Note: Descriptions are shown in the official language in which they were submitted.
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SUSCEPTIBILITY GENE FOR MYOCARDIAL INFARCTION
RELATED APPLICATION
This application claims the benefit of 60/419,432, filed October 17, 2002. The
entire teachings of the above application are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
Myocardial infarction (MI) is one of the most common diagnoses in
hospitalized patients in industrialized countries. Myocardial Infarction
generally
occurs when there is an abrupt decrease in coronary blood flow following a
thrombotic occlusion of a coronary artery previously narrowed by
atherosclerosis.
Infarction occurs when a coronary artery thrombus develops rapidly at a site a
vascular injury, which is produced or facilitated by factors such as cigarette
smoking,
hypertension and lipid accumulation. In most cases, infarction occurs when an
atherosclerotic plaque fissures, ruptures or ulcerates and when conditions
favor
thrombogenesis. In rare cases, infarction may be due to coronary artery
occlusion
caused by coronary emboli, congenital abnormalities, coronary spasm, and a
wide
variety of systemic, particularly inflammatory diseases.
Although classical risk factors such as smoking, hyperlipidemia, hypertension,
2o and diabetes are associated with many cases of coronary heart disease (CHD)
and MI,
many patients do not have involvement of these risk factors. In fact, many
patients
who exhibit one or more of these risk factors do not develop MI. Family
history has
long been recognized as one of the major risk factors. Although some of the
familial
clustering of MI reflects the genetic contribution to the other conventional
risk
factors, a large number of studies have suggested that there are sigiuficant
genetic
susceptibility factors, beyond those of the known risk factors (Friedlander Y,
et al., By~
Heaf-t J. 1985; 53:382-7, Shea S. et al., ,I. Am. Coll. Ca3°diol. 1984;
4:793-801, and
Hopkins P.N., et al., Am. J. Ca~~diol. 1988; 62:703-7). Major genetic
susceptibility
factors have not yet been identified.
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SUMMARY OF THE INVENTION
As described herein, a locus on chromosome 13q12 has been identified as
playing a major role in Myocardial Infarction (MI). The locus, herein after
referred to
as the MI locus, comprises nucleic acid that encodes 5-lipoxygenase activating
protein
(ALOXSAP or FLAP), herein after referred to as FLAP.
The present invention relates to isolated nucleic acid molecules comprising a
portion or the entire human FLAP nucleic acid or a variant thereof. In one
embodiment, the nucleic acid molecule has at least one polymorphism that is
correlated with the incidence of myocardial infarction. The invention also
relates to
pathways targeting for drug delivery. A further embodiment of the invention is
a
method for the diagnosis of MI and a method for identification of
susceptibility to
myocardial infarction, by identifying polymorphisms in the FLAP nucleic acid,
which
identify those at risk. Also, described are haplotypes and SNPs that can be
used to
identify individuals with MI or at risk of developing MI. The polymorphism in
the
FLAP nucleic acid can be indicated by detecting the presence of a haplotype,
comprising one or more of the markers: DGOOAAFIU, SG13S25, DGOOAAJFF,
DGOOAAHII, DGOOAAHID, B SNP_310657, SG13S30, SG13S32, SG13S42, and
SG13S35 at the 13q12 locus comprising a FLAP nucleic acid. The polymorphism
further can comprise at least one of the polymorphisms as indicated in Table
3.
2o Identification of nucleic acids and polymorphisms in the MI locus can pave
the way for a better understanding of the disease process, which in tunz can
lead to
improved diagnostic and therapeutic methods.
The invention further pertains to methods of diagnosing myocardial infarction
or a susceptibility to myocardial infarction, comprising detecting an
alteration in the
expression or composition of a polypeptide encoded by a FLAP nucleic acid in a
test
sample, in comparison with the expression or composition of a polypeptide
encoded
by FLAP in a control sample, wherein the presence of an alteration in
expression or
composition of the polypeptide in the test sample is indicative of myocardial
infarction or a susceptibility to myocardial infarction.
3o The invention also relates to an isolated nucleic acid molecule comprising
a
FLAP nucleic acid, wherein the FLAP nucleic acid has a nucleic acid sequence
of
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SEQ m NO: 1 or SEQ ID NO: 3, or the complement of SEQ ID NO: 1 or SEQ ID
NO: 3, wherein the nucleic acid molecule comprises a polymorphism as indicated
in
Table 3.
In another embodiment, the invention relates to an isolated nucleic acid
molecule having a polymorphism as indicated in Table 3, which hybridizes under
high stringency conditions to a nucleic acid sequence of SEQ ID NO: 1 or SEQ
ID
NO: 3, or the complement of SEQ m NO: 1 or SEQ m NO: 3.
In yet another embodiment, a method for assaying for the presence of a first
nucleic acid molecule in a sample is described, comprising contacting said
sample
to with a second nucleic acid molecule, where the second nucleic acid molecule
comprises a nucleic acid sequence of SEQ )D NO: 1 or SEQ ID NO: 3, and
hybridizes
to the first nucleic acid under high stringency conditions.
The invention also relates to a vector comprising an isolated nucleic acid
molecule of the invention operably linked to a regulatory sequence, as well as
to a
15 recombinant host cell comprising the vector. The invention also provides a
method
for preparing a polypeptide encoded by an isolated nucleic acid molecule
comprising
culturing the recombinant host cell under conditions suitable for expression
of said
nucleic acid molecule.
Also contemplated by the invention is a method of assaying a sample for the
2o presence of a polypeptide encoded by an isolated nucleic acid molecule of
the
invention, comprising contacting the sample with an antibody that specifically
binds
to the polypeptide.
The invention further provides a method of identifying an agent that alters
expression of a FLAP nucleic acid, comprising: contacting a solution
containing a
25 nucleic acid comprising the promoter region of the FLAP nucleic acid
operably linked
to a reporter gene with an agent to be tested; assessing the level of
expression of the
reporter gene; and comparing the level of expression with a level of
expression of the
reporter gene 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
3o significant, from the level of expression in the absence of the agent, then
the agent is
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a.n agent that alters expression of the FLAP nucleic acid. An agent identified
by this
method is also contemplated.
The invention additionally comprises a method of identifying an agent that
alters expression of a FLAP nucleic acid, in which a solution containing a
nucleic acid
described herein or a derivative or fragment thereof is contacted with an
agent to be
tested, and expression of the nucleic acid, derivative or fragment in the
presence of
the agent is assessed and compared with expression of the nucleic acid,
derivative or
fragment in the absence of the agent. If expression of the nucleic acid,
derivative or
fragment in the presence of the agent differs, by an amount that is
statistically
to significant, from the expression in the absence of the agent, then the
agent is an agent
that alters expression of the FLAP 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 more splicing variants) that differ in kind or
in
quantity from the expression of one or more splicing variants) the absence of
the
agent. Agents identified by this method are also contemplated. Representative
agents
include antisense nucleic acid to a FLAP nucleic acid; a FLAP polypeptide; a
FLAP
nucleic acid receptor; a FLAP nucleic acid binding agent; a peptidomimetic; a
fusion
protein; a prodrug thereof; an antibody; and a ribozyme. A method of altering
expression of a FLAP nucleic acid comprising contacting a cell containing a
FLAP
2o nucleic acid with such an agent is also contemplated.
The invention further pertains to a method of identifying a polypeptide which
interacts with a FLAP polypeptide, employing a yeast two-hybrid system that
uses a
first vector which comprises a nucleic acid encoding a DNA binding domain and
a
FLAP 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
FLAP polypeptide.
In a further embodiment, the invention relates to a myocardial infarction
3o therapeutic agent, such as a FLAP nucleic acid or fragment or derivative
thereof; a 5-
lipoxygenase nucleic acid or fragment or derivative thereof; a leulcotriene
synthetase
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nucleic acid or fragment or derivative thereof; a polypeptide encoded by a
FLAP
nucleic acid; a polypeptide encoded by a 5-lipoxygenase nucleic acid; a
polypeptide
encoded by a leukotriene synthetase nucleic acid; a FLAP receptor; a 5-
lipoxygenase
receptor; a leukotriene synthetase receptor; a FLAP nucleic acid binding
agent; a 5-
lipoxygenase binding agent; a leukotriene synthetase binding agent; a FLAP
nucleic
acid binding agent; a 5-liopoxygenase nucleic acid biyding agent; a
leukotriene
synthetase nucleic acid binding agent; a peptidomimetic; a fusion protein; a
prodrug;
an antibody; an agent that alters FLAP nucleic acid expression; an agent that
alters
activity of a polypeptide encoded by a FLAP nucleic acid, a 5-lipoxygenase
nucleic
to acid, or a leukotriene synthetase nucleic acid; an agent that alters
posttranscriptional
processing of a polypeptide encoded by a FLAP nucleic acid, a 5-lipoxygenase
nucleic acid or a leukotriene synthetase nucleic acid; an agent that alters
interaction of
a FLAP nucleic acid with a FLAP nucleic acid binding agent; an agent that
alters
interaction of a 5-lipoxygenase nucleic acid with a 5-lipoxygenase nucleic
acid
binding agent; an agent that alters interaction of a leukotriene synthetase
nucleic acid
with a leukotriene synthetase nucleic acid binding agent; an agent that alters
transcription of splicing variants encoded by a FLAP nucleic acid, a 5-
lipoxygenase
nucleic acid, or a leukotriene synthetase nucleic acid; or ribozymes; and
pharmaceutical compositions comprising at least one myocardial infarction
2o therapeutic agent.
The invention also pertains to a method of treating a disease or condition
associated with FLAP in an individual, comprising administering a myocardial
infarction therapeutic agent to the individual, in a therapeutically effective
amount. In
certain embodiments, the myocardial infarction therapeutic agent is a FLAP
nucleic
acid agonist or a FLAP nucleic acid antagonist.
A transgenic animal comprising a nucleic acid of the invention such as an
exogenous FLAP nucleic acid or a nucleic acid encoding a FLAP polypeptide is
also
contemplated.
In yet another embodiment, the invention relates to a method for assaying a
sample for the presence of a FLAP nucleic acid, by contacting the sample with
a
nucleic acid comprising a contiguous nucleic acid sequence which is at least
partially
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complementary to a part of the sequence of said FLAP nucleic acid, under
conditions
appropriate for hybridization, and assessing whether hybridization has
occurred
between a FLAP nucleic acid and said nucleic acid, wherein if hybridization
has
occurred, a FLAP nucleic acid is present in the nucleic acid. In certain
embodiments,
the contiguous nucleic acid sequence is completely complementary to a part of
the
sequence of said FLAP nucleic acid and in other embodiments; amplification is
of at
least part of said FLAP nucleic acid.
In certain embodiments, the contiguous nucleic acid sequence is 100 or fewer
nucleotides in length and is either: a) at least 80% identical to a contiguous
sequence
of nucleotides of SEQ m NO: 1 or SEQ m NO: 3; b) at least 80% identical to the
complement of a contiguous sequence of nucleotides in of SEQ ID NO: 1 or SEQ m
NO: 3; or c) capable of selectively hybridizing to said FLAP nucleic acid.
The invention also pertains to a reagent for assaying a sample for the
presence
of a FLAP nucleic acid, the reagent comprising a nucleic acid comprising a
contiguous nucleic acid sequence which is at least partially complementary to
a part
of the nucleic acid sequence of said FLAP nucleic acid. The reagent can
comprise a
contiguous nucleotide sequence which is completely complementary to a part of
the
nucleic acid sequence of said FLAP nucleic acid. A reagent kit for assaying a
sample
for the presence of a FLAP nucleic acid is also described, including (e.g., in
separate
2o containers), one or more labeled nucleic acids comprising a contiguous
nucleic acid
sequence which is at least partially complementary to a part of the nucleic
acid
sequence of said FLAP nucleic acid; and reagents for detection of said label.
The
labeled nucleic acid can comprise a contiguous nucleotide sequence which is
completely complementary to a part of the nucleic acid sequence of said FLAP
nucleic acid. Also described herein is a reagent kit for assaying a sample for
the
presence of a FLAP nucleic acid, comprising one or more nucleic acids
comprising a
contiguous nucleic acid sequence which is at least partially complementary to
a part
of the nucleic acid sequence of said FLAP nucleic acid, and which is capable
of acting
as a primer for said FLAP nucleic acid when maintained under conditions for
primer
extension.
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The invention also provides for the use of a nucleic acid for assaying a
sample
for the presence of a FLAP nucleic acid, in which the nucleic acid is 100 or
fewer
nucleotides in length and is either: at least 80% identical to a contiguous
sequence of
nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3; at least 80% identical to the
complement of a contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID
NO: 3; or capable of selectively hybridizing to said FLAP nucleic acid.
In yet another embodiment, the use of a first nucleic acid for assaying a
sample for the presence of a FLAP nucleic acid that has at least one
nucleotide
difference from the first nucleic acid is described, in which the first
nucleic acid is
to 100 or fewer nucleotides in length and which is either: at least 80%
identical to a
contiguous sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 or one of
the
sequences shown in Table 3; at least 80% identical to the complement of a
contiguous
sequence of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 one of the sequences
shown in Table 3; or capable of selectively hybridizing to said FLAP nucleic
acid.
The invention also relates to a method of diagnosing a susceptibility to
myocardial infarction in an individual, comprising determining the presence or
absence in the individual of certain "haplotypes" (combinations of genetic
markers);
the presence of the haplotype is diagnostic of susceptibility to myocardial
infarction.
In one embodiment, a haplotype associated with a susceptibility to myocardial
2o infarction comprises markers DGOOAAFILT, SG13S25, DGOOAAJFF, DGOOAAHII,
SG13S32 and SG13S35 at the 13q12 locus. In one particular embodiment, the
presence of the alleles T, G, G, G, A and G at DGOOAAFIU, SG13S25, DGOOAAJFF,
DGOOAAHII, SG13S32 and SG13S35, respectively (the B6 haplotype), is diagnostic
of susceptibility to myocardial infarction. In another embodiment, a haplotype
associated with a susceptibility to myocardial infarction comprises markers
DGOOAAFILJ, SG13S25, DG00AAHII, SG13S30 and SG13S42 at the 13q12 locus.
In one particular embodiment, the presence of the alleles T, G, G, G and A at
DGOOAAFIU, SG13S25, DGOOAAHII, SG13S30 and SG13S42, respectively (the BS
haplotype), is diagnostic of susceptibility to myocardial infarction. In a
third
3o embodiment, a haplotype associated with a susceptibility to myocardial
infarction
comprises markers SG13S25, DGOOAAHII, SG13S30 and SG13S42 at the 13q12
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locus. In one particular embodiment, the presence of the alleles G, G, G and A
at
SG13S25, DGOOAAHII, SG13S30 and SG13S42 , respectively (the B4 haplotype), is
diagnostic of susceptibility to myocardial infarction. In a fourth embodiment,
a
haplotype associated with a susceptibility to myocardial infarction comprises
markers
DGOOAAFIU, SG13S25, DGOOAAHID, B SNP-310657 and SG13S32 at the 13q12
locus. In one particular embodiment, the presence of the alleles T, G, T, G
and A at
DGOOAAFIU, SG13S25, DGOOAAHID, B SNP_310657 and SG13S32, respectively
(the A5 haplotype), is diagnostic of susceptibility to myocardial infarction.
In a fifth
embodiment, a haplotype associated with a susceptibility to myocardial
infarction
to comprises markers SG13S25, DGOOAAHID, B SNP_310657 and SG13S32 at the
13q12 locus. In one particular embodiment, the presence of the alleles G, T, G
and A
at SG13S25, DGOOAAHID, B SNP 310657 and SG13S32, respectively (the A4
haplotype), is diagnostic of susceptibility to myocardial infarction. The
presence or
absence of the haplotype can be determined by various methods, including, for
example, using enzymatic amplification, restriction fragment length
polymorphism
analysis, sequence analysis or electrophoretic analysis of nucleic acid from
the
individual.
The invention also relates to a method of diagnosing a susceptibility to
myocardial infarction in an individual, comprising: obtaining a nucleic acid
sample
from said individual; and analyzing the nucleic acid sample for the presence
or
absence of a haplotype using markers DGOOAAFIU, SG13S25, DGOOAAJFF,
DGOOAAHII, DGOOAAHID, B SNP_310657, SG13S30, SG13S32, SG13S42, and
SG13S35 , with alleles T, G, G, G, T, G, G, A, A, G, respectively, at the
13q12 locus,
wherein the presence of the haplotype is diagnostic for a susceptibility to
myocardial
infarction.
Also described herein is a method of diagnosing myocardial infarction or a
susceptibility to myocardial infarction in an individual, comprising
determining the
presence or absence in the individual of a haplotype comprising one or more
markers
and/or single nucleotide polymorphisms as shown in Table 3 in the locus on
chromosome 13q12 comprising a FLAP nucleic acid, wherein the presence of the
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haplotype is diagnostic of myocardial infarction or of a susceptibility to
myocariial
infarction.
A method for the diagnosis and identification of susceptibility to miocardial
infarction in an individual is also described, comprising: screening for an at-
risk
haplotype in the FLAP nucleic acid that is more frequently present in an
individual
susceptible to myocardial infarction compared to an individual who is not
susceptible
to myocardial infarction wherein the at-risk haplotype increases the risk
significantly.
In certain embodiments, the significant increase is at least about 20%, and in
other
embodiments, the significant increase is identified as an odds ratio of at
least about
1.2.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the invention will
be apparent from the following more particular description of preferred
embodiments
of the invention.
FIG. 1 shows the multipoint non-parametric LOD scores for a framework
marker map on chromosome 13. A LOD score suggestive of linkage of 2.5 was
found
at marker D13S289. The maker map for chromosome 13 that was used in the
linkage
analysis is show~l in Table 1.
