale, between weighting
each
1o affected pair equally and weighting each family equally. The information
measure
we use is part of the Allegro program output and the information value equals
zero if
the marker genotypes are completely uninformative and equals one if the
genotypes
determine the exact amount of allele sharing by decent among the affected
relatives
(Gretarsdottir et al., Am. J. Hom. Genet, 70:593-603, (2002)). We computed the
P-
1s values two different ways and here report the less significant result. The
first P-
value can be computed on the basis of large sample theory; the distribution of
Z~r =
~(2[loge(10)LODI) approximates a standard normal variable under the null
hypothesis of no linkage (Kong, A. and Cox, N.J. (1997), Am JHutn Genet
61:1179-
88). The second P-value can be calculated by comparing the observed LOD-score
2o with its complete data sampling distribution under the null hypothesis
(e.g.,
Gudbjartsson et al., Nat. Genet. 25:12-3, 2000). When the data consist of more
than
a few families, these two P-values tend to be very similar.
Methods for def°iving mRNA expression data:
2s Real Time (RT)-PCR~ is used to examine RNA levels of MLK-1 in blood
cells and lung tissue of asthma patients and controls. Total RNA is extracted
using
Trizol and purified with Qia RNaeasy spin columns (Qiagen Inc. Valencia, CA).
Two ~g of total RNA is treated with DNaseI and the RNA was reverse transcribed
using the TaqMan Reverse Transcription Reagents kit (N808-0234) and random
3o hexamers. Five ABI SYBR green assays are constructed for estimation of MLK-
1
transcripts (variant A-E, Table A). PCR reactions are carried out on a 384
well plate
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in a total volume of 10,1 on the Applied Biosystems PRISM 7900HT Sequence
Detection System (95°C for 10 minutes followed by 40 cycles of
95°C for 15
seconds, 60°C for 1 minute; with a subsequent dissociation step;
95°C for 15
seconds, 60°C for 15 seconds, 95°C for 15 seconds which
identifies melting
s temperatures of PCR products, thus assuring it specificity). The~reaction
consisted
of lpl of cDNA, 1X SYBR Green PCR Master Mix (part number 4309155) and
900nM primers. All reactions were run in quadruplicates for both the five MLK-
1
isofornns and the housekeeping gene (Beta actin). The RNA levels (copy
numbers)
are determined using sequence specific probes that hybridize to PCR products
of
1o MLK kinases (e.g., MLKl) by employing the 5'-->3' exonuclease activity of
Taq
DNA polymerase on RNA samples that are isolated from cells that-have been
exposed to specific cytokine activators that activate the JNK pathway (such as
ILlb
and TNFa) vs vehicle alone (i.e., no activation). The TaqMan probe consists of
a
site-specific sequence labeled with a fluorescent reporter dye and a
fluorescent
1s quencher dye. During PCR the TaqMan probe hybridizes to its complementary
single strand DNA sequence within the PCR target. When amplification occurs
the
TaqMan probe is degraded due to the 5'-->3' exonuclease activity of Taq DNA
polymerase, thereby separating the quencher from the reporter during
extension.
Due to the release of the quenching effect on the reporter, the fluorescence
intensity
20 of the reporter dye increases. During the entire amplification process this
light
emission increases exponentially, the final level being measured by ,
spectrophotometry after termination of the PCR. In addition to the MLK kinase
(e.g., MLK1) sequence-specific TaqMan probes, SYBR Green are also used as a
fluorescent dye. This dye fluoresces only when bound to double-stranded DNA,
i.e.,
2s when MLK kinase (e.g., MLKl) unique primers bind and successfully allow for
Taq
DNA polymerase extension of DNA fragment representing the MLK kinase (e.g.,
MLK1) gene. The use of primers located in unique exons will ensure that Taq
DNA
polymerase DNA fragments represent the mature RNA structure, furthermore the
use of MLK kinase (e.g., MLK1) sequence-specific TaqMan probes allows for
3o discrimation between different RNA splice variants. Three calibrators are
used to
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correct the quantity of the repeated samples for plate-to-plate variation. All
values
are subsequently normalized to standard corrected housekeeping gene values.
ASSESSMENT OF MAP3K9 GENE DYSREGULATION
s In one aspect, the invention relates to methods of measuring RNA levels of
the MLK kinases (e.g., MLKl) using Real-Time Quantitative PCR assay in which
oligonucleotides specific for members of the MLK kinase family (e.g., MLKl)
are
used to amplify reverse transcribed RNA (c-DNA) on RNA samples that are
isolated
from blood leukocytes or other tissue samples. The method includes obtaining a
1o sample of cells from the patient, and determining RNA levels using sequence
specific probes that hybridize to PCR~products of MLK kinases (e.g., MLK1) by
employing the 5'-->3' exonuclease activity of Taq DNA polymerase on RNA
samples that are isolated from cells that have been exposed to specific
cytokine
activators that activate the JNK pathway (such as ILIb or TNFa) vs vehicle
alone
1s (i.e., no activation). In one aspect, the TaqlVIan probe consists of a site-
specific
sequence labeled with a fluorescent reporter dye and a fluorescent quencher
dye
wherein, during the PCR reaction, the TaqMan probe hybridizes to its
complementary single strand DNA sequence within the PCR target, the final
level
being measured by spectrophotometry after termination of the PCR. In another
2o aspect, MLK kinase expression (e.g., MLK1) is determined using SYBR Green
as a
fluorescent dye. This dye fluoresces only when bound to double-stranded DNA,
i.e.
when MLK kinase (e.g., MLKl) uniquely designed primers bind and allow for
successesful Taq DNA polymerase extension of DNA fragment representing the
MLK kinase (e.g., MLKl) gene. As such, the use of MLK kinase (e.g., ML,Kl)
2s sequence-specific TaqMan probes allows for discrimation between different
RNA
splice variants. In yet another aspect, the invention is directed at methods
that
determine the role of MAP3k9 or its pathway-related genes, by obtaining a
sample
of cells from patients with asthma or other respiratory or inflammatory
disorders,
determining RNA levels of MAP3k9 or its pathway related genes in cells exposed
to
3o pathway specific activators (such as ILlb or TNFa) or vehicle alone (no
activation),
and comparing them with reference RNA levels of the gene in cells isolated
from
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subjects without asthma or other inflammatorylrespiratory disorders. In
another
aspect, the invention relates to methods for predicting efficacy of an
inhibitor drug,
including obtaining a sample of cells from patients with asthma or another
respiratorylinflammatory disorder, determining RNA levels of MAP3k9 or its
pathway related genes in cells isolated from patients who axe taking the drug
compared to those who are not taking the drug. In another aspect, the
invention
relates to methods for predicting efficacy of an inhibitor drug, including
obtaining a
sample of cells from patients with asthma or other respiratory/inflammatory
disorders, determining RNA levels of MAP3k9 or its pathway related genes after
1o exposure of the cells to the inhibitor drug ih vitro.
MONITORING PROGRESS OF TREATMENT
The current invention also pertains to methods of monitoring the response of
an individual, such as an individual in one of the target populations
described above,
us to treatment with a MLK family kinase inhibitor.
Because the level of inflammatory markers can be elevated in individuals
who are in the target populations described above, an assessment of the level
of
inflammatory markers of the individual both before, and during, treatment with
the
MLK family kimase inhibitor may indicate whether the treatment has
successfully
2o decreased production of MLKs in the airway wall (such as in ASM cells) or
in bone-
marrow derived inflammatory cells (such as peripheral blood mononuclear (PBM
cells). For example, in one aspect of the invention, an individual who is a
member
of a target population as described above (e.g., an individual at risk for
asthma, such
as an individual who is at risk due to a MAP3K9 haplotype) can be assessed for
2s response to treatment with a MLK family kinase inhibitor, by examining the
individuals MLK kinase levels in different cells and body fluids. Blood,
serum,
plasma or urinary MLKs kinases (e.g., MLK1), or ex vivo production of MLK
kinases (e.g., MLKl), can be measured before, and during or after treatment
with the
MLK family kinase inhibitor. The MLK or MLK family kinase level before
3o treatrnent is compared with the MLK family kinase level during or after
treatment,
The efficacy of treatment is indicated by a decrease in MLK production: a
level of
MLK family kinase during or after treatment that is significantly lower than
the level
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of MLK family kinase before treatment, is indicative of efficacy. A level that
is
lower during or after treatment can be shown, for example, by decreased serum
or
urinary MLKs, or decreased ex vivo production of MLK family kinases. A level
that
is "significantly lower", as used herein, is a level that is less than the
amount that is
typically found in control individual(s), or is less in a comparison of
disease risk in a
population associated with the other bands of measurement (e.g., the mean or
median, the highest quartile or the highest quintile) compared to lower bands
of
measurement (e.g., the mean or median, the other quartiles; the other
quintiles).
For example, in one aspect of the invention, the level of a MLK family
to kinase is assessed in an individual before treatment with a MLK family
kinase
inhibitor; and during or after treatment with the MLK family kinase inhibitor,
and
the levels are compared. A level of the MLK family kinase during or after
treatment
that is significantly lower than the Ievel of the MLK family kinase before
treatment,
is indicative of efficacy of treatment with the MLK family kinase inhibitor.
In
15 another aspect, production of a MLK family kinase is analyzed in a first
test sample
from the individual, and is also 'determined in a second test sample from the
individual, during or after treatment with the MLK family kinase inhibitor,
and the
level of production in the first test sample is compared with the level of
production
of the MLK family kinase in the second test sample. A level of the MLK family
2o kinase in the second test sample that is significantly lower than the level
of the MLK
family kinase in the first test sample is indicative of efficacy of treatment
with the
MLK family kinase inhibitor.
In another aspect of the invention, an individual who is a member of a target
population of individuals at risk for asthma (e.g., an individual in a target
population
25 described above, can be assessed for response to treatment with a MLK
family
kinase inhibitor, by examining levels of inflammatory markers in the
individual. For
example, levels of an inflammatory marker in an appropriate test sample (e.g.,
serum, plasma or urine) can be measured before, and during or after treatment
with
the MLK family kinase inhibitor. The level of the inflammatory marker before
3o treatment is compared with the level of the inflammatory marker during or
after
treatment. The efficacy of treatment is indicated by a decrease in the level
of the
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inflammatory marker, that is, a level of the inflammatory marker during or
after
treatment that is significantly different (e.g., significantly lower), than
the level of
inflammatory marker before treatment, is indicative of efficacy.
Representative
inflammatory markers include plasma IL-2, IL-2, IL-1 (3 and TNF-a levels and
exhaled nitric oxide (NO).
PHARMACEUTICAL COMPOSITIONS
The present invention also pertains to pharmaceutical compositions
comprising agents described herein, for example, an agent that is a MLK family
1o kinase inhibitor as described herein. For instance, a MLK family kinase
inhibitor
- can be formulated with a physiologically acceptable carrier or excipient to
prepare a
pharmaceutical composition. The carrier and composition can be sterile. The
formulation should suit the mode of administration.
Suitable pharmaceutically acceptable carriers include but are not limited to
1s water, salt solutions (e.g., NaCI), saline, buffered saline, alcohols,
glycerol, ethanol,
gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,
carbohydrates such as lactose, amylose or starch, dextrose, magnesium
stearate, talc,
silicic acid, viscous paraffin, perfume oil, fatty acid esters,
hydroxymethylcellulose,
polyvinyl pyrolidone, etc., as well as combinations thereof. The
pharmaceutical
2o preparations can, if desired, be mixed with auxiliary agents, e.g.,
lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic
pressure, buffers, coloring, flavoring andlor aromatic substances and the like
which
do not deleteriously react with the active agents.
The composition, if desired, can also contain minor amounts of wetting or
25 emulsifying agents, or pH buffering agents. The composition can be a liquid
solution, suspension, emulsion, tablet, pill, capsule, sustained release
formulation, or
powder. The composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can include
standard
carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium
3o stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium
carbonate,
etc. Nebulized~formulation for inhalation can include sodium chloride, sodium
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saccharine or sorbitani trioleas, whereas inhalation via compressed carbonated
formulation in a puffer can include 1, 1, 1, 2-tetrafluoroethanum,
monofluorotrichloromethanum tetrafluorodichloroaethanum or
diflurodichIoromethanum.
Methods of introduction of these compositions include, but are not limited
to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous,
subcutaneous, topical, oral, inhaled and intranasal. Other suitable methods of
introduction can also include gene therapy (as described below), rechargeable
or
biodegradable devices, particle acceleration devices ("gene guns") and slow
release
~o polymeric devices. The pharmaceutical compositions of this invention can
also be
administered as part of a combinatorial therapy with other agents.
The composition can be formulated in accordance with the routine
procedures as a pharmaceutical composition adapted for administration to human
beings. For example, compositions for intravenous administration typically are
15 solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may
also include a solubilizing agent and a local anesthetic to ease pain at the
site of the
injection. Generally, the ingredients are supplied either separately or mixed
together
in unit dosage form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampule or sachette
2o indicating the quantity of active agent. Where the composition is to be
administered
by infusion, it can be dispensed with an infusion bottle containing sterile
pharmaceutical grade water, saline or dextrose/water. Where the composition is
administered by injection, an ampule of sterile water for injection or saline
can be
provided so that the ingredients may be mixed prior to administration.
2s Administration by inhalation includes a mixture of the active drug and the
above
mentioned ingredients.
Far topical application, nonsprayable forms, viscous to semi-solid or solid
forms comprising a carrier compatible with topical application and having a
dynamic viscosity preferably greater than water, can be employed. Suitable
3o formulations include but are not limited to solutions, suspensions,
emulsions,
creams, ointments, powders, enemas, lotions, sots, liniments, salves,
aerosols, etc.,
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which are, if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives,
stabilizers, wetting agents, buffers or salts for influencing osmotic
pressure, etc. The
agent may be incorporated into a cosmetic formulation. For topical
application, also
suitable are sprayable aerosol preparations wherein the active ingredient,
preferably
in combination with a solid or liquid inert carrier material, is packaged in a
squeeze
bottle or in admixture with a pressurized volatile, normally gaseous
propellant, e.g.,
pressurized air.
Agents described herein can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free amino groups
such
to as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc.,
and those formed with free carboxyl groups such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopxopylamine,
triethylamine, 2-
ethylamino ethanol, histidine, procaine, etc. ,
The agents are administered in a therapeutically effective amount. The
Is amount of agents which will be therapeutically effective in the treatment
of a
particular disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques. In addition,
isz
vitro or i~ vivo assays may optionally be employed to help identify optimal
dosage
ranges. The precise dose to be employed in the formulation will also depend on
the
2o route of administration, and the seriousness of the symptoms, and should be
decided
according to the judgment of a practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves derived from in
vitro or animal model test systems.
The invention also provides a pharmaceutical pack or kit comprising one or
2s more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such containers) can
be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
use or sale of pharmaceuticals or biological products, which notice reflects
approval
by the agency of manufacture, use of sale for human administration. The pack
or kit
3o can be labeled with information regarding mode of administration, sequence
of drug
administration (e.g., separately, sequentially or concurrently), or the like.
The pack
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or kit may also include means for reminding the patient to take the therapy.
The
paclc or kit can be a single unit dosage of the combination therapy or it can
be a
plurality of unit dosages. In particular, the agents can be separated, mixed
together
in any combination, present in a single vial or tablet. Agents assembled in a
blister
pack or other dispensing means is preferred. For the purpose of this
invention, unit
dosage is intended to mean a dosage that is dependent on the individual
pharmacodynamics of each agent and administered in FDA approved dosages in
standard time courses.
1o NUCLEIC ACIDS OF THE INVENTION
MAP3K9 Nucleic Acids, Pof~tio~cs ahd Yaf~iants
The full sequence of the MAP3K9 gene is shown in SEQ ID NO: 1 and
FIGS. 7.1 to 7.19. Additional single nucleotide polymorphisms are reported in
Table
5 and may or may not be shown in SEQ ID NO: 1. It should be understood that
the
nucleic acids and their gene products emhraced by the invention include the
nucleotide sequence set forth in SEQ ID NO: 1 and may further comprise at
least
one polymorphism as shown in Table 5. Three novel SNPs have been identified.
Accordingly, the invention pertains to isolated nucleic acid molecules
comprising human MAP3K9 nucleic acid. The term, "MAP3K9 nucleic acid," as
2o used herein, refers to an isolated nucleic acid molecule encoding a MAP3K9
polypeptide (e.g., a MAP3K9 gene, such as shown in SEQ ID NO: 1). The~MAP3K9
nucleic acid molecules of the present invention can be RNA, for example, mRNA,
or DNA, such as cDNA and genomic DNA. DNA molecules can be double-
stranded or single-stranded; single stranded RNA or DNA can be the coding, or
2s sense, strand or the non-coding, or antisense strand. The nucleic acid
molecule can
include all or a portion of the coding sequence of the gene and can further
comprise
additional non-coding sequences such as introns and non-coding 3' and 5'
sequences
(including regulatory sequences, for example).
For example, the MAP3K9 nucleic acid can be the genomic sequence shown
3o in FIGs. 7.1 to 7.19, or a portion or fragment of the isolated nucleic acid
molecule
(e.g., cDNA or the gene) that encodes MAP3K9 polypeptide.
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Additionally, nucleic acid molecules of the invention can be fused to a
marker sequence, for example, a sequence that encodes a polypeptide to assist
in
isolation or purification of the polypeptide. Such sequences include, but are
not
limited to, those that encode a glutathione-S-transferase (GST) fusion protein
and
s those that encode a hemagglutinin A (HA) polypeptide marker from influenza.
An "isolated" nucleic acid molecule, as used herein, is one that is separated
from nucleic acids that normally flank the gene or nucleotide sequence (as in
genomic sequences) and/or has been completely or partially purified from other
transcribed sequences (e.g., as in an RNA library). For example, an isolated
nucleic
to acid of the invention may be substantially isolated with respect to the
complex
cellular milieu in which it naturally occurs, or culture medium when produced
by
recombinant techniques, or chemical precursors ox other chemicals when
chemically
synthesized. In some instances, the isolated material will form part of a
composition
(for example, a crude extract containing other substances), buffer system or
reagent
is mix. In other circumstances, the material may be purified to essential
homogeneity,
for example as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid molecule comprises at least about 50, 80
or 90%
(on a molar basis) of all macromolecular species present. With regard to
genomic
DNA, the term "isolated" also can refer to nucleic acid molecules that are
separated
2o from the chromosome with which the genomic DNA is naturally associated. For
example, the isolated nucleic acid molecule can contain less than about 5 kb
but not
limited to 4 kb, 3 lcb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which
flank the
nucleic acid molecule in the genomic DNA of the cell from which the nucleic
acid
molecule is derived.
25 The nucleic acid molecule can be fused to other coding or regulatory
sequences and still be considered isolated. Thus, recombinant DNA contained in
a
vector is included in the definition of "isolated" as used herein. Also,
isolated
nucleic acid molecules include recombinant DNA molecules in heterologous host
cells, as well as partially or substantially purified DNA molecules in
solution.
30 "Isolated" nucleic acid molecules also encompass in vivo and iu vitro RNA
transcripts of the DNA molecules of the present invention. An isolated nucleic
acid
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molecule can include a nucleic acid molecule or nucleic acid sequence that is
synthesized chemically or by recombinant means. Therefore, recombinant DNA
contained in a vector is included in the definition of "isolated" as used
herein. Also,
isolated nucleic acid molecules include recombinant DNA molecules in
heterologous organisms, as well as partially or substantially purified DNA
molecules
in solution. Ih vivo and ih vits~o RNA transcripts of the DNA molecules of the
present invention are also encompassed by "isolated" nucleic acid sequences.
Such
isolated nucleic acid molecules are useful in the manufacture of the encoded
polypeptide, as probes for isolating homologous sequences (e.g., from other
to mammalian species), for gene mapping (e.g., by ih ,ritu hybridization with
chromosomes), or for detecting expression of the gene in tissue (e.g., human
tissue),
such as by Northern or Southern blot analysis.
The present invention also pertains to nucleic acid molecules which are not
necessarily found in nature but which encode a MAP3K9 polypeptide, or another
15 splicing variant of a MAP3K9 polypeptide or polymorphic variant thereof:
Thus, for
example, the invention pertains to DNA molecules comprising a sequence that is
different from the naturally occurring nucleotide sequence but which, due to
the
degeneracy of the genetic code, encode a MAP3K9 polypeptide of the present
invention. The invention also encompasses nucleic acid molecules encoding
2o portions (fragments), or encoding variant polypeptides such as analogues or
derivatives of a MAP3K9 polypeptide. Such variants can be naturally occurring,
such as in the case of allelic variation or single nucleotide polymorphisms,
or non-
naturally-occurring, such as those induced by various mutagens and mutagenic
processes. Intended variations include, but are not limited to, addition,
deletion and
25 substitution of one or more nucleotides that can result in conservative or
non-
conservative amino acid changes, including additions and deletions. Preferably
the
nucleotide (and/or resultant amino acid) changes are silent or conserved; that
is, they
do not alter the characteristics or activity of a MAP3K9 polypeptide. In one
aspect,
the nucleic acid sequences are fragments that comprise one or more polymorphic
3o microsatellite markers. In another aspect, the nucleotide sequences are
fragments
that comprise one or more single nucleotide polymorphisms in a MAP3K9 gene.
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~ther alterations of the nucleic acid molecules of the invention can include,
for example, labeling, methylation, internucleotide modifications such as
uncharged
linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,
carbamates), charged linleages (e.g., phosphorothioates, phosphorodithioates),
pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,
psoralen),
chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic
acids).
Also included are synthetic molecules that mimic nucleic acid molecules in the
ability to bind to a designated sequence via hydrogen bonding and other
chemical
interactions. Such molecules include, for example, those in which peptide
linkages
Zo substitute for phosphate linkages in the backbone of the molecule.
The invention also pertains to nucleic acid molecules that hybridize under
high stringency hybridization conditions, such as for selective hybridization,
to a
nucleotide sequence described herein (e.g., nucleic acid molecules which
specifically hybridize to a nucleotide sequence encoding polypeptides
described
herein, and, optionally, have an activity of the polypeptide). In one aspect,
the
invention includes variants described herein that hybridize under high
stringency
hybridization conditions (e.g., for selective hybridization) to a nucleotide
sequence
encoding an amino acid sequence or a polymozphic variant thereof. In another
aspect, the variant that hybridizes under high stringency hybridizations has
an
2o activity of a M~4P3K9 polypeptide.
Such nucleic acid molecules can be detected andlor isolated by specific
hybridization (e.g., under high stringency conditions). "Specific
hybridization," as
used herein, refers to the ability of a first nucleic acid to hybridize to a
second
nucleic acid in a manner such that the first nucleic acid does not hybridize
to any
nucleic acid other than to the second nucleic acid (e.g., when the first
nucleic acid
has a higher similarity to the second nucleic acid than to any other nucleic
acid in a
sample wherein the hybridization is to be performed). "Stringency conditions"
for
hybridization is a term of art which refers to the incubation and wash
conditions,
e.g., conditions of temperature and buffer concentration, which permit
hybridization
of a particular nucleic acid to a second nucleic acid; the first nucleic acid
may be
perfectly (i.e., 100%) complementary to the second, or the first and second
may
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share some degree of complementarity which is less than perfect (e.g., 70%,
75%,
85%, 90%, 95%). For example, certain high stringency conditions can be used
which distinguish perfectly complementary nucleic acids from those of less
complementarity. "High stringency conditions", "moderate stringency
conditions"
s and "low stringency conditions" for nucleic acid hybridizations are
explained on
pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Cur~ef~t Protocols in
Moleculaf°
Biology (Ausubel, F.M. et al., "Current Protocols its Molecular Biology", John
Wiley & Sons, (2001)), the entire teachings of which are incorporated by
reference
herein). The exact conditions which determine the stringency of hybridization
1o depend not only on ionic strength (e.g., 0.2X SSC, O.1X SSC), temperature
(e.g.,
room temperature, 42°C, 68°C) and the concentration of
destabilizing agents such as
formamide or denaturing agents such as SDS, but also on factors such as the
length
of the nucleic acid sequence, base composition, percent mismatch between
hybridizing sequences and the frequency of occurrence of subsets of that
sequence
is within other non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while maintaining a
similar
degree of identity or similarity between the two nucleic acid molecules.
Typically,
conditions are used such that sequences at least about 60%, at least about
70%, at
least about 80%, at least about 90%, or at least about 95% or more identical
to each
20 other remain hybridized to one another. By varying hybridization conditions
from a
level of stringency at which no hybridization occurs to a level at which
hybridization
is first observed, conditions which will allow a given sequence to hybridize
(e.g.,
selectively) with the most similar sequences in the sample can be determined.
Exemplary conditions are described in Krause, M.H. and S.A. Aaronson,
2s Methods irZ Efzzynaology 200:546-556 (1991), and in, Ausubel, et al.,
"Cuf°~ent
Protocols ih Molecular Biology", John Wiley ~ Sons, (2001 ), which describes
the
determination of washing conditions for moderate or low stringency conditions.
Washing is the step in which conditions are usually set so as to determine a
minimum level of complementarity of the hybrids. Generally, starting from the
30 lowest temperature at which only homologous hybridization occurs, each
°C by
which the final wash temperature is reduced (holding SSC concentration
constant)
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-76-
allows an increase by 1 % in the maximum extent of mismatching among the
sequences that hybridize. Generally, doubling the concentration of SSC results
in an
increase in Tm of-17°C. Using these guidelines, the washing temperature
can be
determined empirically for high, moderate or low stringency, depending on the
level
s of mismatch sought.
For example, a low stringency wash can comprise washing in a solution
containing 0.2X SSC/0.1% SDS for 10 minutes at room temperature; a moderate
stringency wash can comprise washing in a pre-warmed solution (42°C)
solution
containing 0.2X SSC/0.1% SDS for 15 minutes at 42°C; and a high
stringency wash
1o can comprise washing in pre-warmed (68°C) solution~containing O.1X
SSC/0.1°/aSDS for 15 minutes at 68°C. Furthermore, washes
can be performed
repeatedly or sequentially to obtain a desired result as known in the art.
Equivalent
conditions can be determined by varying one or more of the parameters given as
an
example, as known in the art, while maintaining a similar degree of identity
or
1s similarity between the target nucleic acid molecule and the primer or probe
used.
The percent homology or identity of two nucleotide or amino acid sequences
can be determined by aligning the sequences for optimal comparison purposes
(e.g.,
gaps can be introduced in the sequence of a first sequence for optimal
alignment).
The nucleotides or amino acids at corresponding positions are then compared,
and
2o the percent identity between the two sequences is a function of the number
of
identical positions shared by the sequences (i.e., % identity = # of identical
positions/total # of positions x 100). When a position in one sequence is
occupied by
the same nucleotide or amino acid residue as the corresponding position in the
other
sequence, then the molecules are homologous at that position. As used herein,
25 nucleic acid or amino acid "homology" is equivalent to nucleic acid or
amino acid
"identity". In certain aspects, the length of a sequence aligned for
comparison
purposes is at least 30%, for example, at least 40%, in certain aspects at
least 60%,
and in other aspects at least 70%, 80%, 90% or 95% of the length of the
reference
sequence. The actual comparison of the two sequences can be accomplished by
3o well-known methods, for example, using a mathematical algorithm. A
preferred,
non-limiting example of such a mathematical algorithm is described in Karlin
et al.,
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Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is
incorporated
into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et
al., Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped
BLAST programs, the default parameters of the respective programs (e.g.,
NBLAST) can be used. In one aspect, parameters for sequence comparison can be
set at score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).
Another preferred non-limiting example of a mathematical algorithm utilized
for the comparison of sequences is the algorithm of Myers and Miller, CABIOS
4(1):
11-17 (1988). Such an algorithm is incorporated into the ALIGN program
(version
~ 2.0) which is part of the GCG sequence alignment software package (Accelrys,
Cambridge, UK). When utilizing the ALIGN program for comparing amino acid
sequences, a PAM120 weight residue table, a gap length penalty of 12, and a
gap
penalty of 4 can be used. Additional algorithms for sequence analysis are
known in
the art and include ADVANCE and ADAM as described in Torellis and Robotti,
Conaput. Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson and
Lipman,
Pi°oc. Natl. Acad. Sci. USA 85:2444-8 (1988).
In another aspect, the percent identity between two amino acid sequences can
be accomplished using the GAP program in the GCG software package using either
a BLOSUM63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4
2o and a length weight of 2, 3, or 4. In yet another aspect, the percent
identity between
two nucleic acid sequences can be accomplished using the GAP program in the
GCG software package using a gap weight of 50 and a length weight of 3.
The present invention also provides isolated nucleic acid molecules that
contain a fragment or portion that hybridizes under highly stringent
conditions to a
nucleotide sequence of SEQ ID NO: 1 or the complement of such a sequence, and
also provides isolated nucleic acid molecules that contain a fragment or
portion that
hybridizes under highly stringent conditions to a nucleotide sequence encoding
an
amino acid sequence or polymorphic variant thereof. The nucleic acid fragments
of
the invention are at least about 15, preferably at least about 18, 20, 23 or
25
so nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length.
Longer
fragments, for example, 30 or more nucleotides in length, that encode
antigenic
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polypeptides described herein are particularly useful, such as for the
generation of
antibodies as described below.
Probes and Prirraers
In a related aspect, the nucleic acid fragments of the invention are used as
probes or primers in assays such as those described herein. "Probes" or
"primers"
are oligonucleotides that hybridize in a base-specific manner to a
complementary
strand of nucleic acid molecules. Such probes and primers include polypeptide
nucleic acids, as described in Nielsen et al., Science 254:1497-1500 (1991).
1o A probe or primer comprises a region of nucleotide sequence that hybridizes
to at least about 15, for example about 20-25, and in certain aspects about
40, 50 or
75, consecutive nucleotides of a nucleic acid molecule comprising a contiguous
nucleotide sequence of SEQ ID NO: f or polymorphic variant thereof. In other
aspects, a probe or primer comprises-100 or fewer nucleotides, in certain
aspects
from 6 to 50 nucleotides, for example from 12 to 30 nucleotides. In other
aspects,
the probe or primer is at least 70% identical to the contiguous nucleotide
sequence
or to the complement of the contiguous nucleotide sequence, for example at
least
g0% identical, in certain aspects at least 90% identical, and in other aspects
at least
95% identical, or even capable of selectively hybridizing to the contiguous
2o nucleotide-sequence or to the complement of the contiguous nucleotide
sequence.
v
Often, the probe or primer further comprises a label, e.g., radioisotope,
fluorescent
compound, enzyme, or enzyme co-factor.
The nucleic acid molecules of the invention such as those described above
can be identified and isolated using standard molecular biology techniques and
the
2s sequence information provided herein. For example, nucleic acid molecules
can be
amplified and isolated by the polymerase chain reaction using synthetic
oligonucleotide primers designed based on the sequence of SEQ ID NO: 1 or the
complement of such a sequence, or designed based on nucleotides based on
sequences encoding one or more of the amino acid sequences provided herein.
See
3o generally PCR Technology: Pni~ciples af~d Applications for DNA
Af~zplificatioh (ed.
H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods
a~cd Applications (Eds. Innis et al., Academic Press, San Diego, CA, 1990);
Mattila
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et al., Nucl. Acids Res. 19: 4967 (1991); Eckert et al., PCR Methods arzd
Applications 1:17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and
U.S.
Patent 4,683,202. The nucleic acid molecules can be amplified using cDNA, mRNA
or genomic DNA as a template, cloned into an appropriate vector and
characterized
by DNA sequence analysis.
Other suitable amplification methods include the ligase chain reaction (LCR)
(see Wu and Wallace, Gezzozzzics 4:560 (1989), Landegren et al., Science
241:1077
(1988), transcription amplification (Kwon et al., Proc. Natl. Acad. Sci. USA
86:1173
(1989)), and self sustained sequence replication (Guatelli et al., Pzoc. Nat.
Acad.
1o Sci. USA 87:1874 (1990)) and nucleic acid based sequence amplification
(NASBA).
The latter two amplification methods involve isothermal reactions based on
isothermal transcription, which produce both single stranded RNA (ssRNA) and
double stranded DNA (dsDNA) as the amplification products in a ratio of about
30
or 100,to l, respectively.
is The amplified DNA can be labeled, for example, radiolabeled, and used as a
probe for screening a cDNA library derived from human cells, mRNA in zap
express, ZIPLOX or other suitable vector. Corresponding clones can be isolated
DNA can obtained following ifz vivo excision, and the cloned insert can be
sequenced in either or both orientations by art recognized methods to identify
the
2o correct reading frame encoding a polypeptide of the appropriate molecular
weight.
For example, the direct analysis of the nucleotide sequence of nucleic acid
molecules of the present invention can be accomplished using well-known
methods
that are commercially available. See, for example, Sambrook~et al., Molecular
Clozzizzg, A Labonatofy Manual (2nd Ed., CSHP, New York 1989); Zyskind et al.,
25 Recozzzbizzarzt DNA Laboz°atory Manual, (Acad. Press, 1988)).
Additionally,
fluorescence methods are also available for analyzing nucleic acids (Chen et
al.,
Gez2ozzze Res. 9, 492 (1999)) and polypeptides. Using these or similar
methods, the
polypeptide and the DNA encoding the polypeptide can be isolated, sequenced
and
further characterized.
3o Antisense nucleic acid molecules of the invention can be designed using the
nucleotide sequence of SE(~ ID NO: 1 and/or the complement or a portion, and
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U_
constructed using chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid molecule
(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. Alternatively,
the
antisense nucleic acid molecule can be produced biologically using an
expression
vector into which a nucleic acid molecule has been subcloned in an antisense
orientation (i.e.; RNA transcribed from the inserted nucleic acid molecule
will be of
an antisense orientation to a target nucleic acid of interest).
The nucleic acid sequences can also be used to compare with endogenous
DNA sequences in patients to identify one or more of the disorders described
above,
and as probes, such as to hybridize and discover related DNA sequences or to
subtract out known sequences from a sample. The nucleic acid sequences can
further be used to derive primers for genetic fingerprinting, to raise anti-
polypeptide
antibodies using DNA immunization techniques, and as an antigen to raise anti-
DNA antibodies or elicit immune responses. Portions or fragments of the
nucleotide
sequences identified herein (and the corresponding complete gene sequences)
can be
2o used in numerous ways, such as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a chromosome; and,
thus, locate gene regions associated with genetic disease; (ii) identify an
individual
from a minute biological sample (tissue typing); and (iii) aid in forensic
identification of a biological sample. Additionally, the nucleotide sequences
of the
invention can be used to identify and express recombinant polypeptides for
analysis,
characterization or therapeutic use, or as markers for tissues in which the
corresponding polypeptide is expressed, either constitutively, during tissue
differentiation, or iii diseased states. The nucleic acid sequences can
additionally be
used as reagents in the screening and/or diagnostic assays described herein,
and can
3o also be included as components of kits (e.g., reagent kits) for use in the
screening
and/or diagnostic assays described herein.