2o FIG. 2 shows LOD score results for the families after adding 14 markers to
the candidate region. The inclusion of additional microsatellite markers
increased the
information on sharing by decent from 0.7 to 0.8, around the markers that gave
the
highest LOD scores. The marker map used in the second step of linkage anaysis
is
shown in Table 2.
FIG. 3A shows the results from a haplotype association analysis using 4 and 5
microsatellite markers. Thep-value of the association is plotted on the y-axis
and
position of markers on the x-axis. Only haplotypes that show association with
ap-
value < 10-5 are shown in the figure. The most significant microsatellite
marker
haplotype association is found using markers DG13S1103, DG13S166, DG13S1287,
3o DG13S1061 and DG13S301, with alleles 4, 0, 2, 14 and 3, respectively (p-
value of
1.02 x 10-x). Carrier frequency of the haplotype is 7.3% in affected
individuals and
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0.3% in controls. These results are based on 437 patients and 721 controls.
The area
that is common to all the haploytypes shown in the figure includes only one
gene,
FLAP.
FIG. 3B shows the alleles of the makers defining the most significant
microsatellite marker haplotypes. The area defined with a black square is a
common
area to all the most significantly associated haplotypes. The FLAP nucleic
acid is
located between makers DG13S166 and D13S1238. Two marker haplotype involving
alleles 0 and-2 for markers DG13S166 and S13S1238, respectively, is found in
excess in patients. Carrier frequency of this haploype is 27% in patients and
15.4% in
to controls (p-value 1 X 10-3)
FIG. 4 shows the markers and genes around the FLAP (ALOXSAP) gene.
FIG. 5 shows the relative location of key SNPs and exons of the
ALOXSAPIFLAP gene. Haplotype length varies between 33 to 68 kb.
FIGS. 6A-6Y4 show the genomic sequence of the FLAP gene (SEQ ID NO: 1).
FIG. 7A shows the amino acid sequence of FLAP (SEQ ID N0:2) and the
mRNA of FLAP (SEQ ID NO: 3)
FIGs. 7B-7V show the sequences of the FLAP nucleic acid flanking the SNPs
that were identified by sequencing samples from patients (SEQ ID NOs: 398-
535).
2o DETAILED DESCRIPTION OF THE INVENTION
Extensive genealogical information has been combined with powerful gene
sharing methods to map a locus on chromosome 13q12 that is associated with
myocardial infarction. Patients with myocardial infarction and controls were
initially
genotyped with microsatellite markers with an average spacing between markers
of
less than 100kb over the l2Mb candidate region. An epidemiological study of a
population-based sample of MI patients demonstrated the relative risk for
siblings of a
female MI patient is significantly higher than the relative risk for siblings
of a male
proband (1.59 (CI 1.47 - 1.73) vs. 1.35 (CI 1.28 - 1.42)). The gender
difference in
risk of getting MI (males being more likely to get MI) also suggests somewhat
3o different etiology between males and females, where MI in females might
represent a
more extreme phenotype. This study stratified the population according to sex
to
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determine the genetic causes of MI for males and females. The results of the
genome
wide search of genes that cause MI in Iceland is described. This linkage
analysis
resulted in linkage on chromosome 13q12.
Initial haplotype association analysis using 4 or 5 microsatellite markers
that
extended across the gene and were in excess in patients indicated that FLAP is
a
susceptibility gene for myocardial infarction. A region that is common to all
the
microsatellite haplotypes includes only one gene, the FLAP gene.
The FLAP nucleic acid encodes a 5-lipoxygenase activating protein, which, in
combination with 5-lipoxygenase (5-LO), is required for leukotriene synthesis.
to Inhibitors of its function impede translocation of 5-lipoxygenase from the
cytoplasm
to the cell membrane and inhibit activation of 5-lipoxygenase. One other
member of
the leukotriene pathway, CysLT2 receptor, maps to chromosome 13q14.2 (53 cM on
FIG. 2). The region of this gene shows excess sharing identical by decent (LOD
score=1) in female MI patients. This indicates that CysLT2 receptor might also
play a
role in the pathogenesis of MI.
Mutations andlor polymorphisms within the FLAP nucleic acid show
association with the disease and can be used for methods of diagnosis.
Furthermore,
the FLAP gene and other members of the leukotriene pathway, such as 5-LO,
LTA4,
LTB4, LTC4, LTD4 and CysLT2, are therapeutic targets for myocardial
infarction.
NUCLEIC ACIDS OF THE INVENTION
FLAP Nucleic Acidr, Pof°tiohs afzd Ija~~iahts
Accordingly, the invention pertains to isolated nucleic acid molecules
comprising a human FLAP nucleic acid. The term, "FLAP nucleic acid," as used
herein, refers to an isolated nucleic acid molecule encoding FLAP polypeptide.
The
FLAP 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 either the
coding, or
3o 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 or nucleic acid
and can
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further comprise additional non-coding sequences such as introns and non-
coding 3'
and 5' sequences (including regulatory sequences, for example).
For example, a FLAP nucleic acid can consist of SEQ m NOs: 1 or 3 or the
complement thereof, or to a portion or fragment of such an isolated nucleic
acid
molecule (e.g., cDNA or the nucleic acid) that encodes FLAP polypeptide (e.g.,
a
polypeptide such as SEQ m NO: 2). In a preferred embodiment, the isolated
nucleic
acid molecule comprises a nucleic acid molecule selected from the group
consisting
of SEQ m NOs: 1 or 3, or their complement thereof.
Additionally, the 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
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 nucleic acid 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
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
2o recombinant techniques, or chemical precursors or 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
mix. In other circumstances, the material may be purified to essential
homogeneity,
for example as determined by PAGE or column chromatography such as HPLC. In
certain embodiments, an isolated nucleic acid molecule comprises at least
about 50,
~0 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 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,
3o including but not limited to 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of
nucleotides
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which flank the nucleic acid molecule in the genomic DNA of the cell from
which the
nucleic acid molecule is derived.
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.
"Isolated" nucleic acid molecules also encompass ifa vivo and i~a
vity°o RNA transcripts
of the DNA molecules of the present invention. An isolated nucleic acid
molecule or
l0 nucleic acid sequence 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 nucleotide sequences include recombinant DNA molecules in
heterologous
organisms, as well as partially or substantially purified DNA molecules in
solution.
Iya vivo and in vitYO RNA transcripts of the DNA molecules of the present
invention
are also encompassed by "isolated" nucleotide sequences. Such isolated
nucleotide
sequences are useful in the manufacture of the encoded polypeptide, as probes
for
isolating homologous sequences (e.g., from other mammalian species), for gene
mapping (e.g., by ih situ hybridization with chromosomes), or for detecting
2o expression of the nucleic acid in tissue (e.g., human tissue), such as by
Northern blot
analysis.
The present invention also pertains to nucleic acid molecules which are not
necessarily found in nature but which encode a FLAP polypeptide (e.g., a
polypeptide
having an amino acid sequence comprising an amino acid sequence of SEQ ID NOs:
2), or another splicing variant of a FLAP polypeptide or polymorphic variant
thereof.
Thus, for example, DNA molecules that comprise a sequence that is different
from the
naturally occurring nucleic acid sequence but which, due to the degeneracy of
the
genetic code, encode a FLAP polypeptide of the present invention are also the
subjects of this invention. The invention also encompasses nucleotide
sequences
3o encoding portions (fragments), or encoding variant polypeptides such as
analogues or
derivatives of a FLAP polypeptide. Such variants can be naturally occurring,
such as
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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
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 FLAP polypeptide. In one preferred
embodiment, the
nucleotide sequences are fragments that comprise one or more polymorphic
microsatellite markers. In another preferred embodiment, the nucleotide
sequences
to are fragments that comprise one or more single nucleotide polymorphisms in
a FLAP
nucleic acid (e.g., the single nucleotide polymorphisms set forth in Table 3,
below).
Other 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 linkages (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
2o include, for example, those in which peptide linkages 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
nucleic
acid sequence described herein (e.g., nucleic acid molecules which
specifically
hybridize to a nucleic acid sequence encoding polypeptides described herein,
and,
optionally, have an activity of the polypeptide). In one embodiment, the
invention
includes variants described herein which hybridize under high stringency
hybridization conditions (e.g., for selective hybridization) to a nucleic acid
sequence
comprising a nucleic acid sequence selected from the group consisting of SEQ
ID
3o NOs: 1 or 3 or the complement thereof. In another embodiment, the invention
includes variants described herein which hybridize under high stringency
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hybridization conditions (e.g., for selective hybridization) to a nucleic acid
sequence
encoding an amino acid sequence of SEQ ID NO: 2 or a polymorphic variant
thereof.
In a preferred embodiment, the variant that hybridizes wider high stringency
hybridizations has an activity of a FLAP.
Such nucleic acid molecules can be detected and/or 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
to 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
15 (i.e., 100%) complementary to the second, or the first and second may share
some
degree of complementarity that is less than perfect (e.g., 70%, 75%, 85%,
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" and "low stringency conditions"
for
20 nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages
6.3.1-
6.3.6 in Current Protocols ih Molecular Biology (Ausubel, F.M. et al.,
"Curt°ent
Protocols in Molecular Biology", John Wiley & Sons, (1998), the entire
teachings of
which are incorporated by reference herein). The exact conditions which
determine
the stringency of hybridization depend not only on ionic strength (e.g., 0.2X
SSC,
25 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 within other non-identical sequences. Thus, equivalent
conditions
30 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.
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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 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,
Methods ih Enzymology 200: 546-556 (1991), and in, Ausubel, et al., "Cu~~ent
to Protocols ih Molecular Biology", John Wiley & Sons, (1998), 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 lowest
temperature at which only homologous hybridization occurs, each °C by
which the
final wash temperature is reduced (holding SSC concentration constant) 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 of
mismatch
2o 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 prewarmed solution (42°C)
solution
containing 0.2X SSC/0.1% SDS for 15 minutes at 42°C; and a high
stringency wash
can comprise washing in prewarmed (68°C) solution containing O.1X
SSC/0.1%SDS
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 similarity
between
3o the target nucleic acid molecule and the primer or probe used.
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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 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 axe homologous at that position. As used herein, nucleic acid or
amino
to acid "homology" is equivalent to nucleic acid or amino acid "identity". In
certain
embodiments, the length of a sequence aligned for comparison purposes is at
least
30%, for example, at least 40%, in certain embodiments at least 60%, and in
other
embodiments 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 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., P~oc.
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 embodiment, 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, CABlOS
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 (Acceliys,
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,
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Co~aput. Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson and
Lipman,
Pr~oc. Natl. Acad. Sci. USA 85:2444-8 (1988).
In another embodiment, 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 and a length weight of 2, 3, or 4. In yet another embodiment, 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.
to The present invention also provides isolated nucleic acid molecules that
contain a fragment or portion that hybridizes under highly stringent
conditions to a
nucleic acid sequence comprising SEQ ID NO: 1 or 3 or the complement of SEQ ID
NO: 1 or 3, and also provides isolated nucleic acid molecules that contain a
fragment
or portion that hybridizes under highly stringent conditions to a nucleic acid
sequence
encoding an amino acid sequence of the invention or polyrnorphic variant
thereof.
The nucleic acid fragments of the invention are at least about 15, for
example, at least
about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more
nucleotides in length. Longer fragments, for example, 30 or more nucleotides
in
length, encoding antigenic polypeptides described herein are particularly
useful, such
2o as for the generation of antibodies as described below.
Probes and PYime~s
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.(Sciehce 254:1497-1500 (1991)).
A probe or primer comprises a region of nucleic acid that hybridizes to at
least
about 15, for example about 20-25, and in certain embodiments about 40, 50 or
75,
3o consecutive nucleotides of a nucleic acid of the invention, such as a
nucleic acid
comprising a contiguous nucleic acid sequence of SEQ ID NOs: 1 or 3 or the
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complement of SEQ m Nos: 1 or 3, or a nucleic acid sequence encoding an amino
acid sequence of SEQ m NO: 2 or polymorphic variant thereof. In preferred
embodiments, a probe or primer comprises 100 or fewer nucleotides, in certain
embodiments, from 6 to 50 nucleotides, for example, from 12 to 30 nucleotides.
In
other embodiments, the probe or primer is at least 70% identical to the
contiguous
nucleic acid sequence or to the complement of the contiguous nucleotide
sequence,
for example, at least 80% identical, in certain embodiments at least 90%
identical, and
in other embodiments at least 95% identical, or even capable of selectively
hybridizing to the contiguous nucleic acid sequence or to the complement of
the
l0 contiguous nucleotide sequence. 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
sequence information provided herein. For example, nucleic acid molecules can
be
amplified and isolated using the polymerase chain reaction and synthetic
oligonucleotide primers based on one or more of SEQ m NOs: 1 or 3, or the
complement thereof, or designed based on nucleotides based on sequences
encoding
one or more of the amino acid sequences provided herein. See generally PCR
Techfaology: Principles and Applications foY DNA A~aplificatioya (ed. H.A.
Erlich,
Freeman Press, NY, NY, 1992); PC'R Protocols: A Guide to Methods and
Applications (Eds. Innis et al., Academic Press, San Diego, CA, 1990); Mattila
et al.,.
Nucl. Acids Res. 19:4967 (1991); Eckert et al., PCR Methods and 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, Genomics 4:560 (1989), Landegren et al., Science 241:1077
(1988), transcription amplification (Kwon et al., P~oc. Natl. Acad. Sci. USA
86:1173
(1989)), and self sustained sequence replication (Guatelli et al., Pros. Nat.
Acad. Sci.
USA 87:1874 (1990)) and nucleic acid based sequence amplification (NASBA). The
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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 1,
respectively.
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 in vivo excision, and the cloned insert can be sequenced in
either
or both orientations by art recognized methods to identify the correct reading
frame
encoding a polypeptide of the appropriate molecular weight. For example, the
direct
analysis of the nucleic acid molecules of the present invention can be
accomplished
using well-known methods that are commercially available. See, for example,
Sambroolc et al., Molecular CloyaifZg, A Laboratory Manz~al (2nd Ed., CSHP,
New
York 1989); Zyskind et al., Ree~mbinafat DNA Laboratory Manual, (Acad. Press,
1988)). Using these or similar methods, the polypeptide and the DNA encoding
the
polypeptide can be isolated, sequenced and further characterized.
Antisense nucleic acid molecules of the invention can be designed using the
nucleotide sequences of SEQ m NOs: 1 or 3 and/or the complement of one or more
of SEQ ID NOs: 1 or 3 and/or a portion of one or more of SEQ m NOs: 1 or 3 or
the
2o complement of one or more of SEQ ID NOs: 1 or 3 and/or a sequence encoding
the
amino acid sequences of SEQ m NOs: 2 or encoding a portion of one or more of
SEQ
m NOs: 1 or 3 or their complement. They can be 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
3o using an expression vector into which a nucleic acid molecule has been
subcloned in
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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 related to
FLAP,
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
to herein (and the corresponding complete gene sequences) can be used in
numerous
ways as polynucleotide reagents. For example, these sequences can be used to:
(i)
map their respective genes on a chromosome; and, thus, locate gene regions or
nucleic acid 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 maakers for tissues in which the corresponding
polypeptide is
expressed, either constitutively, during tissue differentiation, or in
diseased states.
The nucleic acid sequences can additionally be used as reagents in the
screening
2o and/or diagnostic assays described herein, and can also be included as
components of
kits (e.g., reagent kits) for use in the screening and/or diagnostic assays
described
herein.
T~ecto~s
Another aspect of the invention pertains to nucleic acid constructs containing
a
nucleic acid molecule of SEQ m NOs: 1 or 3 or the complement thereof (or a
portion
thereof). Yet another aspect of the invention pertains to nucleic acid
constructs
containing a nucleic acid molecule encoding an amino acid of SEQ m NO: 2 or
polymorphic variant thereof. The constructs comprise a vector (e.g., an
expression
3o 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
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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
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
cell, and thereby are replicated along with the host genome. Moreover, certain
io vectors, such as expression vectors, are capable of directing the
expression of genes or
nucleic acids 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.
Preferred recombinant expression vectors of the invention comprise a nucleic
acid molecule of the invention in a form suitable for expression of the
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
2o 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 nucleic acid sequence of interest is linked to the
regulatory
sequences) in a manner which allows for expression of the nucleic acid
sequence
(e.g., in an iya vits~o transcription/translation system or in a host cell
when the vector is
introduced into the host cell). The teen "regulatory sequence" is intended to
include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation
signals). Such regulatory sequences are described, for example, in Goeddel,
"Gene
Expression Technology", Methods i~ Ehzy~aology 185, Academic Press, San Diego,
CA (1990). Regulatory sequences include those which direct constitutive
expression
of a nucleic acid sequence in many types of host cell and those which direct
expression of the nucleic acid sequence only in certain host cells (e.g.,
tissue-specific
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regulatory sequences). It will be appreciated by those 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.
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.,
bacterial cells such as E. coli, insect cells (using baculovirus expression
vectors),
to yeast cells or mammalian cells. Suitable host cells are discussed further
in Goeddel,
supra. Alternatively, the recombinant expression vector can be transcribed and
translated ifz vitf~o, for example using T7 promoter regulatory sequences and
T7
polyrnerase.
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 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
2o 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 tramsfection 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
3o cell, including calcium phosphate or calcium chloride co-precipitation,
DEAE-
dextran-mediated transfection, lipofection, or electroporation. Suitable
methods for
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transforming or transfecting host cells can be found in Sambrook, et al.
(supra), 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 or nucleic acid that encodes a selectable marker
(e.g., for
resistance to antibiotics) is generally introduced into the host cells along
with the gene
or nucleic acid of interest. Preferred selectable markers include those that
confer
resistance to drugs, such as G41 ~, hygromycin and methotrexate. Nucleic acid
to 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. Cells stably transfected with the introduced nucleic acid molecule can
be
identified by drug selection (e.g., cells that have incorporated the
selectable marker
gene or nucleic acid will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic host cell or eukaryotic
host
cell in culture can be used to produce (i. e., express) a polypeptide of the
invention.