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Kits (e.g., reagent kits) useful in the methods of diagnosis comprise
components useful in any of the methods described herein, including for
example,
hybridization probes or primers as described herein (e.g., labeled probes or
primers),
reagents for detection of labeled molecules, restriction enzymes (e.g., for
RFLP
analysis), allele-specific oligonucleotides, antibodies which bind to altered
or to
non-altered (native) MAP3K9 polypeptide, means for amplification of nucleic
acids
comprising a MAP3K9 nucleic acid, or means for analyzing the nucleic acid
sequence of a MAP3K9 nucleic acid or for analyzing the amino acid sequence of
a
MAP3K9 polypeptide as described herein, etc. In one aspect, the kit for
diagnosing
'1o a asthma or a susceptibility to asthma can comprise primers for nucleic
acid
amplification of a region in tie MA.P3K9 nucleic acid comprising an at-risk
haplotype that is more frequently present in an individual having astlnna or
who is
susceptible to asthma. The primers can be designed using portions of the
nucleic
acids flanking SNPs that are indicative of asthma. In a certain aspect, the
primers
are designed to. amplify regions of the MAP3K9 gene associated with an at-risk
haplotype for asthma, as shown in Table 1.
Yecto3s aid Host Cells
Another aspect of the invention pertains to nucleic acid constructs containing
2o a nucleic acid molecules described herein and the complements thereof (or a
portion
thereof). The constructs comprise a vector (e.g., an expression vector) into
which a
sequence of the invention has been inserted in a sense or antisense
orientation. As
used herein, the term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional DNA segments can be ligated. Another type of vector is a viral
vector,
wherein additional DNA segments can be ligated into the viral genome. Certain
vectors are capable of autonomous replication in a host cell into which they
are
introduced (e.g., bacterial vectors having a bacterial origin of replication
and
so episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian
vectors) are integrated into the genome of a host cell upon introduction into
the host
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cell, and thereby are replicated along with the host genome. Expression
vectors are
capable of directing the expression of genes to which they are operably
linked. In
general, expression vectors of utility in recombinant DNA techniques are often
in
the form of plasmids. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g., replication
defective
retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent
functions.
In certain aspects, recombinant expression vectors of the invention comprise
a nucleic acid molecule of the invention in a form suitable for expression of
the
1o nucleic acid molecule in a host cell. This means that the recombinant
expression
vectors include one or more regulatory sequences, selected on the basis of the
host
cells to be used for expression, which is operably linked to the nucleic acid
sequence
to be expressed. Within a recombinant expression vector, "operably linked" or
"operatively linked" is intended to mean that the nucleotide sequence of
interest is
15 linked to the regulatory sequences) in a manner which allows for expression
of the
nucleotide sequence (e.g., in an ih vitro transcription/translation system or
in a host
cell when the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other expression
control
elements (e.g., polyadenylation signals). Such regulatory sequences are
described,
2o for example, in Goeddel, "Gene Expression Technology", Methods ifs
Ehzymology
185, Academic Press, San Diego, CA (1990). Regulatory sequences include those
which direct constitutive expression of a nucleotide sequence in many types of
host
cell and those which direct expression of the nucleotide sequence only in
certain
host cells (e.g., tissue-specific regulatory sequences). It will be
appreciated by those
25 skilled in the art that the design of the expression vector can depend on
such factors
as the choice of the host cell to be transformed and the level of expression
of
polypeptide desired. The expression vectors of the invention can be introduced
into
host cells to thereby produce polypeptides, including fusion polypeptides,
encoded
by nucleic acid molecules as described herein.
3o The recombinant expression vectors of the invention can be designed for
expression of a polypeptide of the invention in prokaryotic or eukaryotic
cells, e.g.,
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bacterial cells such as E. coli, insect cells (using baculovirus expression
vectors),
yeast cells or mammalian cells. Suitable host cells are discussed further in
Goeddel,
supra. Alternatively, the recombinant expression vector can be transcribed and
translated in vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
Another aspect of the invention pertains to host cells into which a
recombinant expression vector of the invention has been introduced. The terms
"host cell" and "recombinant host cell" are used interchangeably herein. It is
understood that such terms refer not only to the particular subject cell but
also to the
to progeny or potential progeny of such a cell. Because certain modifications
may
occur in succeeding generations due to either mutation or environmental
influences,
such progeny may not, in fact, be identical to the parent cell, but are still
included
within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic
acid molecule of the invention can be expressed in bacterial cells (e.g., E.
coli),
insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells
(CHO)
or COS cells). Other suitable host cells are known to those skilled in the
art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized techniques for introducing a foreign nucleic acid molecule (e.g.,
DNA)
into a host cell, including calcium phosphate or calcium chloride co-
precipitation,
DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable
methods for transforming or transfecting host cells can be found in Sambrook,
et al.,
(sups°a), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the expression vector and transfection technique used, only a small fraction
of cells
may integrate the foreign DNA into their genome. In order to identify and
select
these integrants, a gene that encodes a selectable marker (e.g., for
resistance to
3o antibiotics) is generally introduced into the host cells along with the
gene of interest.
Preferred selectable markers include those that confer resistance to drugs,
such as
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6418, hygromycin and methotrexate. Nucleic acid molecules encoding a
selectable
marker can be introduced into a host cell on the same vector as the nucleic
acid
molecule of the invention or can be introduced on a separate vector. Dells
stably
transfected with the introduced nucleic acid molecule can be identified by
drug
selection (e.g., cells that have incorporated the selectable marker gene will
survive,
while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture can be used to produce (z.e., express) a polypeptide of the invention.
Accordingly, the invention further provides methods for producing a
polypeptide
1o using the host cells of the invention. In one aspect, the method comprises
culturing
the host cell of-invention (into which a recombinant expression vector
encoding a
polypeptide of the invention has been introduced) in a suitable medium such
that the
polypeptide is produced. In another aspect, the 'method further comprises
isolating
the polypeptide from the medium or the host cell.
The host cells of the invention can also be used to produce nonhuman
transgenic animals. Fox example, in one aspect, a host cell of the invention
is a
fertilized oocyte or an embryonic stem cell into which a nucleic acid molecule
of the
invention has been introduced (e.g., an exogenous MAP3K9 gene, or an exogenous
nucleic acid encoding a M~IP3K9 polypeptide). Such host cells can then be used
to
2o create non-human transgenic animals in which exogenous nucleotide sequences
have
been introduced into the genome or homologous recombinant animals in which
endogenous nucleotide sequences have been altered. Such animals are useful for
studying the function and/or activity of the nucleotide sequence and
polypeptide
encoded by the sequence and for identifying andlor evaluating modulators of
their
activity. As used herein, a "transgenic animal" is a non-human animal,
preferably a
mammal, more preferably a rodent such as a rat or mouse, in which one or more
of
the cells of the animal include a transgene. Other examples of transgenic
animals
include non-human primates, sheep, dogs, cows, goats, chickens and amphibians.
A
transgene is exogenous DNA which is integrated into the genome of a cell from
3o which a transgenic animal develops and which remains in the genome of the
mature
animal, thereby directing the expression of an encoded gene product in one or
more
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cell types or tissues of the transgenic animal. As used herein, a "homologous
recombinant animal" is a non-human animal, preferably a mammal, more
preferably
a mouse, in which an endogenous gene has been altered by homologous
recombination between the endogenous gene and an exogenous DNA molecule
introduced into a cell of the animal, e.g., an embryonic cell of the animal,
prior to
development of the animal.
Methods for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become conventional in
the
art and are described, for example, in U.S. Patent Nos. 4,736,866 and
4,870,009,
to U.S. Pat. No. 4,873,191 and in Hogan, Mahipulati~g the Mouse Efnbryo (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Methods for
constructing homologous recombination vectors and homologous recombinant
animals are described further in Bradley, Curs~efzt Opifzioh ih BioTechraology
2:823-
829 (1991) and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO
15 9210968, and WO 93/04169. Clones of the non-human transgenic animals
described
herein can also be produced according to the methods described in Wilmut et
al.,
Nature 385:810-813 (1997) and PCT Publication Nos. WO 97107668 and WO
97/07669.
2o RIBONUCLEIC ACID (RNA) OF THE INVENTION:
In one aspect, the invention relates to methods of measuring RNA levels of
the MLK kinases (e.g., MLKI) using Real-Time Quantitative PCR assay in which
oligo nucleotides specific for members ofthe MLK kinase family (e.g., MLK1)
are
used to amplify reverse transcribed RNA (c-DNA) on RNA samples that are
isolated
25 from blood leukocytes or other tissue samples. The method includes
obtaining a
sample of cells from the patient, and determining RNA levels using sequence
specific probes that hybridize to PCR products of MLK kinases (e.g., MLK1) by
employing the 5'-->3' exonuclease activity of Taq DNA polymerase on RNA
samples that are isolated from cells that have been exposed to specific
cytokine
3o activators that activate the JNK pathway (such as ILIb or TNFa) vs vehicle
alone
(i.e., no activation). In one aspect, the TaqMan probe consists of a site-
specific
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sequence labeled with a fluorescent reporter dye and a fluorescent quencher
dye
wherein, during the PCR reaction, the TaqMan probe hybridises to its
complementary single strand DNA sequence within the PCR target, the final
level
being measured by spectrophotometry after termination of the PCR. In another
s aspect, MLK kinase expression (e.g., MLKl) is determined using SYBR Green as
a
fluorescent dye. This dye fluoresces only when bound to double-stranded DNA,
i. e.,
when MLK kinase (e.g., MLKI) uniquely designed primers bind and allow for
discrimation between different RNA splice variants. In yet another aspect the
invention is directed at methods that determine the role of MAP3k9 or its
pathway-
to related genes, by obtaining a sample of cells from patients with asthma or
other
respiratory or inflammatory disorders, determining RNA levels of MAP3k9 or its
pathway related genes in cells exposed to pathway specific activators (such as
ILlb
or TNFa) or vehicle alone (no activation), and comparing them with reference
RNA
levels of the gene in cells isolated from subjects without asthma or other
15 inflammatory/respiratory disorders. In another aspect, the invention
relates to
methods for predicting efficacy of an inhibitor drug, including obtaining a
sample of
cells from patients with asthma or another respiratoryiinflammatory disorder,
determining RNA levels of MAP3k9 or its pathway related genes in cells
isolated
from patients who are taking the drug compared to those who are not taking the
2o drug. In another aspect, the invention relates to methods for predicting
efficacy of
an inhibitor drug, including obtaining a sample of cells from patients with
asthma or
other respiratory/inflammatory disorders, determining RNA levels of MAP3k9 or
its
pathway related genes after exposure of the cells to the inhibitor drug ifs
vita°o.
2s POLYPEPTIDES OF THE INVENTION
The present invention also pertains to isolated polypeptides encoded by
MAP3K9 nucleic acids ("MAP3K9 polypeptides," or "MAP3K9 proteins," such as
the protein shown in SEQ ID NO: 2, FIG. 8, FIG. 9 and NCBI accession number
XM 027237; (mRNA); the entire sequence being incorporated herein by reference)
3o and fragments and variants thereof, as well as polypeptides encoded by
nucleotide
sequences described herein (e.g., other splicing variants). The term
"polypeptide"
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refers to a polymer of amino acids, and not to a specific length; thus,
peptides,
oligopeptides and proteins are included within the definition of a
polypeptide. As
used herein, a polypeptide is said to be "isolated" or "purified" when it is
substantially free of cellular material when it is isolated from recombinant
and non-
recombinant cells, or free of chemical precursors or other' chemicals when it
is
chemically synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell (e.g., in a
"fusion
protein") and still be "isolated" or "purified."
The polypeptides of the invention can be purified to homogeneity. It is
1o understood, however, that preparations in which the polypeptide is not
purified to
homogeneity are useful. The critical feature is that the preparation allows
for the
desired function of the polypeptide, even in the presence of considerable
amounts of
other components. Thus, the invention encompasses various degrees of purity.
In
one aspect, the language "substantially free of cellular material" includes
1s preparations of the polypeptide having less than about 30% (by dry weight)
other
proteins (i. e., contaminating protein), less than about 20% other proteins,
less than
about 10% other proteins, or less than about 5% other proteins.
When a polypeptide is recombinantly produced, it can also be substantially
free of culture medium, i.e., culture medium represents less than about 20%,
less
2o than about 10%, or less than about 5% of the volume of the polypeptide
preparation.
The language "substantially.free of chemical precursors or other chemicals"
includes
preparations of the polypeptide in which it is separated from chemical
precursors or
other chemicals that are involved in its synthesis. In one aspect, the
language
"substantially free of chemical precursors or other chemicals" includes
preparations
25 of the polypeptide having less than about 30% (by dry weight) chemical
precursors
or other chemicals, less than about 20% chemical precursors or other
chemicals, less
than about 10% chemical precursors or other chemicals, or less than about S%
chemical precursors or other chemicals.
In one aspect, a polypeptide of the invention comprises an amino acid
so sequence encoded by a nucleic acid molecule comprising a nucleotide
sequence of
SEQ ID NO: 1; or the complement of such a nucleic acid, or portions thereof,
or a
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_$~_
portion or polymorphic variant thereof. However, the polypeptides of the
invention
also encompass fragment and sequence variants. Variants include a
substantially
homologous polypeptide encoded by the same genetic locus in an organism, i.e.,
an
allelic variant, as well as other splicing variants. Variants also encompass
polypeptides derived from other genetic loci in an organism, but having
substantial
homology to a polypeptide encoded by a nucleic acid molecule comprising a
nucleotide of SEQ ID NO: 1 or a complement of such a sequence, or portions
thereof or polymorphic variants thereof. Variants also include polypeptides
substantially homologous or identical to these polypeptides but derived from
another
organism, i.e., an ortholog. Variants also include polypeptides that are
substantially
homologous or identical to these polypeptides that are produced by chemical -
synthesis. Variants also include polypeptides that are substantially
homologous or
identical to these polypeptides that are produced by recombinant methods.
As used herein, two polypeptides (or a region of the polypeptides) are
is substantially homologous or identical when the amino acid sequences are at
least
about 45-55%, in certain aspects at least about 70-75%, and in other aspects
at least
about 80-85%, and in other aspects greater than about 90% or more homologous
or
identical. A substantially homologous amino acid sequence, according to the
present invention, .will be encoded by a nucleic acid molecule hybridizing to
a
zo nucleic acid of the invention or portion thereof or polymorphic variant
thereof, under
stringent conditions as more particularly described above.
The invention also encompasses polypeptides having a Lower degree of
identity but having sufficient similarity so as to perform one or more of the
same
functions performed by a polypeptide encoded by a nucleic acid molecule of the
25 invention.
Similarity is determined by conserved amino acid substitution where a given
amino acid in a polypeptide is substituted by another amino acid of like
characteristics. Conservative substitutions are likely to be phenotypically
silent.
Typically seen as conservative substitutions are the replacements, one for
another,
3o among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the
hydroxyl
residues Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution
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between the amide residues Asn and Gln, exchange of the basic residues Lys and
Arg and replacements among the aromatic residues Phe and Tyr. Guidance
concerning which amino acid changes are likely to be phenotypically silent are
found in Bowie et al., Science 247:1306-1310 (1990).
A variant polypeptide can differ in amino acid sequence by one or more
substitutions, deletions, insertions, inversions, fusions, and truncations or
a
combination of any of these. Further, variant polypeptides can be fully
functional or
can lack function in one or more activities. Fully functional variants
typically
contain only conservative variation or variation in non-critical residues or
in non-
critical regions. Functional variants can also contain substitution of similar
amino
acids that result in no change or an insignificant change in function.
Alternatively,
such substitutions may positively or negatively affect function to same
degree. Non-
functional variants typically contain one or more non-conservative amino acid
substitutions, deletions, insertions, inversions, or truncation or a
substitution,
1s insertion, inversion, or deletion in a critical residue or critical region.
Amino acids that are essential for function can be identified by methods
known in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis
(Cunningham et al., Science 244:1082-1185 (1989)). The latter procedure
introduces single alanine mutations at every residue in the molecule. The
resulting
2o mutant molecules are then tested for biological activity iYa vita o, or ih
vitro
proliferative activity. Sites that are critical for polypeptide activity can
also be
determined by structural analysis such as crystallization, nuclear magnetic
resonance
or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de
Vos et'
al., Science 255:306-312 (1992)).
2s The invention also includes polypeptide fragments of the polypeptides of
the
invention. Fragments can be derived from a polypeptide encoded by a nucleic
acid
molecule comprising SEQ ID NO: 1 or a complement of such a nucleic acid or
other
variants. However, the invention also encompasses fragments of the variants of
the
polypeptides described herein. As used herein, a fragment comprises at least 6
3o contiguous amino acids. Useful fragments include those that retain one or
more of
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the biological activities of the polypeptide as well as fragments that can be
used as
an immunogen to generate polypeptide-specific antibodies.
Biologically active fragments (peptides which are, for example, 6, 9, 12, 15,
16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) can
comprise a domain, segment, or motif that has been identified by analysis of
the
polypeptide sequence using well-known methods, e.g., signal peptides,
extracellular
domains, one or more transmembrane segments or loops, ligand binding regions,
zinc finger domains, DNA binding domains, acylation sites, glycosylation
sites, or
phosphorylation sites.
1o Fragments can be discrete (not fused to other amino acids ox polypeptides)
or
can be within a larger polypeptide. Further, several fragments can be
comprised
within a single larger polypeptide. In one aspect a fragment designed for
expression
in a host can have heterologous pre- and pro-polypeptide regions fused to the
amino
terminus of the polypeptide fragment and an additional region fused to the
carboxyl
1s terminus of the fragment.
The invention thus provides chimeric or fusion polypeptides. These comprise
a polypeptide of the invention operatively linked to a heterologous protein or
polypeptide having an amino acid sequence not substantially homologous to the
polypeptide.
20 "Operatively linked" indicates that the polypeptide and the heterologous
protein are fused in-frame. The heterologous protein can be fused to the N-
terminus
or C-terminus of the polypeptide. In one aspect the fusion polypeptide does
not
affect function of the polypeptide per se. For example, the fusion polypeptide
can
be a GST-fusion polypeptide in which the polypeptide sequences are fused to
the C
2s terminus of the GST sequences. Other types of fusion polypeptides include,
but are
not limited to, enzymatic fusion polypeptides, for example beta-galactosidase
fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Such
fusion polypeptides, particularly poly-His fusions, can facilitate the
purification of
recombinant polypeptide. In certain host cells (e.g., mammalian host cells),
3o expression and/or secretion of a polypeptide can be increased using a
heterologous
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signal sequence. Therefore, in another aspect, the fusion polypeptide contains
a
heterologous signal sequence at its N-terminus.
EP-A-O 464 533 discloses fusion proteins comprising various portions of
irnmunoglobulin constant regions. The Fc is useful in therapy and diagnosis
and
thus results, for example, in improved pharmacokinetic properties (EP-A 0232
262).
In drug discovery, for example, human proteins have been fused with Fc
portions for
the purpose of high-throughput screening assays to identify antagonists.
Bennett et
al., Journal of Molecular Recognition, 8:52-58 (1995) and Johanson et al., The
.Iournal of Biological Chemistry, 270,16:9459-9471 (1995). Thus, this
invention
~o also encompasses soluble fusion polypeptides containing a polypeptide of
the
invention and various portions of the constant regions of heavy or light
chains of
immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
A chimeric or fusion~polypeptide can be produced by standard recombinant
DNA techniques. For example, DNA fragments coding for the different
polypeptide
sequences are ligated together in-frame in accordance with conventional
techniques.
In another aspect, the fusion gene can be synthesized by conventional
techniques
including automated DNA synthesizers. Alternatively, PCR amplification of
nucleic
acid fragments can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive nucleic acid fragments which
2o can subsequently be annealed and re-amplified to generate a chimeric
nucleic acid
sequence (see Ausubel et al., Current Protocols ire Molecular Biology, 1992).
Moreover, many expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST protein). A nucleic acid molecule encoding
a
polypeptide of the invention can be cloned into such an expression vector such
that
2s the fusion moiety is linked in-frame to the polypeptide.
The isolated polypeptide can be purified from cells that naturally express it,
can be purified from cells that have been altered to express it (recombinant),
or
synthesized using known protein synthesis methods. In one aspect, the
polypeptide
is produced by recombinant DNA techniques. For example, a nucleic acid
molecule
3o encoding the polypeptide is cloned into an expression vector, the
expression vector
introduced into a host cell and the polypeptide expressed in the host cell.
The
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polypeptide can then be isolated from the cells by an appropriate purification
scheme using standard protein purification techniques.
The polypeptides of the present invention can be used to raise antibodies or
to elicit an immune response. The polypeptides can also be used as a reagent,
e.g., a
labeled reagent, in assays to quantitatively determine levels of the
polypeptide or a
molecule to which it binds (e.g., a ligand) in biological fluids. The
polypeptides can
also be used as markers for cells or tissues in which the corresponding
polypeptide is
preferentially expressed, either constitutively, during tissue
differentiation, or in a
diseased state. The polypeptides can be used to isolate a corresponding
binding
1o agent, e.g., ligand or receptor, such as, for example, in an interaction
trap assay, and
to screen for peptide or small molecule antagonists or agonists of the binding
interaction.
ANTIBODIES OF THE INVENTION
1s Polyclonal antibodies andlor monoclonal antibodies that specifically bind
one form of the gene product but not to the other form of the gene product are
also
provided. Antibodies are also provided which bind a portion of either the
variant or
the reference gene product that contains the polymorphic site or sites. The
term
"antibody" as used herein refers to immunoglobulin molecules and
immunologically
2o active portions of immunoglobulin molecules, i.e., molecules that contain
antigen-
binding sites that specifically bind an antigen. A molecule that specifically
binds to
a polypeptide of the invention is a molecule that binds to that polypeptide or
a
fragment thereof, but does not substantially bind other molecules in a sample,
e.g., a
biological sample, which naturally contains the polypeptide. Examples of
2s immunologically active portions of immunoglobulin molecules include Flab)
and
F(ab')2 fragments which can be generated by treating the antibody with an
enzyme
such as pepsin. The invention provides polyclonal and monoclonal antibodies
that
bind to a polypeptide of the invention. The term "monoclonal antibody" or
"monoclonal antibody composition", as used hexein, refers to a population of
3o antibody molecules that contain only one species of an antigen binding site
capable
of immunoreacting with a particular epitope of a polypeptide of the invention.
A
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monoclonal antibody composition thus typically displays a single binding
affinity
for a particular polypeptide of the invention with which it immunoreacts.
Polyclonal antibodies can be prepared as described above by immunizing a
suitable subject with a desired immunogen, e.g., polypeptide of the invention
or a
s fragment thereof. The antibody titer in the immunized subject can be
monitored
over time by standard techniques, such as with an enzyme linked immunosorbent
assay (ELISA) using immobilized polypeptide. If desired, the antibody
molecules
directed against the polypeptide can be isolated from the mammal (e.g., from
the
blood) and further purified by well-known techniques, such as protein A
1o chromatography to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the antibody titers are highest, antibody-producing
cells
can be obtained from the subject and used to prepare monoclonal antibodies by
standard techniques, such as the hybridoma technique originally described by
Kohler
and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique
~s (Kozbor et al., Inanzunol. Today 4: 72 (1983)), the EBV-hybridoma technique
(Cole
et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,l985, Inc., pp.
77-
96) or trioma techniques. The technology for producing hybridomas is well
known
(see generally Current Pf°otocols in Immunology (1994) Coligan et al.,
(eds.) John
Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a
2o myeloma) is fused to lymphocytes (typically splenocytes) from a mammal
immunized with an immunogen as described above, and the culture supernatants
of
the resulting hybridoma cells are screened to identify a hybridoma producing a
monoclonal antibody that binds a polypeptide of the invention.
Any of the many well known protocols used for fusing lymphocytes and
2s immortalized cell lines can be applied for the purpose of generating a
monoclonal
antibody to a polypeptide of the invention (see, e.g., Cuf°rent
Ps°otocols in
Immunology, supra; Galfre et al., Nature 266:55052 (1977); R.H. Kenneth, in
Monoclonal Antibodies: A New DimefZSion In Biological Analyses, Plenum
Publishing Corp., New York, New York (1980); and Lerner, Yale J. Biol. Med.
30 54:387-402 (1981)). Moreover, the ordinarily skilled worker will appreciate
that
there are many variations of such methods that also would be useful.
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Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal antibody to a polypeptide of the invention can be identified and
isolated
by screening a recombinant combinatorial immunoglobulin library (e.g., an
antibody
phage display library) with the polypeptide to thereby isolate immunoglobulin
library members that bind the polypeptide. Kits for generating and screening
phage
display libraries are commercially available (e.g., the Pharnnacia Recombinant
Phage Antibody Systef~a, Catalog No. 27-9400-OI; and the Stratagene SurfZAPTM
Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening antibody
display
~o library can be found in, for example, U.S. Patent No. 5,223,409;
PCT.Publication
No. WO 92/18619; PCT Publication No. WO 9I/17271; PCT Publication No. WO
92120791; PCT Publication No. WO 92115679; PCT Publication No. WO 93101288;
PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT
Publication No. WO 90/02809; Fuchs et al., BiolTechhology 9: 1370-1372 (1991);
Hay et al., Hum. Antibod. Hybridomas 3:81-85 (I992); Huse et al., Sciefzce
246:
1275-1281 (1989); and Griffiths et al., EMBO J. 12:725-734 (1993).
Additionally, recombinant antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made using standard recombinant DNA techniques, are within the scope of the
2o invention. Such chimeric and humanized monoclonal antibodies can be
produced by
recombinant DNA techniques known in the art.
In general, antibodies of the invention (e.g., a monoclonal antibody) can be
used to isolate a polypeptide of the invention by standard techniques, such as
affinity
chromatography or innnunoprecipitation. A polypeptide-specific antibody can
2s facilitate the purification of natural polypeptide from cells and of
recombinantly
produced polypeptide expressed in host cells. Moreover, an antibody specific
for a
polypeptide of the invention can be used to detect the polypeptide (e.g., in a
cellular
lysate, cell supernatant, or tissue sample) in order to evaluate the abundance
and
pattern of expression of the polypeptide. Antibodies can be used
diagnostically to
3o monitor protein levels in tissue as part of a clinical testing procedure,
e.g., to, for
example, determine the efficacy of a given treatment regimen. The antibody can
be
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coupled to a detectable substance to facilitate its detection. Examples of
detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase,
beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic
group
complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an
example of a luminescent material includes luminol; examples of bioluminescent
to materials include luciferase, luciferin, and aequorin, and examples of
suitable
radioactive material include l2sh i3ih ssS or 3H.
DIAGNOSTIC ASSAYS
The nucleic acids, probes, primers, polypeptides and antibodies described
herein can be used in methods of diagnosis of asthma; of a susceptibility to
asthma;
or of a condition associated with a MAP3K9 gene, as well as in kits (e.g.,
useful for
diagnosis of asthma; a susceptibility to asthma; or a condition associated
with a
MAP3K9 gene). In one aspect, the kit comprises primers that can be used to
amplify
the markers of interest.
2o In one aspect of the invention, diagnosis of a disease or condition
associated
with a MAP3K9 gene (e.g., diagnosis of asthma, or of a susceptibility to
asthma) is
made by detecting a polymorphism in a MAP3K9 nucleic acid as described herein.
The polymorphism can be a change in a MAP3K9 nucleic acid, such as the
insertion
or deletion of a single nucleotide, or of more than one nucleotide, resulting
in a
frame shift; the change of at least one nucleotide, resulting in a change in
the
encoded amino acid; the change of at least one nucleotide, resulting in the
generation of a premature stop codon; the deletion of several nucleotides,
resulting
in a deletion of one or more amino acids encoded by the nucleotides; the
insertion of
one or several nucleotides, such as by unequal recombination or gene
conversion,
3o resulting in an interruption of the coding sequence of the gene;
duplication of all or a
part of the gene; transposition of all or a part of the gene; or rearrangement
of all or
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a part of the gene. More than one such change may be present in a single gene.
Such sequence changes cause a difference in the polypeptide encoded by a
.1VI~1P3K9
nucleic acid. Fox example, if the difference is a frame shift change, the
frame shift
can result in a change in the encoded amino acids, andlor can result in the
generation
of a premature stop colon, causing generation of a truncated polypeptide.
Alternatively, a polymorphism associated with a disease or condition or a
susceptibility to a disease or condition associated with a MAP3K9 nucleic acid
can
be a synonymous alteration in one or more nucleotides (i.e., an alteration
that does
not result in a change in the polypeptide encoded by a MAP3K9 nucleic acid).
Such
1o a polymorphism may alter splicing sites, affect the stability or transport
of mRNA,
or otherwise affect the transcription or translation of the gene. A MAP3K9
nucleic
acid that has any of the changes or alterations described above is referred to
herein
as an "altered nucleic acid."
In a first method of diagnosing asthma or a susceptibility to asthma, or
15 another disease or condition associated with a MAP3K9 gene, hybridization
methods, such as Southern analysis, Northern analysis, or irc situ
hybridizations, can
be used (see Cut°rent Ps~otocols ih Molecular Biology, Ausubel, F. et
al., eds, John
Wiley & Sons, including all supplements through 1999). Fox example, a
biological
sample (a "test sample") from a test subject (the "test individual") of
genomic DNA,
2o RNA, or cDNA, is obtained from an individual, such as an individual
suspected of
having, being susceptible to or predisposed for, or carrying a defect for, the
disease
or condition, or the susceptibility to the disease or condition, associated
with a
MAP3K9 gene (e.g., asthma). The individual can be an adult, child, or fetus.
The
test sample can be from any source which contains genomic DNA, such as a blood
2s sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue
sample
from skin, muscle, buccal or conjunctiva) mucosa, placenta, gastrointestinal
tract or
other organs. A test sample of DNA from fetal cells or tissue can be obtained
by
appropriate methods, such as by amniocentesis ox chorionic villus sampling.
The
DNA, RNA, or cDNA sample is then examined to determine whether a
3o polymorphism in a MAP3K9 nucleic acid is present, and/or to determine which
splicing variants) encoded by the MAP3K9 is present. The presence of the
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polymorphism or splicing variants) can be indicated by hybridization of the
gene in
the genomic DNA, RNA, or cDNA to a nucleic acid probe. A "nucleic acid probe",
as used herein, can be a DNA probe or an RNA probe; the nucleic acid probe can
contain, for example, at least one polymorphism in a MAP3K9 nucleic acid
(e.g., as
set forth in Table 2) and/or contain a nucleic acid encoding a particular
splicing
variant of a MAP3K9 nucleic acid. The probe can be any of the nucleic acid
molecules described above (c.g., the gene or nucleic acid, a fragment, a
vector
comprising the gene or nucleic acid, a probe or primer, etc.).
To diagnose asthma, or a susceptibility to asthma; or another condition
o associated with a MA.P3K9 gene, a hybridization sample is formed by
contacting the
test sample containing a MAP3K9 nucleic acid with at least one nucleic acid
probe.
A preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid
probe capable of hybridizing to mRNA or genomic DNA sequences described
herein. The nucleic acid probe can be, for example, a full-length nucleic acid
1s molecule, or a portion thereof, such as an oligonucleotide of at least 15,
30, 50, 100,
250 or 500 nucleotides in length and sufficient to specifically hybridize
under
stringent conditions to appropriate mRNA or genomic DNA. For example, the
nucleic acid probe can be all or a portion of one of SEQ ID NOs: 4-37 or the
complement thereof, or a portion thereof. Other suitable probes for use in the
2o diagnostic assays of the invention are described above (see e.g., probes
and primers
discussed under the heading, "Nucleic Acids of the Invention").
The hybridization sample is maintained under conditions that are sufficient
to allow specific hybridization of the nucleic acid probe to a MAP3K9 nucleic
acid.
"Specific hybridization", as used herein, indicates exact hybridization (e.g.,
with no
2s mismatches). Specific hybridization can be performed under high stringency
conditions or moderate stringency conditions, for example, as described above.
In a
particularly preferred aspect, the hybridization conditions for specific
hybridization
are high stringency.
Specific hybridization, if present, is then detected using standard methods.
If
3o specific hybridization occurs between the nucleic acid probe and MAP3K9
nucleic
acid in the test sample, then the MAP3K9 has the polymorphism, or is the
splicing
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variant, that is present in the nucleic acid probe. More than one nucleic acid
probe
can also be used concurrently in this method. Specific hybridization of any
one of
the nucleic acid probes is indicative of a polymorphism in the MAP3K9 nucleic
acid, or of the presence of a particular splicing variant encoding the MAP3K9
s nucleic acid and is therefore diagnostic for a susceptibility to a disease
or condition
associated with a MAP3K9 nucleic acid (e.g., asthma).
In Northern analysis (see Current Protocols in Molecular Biology, Ausubel,
F. et al., eds., John Wiley ~ Sons, supra) the hybridization methods described
above
are used to identify the presence of a polymorphism or a particular splicing
variant,
o associated with a susceptibility to a disease or condition associated with a
MAP3K9
gene (e.g., asthma). For Northern analysis, a test sample of RNA~is obtained
from
the individual by appropriate means. Specific hybridization of a nucleic acid
probe,
as described above, to RNA from the individual is indicative of a polymorphism
in a
MAP3K9 nucleic acid, or ofthe presence of a particular splicing variant
encoded by
15 a MAP3K9 nucleic acid and is therefore diagnostic for asthma or a
susceptibility to
asthma or a condition associated with a MAP3K9 nucleic acid (e.g., asthma).
For representative examples of use of nucleic acid probes, see, for example,
U.S. Patents No. 5,288,611 and 4,851,330.
Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a
2o nucleic acid probe in the hybridization methods described above. PNA is a
DNA
mimic having a peptide-like, inorganic backbone, such as N-(2-
aminoethyl)glycine
units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen
via a
methylene carbonyl linker (see, for example, Nielsen, P.E. et al.,
Biocorrjugate
Clze~rzistty 5, American Chemical Society, p. 1 (1994). The PNA probe can be
25 designed to specifically hybridize to a gene having a polymorphism
associated with
a susceptibility to a disease or condition associated with a MAP3K9 nucleic
acid
(e.g., asthma). Hybridization of the PNA probe to a MAP3K9 gene is diagnostic
for
asthma or a susceptibility to asthma or a condition associated with a MAP3K9
nucleic acid.