Accordingly, the invention further provides methods for producing a
polypeptide
using the host cells of the invention. In one embodiment, the method comprises
culturing the host cell of invention (into which a recombinant expression
vector
2o encoding a polypeptide of the invention has been introduced) in a suitable
medium
such that the polypeptide is produced. In another embodiment, 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. For example, in one embodiment, 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 FLAP nucleic acid, or an
exogenous nucleic acid encoding a FLAP polypeptide). Such host cells can then
be
used to create non-human transgenic animals in which exogenous nucleotide
sequences have been introduced into the genome or homologous recombinant
animals
3o in which endogenous nucleotide sequences have been altered. Such animals
are
useful for studying the function and/or activity of the nucleic acid sequence
and
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polypeptide encoded by the sequence and for identifying and/or 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 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 cell types or tissues of the transgenic animal. As used herein, an
to "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, U.S.
Pat. No. 4,873,191 and in Hogan, Manipulatifzg the M~use Embryo (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Methods for
constructing
2o homologous recombination vectors and homologous recombinant animals are
described further in Bradley, CuYf°ent Opihiofa iu BioTeclahology 2:823-
829 (1991)
and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, 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 97/07668 and WO 97/07669.
POLYPEPTIDES OF THE INVENTION
The present invention also pertains to isolated polypeptides encoded by FLAP
nucleic acids ("FLAP polypeptides"), and fragments and variants thereof, as
well as
3o polypeptides encoded by nucleotide sequences described herein (e.g., other
splicing
variants). The term "polypeptide" refers to a polymer of amino acids, and not
to a
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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
understood, however, that preparations in which the polypeptide is not
purified to
1o 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
embodiment, the language "substantially free of cellular material" includes
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 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 embodiment, the
language
"substantially free of chemical precursors or other chemicals" includes
preparations 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 5%
chemical precursors or other chemicals.
In one embodiment, a polypeptide of the invention comprises an amino acid
sequence encoded by a nucleic acid molecule comprising a nucleic acid sequence
3o selected from the group consisting of SEQ ID NO: 1 or 3, or the complement
of SEQ
ID NO: 1 or 3, or portions thereof, or a portion or polymorphic variant
thereof.
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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 nucleic acid sequence selected from the group consisting
of
SEQ ID NOs: 1 or 3 or their complement, or portions thereof, or having
substantial
homology to a polypeptide encoded by a nucleic acid molecule comprising a
nucleic
acid sequence selected from the group consisting of nucleotide sequences
encoding
to SEQ >D NO: 2 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
substantially homologous or identical when the amino acid sequences are at
least
about 45-55%, in certain embodiments at least about 70-75%, and in other
embodiments at least about 80-85%, and in others greater than about 90% or
more
2o homologous or identical. A substantially homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic acid molecule
hybridizing to SEQ m NO: 1 or 3 or portion thereof, under stringent conditions
as
more particularly described above, or will be encoded by a nucleic acid
molecule
hybridizing to a nucleic acid sequence encoding SEQ ID NO: 2 or a portion
thereof or
polymorphic variant thereof, under stringent conditions as more particularly
described
thereof.
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
3o invention. Similarity is determined by conserved amino acid substitution.
Such
substitutions are those that substitute a given amino acid in a polypeptide by
another
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_28_
amino acid of like characteristics. Conservative substitutions are likely to
be
phenotypically silent. Typically seen as conservative substitutions are the
replacements, one for another, 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 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
to 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 some degree. Non-
functional variants typically contain one or more non-conservative amino acid
substitutions, deletions, insertions, inversions, or truncation or a
substitution,
insertion, inversion, or deletion in a critical residue or critical region.
2o 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 244:1081-1085 (1989)). The latter procedure
introduces
single alanine mutations at every residue in the molecule. The resulting
mutant
molecules are then tested for biological activity in vitro, or iya 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)).
The invention also includes fragments of the polypeptides of the invention.
3o Fragments can be derived from a polypeptide encoded by a nucleic acid
molecule
comprising SEQ ID NO: 1 or 3, or the complement of SEQ ID NO: 1 or 3 (or other
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variants). However, the invention also encompasses fragments of the variants
of the
polypeptides described herein. As used herein, a fragment comprises at least 6
contiguous amino acids. Useful fragments include those that retain one or more
of 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
to or more transmembrane segments or loops, ligand binding regions, zinc
finger
domains, DNA binding domains, acylation sites, glycosylation sites, or
phosphorylation sites.
Fragments can be discrete (not fused to other amino acids or polypeptides) or
can be within a larger polypeptide. Further, several fragments can be
comprised
within a single larger polypeptide. In one embodiment 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 terminus of the fragment.
The invention thus provides chimeric or fusion polypeptides. These comprise
2o a polypeptide of the invention operatively linked to a heterologous protein
or
polypeptide having an amino acid sequence not substantially homologous to the
polypeptide. "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 embodiment the fusion polypeptide
does not
affect function of the polypeptide pe~~ se. For example, the fusion
polypeptide can be
a GST-fusion polypeptide in which the polypeptide sequences are fused to the C-
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
3o polypeptides, particularly poly-His fusions, can facilitate the
purification of
recombinant polypeptide. In certain host cells (e.g., mammalian host cells),
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expression and/or secretion of a polypeptide can be increased using a
heterologous
signal sequence. Therefore, in another embodiment, the fusion polypeptide
contains a
heterologous signal sequence at its N-terminus.
EP-A-O 464 533 discloses fusion proteins comprising various portions of
immunoglobulin 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.,
.Iournal ofMoleculaf°Recoghitioh, 8:52-58 (1995) and Johanson et al.,
The.Iou~~ual of
to Biological Che~raist~y, 270,16:9459-9471 (1995). Thus, this invention 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 embodiment, 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
can
subsequently be annealed and re-amplified to generate a chimeric nucleic acid
sequence (see Ausubel et al., Cu~refzt Protocols ifz 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 the
fusion
moiety is linked in-frame to the polypeptide.
The isolated polypeptide can be purified from cells that naturally express it,
purified from cells that have been altered to express it (recombinant), or
synthesized
using known protein synthesis methods. In one embodiment, the polypeptide is
3o produced by recombinant DNA techniques. For example, a nucleic acid
molecule
encoding the polypeptide is cloned into an expression vector, the expression
vector
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introduced into a host cell and the polypeptide expressed in the host cell.
The
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
to diseased states. The polypeptides can be used to isolate a corresponding
binding
agent, e.g., ligand, such as, for example, in an interaction trap assay, and
to screen for
peptide or small molecule antagonists or agonists of the binding interaction.
For
example, because members of the leukotriene pathway including FLAP bind to
receptors, the leukotriene pathway polypeptides can be used to isolate such
receptors.
ANTIBODIES OF THE INVENTION
Polyclonal and/or monoclonal aaltibodies that specifically bind one form of
the
polypeptide or nucleic acid product (e.g., a polypeptide encoded by a nucleic
acid
having a SNP as set forth in Table 3), but not to another form of the
polypeptide or
2o nucleic acid product, are also provided. Antibodies are also provided which
bind a
portion of either polypeptide encoded by nucleic acids of the invention (e.g.,
SEQ m
NO: 1 or SEQ ~ NO: 3, or the complement of SEQ ID NO: 1 or SEQ ID NO: 3), or
to a polypeptide encoded by nucleic acids of the invention that contain a
polymorphic
site or sites. The invention also provides antibodies to the polypeptides and
polypeptide fragments of the invention, or a portion thereof, or having an
amino acid
sequence encoded by a nucleic acid molecule comprising all or a portion of SEQ
117
NOs: 1 or 3, or the complement thereof, or another variant or portion thereof.
The
term "antibody" as used herein refers to irrununoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e., molecules
that
3o contain an antigen binding site that specifically binds an antigen. A
molecule that
specifically binds to a polypeptide of the invention is a molecule that binds
to that
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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 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 herein, refers to a population of
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
1o monoclonal antibody composition thus typically displays a single binding
affinity fox
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
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 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 I~ohler and Milstein, Nature
256:495-
497 (.1975), the human B cell hybridoma technique (I~ozbor et al., Irnmunol.
Today
4:72 (1983)); the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies
and
Cancers The~~apy, Alan R. Liss, 1985, Iuc., pp. 77-96); or trioma techniques.
The
technology for producing hybridomas is well known (see generally
Cuf°f~efzt PnotocoZs
in hramunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York,
NY).
Briefly, an immortal cell Iine (typically a myeloma) is fused to lymphocytes
(typically
splenocytes) from a mammal immunized with an immunogen as described above, and
3o the culture supernatants of the resulting hybridoma cells are screened to
identify a
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hybridoma producing a monoclonal antibody that binds a polypeptide of the
invention.
Any of the many well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied for the purpose of generating a
monoclonal
antibody to a polypeptide of the invention (see, e.g., Cufweut Protocols in
Immuf2ology, supra; Galfre et al., Nature 266:55052 (1977); R.H. Kenneth, in
Mofzoclo~zal Antibodies: A New Dimef2sion In Biological Ah.alyses, Plentun
Publishing
Corp., New York, New York (1980); and Lerner, Yale J. Biol. Med. 54:387-402
(1981). Moreover, the ordinarily skilled worker will appreciate that there are
many
to variations of such methods that also would be useful.
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 Pharmacia RecombihafZt Phage
AsZtibody System, Catalog No. 27-9400-O1; and the Stratagene Sm fZAPTM Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods and
reagents
particularly amenable for use in generating and screening antibody display
library can
2o be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO
92118619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791;
PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication
No. WO 90/02809; Fuchs et al., BiolTeclahology 9: 1370-1372 (1991); Hay et
al.,
Hum. Ahtibod. Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281
(1989); Griffiths et al., EMBO.I. 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
invention. Such chimeric and humanized monoclonal antibodies can be produced
by
recombinant DNA techniques known in the art.
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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 immunoprecipitation. A polypeptide-specific antibody can
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
monitor protein levels in tissue as part of a clinical testing procedure,
e.g., to, for
l0 example, determine the efficacy of a given treatment regimen. Detection can
be
facilitated by coupling the antibody to a detectable substance. 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,13-
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
2o materials include luciferase, luciferin and aequorin, and examples of
suitable
radioactive material include l2sh 1311, ssS or 3H.
DIAGNOSTIC ASSAYS
The nucleic acids, probes, primers, polypeptides and antibodies described
herein can be used in methods of diagnosis of MI or diagnosis of a
susceptibility to
MI or to a disease or condition associated with an MI gene, such as FLAP, as
well as
in kits useful for diagnosis of MI or a susceptibility to MI or to a disease
or condition
associated with FLAP. In one embodiment, the kit useful for diagnosis of MI or
susceptibility to MI, or to a disease or condition associated with FLAP
comprises
3o primers as described herein, wherein the primers contain one or more of the
SNPs
identified in Table 3.
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In one embodiment of the invention, diagnosis of MI or susceptibility to MI
(or diagnosis of or susceptibility to a disease or condition associated with
FLAP), is
made by detecting a polymorphism in a FLAP nucleic acid as described herein.
The
polymorphism can be an alteration in a FLAP nucleic acid, such as the
insertion or
s deletion of a single nucleotide, or of more than one nucleotide, resulting
in a frame
shift alteration; 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
to nucleotides, such as by unequal recombination or gene conversion, resulting
in an
interruption of the coding sequence of the gene or nucleic acid; duplication
of all or a
part of the gene or nucleic acid; transposition of all or a part of the gene
or nucleic
acid; or rearrangement of all or a part of the gene or nucleic acid. More than
one such
alteration may be present in a single gene or nucleic acid. Such sequence
changes
15 cause an alteration in the polypeptide encoded by a FLAP nucleic acid. For
example,
if the alteration is a frame shift alteration, 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 disease or condition associated with a FLAP nucleic acid or
a
2o susceptibility to a disease or condition associated with a FLAP 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 FLAP nucleic acid). Such a
polymorphism may alter splicing sites, affect the stability or transport of
mRNA, or
otherwise affect the transcription or translation of the nucleic acid. A FLAP
nucleic
25 acid that has any of the alteration described above is referred to herein
as an "altered
nucleic acid."
In a first method of diagnosing MI or a susceptibility to MI, hybridization
methods, such as Southern analysis, Northern analysis, or iya situ
hybridizations, can
be used (see Curre~zt Ps-otocols in Molecular Biology, Ausubel, F. et al.,
eds., John
3o Wiley & Sons, including all supplements through 1999). For example, a
biological
sample from a test subject (a "test sample") of genomic DNA, RNA, or cDNA, is
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obtained from an individual suspected of having, being susceptible to or
predisposed
fox, or carrying a defect for, a susceptibility to a disease or condition
associated with a
FLAP nucleic acid (the "test individual"). 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 sample, sample of amniotic fluid, sample of cerebrospinal fluid, or
tissue
sample from skin, muscle, buccal or conjunctival 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 or chorionic villus sampling.
The
DNA, RNA, or cDNA sample is then examined to determine whether a polymorphism
to in a nucleic acid is present, and/or to determine which splicing variants)
encoded by
the FLAP is present. The presence of the polymorphism or splicing variants)
can be
indicated by hybridization of the nucleic acid 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 at least one polymorphism in
a
FLAP nucleic acid or contains a nucleic acid encoding a particular splicing
variant of
a FLAP nucleic acid. The probe can be any of the nucleic acid molecules
described
above (e.g., the nucleic acid, a fragment, a vector comprising the nucleic
acid, a probe
or primer, etc.).
To diagnose MI or a susceptibility to MT (or a disease or condition associated
2o with FLAP), the test sample containing a FLAP nucleic acid is contacted
with at least
one nucleic acid probe to form a hybridization sample. 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 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: 1 and 3, or the complement thereof or a portion thereof;
or
can be a nucleic acid encoding all or a portion of one of SEQ ID NO: 2. Other
suitable probes for use in the diagnostic assays of the invention are
described above
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(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 FLAP nucleic acid.
"Specific hybridization", as used herein, indicates exact hybridization (e.g.,
with no
mismatches). Specific hybridization can be performed under high stringency
conditions or moderate stringency conditions, for example, as described above.
hz a
particularly preferred embodiment, the hybridization conditions for specific
hybridization are high stringency.
to Specific hybridization, if present, is then detected using standard
methods. If
specific hybridization occurs between the nucleic acid probe and FLAP nucleic
acid
in the test sample, then the FLAP has the polymorphism, or is the splicing
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
15 acid probes is indicative of a polymorphism in the FLAP nucleic acid, or of
the
presence of a particular splicing variant encoding the FLAP nucleic acid, and
is
therefore diagnostic for a disease or condition associated with FLAP or a
susceptibility to a disease or condition associated with FLAP (e.g., MI).
In Northern analysis (see Cu3"rel2t Protocols in Molecular Biology, Ausubel,
F.
2o 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,
associated with a disease or condition associated with or a susceptibility to
a disease
or condition associated with FLAP (e.g., MI). For Northern analysis, a test
sample of
RNA is obtained from the individual by appropriate means. Specific
hybridization of
25 a nucleic acid probe, as described above, to RNA from the individual is
indicative of a
polymorphism in a FLAP nucleic acid, or of the presence of a particular
splicing
variant encoded by a FLAP nucleic acid, and is therefore diagnostic for the
disease or
condition associated with FLAP, or for susceptibility to a disease or
condition
associated with FLAP (e.g., MI).
3o For representative examples of use of nucleic acid probes, see, for
example,
U.S. Patents No. 5,288,611 and 4,851,330.
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Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a
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 L~ attached to the glycine nitrogen
via a
methylene carbonyl linker (see, for example, Nielsen, P.E. et al.,
Biocoyzjugate
Cheuzi~t~y 5, American Chemical Society, p. 1 (1994). The PNA probe can be
designed to specifically hybridize to a nucleic acid having a polymorphism
associated
with a disease or condition associated with FLAP or associated with a
susceptibility to
a disease or condition associated With FLAP (e.g., MI). Hybridization of the
PNA
l0 probe to a FLAP nucleic acid as described herein is diagnostic for the
disease or
condition or the susceptibility to the disease or condition.
In another method of the invention, mutation analysis by restriction digestion
can be used to detect an altered nucleic acid, or nucleic acids containing a
polymorphism(s), if the mutation or polymorphism in the nucleic acid 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 FLAP 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 C'uY~efzt Protocols izz Molecular Biology, supra). The
digestion pattern
of the relevant DNA fragment indicates the presence or absence of the
alteration or
polymorphism in the FLAP nucleic acid, and therefore indicates the presence or
absence of a disease or condition associated with FLAP or the susceptibility
to a
disease or condition associated with FLAP (e.g., MI).
Sequence analysis can also be used to detect specific polyrnorphisms in the
FLAP 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
nucleic acid,
and/or its flanking sequences, if desired. The sequence of a FLAP 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
3o nucleic acid, nucleic acid fragment, cDNA, cDNA fragment, mRNA, or mRNA
fragment is compared with the known nucleic acid sequence of the nucleic acid,
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cDNA (e.g., one or more of SEQ TD NOs: 1 or 3, andlor the complement of SEQ ID
NO: 1 or 3), or a nucleic acid sequence encoding SEQ m NO: 2 or a fragment
thereof) or mRNA, as appropriate. The presence of a polymorphism in the FLAP
indicates that the individual has disease or a susceptibility to a disease
associated with
FLAP (e. g. , MI).