3o In another method of the invention, alteration analysis by restriction
digestion can be used to detect an altered gene, or genes containing a
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polymorphism(s), if the alteration (mutation) or polymorphism in the gene
results in
the creation or elimination of a restriction site. A test sample containing
genomic
DNA is obtained from the individual. Polymerase chain reaction (PCR) can be
used
to amplify a MAP3K9 nucleic acid (and, if necessary, the flanking sequences)
in the
test sample of genomic DNA from the test individual. RFLP analysis is
conducted
as described (see Curf°etzt Protocols i~ Molecular Biology, supra). The
digestion
pattern of the relevant DNA fragment indicates the presence or absence of the
alteration or polymorphism in the MAP3K9 nucleic acid, and therefore indicates
the
presence or absence of asthma or the susceptibility to a disease or condition
1o associated with a MAP3K9 nucleic acid.
Sequence analysis can also be used to detect specific polymorphisms in a
MAP3K9 nucleic acid. A test sample of DNA or RNA is obtained from the test
individual. PCR or other appropriate methods can be used to amplify the gene
or
nucleic acid, and/or its flanking sequences, if desired. The sequence of a
MAP3K9
1s nucleic acid, or a fragment of the nucleic acid, or cDNA, or fragment of
the cDNA,
or mRNA, or fragment of the mRNA, is determined, using standard methods. The
sequence of the nucleic acid, nucleic acid fragment, cDNA, cD,NA fragment,
mRNA, or mRNA fragment is compared with the known nucleic acid sequence of
the gene or cDNA or mRNA, as appropriate. The presence of a polymorphism in
2o the MAP3K9 indicates that the individual has asthma or a susceptibility to
asthma.
Allele-specific oligonucleotides can also be used to detect the presence of a
polymorphism in a MAP3K9 nucleic acid, through the use of dot-blot
hybridization
of amplified oligonucleotides with allele-specific oligonucleotide (ASO)
probes
(see, for example, Saiki, R. et al., Nature 324:163-166 (1986)}. An "allele-
specific
25 oligonucleotide" (also referred to herein as an "allele-specific
oligonucleotide
probe") is an oligonucleotide of approximately 10-50 base pairs, preferably
approximately 15-30 base pairs, that specifically hybridizes to a MAP3K9
nucleic
acid, and that contains a polymorphism associated with a susceptibility to a
disease
or condition associated with a MAP3K9 nucleic acid. An allele-specific
30 oligonucleotide probe that is specific for particular polymorphisms in a
MAP3K9
nucleic acid can be prepared, using standard methods (see Curs~ent Protocols
ifz
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~llolecular Biology, supra). To identify polymorphisms in the gene that are
associated with a disease or condition associated with a MAP3K9 nucleic acid
or a
susceptibility to a disease or condition associated with a MAP3K9 nucleic acid
a test
sample of DNA is obtained from the individual. PCR can be used to amplify all
or a
s fragment of a MAP3K9 nucleic acid and its flanking sequences. The DNA
containing the amplified MAP3K9 nucleic acid (or fragment of the gene or
nucleic
acid) is dot-blotted, using standard methods (see Current Protocols ih
Molecular
Biology, supra), and the blot is contacted'with the oligonucleotide probe. The
presence of specific hybridization of the probe to the amplified MAP3K9
nucleic
to acid is then detected. Hybridization of an allele-specific oligonucleotide
probe to
DNA from the individual is indicative of a polymorphism in the MAP3K9 nucleic
acid, and is therefore indicative of a disease or condition associated with a
MAP3K9
nucleic acid or susceptibility to a disease or condition associated with a
MAP3K9
nucleic acid (e.g., asthma).
15 The invention further provides allele-specific oligonucleotides that
hybridize
to the reference or variant allele of a gene or nucleic acid comprising a
single
nucleotide polymorphism or to the complement thereof. These oligonucleotides
can
be probes or primers.
An allele-specific primer hybridizes to a site on target DNA overlapping a
2o polymorphism and only primes amplification of an allelic form to which the
primer
exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448
(1989). This primer is used in conjunction with a second primer, which
hybridizes
at a distal site. Amplification proceeds from the two primers, resulting in a
detectable product, which indicates the particular allelic form is present. A
control
2s is usually performed with a second pair of primers,. one of which shows a
single base
mismatch at the polymorphic site and the other of which exhibits perfect
complementarity to a distal site. The single-base mismatch prevents
amplification
and no detectable product is formed. The method works best when the mismatch
is
included in the 3'-most position of the oligonucleotide aligned with the
3o polymorphism because this position is most destabilizing to elongation from
the
primer (see, e.g., WO 93/22456).
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With the addition of such analogs as locked nucleic acids (LNAs), the size of
primers and probes can be reduced to as few as 8 bases. LNAs are a novel class
of
bicyclic DNA analogs in which the 2' and 4' positions in the furanose ring are
joined
via an O-methylene (oxy-LNA), S-methylene (thio-LNA), or amino methylene
s (amino-LNA) moiety. Common to all of these LNA variants is an affinity
toward
complementary nucleic acids, which is by far the highest reported for a DNA
analog.
For example, particular all oxy-LNA nonamers have been shown to have melting
temperatures of 64 ° C and 74 ° C when in complex with
complementary DNA or
RNA, respectively, as oposed to 28 °C for both DNA and RNA for the
1o corresponding DNA nonamer. Substantial increases in Tm axe also obtained
when
LNA monomers are used in combination with standard DNA or RNA monomers.
For primers and probes, depending on where the LNA monomers are included
(e.g.,
the 3' end, the 5'end, or in the middle), the Tm could be increased
considerably.
In another aspect, arrays of oligonucleotide probes that are complementary to
1s target nucleic acid sequence segments from an individual, can be used to
identify
polymorphisms in a MAP3I~9 nucleic acid. For example, in one aspect, an
oligonucleotide array can be used. Oligonucleotide arrays typically comprise a
plurality of different oligonucleotide probes that are coupled to a surface of
a
substrate in different known locations. These oligonucleotide arrays, also
described
2o as "GenechipsTM," have been generally described in the art, for example,
U.S. Pat.
No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092. These
arrays can generally be produced using mechanical synthesis methods or light
directed synthesis methods that incorporate a combination of photolithographic
methods and solid phase oligonucleotide synthesis methods. See Fodor et al.,
25 Science 251:767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854 (see
also PCT
Application No. WO 90/15070) and Fodor et al., PCT Publication No. WO
92110092 and U.S. Pat. No. 5,424,186, the entire teachings of each of which
are
incorporated by reference herein. Techniques for the synthesis of these arrays
using
mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261;
the
3o entire teachings of which are incorporated by reference herein. In another
example,
linear arrays can be utilized.
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Once an oligonucleotide array is prepared, a nucleic acid of interest is
hybridized with the array and scanned for polymorphisms. Hybridization and
scanning are generally carried out by methods described herein and also in,
e.g.,
published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat.
No. 5,424,186, the entire teachings of which are incorporated by reference
herein.
In brief, a target nucleic acid sequence that includes one or more previously
identified polymorphic markers is amplified by well-known amplification
techniques, e.g., PCR. Typically, this involves the use of primer sequences
that are
complementary to the two strands of the target sequence both upstream and
1o downstream from the polymorphism. Asymmetric PCR techniques may also be
used. Amplified target, generally incorporating a label, is then.hybridized
with the
array under appropriate conditions. Upon completion of hybridization and
washing
of the array, the array is scanned to determine the position on the array to
which the
target sequence hybridizes. The hybridization data obtained from the scan is
15 typically in the form of fluorescence intensities as a function of location
on the
array.
Although primarily described in terms of a single detection block, e.g., for
detecting a single polymorphism, arrays can include multiple detection blocks,
and
thus be capable of analyzing multiple, specific polymorphisms: In alternative
2o aspects,it will generally be understood that detection blocks may be
grouped within
a single array or in multiple, separate arrays so that varying, optimal
conditions may
be used during the hybridization of the target to the array. For example, it
may often
be desirable to provide for the detection of those polymorphisms that fall
within G-C
rich stretches of a genomic sequence, separately from those falling in A-T
rich
25 segments. This allows for the separate optimization of hybridization
conditions for
each situation.
Additional uses of oligonucleotide arrays for polymorphism detection can be
found, for example, in U.S. Patents Nos. 5,858,659 and 5,837,832, the entire
teachings of which are incorporated by reference herein. Other methods of
nucleic
so acid analysis can be used to detect polymorphisms in a asthma gene or
variants
encoding by a asthma gene. Representative methods include direct manual
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sequencing (Church and Gilbert, P~oc. Natl. Acad. Sci. USA 81:1991-1995
(1988);
Sanger, F. et al., P~oc. Natl. Acad. Sci. USA 74:5463-5467 (1977); Beavis et
al.,
U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-stranded
conformation polymorphism assays (SSCP); clamped denaturing gel
electrophoresis
(CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V.C. et
al.,
Proc. Natl. Acad. Sci. USA 86:232-236 (1989)), mobility shift analysis (Orita,
M. et
al., Proc. Natl. Acad. Sci. , USA 86:2766-2770 (1989)), restriction enzyme
analysis
(Flavell et al., Cell 15:25 (1978); Geever, et al., P~oc. Natl. Acad. Sci. USA
78:5081
(1981)); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et
al.,
to P~oc. Natl. Acad. Sci. USA 85:4397-4401 (1985)); RNase protection assays
(Myers,
R.M. et al., Scie~zce 230:1242 (1985)); use of polypeptides which recognize_
nucleotide mismatches, such as E, coli mutS protein; allele-specific PCR, for
example.
In one aspect of the invention, diagnosis of a disease or condition associated
15 with a MAP3K9 nucleic acid (e.g., asthma) or a susceptibility to a disease
or
condition associated with a MAP3K9 nucleic acid (e.g., asthma) can also be
made
by expression analysis by quantitative PCR (kinetic thermal cycling). This
technique, utilizing TaqMan~ assays, can assess the presence of an alteration
in the
expression or composition of the polypeptide encoded by a MAP3K9 nucleic acid
or
2o splicing variants encoded by a MAP3K9 nucleic acid. TaqMan ° probes
can also be
used to allow the identification of polymorphisms and whether a patient is
homozygous or heterozygous. Further, the expression of the variants can be
quantified as physically or functionally different.
In another aspect of the invention, diagnosis of asthma or a susceptibility to
25 asthma or a condition associated with a MAP3K9 gene) can be made by
examining
expression and/or composition of a MAP3K9 polypeptide, by a variety of
methods,
including enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. A test sample from an individual
is
assessed for the presence of an alteration in the expression and/or an
alteration in
3o composition of the polypeptide encoded by a MAP3K9 nucleic acid, or for the
presence of a particular variant encoded by a MAP3K9 nucleic acid. An
alteration
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in expression of a polypeptide encoded by a MAP3K9 nucleic acid can be, for
example, an alteration in the quantitative polypeptide expression (i.e., the
amount of
polypeptide produced); an alteration in the composition of a polypeptide
encoded by
a MAP3K9 nucleic acid is an alteration in the qualitative polypeptide
expression
(e.g., expression of an altered MAP3K9 polypeptide or of a different splicing
variant). In a preferred aspect, diagnosis of the disease or condition
associated with
MAP3K9 nucleic acid or a susceptibility to a disease or condition associated
with a
MAP3K9 nucleic acid is made by detecting a particular splicing variant encoded
by
that MAP3K9 nucleic acid, or a particular pattern of splicing variants.
l0 Both such alterations (quantitative and qualitative) can also be present.
The
teen "alteration°' in the polypeptide expression or composition, as
used herein, refers
to an alteration in expression or composition in a test sample, as compared
with the
expression or composition of polypeptide by a MAP3K9 nucleic acid in a control
sample. A control sample is a sample that corresponds to the test sample
(e.g., is
15 from the same type of cells), and is from an individual who is not affected
by a
susceptibility to a disease or condition associated with a MAP3K9 nucleic
acid. An
alteration in the expression or composition of the polypeptide in the test
sample, as
compared with the control sample, is indicative of a susceptibility to a
disease or
condition associated with a MAP3K9 nucleic acid. Similarly, the presence of
one or
2o more different splicing variants in the test sample, or the presence of
signifcantly
different amounts of different splicing variants in the test sample, as
compared with
the control sample, is indicative of a disease or condition associated with a
MAP3K9
nucleic acid or a susceptibility to a disease or condition associated with a
MAP3K9
nucleic acid. Various means of examining expression or composition of the
2s polypeptide encoded by a MAP3K9 nucleic acid can be used, including:
spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and
immunoassays
(e.g., David et al., U.S. Pat. 4,376,110) such as immunoblotting (see also
Current
Ps°otocols ifz Molecular Biology, particularly Chapter 10). For
example, in one
aspect, an antibody capable of binding to the polypeptide (e.g., as described
above),
3o preferably an antibody with a detectable label, can be used. Antibodies can
be
polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment
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thereof (e.g., Fab or F(ab')Z) can be used. The term "labeled", with regard to
the
probe or antibody, is intended to encompass direct labeling of the probe or
antibody
by coupling (i.e., physically linking) a detectable substance to the probe or
antibody,
as well as indirect labeling of the probe or 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 and end-
labeling
a DNA probe with biotin such that it can be detected with fluorescently
labeled
streptavidin.
Western blotting analysis, using an antibody as described above that
1o specifically binds to a polypeptide encoded by an altered MAP3K9 nucleic
acid or
an antibody that specifically binds to a polypeptide encoded by a non-altered
nucleic
acid, or an antibody that specifically binds to a particular splicing variant
encoded
by a nucleic acid, can be used to identify the presence in a test sample of a
particular
splicing variant or of a polypeptide encoded by a polymorphic or altered
MAP3K9
15 nucleic acid, or the absence in a test sample of a particular splicing
variant or of a
polypeptide encoded by a non-polymorphic or non-altered nucleic acid. The
presence of a polypeptide encoded by a polymorphic or altered nucleic acid, or
the
absence of a polypeptide encoded by a non-poIymorphic or non-altered nucleic
acid,
is diagnostic for a disease or condition associated with a MAP3K9 nucleic acid
or a
2o susceptibility to a disease or condition associated with a MAP3K9 nucleic
acid (e.g.,
asthma), as is the presence (or absence) of particular splicing variants
encoded by
the MAP3K9 nucleic acid.
In one aspect of this method, the level or amount of polypeptide encoded by
a MAP3K9 nucleic acid in a test sample is compared with the level or amount of
the
2s polypeptide encoded by the MAP3K9 in a control sample. A level or amount
ofthe
polypeptide in the test sample that is higher or lower than the level or
amount of the
polypeptide in the control sample, such that the difference is statistically
significant,
is indicative of an alteration in the expression of the polypeptide encoded by
the
MAP3K9t nucleic acid, and is diagnostic for a disease or condition associated
with a
3o MAP3K9 nucleic acid or a susceptibility to a disease or condition
associated with
that MAP3K9 nucleic acid (e.g., asthma). Alternatively, the composition of the
,
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polypeptide encoded by a MAP3K9 nucleic acid in a test sample is compared with
the composition of the polypeptide encoded by the MAP3K9 nucleic acid in a
control sample (e.g., the presence of different splicing variants). A
difference in the
composition of the polypeptide in the test sample, as compared with the
composition
of the polypeptide in the control sample, is diagnostic for a disease or
condition
associated with a MAP3K9 nucleic acid or a susceptibility to a disease or
condition
associated with that MAP3K9 nucleic acid (e.g., asthma). In another aspect,
both
the level or amount and the composition of the polypeptide can be assessed in
the
test sample and in the control sample. A difference in the amount or level of
the
to polypeptide in the test sample, compared to the control sample; a
difference in
composition in the test sample, compared to the control sample; or both a
difference
in the amount or level, and a difference in the composition, is indicative of
a disease
or condition associated with a MAP3K9 nucleic acid or a susceptibility to a
disease
or condition associated with that MAP3K9 nucleic acid.
15 The invention further pertains to a method for the diagnosis or
identification
of a susceptibility to asthma in an individual, by identifying an at-risk
haplotype
(e.g., a haplotype comprising a MAP3K9 nucleic acid). The MAP3K9-associated
haplotypes, e.g., those described in the Example section, describe a set of
genetic
markers ("alleles"). In a certain aspect, the haplotype can comprise one or
more
2o alleles, two or more alleles, three or more alleles, four or more alleles,
or five or
more alleles. The genetic markers are particular "alleles" at "polymorphic
sites"
associated with MAP3K9. A nucleotide position at which more than one sequence
is possible in a population. (either a natural population or a synthetic
population, e.g.,
a library of synthetic molecules), is referred to herein as a "polymorphic
site".
25 Where a polymorphic site is a single nucleotide in length, the site is
referred to as a
single nucleotide polymorphism ("SNP"). For example, if at a particular
chromosomal location, one member of a population has an adenine and another
member of the population has a thymine at the same position, then this
position is a
polymorphic site, and, more specifically, the polymorphic site is a SNP.
3o Polymorphic sites can allow for differences in sequences based on
substitutions,
insertions or deletions. Each version of the sequence with respect to the
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polymorphic site is referred to herein as an "allele" of the polymorphic site.
Thus, in
the previous example, the SNP allows for both an adenine allele and a thymine
allele. -
Typically, a reference sequence is referred to for a particular sequence.
Alleles that differ from the reference are referred to as "variant" alleles.
For
example, the reference MAP3K9 sequence is described herein by SEQ ID NO: 1.
The term, "variant MAP3K9", as used herein, refers to a sequence that differs
from
SEQ ID NO: 1 but is otherwise substantially similar. The genetic markers that
make
up the haplotypes described herein are MAP3K9 variants. The variants of MAP3K9
to that are used to determine the haplotypes disclosed herein of the present
invention
are associated with asthma or a susceptibility to asthma.
Additional variants can include changes that affect a polypeptide, e.g., the
MAP3K9 polypeptide. These sequence differences, when compared to a reference
nucleotide sequence, can include the insertion or deletion of a single
nucleotide, or
15 of more than one nucleotide, resulting in a frame shift; the change of at
least one
nucleotide, resulting in a change in the encoded amino acid; the change of at
least
one nucleotide, resulting in the generation of a premature stop codon; the
deletion of
several nucleotides, resulting in a deletion of one or more amino acids
encoded by
the nucleotides; the insertion of one or several nucleotides, such as by
unequal
2o recombination or gene conversion, resulting in an interruption of the
coding
sequence of a reading frame; duplication of all or a part of a sequence;
transposition;
or a rearrangement of a nucleotide sequence, as described in detail above.
Such
sequence changes alter the polypeptide encoded by a MAP3K9 nucleic acid. For
example, if the change in the nucleic acid sequence causes a frame shift, the
frame
25 shift can result in a change in the encoded amino acids, and/or can result
in the
generation of a premature stop codon, causing generation of a truncated
polypeptide.
Alternatively, a polymorphism associated with asthma or a susceptibility to
asthma
can be a synonymous change in one or more nucleotides (i.e., a change that
does not
result in a change in the amino acid sequence). Such a polymorphism can, for
3o example, alter splice sites, afFect the stability or transport of mRNA, or
otherwise
affect the transcription or translation of the polypeptide. The polypeptide
encoded
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by the reference nucleotide sequence is the "reference" polypeptide with a
particular
reference amino acid sequence, and polypeptides encoded by variant alleles axe
referred to as "variant" polypeptides with variant amino acid sequences.
Haplotypes are a combination of genetic markers, e.g., particular alleles at
polymorphic sites. The haplotypes described herein, e.g., having markers such
as
those shown herein, are found more frequently in individuals with asthma than
in
individuals without asthma. Therefore, these haplotypes have predictive value
for
detecting asthma or a susceptibility to asthma in an individual. The
haplotypes
described herein are a combination of various genetic markers, e.g., SNPs and
1o microsatellites. Therefore, detecting haplotypes can be accomplished by
methods
known in the art for detecting sequences at polymorphic sites, such as the
methods
described above.
RNA EXPRESSION LEVELS:
15 In one aspect, the invention relates to methods of measuring RNA levels of
the MLK. kinases (e.g., MLK1) using Real-Time Quantitative PCR. The method
includes obtaining a sample of cells from the patient, and determining RNA
expression levels using sequence specific pxobes that hybridize to PCR
products of
MLK kinases (e.g., MLK1) on RNA samples that are isolated from cells that have
2o been exposed to specific cytokine activators that activate the JNK pathway
(such as
ILlb or TNFa) vs vehicle alone (i.e., no activation). In another aspect the
invention
is directed at methods that determine the role of MAP3k9 or its pathway-
related
genes, by obtaining a sample of cells from patients with asthma or other
respiratory
or inflammatory disorders, determining RNA levels of MAP3k9 or its pathway
25 related genes in cells exposed to pathway specific activators (such as ILlb
or TNFa)
or vehicle alone (no activation), and comparing them with reference RNA levels
of
the gene in cells isolated from subjects without asthma or other
inflammatorylrespiratory disorders. In another aspect, the invention relates
to
methods for predicting efficacy of an inhibitor drug, including obtaining a
sample of
3o cells from patients with asthma or another respiratory/inflarnmatory
disorder,
determining RNA levels of MAP3k9 or its pathway related genes in cells
isolated
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from patients who are taking the drug compared to those who are not taking the
drug. In another aspect, the invention relates to methods for predicting
efficacy of
an inhibitor drug, including obtaining a sample of cells from patients with
asthma or
other respiratorylinflammatory disorders, determining RNA levels of MAP3k9 or
its
pathway related genes after exposure of the cells to the inhibitor drug in
vitro.
SCREENING ASSAYS AND AGENTS IDENTIFIED THEREBY
The invention provides methods (also referred to herein as "screening
assays") for identifying the presence of a nucleotide that hybridizes to a
nucleic acid
of the invention, as well as for identifying the presence of a polypeptide
encoded by
a nucleic acid of the invention. In one aspect, the presence (or absence) of a
nucleic
acid molecule of interest (e.g., a nucleic acid that has significant homology
with a
nucleic acid of the invention) in a sample can be assessed by contacting the
sample
with a nucleic acid comprising a nucleic acid of the invention under stringent
conditions as described above, and then assessing the sample for the presence
(or
absence) of hybridization. In one aspect, high stringency conditions are
conditions
appropriate for selective hybridization. In another aspect, a sample
containing the
nucleic acid molecule of interest is contacted with a nucleic acid containing
a
contiguous nucleotide sequence (e.g., a primer or a probe as described above)
that is
2o at least partially complementary to a part of the nucleic acid molecule of
interest
(e.g., a MAP3K9 nucleic acid), and the contacted sample is assessed for the
presence
or absence of hybridization. In another aspect, the nucleic acid containing a
contiguous nucleotide sequence is completely complementary to a part of the
nucleic
acid molecule of interest.
2s In any of these aspects, all or a portion of the nucleic acid of interest
can be
subjected to amplification prior to performing the hybridization.
In another aspect, the presence (or absence) of a polypeptide of interest,
such
as a polypeptide of the invention or a fragment or variant thereof, in a
sample can be
assessed by contacting the sample with an antibody that specifically
hybridizes to
3o the polypeptide of interest (e.g., an antibody such as those described
above), and
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then assessing the sample for the presence '(or absence) of binding of the
antibody to
the polypeptide of interest.
In another aspect, the invention provides methods for identifying agents
(e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors,
binding
s agents, antibodies, small molecules or other drugs, or ribozymes) that alter
(e.g.,
increase or decrease) the activity of the polypeptides described herein, or
which
otherwise interact with the polypeptides herein. For example, such agents can
be
agents which bind to polypeptides described herein (e.g., MAP3K9 binding
agents);
which have a stimulatory or inhibitory effect on, for example, activity of
to polypeptides of the invention; or which change (e.g., enhance or inhibit)
the ability
of the polypeptides of the invention to interact with MAP3K9 binding agents
(e.g.,
receptors or other binding agents); or which alter posttranslational
processing of the
MAP3K9 polypeptide (e.g., agents that alter proteolytic processing to direct
the
polypeptide from where it is normally synthesized to another location in the
cell,
i5 such as the cell surface; agents that alter proteolytic processing such
that more
polypeptide is released from the cell, etc.
In one aspect, the invention provides assays for screening candidate or test
agents that bind to or modulate the activity of polypeptides described herein
(or
biologically active portions) thereof), as well as agents identifiable by the
assays.
20 Test agents can be obtained using any of the numerous approaches in
combinatorial
library methods known in the art, including: biological libraries; spatially
addressable parallel solid phase or solution phase libraries; synthetic
library methods
requiring deconvolution; the 'one-bead one-compound' library method; and
synthetic library methods using affinity chromatography selection. The
biological
25 library approach is limited to polypeptide libraries, while the other four
approaches
are applicable to polypeptide, non-peptide oligomer or small molecule
libraries of
i
compounds (Lam, K.S., Ahticance~ Drug Des. 12:145 (1997)).
In one aspect, to identify agents which alter the activity of a MAP3K9
polypeptide, a cell, cell lysate, or solution containing or expressing a
MAP3K9
3o polypeptide, or another splicing variant encoded by a MAP3K9 gene or a
fragment
or derivative thereof (as described above), can be contacted with an agent to
be
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tested; alternatively, the polypeptide can be contacted directly with the
agent to be
tested. The level (amount) of MAP3K9 activity is assessed (e.g., the level
(amount)
of MAP3K9 activity is measured, either directly or indirectly), and is
compared with
the level of activity in a control (i.e., the level of activity of the MAP3K9
polypeptide or active fragment or derivative thereof in the absence of the
agent to be
tested). If the level of the activity in the presence of the agent differs, by
an amount
that is statistically significant, from the level of the activity in the
absence of the
agent, then the agent is an agent that alters the activity of a MAP3K9
polypeptide.
An increase in the level of MAP3K9 activity relative to a control sample
indicates
that the agent is an agent that enhances (is an agonist of) MAP3K9 activity.
Similarly, a decrease in the level of MAP3K9 activity relative to a control
indicates
that the agent is an agent that inhibits (is an antagonist of) MAP3K9
activity. In
another aspect, the level of activity of a MAP3K9 polypeptide or derivative or
fragment thereof in the presence of the agent to be tested, is compared with a
control
level that has previously been established. A level of the activity in the
presence of
the agent that differs from the control level by an amount that is
statistically
significant indicates that the agent alters MAP3K9 activity.
The present invention also relates to an assay for identifying agents which
alter the expression of a MAP3K9 nucleic acid (e.g., antisense nucleic acids,
fusion
2o proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding
agents,
antibodies, small molecules or other drugs, or ribozymes) which alter (e.g.,
increase
or decrease) expression (e.g., transcription or translation) of the gene or
which
otherwise interact with the nucleic acids described herein, as well as agents
identifiable by the assays. For example, a solution containing a nucleic acid
encoding a MAP3K9 polypeptide (e.g., a MAP3K9 gene or nucleic acid) can be
contacted with an agent to be tested. The solution can comprise, for example,
cells
containing the nucleic acid or cell lysate containing the nucleic acid;
alternatively,
the solution can be another solution that comprises elements necessary for
transcription/translation of the nucleic acid. Cells not suspended in solution
can also
be employed, if desired. The level andlor pattern of MAP3K9 expression (e.g.,
the
level and/or pattern of mRNA or of protein expressed, such as the level and/or
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pattern of different splicing variants) is assessed, and is compared with the
level
and/or pattern of expression in a control (i. e., the level and/or pattern of
the
MAP3K9 expression in the absence of the agent to be tested). If the level
andlor
pattern in the presence of the agent differs by an amount or in a manner that
is
statistically significant, from the level andlor pattern in the absence of the
agent,
then the agent is an agent that alters the expression of a asthma gene.
Enhancement
of MAP3K9 expression indicates that the agent is an agonist of MAP3K9
activity.
Similarly, inhibition of MAP3K9 expression indicates that the agent is an
antagonist
of MAP3K9 activity. In another aspect, the level and/or pattern of MAP3K9
to polypeptide(s) (e.g., different splicing variants) in the presence of the
agent to be
tested, is compared with a control level and/or pattern that have previously
been
established. A level and/or pattern in the presence of the agent that differs
from the
control level and/or pattern by an amount or in a manner that is statistically
significant indicates that the agent alters MAP3K9 expression.
In another aspect of the invention, agents which alter the expression of a
MAP3K9 nucleic acid or which otherwise interact with the nucleic acids
described
herein, can be identified using a cell, cell lysate, or solution containing a
nucleic acid
encoding the promoter region of the MAP3K9 gene or nucleic acid operably
linked
to a reporter gene. After contact with an agent to be tested, the level of
expression
of the reporter gene (e.g., the level of mRNA or of protein expressed) is
assessed,
and is compared with the level of expression in a control (i.e., the level of
the
expression of the reporter gene in the absence of the agent to be tested). If
the level
in the presence of the agent differs, by an amount or in a manner that is
statistically
significant, from the level in the absence of the agent, then the agent is an
agent that
alters the expression of the MAP3K9, as indicated by its ability to alter
expression of
a gene that is operably linked to the MAP3K9 gene promoter. Enhancement of the
expression of the reporter indicates that the agent is an agonist of MAP3K9
activity.
Similarly, inhibition of the expression of the reporter indicates that the
agent is an
antagonist of MAP3K9 activity. In another aspect, the level of expression of
the
3o reporter in the presence of the agent to be tested is compared with a
control level
that has previously been established. A level in the presence of the agent
that differs
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from the control level by an amount or in a manner that is statistically
significant
indicates that the agent alters expression.
Agents which alter the amounts of different splicing variants encoded by a
MAP3K9 nucleic acid (e.g., an agent which enhances activity of a first
splicing
variant, and which inhibits activity of a second splicing variant), as well as
agents
which are agonists of activity of a first splicing variant and antagonists of
activity of
a second splicing variant, can easily be identified using these methods
described
above.
In other aspects of the invention, assays can be used to assess the impact of
a
1o test agent on the activity of a polypeptide in relation to a MAP3K9 binding
agent.
For example, a cell that expresses a compound that interacts with a MAP3K9
polypeptide (herein referred to as a "MAP3K9 binding agent", which can be a
polypeptide or other molecule that interacts with a MAP3K9 polypeptide, such
as a
receptor) is contacted with a MAP3K9 in the presence of a test agent, and the
ability
1s of the test agent to alter the interaction between the MAP3K9 and the
MAP3K9
binding agent is determined. Alternatively, a cell lysate or a solution
containing the
MAP3K9 binding agent, can be used. An agent that binds to the MAP3K9 or the
MAP3K9 binding agent can alter the interaction by interfering with, or
enhancing
the ability of the MAP3K9 to bind to, associate with, or otherwise interact
with the
2o MAP3K9 binding agent. Determining the ability of the test agent to bind to
a
MAP3K9 nucleic acid or a MAP3K9 binding agent can be accomplished, for
example, by coupling the test agent with a radioisotope or enzymatic label
'such that
binding of the test agent to the polypeptide can be determined by detecting
the
labeled with 12$h 355, iaC or 3H, either directly or indirectly, and the
radioisotope
2s detected by direct counting of radioemmission or by scintillation counting.
Alternatively, test agents 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. It is
also within the scope of this invention to determine the ability of a test
agent to
3o interact with the polypeptide without the labeling of any of the
interactants. For
example, a microphysiometer can be used to detect the interaction of a test
agent
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with a MAP3K9 polypeptide or a MAP3K9 binding agent without the labeling of
either the test agent, MAP3K9 polypeptide, or the MAP3K9 binding agent.
McConnell, H.M. et al., Sciefzce 257:1906-1912 (1992). As used herein, a
"microphysiometer" (e.g., CytosensorTM) is an analytical instrument that
measures
the rate at which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate can be used
as an
indicator of the interaction between ligand and polypeptide.
Thus, these receptors can be used to screen for compounds that are agonists
or antagonists, for use in treating a susceptibility to a disease or condition
associated
1o with a MAP3K9 gene or nucleic acid, or for studying a susceptibility to a
disease or
condition associated with a MAP3K9 (e.g., asthma). Drugs could be designed to
regulate MAP3K9 activation that in turn can be used to regulate signaling
pathways
and transcription events of genes downstream.
In another aspect of the invention, assays can be used to identify
polypeptides that interact with one or more MAP3K9 polypeptides, as described
herein. For example, a yeast two-hybrid system such as that described by
Fields and
Song (Fields, S. and Song, O., Nature 340:245-246 (1989)) can be used to
identify
polypeptides that interact with one or more MAP3K9 polypeptides. In such a
yeast
two-hybrid system, vectors are constructed based on the flexibility of a
transcription
2o factor that has two functional domains (a DNA binding domain and a
transcription
activation domain). If the two domains are separated but fused to two
different
proteins that interact with one another, transcriptional activation can be
achieved,
and transcription of specific markers (e.g., nutritional markers such as His
and Ade,
or color markers such as lacZ) can be used to identify the presence of
interaction and
2s transcriptional activation. For example, in the methods of the invention, a
first
vector is used which includes a nucleic acid encoding a DNA binding domain and
also a MAP3K9 polypeptide, splicing variant, or fragment or derivative
thereof, and
a second vector is used which includes a nucleic acid encoding a transcription
activation domain and also a nucleic acid encoding a polypeptide which
potentially
3o may interact with the MAP3K9 polypeptide, splicing variant, or fragment or
derivative thereof (e.g., a MAP3K9 polypeptide binding agent or receptor).
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Incubation of yeast containing the first vector and the second vector under
appropriate conditions (e.g., mating conditions such as used in the
MatchmakerTM
system from Clontech (Palo Alto, California, USA)) allows identification of
colonies that express the markers of interest. These colonies can be examined
to
identify the polypeptide(s) that interact with the MAP3K9 polypeptide or
fragment
or derivative thereof. Such polypeptides may be useful as agents that alter
the
activity of expression of a MAP3K9 polypeptide, as described above.
In. more than one aspect of the above assay methods of the present
invention, it may be desirable to immobilize either the MAP3K9 gene or nucleic
so acid, the MAP3K9 polypeptide, the MAP3K9 binding agent, or other components
of
the assay owa solid support, in order to facilitate separation of complexed
from
uncomplexed forms of one or both of the polypeptides, as well as to
accommodate
automation of the assay. Binding of a test agent to the polypeptide, or
interaction of
the polypeptide with a binding agent in the presence and absence of a test
agent, can
be accomplished in any vessel suitable for containing the reactants. Examples
of
such vessels include microtitre plates, test tubes, and micro-centrifuge
tubes. In one
aspect, a fusion protein (e.g., a glutathione-S-transferase fusion protein)
can be
provided which adds a domain that allows a MAP3K9 nucleic acid, MAP3K9
polypeptide, or a MAP3K9 binding agent to be bound to a matrix or other solid
support.
In another aspect, modulators of expression of nucleic acid molecules of the
invention are identified in a method wherein a cell, cell lysate, or solution
containing
a MAP3K9 nucleic acid is contacted with a test agent and the expression of
appropriate mRNA or polypeptide (e.g., splicing variant(s)) in the cell, cell
lysate, or
solution, is determined. The level of expression of appropriate mRNA or
polypeptide(s) in the presence of the test agent is compared to the level of
expression of mRNA or polypeptide(s) in the absence of the test agent. The
test
agent can then be identified as a modulator of expression based on this
comparison.