Allele-specific oligonucleotides can also be used to detect the presence of
polymorphism(s) in the FLAP nucleic acid, through the use of dot-blot
hybridization
of amplified oligonucleotides with allele-specific oligonucleotide (ASO)
probes (see,
for example, Sail~i, R. et al., Natuf°e 324:163-166 (1986)). An "allele-
specific
l0 oligonucleotide" (also referred to herein as an "allele-specific
oligonucleotide probe")
is an oligonucleotide of approximately 10-50 base pairs, for example,
approximately
15-30 base pairs, that specifically hybridizes to a FLAP nucleic acid, and
that contains
a polymorphism associated with a disease or condition associated with FLAP or
a
susceptibility to a disease or condition associated with FLAP (e.g., Ml). An
allele-
specific oligonucleotide probe that is specific for particular polymorphisms
in a FLAP
nucleic acid can be prepared, using standard methods (see Cuy~y-efzt Protocols
iyi
Molecular Biology, supra). To identify polymorphisms in the nucleic acid
associated
with disease or susceptibility to disease, a test sample of DNA is obtained
from the
individual. PCR can be used to amplify all or a fragment of a FLAP nucleic
acid, and
its flanking sequences. The DNA containing the amplified FLAP nucleic acid (or
fragment of the nucleic acid) is dot-blotted, using standard methods (see
Cur~e~zt
Protocols in. Molecular Biology, supy~a), and the blot is contacted with the
oligonucleotide probe. The presence of specific hybridization of the probe to
the
amplified FLAP is then detected. Specific hybridization of an allele-specific
oligonucleotide probe to DNA from the individual is indicative of a
polymorphism in
the FLAP, and is therefore indicative of a disease or condition associated
with FLAP
or a susceptibility to a disease or condition associated with FLAP (e.g., MI).
An allele-specific primer hybridizes to a site on target DNA overlapping a
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
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distal site. Amplification proceeds from the two primers, resulting in a
detectable
product which indicates the particular allelic form is present. A control is
usually
performed with a second pair of primers, one of which shows a single base
mismatch
at the polyrnorphic 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 polymorphism because
this
position is most destabilizing to elongation from the primer (see, e.g., WO
93/22456).
In another embodiment, arrays of oligonucleotide probes that are complementary
to
to target nucleic acid sequence segments from an individual, can be used to
identify
polymorphisms in a FLAP nucleic acid. For example, in one embodiment, 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 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 WO 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.,
Science
251:767-777 (1991); Pirrung et al., U.S. Pat. 5,143,854; (see also PCT
Application
WO 90/15070); Fodor et al., PCT Publication WO 92/10092; and U.S. Pat.
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. 5,384,261, the entire teachings of which are
incorporated
by reference herein. In another example, linear arrays can be utilized.
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
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polyrnorphic markers is amplified using 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 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 typically in the
form of
fluorescence intensities as a function of location on the array. In a reverse
method, a
1o probe, containing a polymorphism, can be coupled to a solid surface and PCR
amplicons are then added to hybridize to these probes.
Although primarily described in terms of a single detection block, e.g.,
detection of a single polymorphism arrays can include multiple detection
blocks, and
thus be capable of analyzing multiple, specific polymorphisms. It will
generally be
i5 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 polyrnorphisms that fall within G-C rich
stretches of
a genomic sequence, separately from those falling in A-T rich segments. This
allows
2o for the separate optimization of hybridization conditions for each
situation.
Additional uses of oligonucleotide arrays for detection of polymorphisms 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
acid analysis can be used to detect polymorphisms in a nucleic acid described
herein,
25 or variants encoded by a nucleic acid described herein. Representative
methods
include direct manual sequencing (Church and Gilbert, Proc. 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
30 electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE)
(Sheffield,
V.C. et al., P~oc. Natl. Acad. Sci. USA 86:232-236 (1989)), mobility shift
analysis
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(Orita, M. et al., Py~oc. Natl. Acad. Sci. USA 86:2766-2770 (1989)),
restriction enzyme
analysis (Flavell et al., Cell 15:25 (1978); Geever, et al., Pf~oc. Natl.
Acad. Sci. USA
78:5081 (1981)); heteroduplex analysis; chemical mismatch cleavage (CMC)
(Cotton
et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)); RNase protection
assays
(Myers, R.M. et al., Scieface 230:1242 (1985)); use of polypeptides which
recognize
nucleotide mismatches, such as E. coli mutS protein; allele-specific PCR, for
example.
In one embodiment of the invention, diagnosis of a disease or condition
associated with FLAP (e.g., MI) or a susceptibility to a disease or condition
associated
to with FLAP (e.g., MI) can also be made by expression analysis by
quantitative PCR
(kinetic thermal cycling). This technique utilizing TaqMan ~ can be used to
allow the
identification of polymorphisms and whether a patient is homozygous or
heterozygous. The technique can assess the presence of an alteration in the
expression or composition of the polypeptide encoded by a FLAP nucleic acid or
splicing variants encoded by a FLAP nucleic acid. Further, the expression of
the
variants can be quantified as physically or functionally different.
In another embodiment of the invention, diagnosis of MI or a susceptibility to
MI (or of another disease or condition associated with FLAP) can also be made
by
examining expression and/or composition of a FLAP polypeptide, by a variety of
2o 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
composition of the polypeptide encoded by a FLAP nucleic acid, or for the
presence
of a particular variant encoded by a FLAP nucleic acid. An alteration in
expression of
a polypeptide encoded by a FLAP 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 FLAP nucleic acid
is an
alteration in the qualitative polypeptide expression (e.g., expression of an
altered
FLAP polypeptide or of a different splicing variant). In a preferred
embodiment,
diagnosis of disease or condition associated with FLAP or a susceptibility to
a disease
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or condition associated with FLAP is made by detecting a particular splicing
variant
encoded by that FLAP variant, or a particular pattern of splicing variants.
Both such alterations (quantitative and qualitative) can also be present. An
"alteration" in the polypeptide expression or composition, refers to an
alteration in
expression or composition in a test sample, as compared with the expression or
composition of polypeptide by a FLAP nucleic acid in a control sample. A
control
sample is a sample that corresponds to the test sample (e.g., is from the same
type of
cells), and is from an individual who is not affected by the disease or a
susceptibility
to a disease or condition associated with a FLAP nucleic acid. An alteration
in the
to expression or composition of the polypeptide in the test sample, as
compared with the
control sample, is indicative of disease or condition associated with FLAP or
a
susceptibility to a disease or condition associated with FLAP (e.g., MI).
Similarly,
the presence of one or more different splicing variants in the test sample, or
the
presence of significantly different amounts of different splicing variants in
the test
sample, as compared with the control sample, is indicative of a susceptibility
to a
disease or condition associated with a FLAP nucleic acid. Various means of
examining expression or composition of the polypeptide encoded by a FLAP
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
2o irmnunoblotting (see also Cur rent Protocols ifZ Molecular Biology,
particularly
Chapter 10). For example, in one embodiment, an antibody capable of binding to
the
polypeptide (e.g., as described above), preferably an antibody with a
detectable label,
can be used. Antibodies can be polyclonal, or more preferably, monoclonal. An
intact antibody, or a fragment thereof (e.g., Fab or F(ab')a) 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
3o secondary antibody and end-labeling of a DNA probe with biotin such that it
can be
detected with fluorescently labeled streptavidin.
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Western blotting analysis, using an antibody as described above that
specifically binds to a polypeptide encoded by an altered FLAP (e.g., by a
FLAP
having a SNP as shown in Table 3), 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 FLAP, or the absence in a test sample of a
particular splicing variant or of a polypeptide encoded by a non-polyrnorphic
or non-
altered nucleic acid. The presence of a polypeptide encoded by a polymorphic
or
to altered nucleic acid, or the absence of a polypeptide encoded by a non-
polymorphic or
non-altered nucleic acid, is diagnostic for disease or condition associated
with FLAP
or a susceptibility to a disease or condition associated with, as is the
presence (or
absence) of particular splicing variants encoded by the FLAP nucleic acid.
In one embodiment of this method, the level or amount of polypeptide
encoded by a FLAP nucleic acid in a test sample is compared with the level or
amount
of the polypeptide encoded by the FLAP in a control sample. A level or amount
of
the 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
2o by the FLAP, and is diagnostic for disease or condition, or for a
susceptibility to a
disease or condition, associated with that FLAP. Alternatively, the
composition of the
polypeptide encoded by a FLAP nucleic acid in a test sample is compared with
the
composition of the polypeptide encoded by the FLAP 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, or for a
susceptibility to
a disease or condition, associated with that FLAP. In another embodiment, 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
3o 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
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in the amount or level, and a difference in the composition, is indicative of
a disease
or condition, or a susceptibility to a disease or condition, associated with
FLAP (e.g.,
MI).
The invention further pertains to a method for the diagnosis and
identification
of susceptibility to myocardial infarction in an individual, by identifying an
at-risk
haplotype in FLAP. In one embodiment, the at-risk haplotype is one which
confers a
significant risk of MI. In one embodiment, sig~lificance associated with a
haplotype is
measured by an odds ratio. In a further embodiment, the significance is
measured by
a percentage. In one embodiment, a significant risk is measured as an odds
ratio of at
to 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 embodiment, an odds ratio of at least 1.2 is significant. In a
further
embodiment, an odds ratio of at least about 1.5 is significant. In a further
embodiment, a significant increase in risk is at least about 1.7 is
significant. In a
further embodiment, a significant increase in risk is at least about 20%,
including but
riot hmlted tO about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, and 98%. In a further embodiment, a significant increase
in
risk is at least about 50%. It is understood however, that identifying whether
a risk is
medically significant may also depend on a variety of factors, including the
specific
disease, the haplotype, and often, environmental factors.
The invention also pertains to methods of diagnosing myocardial infarction or
a susceptibility to myocardial infarction in an individual, comprising
screening for an
at-risk haplotype in the FLAP nucleic acid that is more frequently present in
an
individual susceptible to myocardial infarction (affected), compared to the
frequency
of its presence in a healthy individual (control), wherein the presence of the
haplotype
is indicative of myocardial infarction or susceptibility to myocardial
infarction.
Standard techniques for genotyping for the presence of SNPs and/or
microsatellite
markers that are associated with myocardial infarction can be used, such as
fluorescent based techniques (Chen, et al., Geraonae Res. 9, 492 (1999), PCR,
LCR,
Nested PCR and other techniques for nucleic acid amplification. In a preferred
embodiment, the method comprises assessing in an individual the presence or
frequency of SNPs and/or microsatellites in the FLAP nucleic acid that are
associated
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with myocardial infarction, wherein an excess or higher frequency of the SNPs
and/or
microsatellites compared to a healthy control individual is indicative that
the
individual has myocardial infarction or is susceptible to myocardial
infarction. See
Table 3 that sets forth SNPs and markers for use as screening tools.
In one embodiment, the at-risk haplotype is characterized by the presence of
polyrnorphism(s) represented in Table 3. For example, DGOOAAFICT at position
256047, where the SNP can be a "C" or a "T"; SG13S25 at position 283477, where
the SNP can be a "G" or an "A"; DGOOAAJFF at position 287889, where the SNP
can be a "G" or an "A"; DGOOAAHII at position 294503, where the SNP can be a
to "G" or an "A"; DGOOAAHID at position 296020, where the SNP can be a "T" or
an
"A"; B SNP-310657 at position 310657, where the SNP can be a_ "G" or an "A";
SG13S30 at position 312056, where the SNP can be a "G" or a "T"; SG13S32 at
position 316763, where the SNP can be a "C" or an "A"; SG13S42 at position
320393, where the SNP can be a "G" or an "A"; and SG13S35 at position 324333,
where the SNP can be a "G" or an "A". 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) FLAP polypeptide, means for
amplification
of nucleic acids comprising a FLAP, or means for analyzing the nucleic acid
sequence
of a nucleic acid described herein, or for analyzing the amino acid sequence
of a
polypeptide as described herein, etc. In one embodiment, a kit for diagnosing
MI or
susceptibility to MI can comprise primers for nucleic acid amplification of a
region in
the FLAP nucleic acid comprising an at-risk haplotype that is more frequently
present
in an individual having MI or susceptible to MI. The primers can be designed
using
portions of the nucleic acids flanking SNPs that are indicative of MI. In a
particularly
preferred embodiment, the primers are designed to amplify regions of the FLAP
nucleic acid associated with an at-risk haplotype for MI, or more particularly
the
3o haplotypes defined by the following SNPs: DG00AAFIU, SG13S25, DGOOAAJFF,
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DGOOAAHII, DGOOAAHID, B SNP_310657, SG13S30, SG13S32, SG13S42, and
SG13S35 , at the locus on chromosome 13q12.
SCREENING ASSAYS AND AGENTS IDENTIFIED THERBY
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 embodiment, the presence (or absence) of
a
nucleic acid molecule of interest (e.g., a nucleic acid that has significant
homology
to 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 (e.g., a
nucleic
acid having the sequence of one of SEQ ID NOs: f or 3 or the complement
thereof, or
a nucleic acid encoding an amino acid having the sequence of SEQ ID NO: 2, or
a
fragment or variant of such nucleic acids), under stringent conditions as
described
i5 above, and then assessing the sample for the presence (or absence) of
hybridization.
In a preferred embodiment, high stringency conditions are conditions
appropriate for
selective hybridization. In another embodiment, a sample containing a nucleic
acid
molecule of interest is contacted with a nucleic acid containing a contiguous
nucleic
acid sequence (e.g., a primer or a probe as described above) that is at least
partially
2o complementary to a part of the nucleic acid molecule of interest (e.g., a
FLAP nucleic
acid), and the contacted sample is assessed for the presence or absence of
hybridization. In a preferred embodiment, the nucleic acid containing a
contiguous
nucleic acid sequence is completely complementary to a part of the nucleic
acid
molecule of interest.
25 In any of these embodiments, all or a portion of the nucleic acid of
interest can
be subjected to amplification prior to performing the hybridization.
In another embodiment, 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
30 to 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 embodiment, the invention provides methods for identifying agents
(e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors,
binding
agents, antibodies, small molecules or other drugs, or ribozymes which 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., binding agent for
members
of the leukotriene pathway, such as FLAP binding agents); which have a
stimulatory
to or inhibitory effect on, for example, activity of polypeptides of the
invention; or
which change (e.g., enhance or inhibit) the ability of the polypeptides of the
invention
to interact with members of the leukotriene pathway binding agents (e.g.,
receptors or
other binding agents); or which alter posttranslational processing of the
leukotriene
pathway member polypeptide, such as a FLAP polypeptide (e.g., agents that
alter
proteolytic processing to direct the polypeptide from where it is normally
synthesized
to another location in the cell, such as the cell surface; agents that alter
proteolytic
processing such that more polypeptide is released from the cell, etc.)
In one embodiment, the invention provides assays for screening candidate or
test agents that bind to or modulate the activity of polypeptides described
herein (or
2o biologically active portions) thereof), as well as agents identifiable by
the assays.
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 library
approach is
limited to polypeptide libraries, while the other four approaches are
applicable to
polypeptide, non-peptide oligomer or small molecule libraries of compounds
(Lam,
K.S.,Anticahce~DrugDes. 12:145 (1997)).
In one embodiment, to identify agents which alter the activity of a FLAP
polypeptide, a cell, cell lysate, or solution containing or expressing a FLAP
polypeptide (e.g., SEQ ID NO: 2 or another splicing variant encoded by a FLAP
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nucleic acid, such as a nucleic acid comprising a SNP as shown in Table 3), or
a
fragment or derivative thereof (as described above), can be contacted with an
agent to
be tested; alternatively, the polypeptide can be contacted directly with the
agent to be
tested. The level (amount) of FLAP activity is assessed (e.g., the level
(amount) of
FLAP 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 FLAP
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 FLAP polypeptide. An
increase in the
level of FLAP activity in the presence of the agent relative to the activity
in the
absence of the agent, indicates that the agent is an agent that enhances (is
an agonist
of) FLAP activity. Similarly, a decrease in the level of FLAP activity in the
presence
of the agent, relative to the activity in the absence of the agent, indicates
that the agent
is an agent that inhibits (is an antagonist of) FLAP activity. In another
embodiment,
the level of activity of a FLAP 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 statistically significant difference in the level of the
activity in
the presence of the agent from the control level indicates that the agent
alters FLAP
activity.
The present invention also relates to an assay for identifying agents which
alter the expression of a FLAP nucleic acid (e.g., antisense nucleic acids,
fusion
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 nucleic acid
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 FLAP polypeptide (e.g., a FLAP 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
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acid. Cells not suspended in solution can also be employed, if desired. The
level
and/or pattern of FLAP expression (e.g., the level and/or pattenl of mRNA or
of
protein expressed, such as the level and/or 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 FLAP expression in the absence of the agent to
be
tested). If the level and/or pattern in the presence of the agent differ, by
an amount or
in a manner that is statistically significant, from the level and/or pattern
in the absence
of the agent, then the agent is an agent that alters the expression of the
FLAP nucleic
acid. Enhancement of FLAP expression indicates that the agent is an agonist of
to FLAP activity. Similarly, inhibition of FLAP expression indicates that the
agent is an
antagonist of FLAP activity.
In another embodiment, the level and/or pattern of FLAP 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 FLAP expression.
In another embodiment of the invention, agents which alter the expression of a
FLAP nucleic acid or which otherwise interact with the nucleic acids described
2o herein, can be identified using a cell, cell lysate, or solution containing
a nucleic acid
encoding the promoter region of the FLAP 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 mariner 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 FLAP nucleic acid, as indicated by its ability to alter expression of a
nucleic
acid that is operably linked to the FLAP nucleic acid promoter.
3o Enhancement of the expression of the reporter indicates that the agent is
an
agonist of FLAP activity. Similarly, inhibition of the expression of the
reporter
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indicates that the agent is an antagonist of FLAP activity. In another
embodiment, the
level of expression of the reporter in the presence of the test agent, is
compared with a
control level that has previously been established. A level in the presence of
the agent
that differs 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
FLAP nucleic acid (e.g., an agent which enhances activity of a first splicing
variant,
and wluch inhibits activity of a second splicing variant), as well as agents
which are
agonists of activity of a first splicing variamt and antagonists of activity
of a second
to splicing variant, can easily be identified using these methods described
above.