For example, when expression of mRNA or polypeptide is greater (statistically
3o significantly greater) in the presence of the test agent than in its
absence, the test
agent is identified as a stimulator or enhancer of the mRNA or polypeptide
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expression. Alternatively, when expression of the mRNA or polypeptide is less
(statistically significantly less) in the presence of the test agent than in
its absence,
the test agent is identified as an inhibitor of the mRNA or polypeptide
expression.
The level of mRNA or polypeptide expression in the cells can be determined by
methods described herein for detecting mRNA or polypeptide.
This invention further pertains to novel agents identified by the above-
described screening assays. Accordingly, it is within the scope of this
invention to
further use an agent identified as described herein in an appropriate animal
model.
For example, an agent identified as described herein (e.g., a test agent that
is a
to modulating agent, an antisense nucleic acid molecule, a specific antibody,
or a
polypeptide-binding agent) can be used in an animal model to determine the
efficacy, toxicity, or side effects of treatment with such an agent.
Alternatively, an
agent identified as described herein can be used in an animal model to
determine the
mechanism of action of such an agent.
15 Furthermore, this invention pertains to uses of novel agents identified by
the
above-described screening assays for treatments as described herein. In
addition, an
agent identified as described herein can be used to alter activity of a
polypeptide
encoded by a MAP3K9 nucleic acid, or to alter expression of a MAP3K9 nucleic
acid, by contacting the polypeptide or the nucleic acid (or contacting a cell
2o comprising the polypeptide or the nucleic acid) with the agent identified
as
described herein.
NUCLEIC ACID PHARMACEUTICAL COMPOSITIONS
The present invention also pertains to pharmaceutical compositions
25 comprising nucleic acids described herein, particularly nucleotides
encoding the
polypeptides described herein (e.g., a MAP3K9 polypeptide); comprising
polypeptides described herein andlor comprising other splicing variants
encoded by
a MAP3K9 nucleic acid; and/or an agent that alters (e.g., enhances or
inhibits)
MAP3K9 nucleic acid expression or MAP3K9 polypeptide activity as described
3o herein. For instance, a polypeptide, protein (e.g., a MAP3K9 nucleic acid
receptor),
an agent that alters MAP3K9 nucleic acid expression, or a MAP3K9 binding agent
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or binding partner, fragment, fusion protein or pro-drug thereof, or a
nucleotide or
nucleic acid construct (vector) comprising a nucleotide of the present
invention, or
an agent that alters MAP3I~9 polypeptide activity, can be formulated with a
physiologically acceptable carrier or excipient to prepare a pharmaceutical
s composition. The carrier and composition can be sterile. The formulation
should
suit the mode of administration.
Suitable pharmaceutically acceptable carriers include but are not limited to
water, salt solutions (e.g., NaCI), saline, buffered saline, alcohols,
glycerol, ethanol,
gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,
to carbohydrates such as lactose, amylose or starch, dextrose, magnesium
stearate, talc,
silicic acid, viscous paraffin, perfume oil, fatty acid esters,
hydroxymethylcellulose,
polyvinyl pyrolidone, etc., as well as combinations thereof. The
pharmaceutical
preparations can, if desired, be mixed with auxiliary agents, e.g.,
lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic
15 pressure, buffers, coloring, flavoring and/or aromatic substances and the
like which
do not deleteriously react with the active agents.
The composition, if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. The composition can be a liquid
solution, suspension, emulsion, tablet, pill, capsule, sustained release
formulation, or
2o powder. The composition can be formulated as a suppository, with
traditional
binders and carriers such as triglycerides. Oral formulation can include
standard
carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium
carbonate,
etc.
25 Methods of introduction of these compositions include, but are not limited
to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous,
subcutaneous, topical, oral and intranasal. Other suitable methods of
introduction
can also include gene therapy (as described below), rechargeable or
biodegradable
devices, particle acceleration devises ("gene guns") and slow release
polymeric
3o devices. The pharmaceutical compositions of this invention can also be
administered as part of a combinatorial therapy with other agents.
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The composition can be formulated in accordance with the routine
procedures as a pharmaceutical composition adapted for administration to human
beings. For example, compositions for intravenous administration typically are
solutions in sterile isotonic aqueous buffer. Where necessary, the composition
may
s also include a solubilizing agent and a local anesthetic to ease pain at the
site of the
injection. Generally, the ingredients are supplied either separately or mixed
together
in unit dosage form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampule or sachette
indicating the quantity of active agent. Where the composition is to be
administered
1o by infusion, it can be dispensed with an infusion bottle containing sterile
pharmaceutical grade water, saline or dextrose/water. Where the composition is
administered by injection, an ampule of sterile water for injection or saline
can be
provided so that the ingredients may be mixed prior to administration.
For topical application, nonsprayable forms, viscous to semi-solid or solid
is forms comprising a carrier compatible with topical application and having a
dynamic viscosity preferably greater than water, can be employed. Suitable
formulations include but are not limited to solutions, suspensions, emulsions,
creams, ointments, powders, enemas, lotions, sots, liniments, salves,
aerosols, etc.,
which are, if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives,
2o stabilizers, wetting agents, buffers or salts for influencing osmotic
pressure, etc. The
agent may be incorporated into a cosmetic formulation. For topical
application, also
suitable are sprayable aerosol preparations wherein the active ingredient,
preferably
in combination with a solid or liquid inert carrier material, is packaged in a
squeeze
bottle or in admixture with a pressurized volatile, normally gaseous
propellant, e.g.,
2s pressurized air.
Agents described herein can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free amino groups
such
as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc.,
and those formed with free carboxyl groups such as those derived from sodium,
3o potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-
ethylamino ethanol, histidine, procaine, etc.
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The agents are administered in a therapeutically effective amount. The
amount of agents which will be therapeutically effective in the treatment of a
particular disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques. In addition,
in
s vitro or ifz vivo assays may optionally be employed to help identify optimal
dosage
ranges. The precise dose to be employed in the formulation will also depend on
the
route of administration, and the seriousness of the symptoms, and should be
decided
according to the judgment of a practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves derived from i~
1o vit~~o or animal model test systems.
The invention also provides a pharnnaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such containers) can
be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
15 use or sale of pharmaceuticals or biological products, which notice
reflects approval
by the agency of manufacture, use of sale for human administration. The pack
or kit
can be labeled with information regarding mode of administration, sequence of
drug
administration (e.g., separately, sequentially or concurrently), or the like.
The pack
or kit may also include means for reminding the patient to take the therapy.
The
2o pack or kit can be a single unit dosage of the combination therapy or it
can be a
plurality of unit dosages. In particular, the agents can be separated, mixed
together
in any combination, present in a single vial or tablet. Agents assembled in a
blister
pack or other dispensing means is preferred. For the purpose of this
invention, unit
dosage is intended to mean a dosage that is dependent on the individual
2s pharmacodynamics of each agent and administered in FDA approved dosages in
standard time courses.
The present invention is now illustrated by the following Exemplification,
which is not intended to be limiting in any way. All references cited herein
are
incorporated by reference in their entirety.
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EXAMPLES
EXAMPLE 1
Patient Population
The original patient list contained the names of over 7,000 patients who
attended the private clinics or outpatient clinics of allergists practicing at
the
Allergy/Pulmonary Divisions of the National University Hospital of Iceland
during
the years 1977 to 2001 (ECRHSG; 1997). For this study, patients were selected
with
physician-diagnosed asthma who were being treated with asthma drugs and who
were
related to at least one other patient within and including 6 meiotic events (6
meiotic
to events separate 2nd cousins) as revealed by a computerized genealogy
database. Ages
ranged from 12-70 years (mean 39.3 yrs) and 62 % were females. Information
regarding the age at diagnosis, medications, hospital admissions, and family
history of
atopy and asthma were gathered. The diagnosis of asthma and atopy were
clinically
re-confirmed in the study by a new physical examination, measurements of skin
test
15 reactivity to 12 aeroalIergens (including birch, grass, Rumex c~ispus, cat,
dog, horse,
Cladospof°ium, Mucor, Altef~naYia, Des°matophagoides
pteronyssiJZUS, D. fariyzae, and
Lepidoglyphus destYUCto~°), total IgE levels, and pulmonary function
tests (PFT).
Unless a baseline forced expiratory volume in 1 s (FEV1) was < 70% of
predicted
value (based on sex, height, and race), a methacholine (MCh)- challenge test
was
2o performed. The phenotype assessments, PFTs, and methacholine tests were
performed according to ATS guidelines (Cockcroft, et al., 1977; Palmquist, et
al.,
1988). Patients were considered as being atopic if their skin prick test
reaction was
positive {i.e., >_ 3 mm or >_ SO% of the histamine positive control response).
The
diagnosis of asthma in Iceland is based on the diagnostic criteria outlined by
the
2s NHLB and the American Thoracic Society (National Institutes of Health 1997;
American Thoracic Society 1995) and includes any of the following measures:
~ Patient having recurrent symptoms of cough and wheezing for more
than 2 years and demonstrating clinical response to bronchodilator therapy (as
measured by > 15% increase in FEVl following bronchodilator treatment).
30 ~ Patient having reduced FEV1 (FEVI< g0%) at baseline prior to
bronchodilator therapy and showing >15% improvement in FEV1 following
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bronchodilator therapy.
~ Patient having recurrent symptoms of cough and wheezing and on
methacholine challenge test, performed in accordance to ATS guidelines
(American Thoracic Society 1995), has > 20% drop in FEV 1 at methacholine
concentrations <8 mg/L.
Severity of asthma was determined by the combination of signs and
symptoms, PFTs, MCh values, and requirements for therapy. On the basis of
these
criteria, approximately 90% of the patients were scored as having mild to
moderate
asthma and 10% with severe asthma. FEV1 > 80% predicted was considered normal.
1o Before blood samples were obtained, all patients included in the study were
re-
examined by the same two allergists who confirmed the asthma phenotype and
severity level and supervised the measurements of total IgE levels, spirometry
and
skin testing prior to obtaining blood samples. In this study, a reduction in
FEV 1 of
20% or more at a MCh concentration of 8 mg/L or less is considered positive
1s challenge test.
The patient participation rate for the study exceeded 90%. A11 patients
signed informed consent, donated blood samples, and completed a detailed
medical
questionnaire and all tests necessary for proper phenotyping. The study was
approved
by the Icelandic Data Protection Commission and the National Bioethics
Committee.
2o Personal identities of the patients and their family members were
subsequently
encrypted by the Data Protection Commission of Iceland (Gulcher et al., 2000).
All
blood and DNA samples were also coded in the same way. All participants were
asked, by questionnaire, whether they had been diagnosed with asthma and/or
atopy
and whether they were receiving drugs to treat if they were being treated. The
names
25 of the drugs were recorded and confirmed to be anti-asthma and/or allergy
medications. Blood was also collected from close relatives of the index cases
to
increase the information available for the linkage analysis. All participants
had their
lung function measured at the time the blood was drawn for the study.
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Pedig~~ees
deCODE has built a computerized genealogy database with over 650,000
names that includes all 285,000 living Icelanders and most of their ancestors
(Gulcher
and Stefanssori 1998). The database has a connectivity of over 95% in the 20~'
century and 86% in the 19~' century. Its maternal connections are 99.3%
accurate as
measured by mitochondria) polymorphisms of maternally linked individuals
(Helgason et al., 2000). The genealogy database was used to cluster the
patients in
pedigrees. The genealogy database is reversibly encrypted by the Data
Protection
Commission of Iceland before it is used in our laboratory (Gulcher and
Stefansson
1998). Recursive algorithms are used with the encrypted personal identifiers
to find
all ancestors in the database who are related to any member on the patient
list within a-
given number of generations back. The cluster function then identifies
ancestors who
are common to any two or more members of the patient list.
Genotyping
Five hundred ninety-six patients were genotyped with asthma belonging to 175
families, in which each patient was related to at least one other patient
within and
including 6 meiotic events. DNA samples from all 596 patients and 538
relatives
were successfully genotyped using 976 specific fluorescently labelled primers
with an
2o initial average spacing of 3-4 cM genome-wide. A-microsatellite screening
set was
developed based in part on the ABI Linkage Marker (v2) screening set and the
ABI
Linkage Marker (v2) intercalating set in combination with over 500 custom-made
markers. All markers were extensively tested for multiplex PCR reactions. The
PCR
amplifications were set up and pooled using Cyberlab robots. The reaction
volume
used was 5 pl and for each PCR reaction 20 ng of genomic DNA was amplified in
the
presence of 2 pmol of each primer, 0.25 U AmpliTaq Gold, 0.2 mmol/L dNTPs and
2.5 mmol/L MgCl2. .The PCR conditions used were 95°C for 10 minutes,
then 37
cycles of 15 s at 94°C, 30s at 55°C and 1 min at 72°C.
The PCR products were
supplemented with the internal size standard and the pools were separated and
3o detected on Applied Biosystems model 3700 Sequencer using Genescan v3.0
peak
calling software. Alleles were called automatically with the DAC program (Fj
alldal et
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al., 2001), and the program, DecodeGT, was used to fractionate according to
quality
and edit the called genotypes (Palsson et al., 1999).
In regions demonstrating linkage using the framework marker set, marker
density was
further increased (i.e., fine mapping of locus) by additional microsatellite
markers to
obtain coverage of 0.2 cM on the average in these regions.
Statistical flnalyses
A genome-wide linkage scan was performed using a framework map of 976
microsatellite markers. The data was analyzed using the Allegro program
io (Gudbjartsson et al., 2000) and determined statistical significance by
applying
affecteds-only allele-sharing-methods (not specifying any particular
inheritance
model). The Allegro program, a linkage program developed at deCODE genetics,
calculates LOD scores based on multipoint calculations (Gudbjartsson et al.,
2000;
Kruglyak et al., 1996;.Kong and Cox 1997) and is available for free for non-
15 commercial use by sending e-mail to allegro@decode.is. The linkage analysis
approach uses the Spa; scoring function (Kruglyak et al., 1996; Whittemore and
Halpern 1994), the exponential allele-sharing model (Kong and Cox 1997), and a
family weighting scheme that is halfway, on the log scale, between weighting
each
affected pair equally and weighting each family equally. All genotyped
individuals
2o who are unaffected are treated as "unknown". P values are calculated based
on the
large sample theory; Zlr = ~(2 loge (10) LOD) is approximately distributed as
a
standard normal distribution under the null hypothesis of no linkage (Kong and
Cox
1997) and the observed LOD score is compared to its complete data sampling
distribution under the null hypothesis (Gudbjartsson et al., 2000). The
information
25 measure we use is part of the Allegro program output (Nicolae 1999) and
closely
related to a classical measure (Dempster et al., 1977). Information equals
zero if the
marker genotypes are completely uninformative and equals one if the genotypes
determine the exact amount of allele sharing by descent among the affected
relatives.
The marker order and positions for the framework mapping set were obtained
using a
so high-density genetic map developed at deCODE. Data from 146 Icelandic
nuclear
families (sibships with genotypes for two to seven siblings and both parents)
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providing 1257 meioses were analyzed to estimate the genetic distances. By
comparison, distances in the Marshfield genetic map were estimated based on
188
meioses. Inter-marker distances in the peak region after enrichment with 4
markers
were estimated using an adaptation of the EM algorithm (Dempster 1977) within
Allegro.
Linkage Afzalysis
A list of over 7,000 patients from the National University Hospital of Iceland
were cross-matched with the genealogy database. In the present study, patients
with
1o physician-diagnosed asthma who were related by 6 or fewer meiotic events to
other
patients with asthma (6 meiotic events separate 2"d cousins) were included.
These
were 596 patients in 175 families with asthma. In the present study, over
thirty
families had at least 6 affected members each. Two of these families used in
the
analysis are displayed in FIG. 2. The patients' demographic data, geometric
mean IgE
15 values, lung function tests (including % predicted FEV1, and FEV1/FVC
ratio),
methacholine challenge test and skin test results to the most common
aeroallergens in
Iceland are shown in FIG. 4. Seventy-three percent of the patients were atopic
as
defined by a positive skin test reaction.
More than'400 of the study patients were tested for airway reactivity using
2o MCh challenge. As shown in FIG. 4, although 67% of the patients tested had
more
than 20% drop in FEV 1 at a MCh concentration of less than 2 mg/L, over 90% of
the
patients tested positive at a MCh concentration of 8 mg/L or less. This would
be
consistent with moderate to severe airway hyperresponsiveness in 2/3 of our
asthma
study population. The spirometric values reported in FIG. 4 are those obtained
during
25 the study at which time the majority of the patients had stable asthma and
were on full
therapy; however, all these patients have previous spirometric values with FEV
1
<80% predicted at one or more earlier time points in their medical charts
(data not
shown). Bronchodilator reversibility was tested in selective cases including
those
patients who had negative results of a MCh challenge and from whom clinician
3o determined the test to be necessary to support the asthma phenotype.
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Thirty-three percent of the patients gave a history of having smoked for more
than 1 pack-year. Of those, 47 percent had smoked for fewer than 10 pack-
years. The
possibility that some or few of the study participants who are smokers had
mild co-
existing COPD cannot be excluded; however, only 0.5 % of study the patients
who
were 55 years or older had smoked for more than 20 pack years (FIG. 4). In
contrast,
all study subjects have asthma as defined by the ATS criteria (American
Thoracic
Society; 1995); which is the phenotype used for this study thereby minimizing
the
potential of confounding effects from COPD.
Five hundred ninety-six patients and 538 of their unaffected (i.e., non-
1o asthmatic) relatives were genotyped using 976 microsatellite markers in a
genome-
wide linkage scan. The disease status of relatives was obtained by a
questionnaire.
The data was analyzed and determined statistical significance by applying
affecteds-
only, allele-sharing methods (which do not specify any particular inheritance
model)
(Gulcher et al., 2001). Since the linkage scan was performed on affected
status only,
15 there is no possible confounding effect from the relatives even if an
affected relative
in the group was not identified. The genomic region that showed the most
evidence
for linkage to the asthma phenotype was chromosome 14q24 with a lod score of
2.66
(single test p = 2.16 x 10-4). Given that the information content on identity
by descent
sharing in the region was less than 85% (i.e., 0.77), which is less than
preferred,
2o another 34 microsatellite markers under this peak were added to ensure that
the results
are a true reflection of the information contained in the material. This
increased the
information content at the peak to over 95% and produced a lod score of 4.00
(single
test p = 8.70 x 10'6). This locus is significant on a genome-wide basis, even
allowing
for the multiple marker testing, and it corresponds to a genome-wide adjusted
P-value
2s of less than 0.05 (Krugylak and Lander 1996). This locus was designated as
asthma
locus one (ASI). The locus peals is centered on markers D14S588 and D14S603,
which are spaced 84 kb apart. The locus, defined by a drop of approximately
1.0 in
the LOD score, is between markers D14S1069 and D14S289 centromeric, and
telomeric respectively. The segment with a 1-LOD drop is around 3.9
centimorgans
3o and is estimated to correspond to around 3.0 million bases.
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The genome scan shows for the first time significant linkage of asthma to
markers on
chromosome 14q24. It should be noted that a suggestive linkage to this locus
has
been previously demonstrated by investigators in the United Kingdom (Mansur
1999)
and the United States (The Collaborative Study of the Genetics of Asthma
1997).
Chromosome 14q24 contains many genes that could contribute to the
susceptibility to
asthma, including genes that encode for the EGF-response factor 1,
phosphatidylinositol glycan class H, secreted modular calcium-binding protein
1, a
disintegrin and metalloproteinase domain-20 and-21, and RNA polymerase II
transcriptional regulation mediator, to name a few.
EXAMPLE-2
Assessment of MAP3K9 expression in human airway tissue:
Lung tissue from 2 asthma patients and 2 controls (all 4 are smokers who
developed lung cancer and needed resection) were studied. Airway tissue from a
non-cancerous (healthy) part of their small airways was isolated and examined
for
expression of MAP3K9 using RT-PCR. A 10 fold increased expression of the b-
isofonn of MAP3K9 was observed in these 2 patients (see FIG. 9) compared to
controls.
2o EXAMPLE 3.
Assessment of MAP3K9 expression in peripheral blood mononuclear (PBM) cells:
The expression of MAP3K9 in PBM cells from asthma patients vs controls
was examined by the above described methods and significantly enhanced
expression was seen for the b-isoform (variant b) of the gene in patients
compared to
control (numbers are listed in FIG.10).
In conclusion, evidence from the linkage and case-control association studies
demonstrate that the MAP3k9 gene is the culprit asthma gene, as judged by
positional
cloning. In addition, evidence from both asthmatic airway tissue and PBM cells
from
3o patients with asthma show that expression of the b-isoform (variant b) of
the
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MAP3K9 gene is enhanced relative to control samples. Collectively, this
evidence
supports the role of the MAP3k9 gene as a therapeutic target for asthma.
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Table 1
Haplotype Analysts
haplotypemarker type Haplotype
allele
hapl DG14S205 microsatellite-4
hapl DG14S428 microsatellite0
hapl D14S1002 microsatellite10
hapl DG14399 znicrosatellite13
hapl DG14S404 microsatellite0
hap2 DG14S428 microsatellite0
hap2 D14S1002 microsatellite10
hap2 DG14S399 microsatellite13
hap2 DG14S404 microsatellite0
hap3 D14S251 microsatellite2
hap3 DG14S1300microsatellite0
hap3 DG14S420 microsatellite4
hap3 DG14S1266microsatellite2
hap4 DG14S1266microsatellite-2
hap4 DG14S462 microsatellite6
hap4 DG14S448 microsatellite14
hap4 DG14S205 microsatellite4
hap5 D 14S microsatellite10
1002
hap5 DG14S399 microsatellite13
hap5 DG14S404 microsatellite0
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hap6 DG14S399 microsatellite13
hap6 DG14S404 microsatellite0
hap6 DG14S406 microsatellite4
hap7 DG14S399 microsatellite13
hap7 DG14S404 microsatellite4
Alleles #'s: For microsatellite alleles: the CEPH samples (CEPH
genomics repository #1347-02) are used as references, the lower allele of
each microsatellite in this sample is set at 0 and all other alleles in other
samples are numbered in relation to this reference. Thus allelel is 1 by
longer than the lower allele in the CEPH sample, allele 2 is 2 by longer than
the lower allele in the CEPH sample, allele 3 is 3 by longer than the lower
allele in the CEPH sample, allele 4 is 4 by longer than the lower allele in
the
CEPH sample, allele -1 is 1 by shorter than the lower allele in the CEPH
to sample, allele -2 is 2 by shorter than the lower allele in the CEPH sample,
and so on.
1s
25
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Table 2
Allele frequencies and significance of haplotypes that capture the relative
risk of the asthma gene, MAP3K9.
Hap p-val #a aff.freq#co con. HO.freqX2 info
# r ff n fre
hap 0.0015.913 169 0.067 134 0.0120.042 10.340.813
1
hap 0.000310.400 179 0.062 135 0.0060.039 12.950.810
2 4
hap 0.001811598.50111 0.032 120 0.0000.013 9.7440.939
3 0
hap 0.00249819.480162 0.028 107 0.0000.017 9.2030.999
4
hap 0.0001150.592 185 0.060 139 0.0000.040 14.970.606
9
hap 0.00122.784 184 0.135 138 0.0530.103 10.470.832.
6 8
hap 0.00062.83183 196 0.14582141 0.0560.1103711.930.8293
7 6 8588 7 52 11
s Hap# - Haplotype number
p-val - p-value
r - relative risk
#aff - number of affecteds (patients)
aff.freq - frequency (of haplotype) in affecteds
io #con - number of controls
con.freq - frequency (of haplotype) in controls
HO.freq - frequency (of the haplotype) under the null hypothesis (in
affecteds and controls)
X2 - chi squared
Info - information content
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Table 3
MarkersSeq. Pos. in NCBI Primer Pair SEQ
Build 33
NO:
DG14S205chrl4: 69.250345-69.250499F: CATGGGTAAGAGAAAGGGAACA3
R:AGCTCCCAGCATAGTTCCAG 4
DG14428chrl4: 69.267406-69.267644F: GGCAACGTTGACTTGCAGTA 5
R:CAGCCCAGAGTTCAAGACG 6
D14S1002chr: 69.291050-69.291217F: AGATTTTGGATGTATCAGGG 7
R:CAGAAGCAATAGGATGGATG 8
DG14S399chr: 69.316671-69.317039F: GTGTCAGGAACTGCACGATT 9
R:GGCATGGTGGTACATGTCTG 10
DG14S404Chrl4: 69.3400520-F: AAAGCTCAGCCAGAGTCTCAA 11
69.340691 R: GGCCTTACAGTGCCTAGCAA 12
D14S251CHR14: 69.115621- F: AAAGGATGAACTATTGGTGC 13
69.115940 R: 14
TTTACTTGTACCCAGTATGTNTCTG
DG14S130Chrl4: 69.134571-69.134971F: TGAAAGGGAGCCTACGTCTG 15
0 R: TGAATGCGGGAGTAAATAAATG16
DG14S420Chr: 69.167270-69.167488F: AGGGTGAGAGACTGCTCTGG 17
R:GGAGGGAGGGAAGAAAGAGA 18
DG14S126Chrl4: 69.182501-69.182636F: GCCAAAGAGAGAGGCAGGTA 19
6 R:CCAGATTGCTTCCCTTGGT 20
DG14S462Chrl4: 69.196223-69.196387F: CCAGGGAAACTAATTCATTGACA21
R: 22
TCCTAAGGAAACTTGCCATATACT
T
DG14S448Chr14:69.216835-69217046F: 23
CAAAGGATTAGCTTTACGGTATGT 24
R:TCATGGCTGTGGGACAGTAG
DG14S406Chr14:69.216835-69.217046F: 25
CAAAGGATTAGCTTTACGGTATGT 26
R:TCATGGCTGTGGGAGAGTAG
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Table 4
CEPH SEQ ID NO:
(bp) Amplimer
165/165 27 CATGGGTAAGAGAAAGGGAACATTTACATTTTGGGATCACGTAAGTAAAC
CAGTTCACATGATATttcattcattcattcattcattcattcattcCTTT
TATCTATacatttattgaggaccctactatgcacctggaactatgctggg
agct
244/244 2g GGCAACGTTGACTTCCAGTAAGAAAGGTTCTCTGATCTTTTTTTCTCTTT
TCTTTTCTTTTCTTTTCTTTTCGTTTCTTTTCTTTTCTTTTCTTCCAGGG
TCTCAGTTTGTTGTCCAGGCCAGAGTGCAGTGGCGCAATCTCGGCTCACT
GCAGCCTCAACATCCTAGGCTCAAGCCATCCTCCCACCTCACCCTCCCGA
GTAGCTGGAAGTACAGGCTCGTCTTGAACTCTGGGCTG
1571157 - 29 AGCTCTNCCTGGTTCACTAGCAGACTGTCTTTGGACTTGAACTGAAACCC
TTTCCTACATCTCTGGCCAGTNAGCCTNCCGTGTCAGATTTTGGATGTAT
CAGGCTTCCACAATTGGGTGAGGCGATTCCTTTAGAATAAATCTCTTTTA
CACACACACACACACACACACACGTGTACCCAGACACACGTGCATATGCA
CACACACACACGNACACATCCATCCTATTGCTTCTGTTTCTNTGGAGANC
CCTNATACAAATTNGCATGTCATT'AAATATCACTAGTGTTGTAGCT
375/379 30 gtgtcagcaactgcacgatttctccctattcagccagtagggaatctaga
gcaattctattatctagtacaactttagcaagagaatttaaagtctgtta
tgcaaccatagcctttgcaatagaatctgctatagagatgattataagga
atttccttccttccttccttccttccttccttccttccttccttctttct
ttctttctttctttctttctttctttctttctttctttctttcttctttc
tttttctttctttctttccttctttGAGACAGGGTCTTGctcaacctccc
aggctcaagcaattctccctacctcagtctcccaagtggctgggagtaca
gacatgtaccaccatgcc
174/178 31 AAAGCTCAGCCAGAGTCTCAATTCCTATAACCTCCTTCccatccatccat
tcatccatccatctatccatccatccatccatccatccatccatccatcc
aGCTGATGGAttaataaatttatatcaagcacttactctgagcaaggcat
tttgctaggcactgtaaggcc
298!300 32 CTACAGAGCAATTAAAAAAGGATGAACTATTGGTGCATTTAACAACTTGG
ATGCATTGCAAGGAAATTATGCTGAGTGTAAAAAGCGCCTTCCAAAGGAT
TTCATGCTATGTGATTCCATTTATATAATATTTTCAAAATGACAAAATTT
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TAAAAATAGAGAACAGGTTAGCAGTTGTCAGGGGTTAAAGAGGAAGTGGT
GGGACAGGAAGGAGATGATAGAGGAACCTACACACGATAAAACTGTATAT
AACTAAACACACACACGCACACACACACACACACACACANACAGANACAT
ACTGGGTACAAGTAAAGCT
407/407 33 TGAAAGGGAGCCTACGTCTGACCCCTAGACTTGCCTTGTTTTCCAAGTCt
cacagaatattagggctggacagaagcttgaagctcagtagttcaaccct
cttttctttcagatgagagatcaagtccagagatgattaagagatttatt
caaggtcacacagatagtaagcagcaaatctgaactggaactcaggtcag
tttcctgacaacaaacaatactctttccactgaaccaAAGTATACTTCCA
AAatatatatacacacatatatgtatatgtgtgtacgtgtgtgtatatat
atatatatgtgtgtgtgtgtgtatatatatatatGCTGTAAAATTGTTCT
CTTCTAAAGAAAGAAGGAGCATAGTACTCATTTATTTACTCCCGCATTCA
243/243 34 - AGGGTGAGAGACTGCTCTGGAGCTCAGTGAGGGACCTCAGGGTTACACAA
GAAGGGACTCTGCGCTGGGTTCCAGGC-CTGATTCTTTTATTTTCTCTTTT
TCTTTCTTTCTCTTTCTTTTTCTTTCTTTCTTTTCTTTCTTTCTTTCTTT
CTTTCTTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTCTC
TCTTTCTTCCCTCCCTCC
144/144 35 GCCAAAGAGAGAGGCAGGTACAttagggtgaaccatatgaaatagctgat
attcgactgtttttgacgtaaaaCCGTACGTATATTTcacacacacacac
acacacacTTTTCTGCACCAAGGGAAGCAATCTGG
171/171 3b CCAGGGAAACTAATTCATTCACATATAGGTGTATGTTCACACACACACAC
ACACACACACACTTACATGTATGTGTGAGTATTTTTTTTAAGCAAATACT
GTAGCAAAGATAAAATTGGTCCAGATTTGGCTTAT'TATAAAGTATATGGC
AAGTTTCCTTAGGA
210/210 37 CAAAGGATTAGCTTTACGGTATGTGAATAATAGCTCAATAAAGCTATTAT
TTAAAATAAACGTGCACACACACACACACACACACACACACACACATACA
CAGCTCACTTTACAACACATCAGGACAATAACTCCATGGACTGATATATA
ATGATAAACTACAGTCCTTTCTTTTGTATACTAAGGTGATGCTACTGTCC
CACAGCCATGA
120/124 3$ CAAAGGATTAGCTTTACGGTATGTGAATAATAGCTCAATAAAGCTATTAT
TTAAAATAAACGTGCACACACACACACACACACACACACACACACATACA
CAGCTCACTTTACAACACATCAGGACAATAACTCCATGGACTGATATATA
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ATGATAAACTACAGTCCTTTCTTTTGTATACTAAGGTGATGCTACTGTCC
CACAGCCATGA
Table 5
MAP3K9 SNPs in Chromosome 14q24.2
Position) Marker Name Public Name
69186428rs2286054 rs2286054
69186468SG14S94 rs2286053
69187796SNPI4MAP3K9MD174E1213 rs1476610
69189493SNP14MAP3K9Y609E805 rs3829955
69189852rs3814874 rs3814874
69190615SNP14MAP3K9MD59E41 nets
69189871rs3814873 rs3814873
69191845rs4899367 rs4899367
69192691SNP14MAP3K9RU28E68 rs4141095
'
69193272rs1859465 rsi859465
69197135SG14S93 rs2286052
69199037SNP14MAP3K9YU72E240 rs4899368
69199066SNP14MAP3K9KU43E240 rs4902843
69199320rs1990032 rs1990032
69199696rs3081458 rs3081458
69202427rs4902844 rs4902844
69202855SG14S92 rs2269946
69203912rs2332455 rs2332455
69204821rs4902845 rs4902845
69204879rs4899369 rs4899369
69204881rs4899370 rs4899370
69205272rs2332456 rs2332456
69205421SNP14MAP3K9MU166E175 rs2332457
69205568rs5809495 rs5809495
69205576rs5809496 rs5809496
69205770SNP14MAP3K9YD8E175 new'
69207818rs1160880 rs1160880
69209251rs4902846 rs4902846
69210078rs4902847 rs4902847
69210257rs4902848 rs4902848
69212106rs1476609 rs1476609
69212484rs2877693 rs2877693
69217954SNP14MAP3K9KD14E180 rs3814872
69217954rs3814872 rs3814872
69218041SNP14MAP3K9YDi01E180 new'
69219322rs886600 rs886600
69219322rs4612997 rs4612997
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69220250 rs3081478 rs3081478
69220761 rs3889682 rs3889682
69220771 rs3889683 rs3889683
69220869 rs1142243 rs1142243
69221773 rs4902849 rs4902849
69222957 rs4902852 rs4902852
69223699 rs2332458 rs2332458
6922463? rs1548585 rs1548585
69224705 SG14S83 rs1548584
69235648 rs4902853 rs4902853
69236948 rs2107666 rs2107666
69237009 rs2158531 rs2158531
69237036 rs2158530 rs2158530
69238432 rs4902854 rs4902854
69244156 rs1987652 rs1987652
69244739 rs1034769 rs1034769
69244995 rs4902855 rs4902855
69249713 rs2107665 rs2107665
69249922 SG14S90 rs2158529
69251671 rs4528504 rs4528504
69253468 rs2051857 rs2051857
69254165 rs4902856 rs4902856
69257386 SNP14MAP3K9RU39E413 rs4902857
69259152 rs4899371 rs4899371-
69261096 SG14S89 rs2023955
69261390 rs731571 rs731571
69261417 rs2023954 rs2023954
69261451 rs5809497 rs5809497
69261452 rs3081535 rs3081535
69262833 rs4902858 rs4902858
69265364 SNP14MAP3K9YU160E201 rs4902859
69265815 rs3832971 rs3832971
' Position numbering relative to Build33' (Database name)
Z Detected by sequencing
3 Genotyped in Taqman
15
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The teachings of all publications cited herein are incorporated herein by
reference in their entirety. While this invention has been particularly shown
and
described with references to preferred aspects thereof, it will be understood
by those
skilled in the art that various changes in form and details may be made
therein
without departing from the scope of the invention encompassed by the appended
claims.