In other embodiments of the invention, assays can be used to assess the impact
of a test agent on the activity of a polypeptide relative to a FLAP binding
agent. For
example, a cell that expresses a compound that interacts with a FLAP nucleic
acid
(herein referred to as a "FLAP binding agent", which can be a polypeptide or
other
molecule that interacts with a FLAP nucleic acid, such as a receptor, or
another
molecule, such as 5-LO) is contacted with a FLAP in the presence of a test
agent, and
the ability of the test agent to alter the interaction between the FLAP and
the FLAP
binding agent is determined. Alternatively, a cell lysate or a solution
containing the
FLAP binding agent, can be used. An agent which binds to the FLAP or the FLAP
2o binding agent can alter the interaction by interfering with, or enhancing
the ability of
the FLAP to bind to, associate with, or otherwise interact with the FLAP
binding
agent. Determining the ability of the test agent to bind to a FLAP nucleic
acid or a
FLAP nucleic acid 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 l2sh 3sS, i4C
or 3H,
either directly or indirectly, and the radioisotope 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
3o 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 interact with the
polypeptide
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without the labeling of any of the interactants. For example, a
microphysiometer can
be used to detect the interaction of a test agent with a FLAP or a FLAP
binding agent
without the labeling of either the test agent, FLAP, or the FLAP 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
1 o for use in treating a disease or condition associated with FLAP or a
susceptibility to a
disease or condition associated with FLAP, or antagonists for studying a
susceptibility
to a disease or condition associated with FLAP (e.g., MI). Drugs can be
designed to
regulate FLAP activation, that in turn can be used to regulate signaling
pathways and
transcription events of genes downstream or of proteins or polypeptides
interacting
with FLAP (e.g., 5-LO).
In another embodiment of the invention, assays can be used to identify
polypeptides that interact with one or more FLAP 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
2o polypeptides that interact with one or more FLAP polypeptides. In such a
yeast two-
hybrid system, vectors are constructed based on the flexibility of a
transcription 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
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
FLAP polypeptide, splicing variant, or fragment or derivative thereof, and a
second
3o vector is used which includes a nucleic acid encoding a transcription
activation
domain and also a nucleic acid encoding a polypeptide which potentially may
interact
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with the FLAP polypeptide, splicing variant, or fragment or derivative thereof
(e.g., a
FLAP polypeptide binding agent or receptor). 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
FLAP
polypeptide or fragment or derivative thereof. Such polypeptides may be useful
as
agents that alter the activity of expression of a FLAP polypeptide, as
described above.
In more than one embodiment of the above assay methods of the present
i0 invention, it may be desirable to immobilize either the FLAP, the FLAP
binding
agent, or other components of the assay on a 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 embodiment, a fusion protein (e.g., a glutathione-S-
transferase fusion protein) can be provided which adds a domain that allows a
FLAP
nucleic acid or a FLAP binding agent to be bound to a matrix or other solid
support.
2o In another embodiment, modulators of expression of nucleic acid molecules
of
the invention are identified in a method wherein a cell, cell lysate, or
solution
containing a nucleic acid encoding a FLAP 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
significantly greater) in the presence of the test agent than in its absence,
the test agent
3o is identified as a stimulator or enhancer of the mRNA or polypeptide
expression.
Alternatively, when expression of the mRNA or polypeptide is less
(statistically
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significantly less) in the presence of the test agent than in its absence, the
test agent is
identified as an inhibitor of the inRNA 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.
In yet another embodiment, the invention provides methods for identifying
agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs,
receptors,
binding agents, antibodies, small molecules or other drugs, or ribozymes)
which alter
(e.g., increase or decrease) the activity of a member of leukotriene pathway
binding
agent, such as a FLAP binding agent (e.g., 5-LO), as described herein. For
example,
to such agents can be agents which have a stimulatory or inhibitory effect on,
for
example, the activity of a member of leukotriene pathway binding agent, such
as a
FLAP binding agent; which change (e.g., enhance or inhibit) the ability a
member of
leukotriene pathway binding agents, (e.g., receptors or other binding agents)
to
interact with the polypeptides of the invention; or which alter
posttranslational
processing of the member of leukotriene pathway binding agent, (e.g., agents
that
alter proteolytic processing to direct the member of the leukotriene pathway
binding
agent from where it is normally synthesized to another location in the cell,
such as the
cell surface; agents that alter proteolytic processing such that more active
binding
agent is released from the cell, etc.).
2o For example, the invention provides assays for screening candidate or test
agents that bind to or modulate the activity of a member of the leukotriene
pathway
(or enzymatically active portions) thereof), as well as agents identifiable by
the
assays. As described above, 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 library approach is limited to polypeptide
libraries, while
the other four approaches are applicable to polypeptide, non-peptide oligomer
or
small molecule libraries of compounds (Lam, K.S. Afatieayace~ Df°ug
Des., 12:145
( 1997)).
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In one embodiment, to identify agents which alter the activity of a member of
the leukotriene pathway (such as a FLAP binding agent), a cell, cell lysate,
or solution
containing or expressing a binding agent (e.g., 5-LO, or a leukotriene pathway
member receptor), or a fragment (e.g., an enzymatically active fragment) or
derivative
thereof, can be contacted with an agent to be tested; alternatively, the
binding agent
(or fragment or derivative thereof) can be contacted directly with the agent
to be
tested. The level (amount) of binding agent activity is assessed (either
directly or
indirectly), and is compared with the level of activity in a control (i.e.,
the level of
activity in the absence of the agent to be tested). If the level of the
activity in the
to 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 the member of the leukotriene pathway. An increase in the
level of the
activity relative to a control, indicates that the agent is an agent that
enhances (is an
agonist of) the activity. Similarly, a decrease in the level of activity
relative to a
control, indicates that the agent is an agent that inhibits (is an antagonist
of) the
activity. W another embodiment, the level of activity 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
the activity.
2o 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
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.
Furthermore, this invention pertains to uses of novel agents identified by the
3o 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
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encoded by a FLAP nucleic acid, or to alter expression of a FLAP nucleic acid,
by
contacting the polypeptide or the nucleic acid (or contacting a cell
comprising the
polypeptide or the nucleic acid) with the agent identified as described
herein.
PHARMACEUTICAL COMPOSITIONS
The present invention also pertains to pharmaceutical compositions
comprising nucleic acids described herein, particularly nucleotides encoding
the
polypeptides described herein; comprising polypeptides described herein (e.g.,
one or
more of SEQ ID NO: 1 or 3 or the complement thereof, and/or comprising other
l0 splicing variants encoded by a FLAP nucleic acid; and/or an agent that
alters (e.g.,
enhances or inhibits) FLAP nucleic acid expression or FLAP polypeptide
activity as
described herein. For instance, a polypeptide, protein (e.g., a FLAP
receptor), an
agent that alters FLAP nucleic acid expression, or a FLAP nucleic acid binding
agent
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 FLAP polypeptide activity, 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.
2o Suitable pharmaceutically acceptable Garners include but are not limited to
water, salt solutions (e.g., NaCl), 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
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 and/or aromatic substances and the like
which do
not deleteriously react with the active agents.
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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
powder.
The composition can be formulated as a suppository, with traditional binders
and
carriers such as triglycerides. Oral formulation can include standard Garners
such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,
polyvinyl
pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
Methods of introduction of these compositions include, but are not limited to,
intradermal, intrasnuscular, intraperitoneal, intraocular, intravenous,
subcutaneous,
io topical, oral 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 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 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.
2o 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 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
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,
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powders, enemas, lotions, sots, liniments, salves, aerosols, etc., 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
i
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.
to Pharmaceutically acceptable salts include those fornzed 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,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-
ethylamino ethanol, histidine, procaine, etc.
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, ih vitYO
or is~ vivo
assays may optionally be employed to help identify optimal dosage ranges. The
2o 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 ih vitro or animal
model
test systems.
The invention also provides a pharmaceutical 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,
use or sale of pharmaceuticals or biological products, which notice reflects
approval
3o 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
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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 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 pharmacodynamics
of
each agent and administered in FDA approved dosages in standard time courses.
to METHODS OF THERAPY
The present invention also pertains to methods of treatment (prophylactic
and/or therapeutic) for MI or a susceptibility to MI, using an MI therapeutic
agent.
An "MI therapeutic agent" is an agent that alters (e.g., enhances or inhibits)
FLAP
polypeptide activity and/or FLAP nucleic acid expression, as described herein
(e.g., a
nucleic acid agonist or antagonist). MI therapeutic agents can alter FLAP
polypeptide
activity or nucleic acid expression by a variety of means, such as, for
example, by
providing additional FLAP polypeptide or upregulating the transcription or
translation
of the FLAP nucleic acid; by altering posttranslational processing of the FLAP
polypeptide; by altering transcription of FLAP splicing variants; or by
interfering with
2o FLAP polypeptide activity (e.g., by binding to a FLAP polypeptide), or by
downregulating the transcription or translation of a FLAP nucleic acid.
Representative MI therapeutic agents include the following:
nucleic acids or fragments or derivatives thereof described herein,
particularly
nucleotides encoding the polypeptides described herein and vectors
comprising such nucleic acids (e.g., a gene, nucleic acid, cDNA, and/or
mRNA, such as a nucleic acid encoding a member of the leukotriene
pathway, such as a FLAP polypeptide or active fragment or derivative thereof,
or an oligonucleotide; for example, one of SEQ ID Nos. 1 or 3 or the
3o complement thereof, or a nucleic acid encoding SEQ ID NO: 2, or fragments
or derivatives thereof);
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polypeptides described herein and/or other splicing variants encoded by a
FLAP nucleic acid, or fragments or derivatives thereof);
other polypeptides (e.g., receptors of members of the leukotriene pathway,
such as LTB4 receptors, LTC4 receptors, LTD4 receptors, Cys LT1 receptors,
Cys LT2 receptors); binding agents of the leukotriene pathway, such as FLAP
binding agents (e.g., 5-LO); peptidomimetics; fusion proteins or prodrugs
thereof; antibodies (e.g., an antibody to an altered FLAP polypeptide, or an
to antibody to a non-altered FLAP polypeptide, or an antibody to a particular
splicing variant encoded by a FLAP nucleic acid, as described above);
ribozymes; other small molecules; and
other agents that alter (e.g., enhance or inhibit) a member of the leukotriene
15 pathway gene expression, such as FLAP nucleic acid expression or
polypeptide activity, or that regulate transcription of FLAP splicing variants
(e.g., agents that affect which splicing variants are expressed, or that
affect the
amount of each splicing variant that is expressed.
2o More than one MI therapeutic agent can be used concurrently, if desired.
An MI nucleic acid therapeutic agent that is a nucleic acid is used in the
treatment of a susceptibility to MI. The term, "treatment" as used herein,
refers not
only to ameliorating symptoms associated with the disease, but also preventing
or
25 delaying the onset of the disease, and also lessening the severity or
frequency of
symptoms of the disease. The therapy is designed to alter (e.g., inhibit or
enhance),
replace or supplement activity of a FLAP polypeptide in an individual. For
example,
an MI nucleic acid therapeutic agent can be administered in order to
upregulate or
increase the expression or availability of the FLAP nucleic acid or of
specific splicing
3o variants of FLAP nucleic acid, or, conversely, to downregulate or decrease
the
expression or availability of the FLAP nucleic acid or specific splicing
variants of the
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FLAP nucleic acid. Upregulation or increasing expression or availability of a
native
FLAP nucleic acid or of a particular splicing variant could interfere with or
compensate for the expression or activity of a defective nucleic acid or
another
splicing variant; downregulation or decreasing expression or availability of a
native
FLAP 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 MI therapeutic agents) are administered in a therapeutically effective
amount (i.e., an amount that is sufficient to treat the disease, such as by
ameliorating
1o symptoms associated with the disease, preventing or delaying the onset of
the disease,
and/or also lessening the severity or frequency of symptoms of the disease).
The
amount which will be therapeutically effective in the treatment of a
particular
individual's disorder or condition will depend on the symptoms and severity of
the
disease, and can be determined by standard clinical techniques. In addition,
ih vitYo
or ih vivo assays may optionally be employed to help identify optimal dosage
ranges.
The precise dose to be employed in the fornmlation 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 doses may be extrapolated from dose-response curves derived from in
vitro
or animal model test systems.
In one embodiment, a nucleic acid of the invention (e.g., a nucleic acid
encoding a FLAP polypeptide, such as one of SEQ ID NO: 1 or 3 or the
complement
thereof; or another nucleic acid that encodes a FLAP polypeptide or a splicing
variant,
derivative or fragment thereof, such as a nucleic acid encoding SEQ ID NO: 2,
can be
used, either alone or in a pharmaceutical composition as described above. For
example, a FLAP nucleic acid or a cDNA encoding a FLAP polypeptide, either by
itself or included within a vector, can be introduced into cells (either iya
vitro or ih
vivo) such that the cells produce native FLAP polypeptide. If necessary, cells
that
have been transformed with the nucleic acid or cDNA or a vector comprising the
3o nucleic acid or cDNA can be introduced (or re-introduced) into an
individual affected
with the disease. Thus, cells which, in nature, lack native FLAP expression
and
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activity, or have altered FLAP expression and activity, or have expression of
a
disease-associated FLAP splicing variant, can be engineered to express the
FLAP
polypeptide or an active fragment of the FLAP polypeptide (or a different
variant of
the FLAP polypeptide). In a preferred embodiment, nucleic acid encoding a FLAP
polypeptide, or an active fragment or derivative thereof, can be introduced
into an
expression vector, such as a viral vector, and the vector can be introduced
into
appropriate cells in an animal. Other nucleic acid transfer systems, including
viral
and nonviral transfer systems, can be used. Alternatively, nonviral nucleic
acid
transfer methods, such as calcium phosphate coprecipitation, mechanical
techniques
to (e.g., microinjection); membrane fusion-mediated transfer via liposomes; or
direct
DNA uptake, can also be used.
Alternatively, in another embodiment of the invention, 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 (e.g., an oligonucleotide as described below),
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 an MI nucleic acid
is
administered or generated ih situ. The antisense nucleic acid that
specifically
hybridizes to the mRNA and/or DNA inhibits expression of the FLAP polypeptide,
e.g., by inhibiting translation and/or transcription. Binding of the antisense
nucleic
2o acid can 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 of the present invention 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 FLAP polypeptide. 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 and/or genomic DNA of the
FLAP. In one embodiment, the oligonucleotide probes are modified
oligonucleotides
3o that are resistant to endogenous nucleases, e.g., exonucleases and/or
endonucleases,
thereby rendering them stable ifa vivo. Exemplary nucleic acid molecules for
use as
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antisense oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate analogs ofDNA (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.
(Biotechfaiques 6:958-976 (1988)); and Stein et al. (Cahce~ Res. 48:2659-2668
(1988)). With respect to antisense DNA, oligodeoxyribonucleotides derived from
the
translation initiation site are preferred.
To perform antisense therapy, oligonucleotides (mRNA, cDNA or DNA) are
designed that are complementary to mRNA encoding the FLAP. The
to antisense oligonucleotides bind to FLAP mRNA 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 hybridizing nucleic acid, the more base mismatches with an RNA
it
may contain and still form a stable duplex (or triplex, as the case may be).
One
2o 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
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 in vivo), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger et al., Ps°oc. Natl.
Acad. Sci. USA
86:6553-6556 (1989); Lemaitre et al., PYOC. Natl. Acad. Sci. USA 84:648-652
(1987);
3o PCT International Publication No. WO 88/09810) or the blood-brain barrier
(see, e.g.,
PCT International Publication No. WO 89110134), or hybridization-triggered
cleavage
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agents (see, e.g., I~rol et al., BioTec7araiques 6:958-976 (1988)) or
intercalating agents.
(See, e.g., Zon, Pharm.Res. 5: 539-549 (1988)). To this end, the
oligonucleotide may
be conjugated to another molecule (e.g., a peptide, hybridization triggered
cross-
linking agent, transport agent, hybridization-triggered cleavage agent).
The antisense molecules are delivered to cells that express FLAP ih 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
to surface) can be administered systematically. Alternatively, in a preferred
embodiment, a recombinant DNA construct is utilized in 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
15 complementary base pairs with the endogenous FLAP transcripts and thereby
prevent
translation of the FLAP mRNA. For example, a vector can be introduced iya 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 it
can be
transcribed to produce the desired antisense RNA. Such vectors can be
constructed
2o 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).
25 Endogenous FLAP expression can also be reduced by inactivating or
"knocking out" FLAP 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 FLAP (or a completely unrelated DNA sequence) flanked by DNA
3o homologous to the endogenous FLAP (either the coding regions or regulatory
regions
of FLAP) can be used, with or without a selectable marker and/or a negative
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selectable marker, to transfect cells that express the FLAP ira vivo.
Insertion of the
DNA construct, via targeted homologous recombination, results in inactivation
of the
FLAP. The recombinant DNA constructs can be directly administered or targeted
to
the required site ih. vivo using appropriate vectors, as described above.
Alternatively,
expression of non-altered FLAPs can be increased using a similar method:
targeted
homologous recombination can be used to insert a DNA construct comprising a
non-
altered functional FLAP, or the complement thereof, or a portion thereof, in
place of
an altered FLAP in the cell, as described above. In another embodiment,
targeted
homologous recombination can be used to insert a DNA construct comprising a
to nucleic acid that encodes an MI polypeptide variant that differs from that
present in
the cell.
Alternatively, endogenous FLAP expression can be reduced by targeting
deoxyribonucleotide sequences complementary to the regulatory region of a FLAP
(i.e., the FLAP promoter and/or enhancers) to form triple helical structures
that
prevent transcription of the FLAP in target cells in the body. (See generally,
Helene,
C., AtzticahceY Drug Des., 6(6):569-84 (1991); Helene, C. et al., AsZf2. 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 FLAP proteins, can be used in the maupulation of tissue, e.g.,
tissue
2o differentiation, both ih vivo and fog ex vivo tissue cultures. Furthermore,
the anti-
sense techniques (e.g., microinjection of antisense molecules, or transfection
with
plasmids whose transcripts are anti-sense with regard to an MI nucleic acid
RNA or
nucleic acid sequence) can be used to investigate the role of FLAP in normal
cellular
function. Such techniques can be utilized in cell culture, but can also be
used in the
creation of transgenic animals.
In yet another embodiment of the invention, other MI therapeutic agents as
described herein can also be used in the treatment or prevention of a
susceptibility to a
disease or condition associated with FLAP. The therapeutic agents can be
delivered
in a composition, as described above, or by themselves. They can be
administered
3o systemically, or can be targeted to a particular tissue. The therapeutic
agents can be
produced by a variety of means, including chemical synthesis; recombinant
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production; irZ vivo production (~,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.
A combination of any of the above methods of treatment (e.g., administration
of non-altered FLAP polypeptide in conjunction with antisense therapy
targeting
altered FLAP mRNA; administration of a first splicing variant encoded by a
FLAP in
conjunction with antisense therapy targeting a second splicing encoded by a
FLAP)
can also be used.
The present invention is now illustrated by the following Exemplification,
to which is not intended to be limiting in any way.
EXEMPLIFICATION
SUBJECTS AND METHODS
Study population
Patients entering the study were defined from an infarction registry that
includes all MIs (over 8,000 patients) in Iceland 1981-2000. This registry is
a part of
the World Health Organization MONICA Project (The World Health Organization
MONICA Project (monitoring trends and determinants in cardiovascular disease):
a
major international collaboration. WHO MONICA Project Principal Investigators.
J
ClifZ. Epidemiol. 1988; 41:105-14). Diagnosis of all patients in the registry
follow
strict diagnostic rules based on symptoms, electrocardiograms, cardiac
enzymes, and
necropsy findings.
Blood samples from 1342 MI patients, both cases with a family history and
sporadic cases were collected. For each patient that participated, blood was
collected
from 2 relatives (unaffected or affected). Their genotypes were used to help
with
construction of haplotypes. In addition, blood samples from 624 unrelated
controls
were collected.
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Lizzlzage an.al~sis
Extended families (pedigrees) by clustering related female MI patients were
constructed into families such that each patient is related to at least one
other patient
within and including six meiotic events. The information regarding the
relatedness of
patients was obtained from an encrypted genealogy database that covers the
entire
Icelandic nation (Gulcher et al., Euz°. J. Hum. Genet. 8: 739=742
(2000)). A
genomewide scan was performed using a framework map of 1000 microsatellite
markers, using protocols described elsewhere (Gretarsdottir S., et al. Am. J.
Huzzz.
to Gezzet., 70: 593-603, 2002)). The marker order and positions where obtained
from a
modified version of the Marshfield genetic map (Center for Medical Genetics,
Marshfield Medical Research Foundation), using genetic mapping based on our
own
data, and from deCODE genetics high resolution genetic map (Kong A., et al.,
Nat.
genet., 31: 241-247 (2002)). The population-based allele frequencies were
constructed from a cohort of more than 30,000 Icelanders who have participated
in
genetic studies of various disease projects. Additional markers were genotyped
within the locus on chromosome 13 to increase the information on identity by
descent
within the families. For those markers at least 180 Icelandic controls were
genotyped
to derive the population allele frequencies.
2o For statistical analysis, multipoint, affected only allele-sharing methods
were
used to assess evidence for linkage. All results, both the LOD and the non-
parametric
linkage (NPL) score, were obtained using the program ALLEGRO (Gudbjartsson
D.F., et al., Nat Genet., 25: 12-13(2000)). The baseline linkage analysis
(Gretarsdottir S., et al., Am. J. Hum. Genet. 70: 593-603, (2002)) uses the
Spairs
scoring function (Whittermore AS, and Haplern J A., Biometrics 50: 118-127
(1994))
and Kruglyak et al., Am. J. Hum. Genet., 58:1347-1363 (1996)) the exponential
allele-
sharing model (Kong A., and Cox N.J., Azzz. J. Hum. Genet. 61:1179-1188
(1997)),
and a family weighting scheme which is halfway, on the log-scale, between
weighing
each affected pairs equally and weighing each family equally.
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Ultf~a-fine mapping anal haplotype analysis
A candidate susceptibility locus was defined as the region under the LOD
score curve where the score was one lower than the highest lod score ((peak
lod score
-1)\one lod drop). This region (approx. l2Mb) was ultra-fmemapped 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 were used. In addition, microsatellite markers identified within
the
deCODE genetics sequence assembly of the human genome were used.
Haplotype analysis
The frequencies of haplotypes in the patient and the control groups using an
expectation-maximization algorithm were estimated (Dempster A.P. et al., J. R.
Stat.
Soc. B. 39: 1-389 (1977)). An implementation of this algorithm that can handle
missing genotypes and uncertainty with the phase was used. Under the null
hypothesis, the patients and the controls are assumed to have identical
frequencies.
Using a likelihood approach, an alternative hypothesis where a candidate at-
risk
haplotype is allowed to have a higher frequency in patients than controls,
while the
ratios of the frequencies of other haplotypes are assumed to be the same in
both
groups was tested. Likelihoods are maximized separately under both hypothesis
and a
corresponding 1-df likelihood ratio statistics is used to evaluate the
statistic
significance.
To look for at-risk-haplotypes in the 1-lod drop, association of all possible
combinations of genotyped marlcers was studied, provided those markers spanned
a
region of size less than 1000 kb. Due to a certain amount of testing, thep-
values were
adjusted using simulations. The combined patient and control groups were
randomly
divided into two sets, equal in size to the original group of patients and
controls. The
haplotype analysis was then repeated and the most significantp-value
registered was
observed. This randomisation scheme was repeated over 100 times to construct
an
empirical distribution ofp-values.
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SNP haplotype association to MI
In an effort to identify SNP haplotypes that associate with MI we have typed
SNPs identified mainly by sequencing the FLAP gene and the region flanking the
gene. We genotyped a total number of 45 SNPs in 1343 patients and 624
unrelated
controls. The largest subset of unrelated patients (related no closer than 4
meioses)
was 921. We observed two correlated series of SNP haplotypes in excess in
patients,
denoted as A and B in Table 7. The length of the haplotypes varies between 33
and
69 I~b and cover one or two blocks of linkage disequilibrium. Both series of
haplotypes contain the common allele 2 of the SNP SG13S25. All haplotypes in
the
to A series contain the SNP DGOOAAHID, while all haplotypes in the B series
contain
the SNP DGOOAAHII. In the B series, the haplotypes B4, B5, and B6 have a
relative
risk (RR) greater than 2 and allelic frequencies above 10% (Table 1). The
haplotypes
in the A series have slightly lower RR and p-values, but higher allelic
frequency (15-
16%), and as such we also consider them interesting. The haplotypes in series
B and
A are strongly correlated, i.e. the B haplotypes define a subset of the A
haplotypes.
Hence, B haplotypes are more specific than A haplotypes. However, A haplotypes
are more sensitive, i. e. they capture more individuals with the putative
mutation, as is
observed in the population attributable risk which is less for B than for A.
Furthermore, these haplotypes show similar risk ratios and allelic frequency
for early-
onset patients (defined as onset of first MI before the age of 55). In
addition,
analyzing various groups of patients with known risk factors, such as
hypertension,
high cholesterol, smoking and diabetes, did not reveal any significant
correlation with
these haplotypes.
In conclusion, we have identified a series of correlated MI disease risk
haplotypes, consisting of 4-6 SNPs, with relative risk greater than 2 and
allelic
frequency in MI patients greater than 10%. The length of the haplotypes varies
between 39-68 kb. These haplotypes are carried by 19% (B5) to 29% (A4) of MI
patients. Our results suggest that the 'at risk' haplotypes in the FLAP gene
represent
a new major independent risk factor for MI.
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Discussion
In a genome wide search for susceptibility nucleic acids for MI, a locus to
13q12 was mapped. This locus was ultra-fine mapped with microsatellite
markers.
Haplotype analysis strongly suggested a nucleic acid for FLAP (ALOXSAP), as a
susceptibility gene for MI.
The FLAP gene encodes for a protein that is required for leukotriene
s5nlthesis
(LTA4, LTB4, LTC4, LTD4). Inhibitors of its function impede translocation of 5-
lipoxygenase from the cytoplasm to the cell membrane and inhibit activation of
5-
to lipoxygenase. The leukotrienes are potent inflammatory lipid mediators
derived from
arachidonic acid that can potentially contribute to development of
atherosclerosis and
destabilization of atherosclerotic plaques through lipid oxidation and/or
proinflammatory effects. Allen et al., (Circulation. 97: 2406-2413(1998))
described a
novel mechanism in which atherosclerosis is associated with the appearance of
a
leukotriene receptors) capable of inducing hyperreactivity of human epicardial
coronary arteries in response to LTC4 and LTD4. Allen et al. show a
photomicrograph of a section of human atherosclerotic coronary artery a
positive
staining of a number of members of the leukotriene pathway, including FLAP.
Mehrabian et al. described the identification of 5-Lipoxygenase (5-LO) as a
major
2o gene contributing to atherosclerosis susceptibility in mice. Mehrabian et
al. described
that heterozygous deficiency for the enzyme in a knockout model decreased the
atherosclerotic lesion size in LDL-/- mice by about 95%. Mehrabian et al show
that
the enzyme is expressed abundantly in macrophage-rich regions of
atherosclerotic
lesions, and suggested that 5-LO and/or its products might act locally to
promote
lesion development (Mehrabian et al., Circulation Research. 91:120 (2002)).
Studies of FLAP inhibition in animal models of atheroscerosis are scarce.
However, in a rabbit model of acute MI assesssed 72 hours after coronary
artery
ligation the FLAP-inhibitor BAYx1005 madedly reduced mortality, from 65% to
25%, and blocked the increase in CPIs and neutrophil accumulation as well as
the
3o ECG-changes observed in sham treated animals (J. Plzai°fuacol. Exp.
They., 276:332
(1996)).
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Mutations and Jor polymorphisms within the FLAP nucleic acid, and other
members of the same pathway (i.e., 5-lipoxygenase, LTA4, LTB4, LTC4, and
CysLT2 receptor), that show association with the disease, can be used as a
diagnostic
test. The members of the 5-LO pathway in particular are valuable therapeutic
targets
for myocardial infarction.
Table 1 The marker map for chromosome 13 used in the linkage analysis.
Location Marker Location (cM) Marker
(cM)
6 D13S175 63.9 D13S170
9.8 D13S1243 68.7 D13S265
13.5 D13S1304 73 D13S167
17.2 D13S217 76.3 D13S1241
21.5 D13S289 79.5 D13S1298
25.1 D13S171 81.6 D13S1267
28.9 D13S219 84.7 D13S1256
32.9 D13S218 85.1 D13S158
38.3 D13S263 87 D13S274
42.8 D13S326 93.5 D13S173
45.6 D 13 S 153 96.7 D 13 5778
49.4 D13S1320 102.7 D13S1315
52.6 D13S1296 110.6 D13S285
55.9 D13S156 115 D13S293
59.8 D13S1306
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Table 2 Marker Map for the second step of Linkage Analysis
Location Marker Location (cM) Marker
(cM)
1.758 D13S175 42.585 D13S1248
9.235 D13S787 44.288 D13S1233
11.565 D13S1243 44.377 D13S263
16.898 D13S221 45.535 D13S325
17.454 D13S1304 45.536 D13S1270
18.011 D13S1254 45.537 D13S1276
18.59 D13S625 49.149 D13S326
19.308 D13S1244 49.532 D13S1272
19.768 D13S243 52.421 D13S168
22.234 D13S1250 52.674 D13S287
22.642 D13S1242 60.536 D13S1320
22.879 D13S217 64.272 D13S1296
25.013 D13S1299 71.287 D13S156
28.136 D13S289 76.828 D13S1306
28.678 D13S290 77.86 D13S170
29.134 D13S1287 82.828 D13S265
30.073 D13S260 91.199 D13S1241
31.98 D13S171 93.863 D13S1298
32.859 D13S267 97.735 D13S779
33.069 D13S1293 100.547 D13S1256
33.07 , D13S620 102.277 D13S274
34.131 D13S220 111.885 D13S173
36.427 D13S219 112.198 D13S796
39.458 D13S1808 115.619 D13S778
40.441 D13S218 119.036 D13S1315
41.113 D13S1288 126.898 D13S285
41.996 D13S1253 131.962 D13S293
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Table 3 shows the five exons with positions that encode the FLAP protein,
marl~ers
and SNPs identified within the genomic sequence by the methods described
herein.
Of the six SNPs, one SNP, B SNP_302465, is in the coding region. The
polymorphism, SNP 302465, does not change the amino acid sequence in the
protein.
Table 3
Size(bp)
Exons/markers/SNPs Position(bp) SNPs
249198- 248
D13S289 249445
249855- 395
DG13S166 250249
293667- 70
Exon1 293736
302413- 100
Exon2 302512
B SNP 302465 302465 ~ hetero ous C-T
3%
1 heterozygous
A-C
B SNP 302524 302524 55%
homozygous A-A
(22.5%)
homozygous C-C
(22.5%)
B SNP 302560 302560 1 heteroz ous A-G
2%
1 heterozygous
C-T
B SNP 302617 302617 37%
homoz ous T-T
59%
homoz ous C-C
4%
310405- 71
Exon3 310475
B SNP 310657 310657 1 hetero ous A-G
6%
314297- 82
Exon4 314378
1 heterozygous
G-C
B SNP 314500 314500 24%
homoz ous C-C
6%
Homozygous G-G
70%
322297- 163
ExonS 322459
330669- 218
DG13S164 330886
330679- 153
D13S1238 330831
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363743- 162
DG13S163 363904
SNP13B 81028729 ' 1
rs1028729) 145600 homozygousC-C
(11 %),
heteroz gous
C-T 41
homoz gous T-T
47%
SNP13B Y1323898 1
rs1323898 * 151047 homozygousG-G(38%)
heteroz gous
G-A 47%
homoz ous A-A
15%
SNP13B K912392 1
rs912392 * 193119 homozygousC-C
(13I)
heteroz gous
C-T 46%
homoz ous T-T
41
DGOOAAFQR 1
rs1556428 * 117676 homozygousG-G(1%)
heteroz ous
G-A 18%
homoz ous A-A
80%
DGOOAAFIV 1
rs22485654 * 227629 homozygousT-T
(75%)
heteroz ous
T-A 23%
homoz gous A-A
2%
DG00AFJT 293754 1 Homoz ousC-C
45% ,
heterozygous
C-
A 45%),
homoz ous A-A
10%
DGOOAAHII 294503 1 homoz oust-G
44% ,
heterozygous
G-
A 46% ,
homoz ous A-A
10%
DG00AAHID 296020 1 homoz ousT-T
43% ,
heteroz ous
T-A 45% ,
homoz ous A-A
12%
DGOOAAHIJ 298098 i homoz oust-G
60% ,
heterozygous
G-
A(35% ,
homoz ous A-A
6%
DGOOAAHIH 298188 1 homoz oust-G
32% ,
heterozygous
G-
A 48% ,
-, h omozygous A-A
(19%)
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1
~DGOOAAHIE homozygous C-C
I rs3885907 * 298379 (23%),
heterozygous
C-
A 48% ,
homoz gous A-A
29%
DGOOAAHIG 304334 1 homoz ousC-C
21% ,
heterozygous
C-
T 49% ,
homoz ous T-T
31
DGOOAAHIF 324849 1 homoz oust-G
54%),
heterozygous
G-
C 39%),
homoz gous C-C
7%
DGOOAAHOI 325651 1 homoz gousG-G
59% ,
heterozygous
G-
A(36%),
homoz ous A-A
5%
FLA267479 267479 1
FLA267696 267696 1
FLA267853 267853 1
FLA270742 270742 1
FLA270830 270830 1
FLA273407 273407 1
FLA274084 274084 1
FLA275784 275784 1
FLA275952 275952 1
FLA277478 277478 1
FLA277678 277678 1
FLA278185 278185 1
FLA278492 278492 1
FLA278845 278845 1
FLA280183 280183 1
FLA280923 280923 1
FLA283400 283400 1
FLA283477/SG13S25 283477 1
FLA284410 284410 1
FLA284815 284815 1
FLA284903 284903 1
FLA290195 290195 1
FLA290553 290553 1
FLA290570 290570 1
FLA292253 292253 1
FLA292576 292576 1
FLA295036 295036 1
FLA296102 296102 1
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FLA298098 298098 1
FLA298188 298188 1
FLA298230 298230 1
~FLA298379 298379 1
FLA298507 298507 1
FLA298604 298604 1
FLA298987 298987 1
FLA299063 299063 1
FLA299772 299772 1
FLA299843 299843 1
FLA299980 299980 1
FLA300662 300662 1
FLA300864 300864 1
FLA302094 302094 1
FLA302465 302465 1
FLA302524 302524 1
FLA303769 303769 1
FLA303796 303796 1
FLA303957 303957 1
FLA303967 303967 1
FLA304170 304170 1
FLA304334 304334 1
FLA304512 304512 1
FLA304583 304583 1
FLA305089 305089 1
FLA305505 305505 1
FLA305678 305678 1
FLA305956 305956 1
FLA306447 306447 1
FLA307155 307155 1
FLA307165 307165 1
FLA308514 308514 1
FLA308527 308527 1
FLA309851 309851 1
FLA310657 310657 1
FLA311122 311122 1
FLA311248 311248 1
FLA311737 311737 1
FLA312038 312038 1
FLA3120561SG13S30 312056 1
FLA314500 314500 1
FLA314532 314532 1
FLA315014 315014 1
FLA315232 315232 1
FLA315355 315355 1
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FLA315611 315611 1
FLA316131 316131 1
FLA316408 316408 1
FLA316472 316472 1
FLA316515 316515 1
FLA316569 316569 1
FLA316607 316607 1
FLA316763/SG13S32 316763 1
FLA317496 317496 1
FLA317619 317619 1
FLA317620 317620 1
FLA317647 317647 1
FLA317733 317733 1
FLA317744 317744 1
FLA317815 317815 1
FLA318219 318219 1
FLA319969 319969 1
FLA320261 320261 1
FLA320393iSG13S42 320393 1
FLA320595 320595 1
FLA321774 321774 1
FLA321966 321966 1
FLA322025 322025 1
FLA322093 322093 1
FLA323013 323013 1
FLA323316/SG13S34 323316 1
FLA323366 323366 1
FLA324591 324591 1
FLA324601 324601 1
FLA324849 324849 1
FLA325369 325369 1
FLA326187 326187 1
FLA326657 326657 1
FLA327265 327265 1
FLA328964 328964 1
FLA330265 330265 1
FLA330455 330455 1
FLA331234 331234 1
FLA331374 331374 1
FLA331395 331395 1
FLA331473 331473 1
FLA331517 331517 1
FLA331526 331526 1
FLA331651 331651 1
IFLA331841 331841 1
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FLA287889/DGOOAAJFF 287889 1
DGOOAAFIU/SNP 13 256047 1
Y1323892
SG 13535/FLA324333 324333 1
SG13S86 305031 1
* indicates a ublicly
available SNP.
Table 4
Most significant 4 microsatellite marker haplotypes. Length=length of
haplotype in Mb. P-val=p-value. RR=Relative,risk. N a~Number of patients. P
al=allelic frequency of haplotype. P ca =carrier frequency of haplotype. N ct=
number
of controls. Alleles= alleles in the haplotype. Markers= markers in the
haplotype.
4
markers: pos.rr-frqgt1
pert
Allele
lengthp-val RR N P P N P P s Markers
of al ca ct al ca
DG13S80
DG13S83
DG13S1110
0.88 4.71 6.23428 0.0650.125721 0.0110.0220 -12-6O DG13S163
E-06
DG13S111
1
DG13S1103
D13S1287
0.82 8.60E-061NF438 0.0320.062720 0 0 0 4 2 14 DG13S1061
DG13S1103
DG13S163
1 D13S290
g_g
0.67 6.98E-061 435 0.030.059721 0.0020.0038 6 0 8 DG13S1061
DG13S1101
DG13S166
26.7 D13S1287
0.7674.85E-062 436 0.0480.094721 0.0020.0040 0 2 12 DG13S1061
DG13S166
DG13S163
D13S290
0.5151.93E-061NF422 0.0480.094721 0 0 2 0 0 6 DG13S1061
DG13S166
DG13S163
- DG13S1061
0.8641.68E-061NF424 0.0240.048717 0 0 0 2 0 16 DG13S293
DG13S1103
D13S1287
DG13S1061
0.9275.38E-061NF435 0.0340.067720 0 0 4 2 143 DG13S301
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Table 5
Most significant 5 microsatellite marker haplotypes. Length=length of
haplotype in Mb. P-val=p-value. RR=Relative risk. N a~Number of patients. P
al=allelic frequency of haplotype. P ca =carrier frequency of haplotype. N ct=
number
of controls. Alleles= alleles in the haplotype. Markers= markers in the
haplotype
5markers pos.rr-frqgt1
perc
length p-valRR N P P N P P Alleles Markers
of al ca ct al ca
DG13579
D1351299
DG 13587
D1351246
0.851 7.45E-0615.43413 0.0340.067715 0.0020.0050 180 0 DG13S166
0
DG 13579
DG13583
DG13S1104
DG1351103
0.964 8.07E-061NF437 0.0230.045721 0 0 0 -126 8 DG13S163
6
DG13579
DG13S1104
DG135172
DG1351103
0.964 2.38E-061NF437 0.0260.052720 0 0 0 6 0 8 DG135163
6
DG13579
DG1351110
DG135175
DG13S166
0.931 7.05E-065.8429 0.0680.131721 0.0120.0250 -600 -2D1351238
DG13579
DG1351098
DG1351103
DG135166
0.964 8.13E-061NF434 0.0210.041721 0 0 0 3 82 6 DG13S163
DG1351110
DG13589
DG135175
DG135166
0.597 9.78E-064.58428 0.0740.143717 0.0170.034-6 0 00 -2D1351238
DG13583
DG1351110
DG13S166
D13S1238
0.896 6.92E-06NF 428 0.0260.051721 0 0 -12 -60-2 2 D135290
I
DG1351110
D135289
DG135166
D13S1238
0.722 2.18E-061NF 453 0.0260.051738 0 0 -6 0 0-2 2 D135290
DG13587
DG13S175
DG1351103
D13S1287
0.982 7.88E-06NF 437 0.0280.055721 0 0 0 0 42 14DG1351061
I
DG13S89
DG1351111
DG1351103
0.841 8.88E-06NF 438 0.0320.062720 0 0 0 0 42 14D13S1287
I
CA 02502359 2005-04-14
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DG13S1061
DG13S89
DG13S1103
DG13S163
D13S290
0.841 9.67E-07NF 435 0.0290.057721 0 0 0 8 6 0 8 DG13S1061
I
- DG13S87
DG13S1103
DG13S166
D13S1287
0.982 7.90E-0618.63437 0.0260.052721 0.0010.0030 4 0 2 14DG13S1061
DG13S89
DG13S1101
DG13S166
D13S1287
0.841 3.52E-0628.52436 0.0480.094721 0.0020.0040 0 0 2 12DG13S1061
DG13S175
DG13S1103
DG13S163
D13S290
0.705 5.28E-061NF435 0.0270.053721 0 0 0 8 6 0 8 DG13S1061
DG13S89
DG13S166
DG13S163
D13S290
0.841 A..21E-061NF422 0.0480.093721 0 0 0 2 0 0 6 DG13S1061
DG13S1101
DG13S175
DG13S166
D13S1287
0.767 4.02E-0628.11436 0.0490.095721 0.0020.0040 0 0 2 12DG13S1061
DG13S1101
DG13S172
DG13S166
D13S1287
0.767 1.29E-0631.07436 0.0470.092721 0.0020.0030 0 0 2 12DG13S1061
DG13S175
DG13S166
DG13S163
D13S290
0.705 4.25E-071NF422 0.0480.093721 0 0 0 2 0 0 6 DG13S1061
DG13S172
DG13S1103
DG13S166
D13S1287
0.683 6.58E-061NF437 0.0290.056721 0 0 0 4 0 2 14DG13S1061
DG13S1101
DG13S166
D13S290
D13S1287
0.767 2.85E-0632.43436 0.0440.087721 0.0010.0030 0 0 2 12DG13S1061
D13S289
DG13S166
DG13S163
D13S1287
0.8 9.58E-0618.39451 0.0230.045739 0.0010.0030 0 2 2 -16DG13S293
65
_ D13S289
DG13S166
DG13S163
DG13S1061
0.865 5.08E-061NF453 0.0190.038739 0 0 0 0 2 0 -16DG13S293
DG13S1103
DG13S166
D13S1287
DG13S1061
0.927 1.02E-0727.65437 0.0370.073721 0.0010.0034 0 2 143 DG13S301
CA 02502359 2005-04-14
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Table 6
Position (Mb) of microsatellite markers sequence assembly (SAS), primers
and size of the markers.
mb marker orward r everse size
f
25.09 ACGGTGATGACGCCTACATT CACATGGACCAATTACCTAGA
T
2042DG13S2101SEQ ID NO:. 4 A SEO ID NO: 5) 188
25.09 CAAATTTCAGATGTGCCAACC ACGGTGATGACGCCTACATT(S
2042DG13S48 SEQ ID NO: 6 EQ ID NO: 7 214
25.39 ACCAGCCTTTGCTTAGGA(SEQ ACATTCTAGTGCTACAGGGTA
6504D13S1304D NO: 8) CTC SEQ ID NO: 9 133
I
25.39 TGTTCTGCACACGAACATTCT(SETCCTGAGTCCTCTCCACCTG(S
6535DG13S2105Q ID NO: 10 EQ ID NO: 11 ) 104
25.44 TGGGAATTAATGAAGAACAACAACATGTTTCGAAGAACTCAAGA
5511DG13S2106A SEQ ID NO: 12 GG SEQ ID NO: 13 428
25.54 AAATTACTTCATCTTGACGATAACCTATTGGGGACTGCAGAGAG
4920D13S1254A SEQ ID NO: 14 SEQ ID NO: 15 218
25.54 GGGACTGCAGAGAGCAGAAG CAAGAAGGGAAATTCCTACGC
4925DG13S2107SEQ ID NO: 16 SEQ ID NO: 17 95
25.56 AGCCAGTGTCCACAAGGAAG GAGGGTGAGACACATCTCTGG
5956DG13S55 SEQ ID NO: 18) SEQ ID NO: 19 283
25.60 AATCGTGCCTCAGTTCCATC CCACCAGGAACAACACACAC
5793DG13S54 SEQ 1D NO: 20 SEQ ID NO: 21 156
25.61 TTGCTCTCCAGCCTGGGC (SEQTTCCTCTGGCTGCCTGCG
9693D13S625 ID NO: 22 ( SEQ ID NO: 23) 185
25.68 TTTGATTCCGTGGTCCATTA TTATTTGGTCGGTGCACCTTT
7422DG13S1479(SEQ ID NO: 24) SEQ ID N0.25) 339
25.74 GGTAGGTTGAAATGGGCTAACA TCATGACAAGGTGTTGGATTT
9344DG13S1440(SEQ ID NO: 26) SEQ ID NO: 27) 153
25.90 CCTCCTCTGCCATGAAGCTA CTATTTGGTCTGCGGGTfGT
1212DG13S1890(SEQ ID NO: 28) SEQ ID NO: 29) 418
25.92 TTTGAGCCCAGATCTAAGCAA AAATGTTAATGTCACCGACAAA
8081DG13S1879SEQ ID NO: 30 SEQ ID NO: 31 443
25.93 TACTGGGTTATCGCCTGACC CCAATGGACCTCTTGGACAT
2609DG13S1540SEQ lD NO: 32 SEQ ID NO: 33 152
25.94 TTTGAATGTTCATATATTTGTGGTCCCTCGTAATGAAACCTATTTG
6743DG13S1889G SEQ ID NO: 34 A SEQ ID NO: 35 222
25.94 TTTCGGCACAGTCCTCAATA CAGGGTGTGGTGACAT (SEQ
8679DG13S59 SEQ ID NO: 36 ID NO: 37 228
25.95 TGTTTCTTTCTTTCTCTCTCTCTTAAATGAGTTCAATGAGTTGTGG
2347DG13S1894TC SEQ ID NO: 38 TT SEQ ID NO: 39 209
25.98 CAGAGAGGAACAGGCAGAGG AGTGGCTGGGAAGCCTTATT
8360DG13S1545SEQ ID NO: 40 SEQ ID N0:41 394
26.07 AGGTGAGAGAACAAACCTGTCTTGCCTTCCTTCTAAGGCCAAC
1866DG13S1524SEQ ID NO: 42 SEQ ID NO: 43 115
26.18 TGTTATACATTTCAATTTCACCTCGTACTCCAGCCGGGCAAC
3492DG13S1491A SEQ ID NO: 44 SEQ ID NO: 45 286
26.23 TTGTTCAGTGCTCTATAGTTACAAGGTCACAAAGCTATGCGATTA
6289DG13S62 AGT SEQ ID NO: 46 SEQ lD NO: 47 158
26.27D13S1244TCAACAAGTGGATTAAGAAACTGCTGTTTATGGCTGAGAAGTATG~
86
CA 02502359 2005-04-14
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-82-
3463 TG C
(SEQ (SEQ
ID ID
NO: N0:49)
48)
26.28 TAGCAGGGTGCAGTCTA ACCATACCACCACCACCATC
(SEQ
ID
6935DG13S64 N0:50 SEQ 247
ID
NO:
51
26.31 ACTGTACTTCTGCCTGGGC TTTTGTAATGCCTCAACCATG
(SEQ
4501D13S243 ID SEQ 147
NO: ID
52 NO:
53
26.32 CTGTAGACTTTATCCCTGACTTA
CAATGAATGATGAAGATTCCAC
7184DG13S1529CTG SEQ ID NO: 54 TC 132
SEQ
ID
NO:
55
26.33 TGACACCATGTCTTACTGTTTGC
GAGGATACAATGAGAACCAAA
8767DG13S1908SEQ TCTC 224
ID SEQ
NO: ID
56 NO:
57
26.38 CCACAGAATGCTCCAAAGGT GAGTTCAAGTGATGGATGACG
8034DG13S1546SEQ A 357
ID SEQ
NO: ID
58 N0.59
26.43 CAGATAGATGAATAGGTGGATGG
CACTGTTCCAAGTGCTTTGC
5811DG13S1444A SEQ ID NO: 60 SEQ 193
ID
NO:
61
26.48 GCAGGGCAAACTGCCTTAT TTTGGTGAAATGTCTGTTTATG
(SEQ
6657DG13S1458ID N0:62 G 402
SEQ
ID
NO:
63)
26.50 CTCAACCTGGCTTCTACT(SEQ TACTCCTTAATAAACTCCCC
4545D13S252 ID (SEQ 338
NO: ID
64 NO:
65
26.50 TATGCGTTGTGTGTGTG GGGCCTTAGATTCTTGTAGTG
(SEQ
ID
8231DG13S66 NO:66 G 217
SEQ
ID
NO:
67)
27.11 CTCGCATCTCGCTTCTCACT CTCAAGGGTCCAGTGGTTTG
5120DG13S1554SEQ SEQ 420
ID ID
NO: NO:
68) 69)
27.14 TGTCCAGACTGCCTCCTACA TGCAACACCTGGTTCACAAT
0675DG13S1907SEQ (SEQ 131
ID ID
NO:70) NO:
71)
27.14 CACAGTGAGACTCTATCTCAAAA
TCAGACTGGCTTAGACTGTGG
5842D13S802 A (SEQ 150
SEQ ID
ID NO:
NO: 73
72
27.24 AAATTCCAAAGGCCAGGTG CCATACAGTTTCCTAGGTTCTG
(SEQ
0616DG13S1892D NO: 74 G 373
I SEQ
ID
NO:
75
27.25 CACCTGGCCAAATGTTTGTT TGCTTGAATCCAGAGACTGC
3452DG13S1849SEQ SEQ 190
ID ID
NO: NO:
76 77)
27.27 TTTGCGAGTCCTTGTGGAGT ACAGTCCGCTCCCTCCTAAT
3860DG13S68 SEQ SEQ 238
ID ID
NO: NO:
78 79
27.28 ATGCTTGGCCCTCAGTTT TTGGCAACCCAAGCTAATATG
(SEQ
0461DG13S69 ID (SEQ 296
NO: ID
80 N0:81
27.48 CTCCACAGTGACAGTGAGG GAGAGGTTCCCAATCCC
(SEQ
3799D13S1250SEQ ID 160
ID NO:
N0:82 83)
27.61 CATCAACCTCCCCACCAC(SEQ
TATTTTTTCAGTCCCACAGTTA
0406D13S1448ID GC 227
NO: SEQ
84 ID
N0:85
27.61 CAGCTCCTGGCCATATTTCT GAGCCATTTCTCTGGGTCTG
5814DG13S574SEQ SEQ 153
ID ID
NO: N0:87
86
27.64 GGTCCGTGTCAACCCTTAGA CAGGTTGATGGGAGGGAAA
1211DG13S73 SEQ SEQ 198
ID ID
NO: NO:
88 89
27.66 CGGGAAATGACAGTGAGACC TGCCTAGATTCTCCCGTAAG
1507DG13S1532SEQ SEQ 163
ID ID
NO: NO:
90 91
27.70 GTGCCCAGCCAGATTC GCCCCCAGTCAGGTTT
(SEQ (SEQ
ID
5347D13S1242NO: ID 198
92 NO:
93
27.88 TTTCTCTCTCCACGGAATGAA AACCCATTCTCACAGGGTGTA
3872DG13S576SEQ SEQ 199
ID ID
NO:94 NO:
95
27.89 AGGAGTGTGGCAGCTTTGAG TGGATTCCCGTGAGTACCAG
7365DG13S1917SEQ SEQ 165
ID ID
NO: NO:
96 97)
27.93 ATGCTGGGATCACAGGC AACCTGGTGGACTTTTGCT
(SEQ
ID
2154D13S217 NO: SEQ 170
98) ID
NO:
99)
28.08~DG13S581IAGCATTTCCAATGGTGCTTT CATGTTGATATGCCTGAAGGA
367
CA 02502359 2005-04-14
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-83-
0632( SEQ ID NO: 100) ( SEQ ID N0:101 )
28.16 CACTGTCTGCTGCCACTCAT AGAGATTATGTGATGTACCCTC
5348DG13S1471SEQ ID N0:102 TCTAT SEQ ID N0:103 267
28.30 CAAGCCTGGGACACAGAAAT TTTGCAGACACCACAACACA
3252DG13S583SEQ ID NO: 104 ( SEQ ID NO: 105 264
28.30 ATGACCTAGAAATGATACTGGC CAGACACCACAACACACATT
3256D13S120 SEQ ID NO: 106 SEQ ID NO: 107 175
(
28.38 TGGTTTAAAAACCTCATGGG ATCCCAAACTCTGTACTTATGT
5566D13S1486SEQ ID NO: 108 AGG SEQ ID NO: 109 151
28.41 TTTGCACATACACATAAGCGAACCACAAATCCCGTGCACTAAA
5530DG13S1024SEQ ID NO: 110 SEQ ,ID NO: 111 139
28.41 ATTCCTGGGCTCATGGTACA TGCCGTCATCTGCTTTAGAA
5530DG13S1510SEQ ID NO: 112) SEQ 1D NO: 113 390
28.43 CCTTGGCTGTTGTGACTGGT CACTCAGGTGGGAGGATCAC
0308DG13S1495SEQ ID N0:114 SEQ ID NO: 115 285
28.51 GCTGTTTCCTTGGCTTCTTCT GCGATACTTGAGATGACCATG
7541DG13S1482SEQ ID NO: 116 A SEQ ID NO: 117) 291
28.55 CACTTTGCCAGTAGCCTTGA TTGGGAAAGTTAACCCAGAGA
1060DG13S1845SEQ ID N0:118 SEQ ID NO: 119 284
28.63 TTTGGGAAGAGCCATGAGAC CTCTGGGCATTGGAGGATTA
4903DG13S1030(SEQ ID NO: 120) (SEQ ID NO: 121 ) 354
28.63 TTTGGGAAGAGCCATGAGAC AATGCCCATGTGCACTGTAG
4903DG13S1467(SEQ ID NO: 122) (SEQ ID NO: 123) 231
28.68 GGGAGACAAGTCAGGTGAGG CTGAGTATGGAGTCTTCATCAT
6607DG13S584SEQ ID NO: 124) TATC (SEQ ID NO: 125)151
28.79 TCGTCTCGAAGAAAGAAAGAAGACACCATGGGTTAATTGCACA
4032DG13S1519SEQ ID N0:126 SEQ ID NO: 127) 286
28.87 TGACGTGGGTTCAGGTTGTA AGTGCATTGGTGCCTTCTCT
6156DG13S77 (SEQ ID NO: 128 SEQ fD NO: 129 220
28.97 GGACTGCCAATTCTACAGCA TTTCCATGGGAAATTTGGTC
0723DG13S586SEQ lD NO: 130 SEQ ID NO: 131 151
28.97 TGCTACTAGATTTGACCAACCA GACTTGTAAAGGATTTAGTGAT
5641DG13S79 SEQ ID NO: 132 TTCG SEQ ID NO: 133 128
29.05 GTGGAAGGCCTCTCTTG TGCTTCTTGAGGGAAAGCAT
9394DG13S80 SEQ ID NO: 134) SEQ ID NO: 135 233
29.12 CACGTGGTTCACCTCTCTAGG TTGGCCACTTATTTGTG
6152DG13S82 SEQ ID NO: 136 SEQ ID NO: 137 302
29.15 CGATGAGTGACAGGGCT (SEQ CCTCGTGGGTGGAATAA
ID
4691D13S1299NO: 138 SEQ ID NO: 139 225
29.15 TTGGCCATTAGCAATTAGCA CGTGGGTGGAATAAATCAGG
4737DG13S85 SEQ ID NO: 140 SEQ ID NO: 141 '153
29.15 GTTGAGGCAAGAGAATCACT GCACATTTACACCAGGGTG
8462D13S629 SEQ ID NO: 142 SEQ ID N0.143 145
29.22 CCTTCAGAGGATTTCCCTTTC CTGGTTTGACTCCAGCTTCA
4060DG13S1934SEQ ID NO: 144 SEQ ID NO: 145 431
29.24 TGTTCAAACCTAAGGTGCTTCA GAAACAACAACAACAACAACAA
5462DG13S1098SEQ ID NO: 146 CA SEQ ID NO: 147 416
29.25 CCTGGCACGGAATAGACACT GGCCTCCTTTGCTCTGAAG
9840DG13S1104SEQ ID NO: 148 SEQ 1D NO: 149 378
29.29 CATCCCTGTGGCTGATTAAGA AACAGTTCCAGCCCGTTCTA
4436DG13S1097SEQ 1D NO: 150) (SEQ ID NO: 151) 162
29.30DG13S1110TTTCAAAGGAATATCCAAGTGC TGGCGTACCATATAAACAGTTC265
CA 02502359 2005-04-14
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-84-
9700( SEQ ID NO: 152) TC (SEQ ID NO: 153)
29.30 TTTCAAAGGAATATCCAAGTGC AAACGTGACACTTCCACACA
9909DG13S86 SEQ ID NO: 154 SEQ ID NO: 155 177
29.35 TTCAATGAAGGTGCCGAAGT TGTCTATCCCAAAGCAA(SEQ
9961DG13S87 SEQ ID NO: 156 I D NO: 157 218
29.52 GCAAGACTCTGTTGAAGAAGAAGTCCCTCTGTTTGAGTTTCTCG
2443DG13S1111A SEQ ID NO: 158 SEQ ID NO: 159 110
29.57 AGGCACAGTCGCTCATGTC AAACTTTAGCTAATGGTGGTCA
(SEQ
4665DG13S1101D NO: 160 AA SEQ ID NO. 161 333
I
29.62 TGTGATTCCAGGGAGCTATCA TAGGTGTGTGGAGGACAGCA
2755DG13S1106SEQ ID NO. 162 SEQ ID NO. 163 416
29.65 CCAGTTTCAGTTAGCCAAGTCTGGAGAGGGAATGAATGCAGGA
8910DG13S172SEQ ID NO: 164 SEQ ID NO: 165 267
29.66 GAGCATGTGTGACTTTCATATTCAGTGGCTATTCATTGCTACAG
5709D13S1246AG SEQ ID NO: 166 G SEQ ID NO: 167 177
29.67 TTGCTGGATGCTGGTTTCTA(SEQAAAGAGAGAGAGAAAGAGAAA
2561DG13S1103D NO: 168 GAAAGA SEQ ID NO: 264
I 169
29.82 CTGGTTGAGCGGCATT(SEQ TGCAGCCTGGATGACA(SEQ
ID
5975D13S289 NO: 170) ID NO: 171 260
29.82 CCTATGGAAGCATAGGGAAGAA(CCCACTTCTGAGTCTCCTGAT(
6631DG13S166SEQ ID NO: 172) SEQ ID NO: 173) 395
29.90 GGGATGCAGAAAGGATGTGT(SEAAGAATGCTGGCCAACGTAA(S
6689DG13S164Q ID NO: 174) EQ ID NO: 177 218
29.90 CTCTCAGCAGGCATCCA(SEQ GCCAACGTAATTGACACCA(SE
ID
6700D13S1238NO: 178) Q ID N0:179) 129
30.03 CCTTAGGCCCCATAATCT(SEQ CAAATTCCTCAATTGCAAAAT(S
ID
1378D13S290 NO: 180 EQ ID N0:181 176
30.08 GGTCATTCAGGGAGCCATTC(SECCATTATATTTCACCAAGAGGC
6303D13S1229Q ID NO: 182 TGC SEQ ID NO: 183 119
30.19 TGCCTGGTCATCTACCCATT(SEQTCTACTGCAGCGCTGATCTT(S
2847DG13S1460ID NO: 184) EQ ID NO: 185 264
30.21 CATTTATGAATGGAGGTGAAGC(ATGGGAGCTCAAAGGGAAAT(S
7670DG13S1933SEQ ID NO: 186 EQ ID NO: 187 186
30.30 CAGCAGGAAGATGGACAGGT(SECACACTGCATCACACATACCC(
3213DG13S1448Q ID NO: 188 SEQ ID NO: 189 136
30.31 TATGCCAGTATGCCTGCT(SEQ GTCACATCAGTCCATTTGC(SE
1D
7871D13S1287NO: 190 Q ID NO: 191 232
30.34 CCAAAGCAAGTAACCTCCTCA(SAAACAATCACTGCCCTCTGG(S
2102DG13S1061EQ ID NO: 192 EQ ID NO. 193 227
30.57 TGATGAAATTGCCTAGTGATGC(SGGATCCAATCGTACGCTACC(S
1837DG13S1904EQ ID NO: 194 EQ ID NO. 195 136
30.64 CGAATGGGTGACTAACAGCA(SECTGGAGTGCAGGGACATGA(S
3438DG13S882Q ID NO: 196 EQ ID NO: 197 378
30.66 AAAGAAATATTCCAAGAAGAAAGTTGCACAACTTTGTGTAGAGCA
5937DG13S295AAA SEO ID NO: 198 T SEQ ID NO: 199 279
30.67 GGGTATGTCTTTATTCTCGGCAGGTGCATTCACAGACCAGTCATT
4468D13S1226TA SEQ ID NO: 200 (SEQ ID NO: 201 219
30.69 GGGCTTGAAGGCACTAAATGT(SCCAAGCAGTAATTCCTTCCTCA
0959DG13S293EQ ID NO: 202 SEQ ID N0:203) 313
30.71 ACCTAAACACCACGGACTGG(SECAGGTATCGACATTCTTCCAAA
2468DG13S1490Q ID NO: 204) (SEQ ID NO: 205 418
30.82DG13S93 TGGGAAGCCAGTAAAGTAGGAA(AAAGAGACTCCACACATCCATT190
CA 02502359 2005-04-14
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-85-
448 3 SEQ ID NO: 206) T(SEQ ID NO: 207)
30.82 AGGGCTATTCCTCAAGGTGTT(STGCTAACACTACCCTCGCAAT(
4859DG13S94 EQ ID NO: 208 SEQ ID NO: 209) 332
30.92 GGGCAGGAATCTCTGAAGTG CTCCACTGAGAAGCCAAGGA(S
8429DG13S1534SEQ ID NO: 210 EQ ID NO. 211 382
30.94 AGGCCAAGCTGGTCCATAG(SEQTCTCTCAAAGCCTCGCTCTC(S
0369DG13S95 ID N0:212 EQ ID NO: 213 126
30.97 CCTTTGAGGCTGGATCTGTT(SETTTCCTTATCATTCATTCCCTC
0238DG13S96 Q ID NO: 214 A SEQ ID NO: 215 218
31.03 AGATATTGTCTCCGTTCCATGA(SCCCAGATATAAGGACCTGGCT
8874D13S260 EQ ID NO: 216 A SEQ ID NO: 217 163
31.09 TTTAAGCCCTGTGGAATGTATTT(GACATTGCAGGTCAAGTAGGG
2294DG13S17 SEQ ID NO: 218 SEQ ID NO: 219 157
31.20 TGCATAAGGCTGGAGACAGA(SECACAGCAGATGGGAGCAAA(S
7844DG13S306 Q ID NO: 220 EQ ID N0:221 158
31.26 GTGCATGTGCATACCAGACC(SEGGCAAGATGACCTCTGGAAA(S
0521DG13S18 Q ID NO: 222 EQ ID NO: 223 31g
31.29 GTCCACTGCAGCACACAGAG(SEGCACTGGTAGATACATGCTAA
9720DG13S1905Q ID NO: 224 CG SEQ ID NO: 225 383
31.35 GGGTATCTTGGCCAGGTGT(SEQTGGCTAAGCACAATCCCTTT(S
3230DG13S307 ID NO: 226) EQ ID NO: 227 403
31.35 TTTGTGTTCCAGGTGAGAATTG(SGAACCATATCCCAAGGCACT(S
5135DG13S1062EQ ID NO: 228 EQ ID NO: 229) 120
31.41 AACCCAAATCAACAAACCAGA(SEAATGAATTCTGGGTCACATGC(
4329DG13S1874Q ID NO: 230) SEQ ID NO: 231) 404
31.42 TTGTTCCCACATTCATTCTACA(STTAAACTCGTGGCAAAGACG(S
9562DG13S1093EQ ID NO: 232 EQ ID NO: 233 273
31.62 CACCATGCCTGGCTCTTT(SEQAACTTCTCCAGTTGTGTGGTTG
ID
6502DG13S1059NO: 234 (SEQ ID NO: 235) 330
31.72 AGCTGAGCTCATGCCACT(SEQCAAGACCTTGTGCATTTGGA(S
3749DG13S1086ID NO: 236 EQ ID NO: 237 155
31.74 AGCCAGACATGGTAGTGTGC(SEGCAATAACTCACACATCAGCAA
6074DG13S1515Q ID NO: 238 SEQ ID N0:239) 417
31.85 CCTACCATTGACACTCTCAG(SETAGGGCCATCCATTCT(SEQ
ID
5732D13S171 Q ID NO: 240 NO: 241 231
31.91 ACCAAGATATGAAGGCCAAA(SECCTCCAGCTAGAACAATGTGA
7332DG13S1092Q ID NO: 242 A SEQ ID NO: 243 176
32.00 TGTCCATAGCTGTAGCCCTGT(SCTCAATGGGCATCTTTAGGC(S
2852DG13S1449EQ ID NO: 244 EQ ID NO: 245 27g
32.07 TGTAATTCAACGACTGGTGTCC(SAGCTTCTGATGGTTGCTGGT(S
2957DG13S1489EQ ID NO: 246 EQ ID NO: 247 130
32.08 CAAACAAACAAACAAGCAAACC(TGGACGTTTCTTTCAGTGAGG(
3989DG13S312 SEQ ID NO: 248 SEQ ID NO: 249 349
32.12 TGATAACTTACCAGCATGTGAGCTCACCTCACCTAAGGATCTGC(
5177DG13S1511SEQ ID NO: 250 SEQ ID NO: 251 314
32.18 CATGCAATTGCCCAATAGAG(SETTGGGCTTGTCTACCTAGTTCA
3547DG13S314 Q ID NO: 252 SEQ ID NO: 253 335
32.19 TGGGTTCCTCATACTGGAGTG(SGCCTGAGCTCCAAGCTCTTT(S
5358DG13S1090EQ ID NO: 254 EQ ID NO: 255 169
32.25 GCTGCACGTATTTGTTGGTG(SEAAACAGCAGAAATGGGAACC(S
1038DG13S1071Q ID NO: 256 ) EQ ID NO: 257 239
~ DG13S1068CCGTGGGCTATCAATTTCTG(SEAAGATGCAATCTGGTTTCCAA(238
32.35~~ ~
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WO 2004/035746 PCT/US2003/032805
-86-
6895 Q ID NO: 258 ) SEQ ID NO: 259 )
32.37 CCCAAGACTGAGGAGGTCAA(SEGCTGACGGAGAGGAAAGAGA(
3040DG13S1077Q ID NO: 260 SEQ ID NO: 261 374
32.42 TGACAAGGGTGTGGTTATGG CCGCACTTTCTCTTCTGGAC
2780DG13S1906SEQ ID NO: 262 SEQ ID N0:263 425
32.51 TGAGAAGCCTGGGCATTAAG ACAAGCTCATCCAGGGAAAG
1590DG13S316SEQ ID NO: 264 SEQ ID NO: 265 243
32.61 TTGGAAAGGAAGAAAGGAAGG TTGAAACCTAAATGCCACCTG
0517DG13S317SEQ ID NO: 266 ( SEQ ID N0:267 215
32.61 ACCTGTTGTATGGCAGCAGT GGTTGACTCTTTCCCCAACT
0713D13S1493SEQ ID NO: 268 SEQ ID NO: 269 248
(
32.78 AGAGCTGATCTGGCCGAAG GGTGGACACAGAATCCACACT
9894DG13S1558SEQ ID N0:270 SEQ ID NO: 271 399
(
32.86 GGCCTGAAAGGTATCCTC (SEQTCCCACCATAAGCACAAG
5950D13S267 D N0:272 SEQ ID NO: 273 160
I
32.96 TCAACCTAGGATTGGCATTACA TCTAGGATTTGTGCCTTTCCA
1410DG13S1478SEQ ID NO: 274 SEQ ID NO: 275) 387
33.00 GACGTCTTAGGATTGACTTCTGCCCAAATACACATTCTTAAAGGG
9922DG13S1513SEQ ID NO: 276 AAA SEQ ID NO: 277 173
33.12 GACTGCAGATCGTGGGACTT TTCTCCAGAGAAACCAAACCA
5696DG13S1461(SEQ ID NO: 278) (SEQ ID NO: 279 148
33.16 ATTCGTGCAGCTGTTTCTGC GCATGACATTGTAAATGGAGG
8468DG13S1551(SEQ ID NO. 280) A SEQ ID N0:281 263
33.25 GGTGGGAATGTGTGACTGAA CCAGGTACAACATTCTCCTGAT
4989DG13S1884(SEQ ID NO. 282) (SEQ ID N0:283) 123
33.34 TGCAGGTGGGAGTCAA (SEQ AAATAACAAGAAGTGACCTTCC
ID
0124D13S1293NO. 284 TA SEQ ID NO: 285 129
33.34 TGTTCTCCTCACCCTGCTCT TTTCAGGCTAGGAAGATCCTTT
6908DG13S326SEQ ID NO: 286 (SEQ ID NO: 287 261
33.39 AAAGGATGCATTCGGTTAGAG ACTGTCCTGTGCCTGTGCTT
2629DG13S1518SEQ ID NO: 288 SEQ ID NO: 289 375
33.40 CCTGAATAGGTGGAATTAAGATCTCAAGGAGCATACACACACAC
5527DG13S23 AA SEQ ID NO: 290 A SEQ ID NO: 291 107
33.43 GTCCACCTAATGGCTCATTC CAAGAAGCACTCATGTTTGTG
1536D13S620 SEQ ID NO: 292 SEQ ID NO: 293 185
33.43 AGCCTGTGATTGGCTGAGA GGCTTACAGCTGCCTCCTTT
(SEQ
7092DG13S1866ID NO: 294 SEQ ID NO: 295 410
33.49 CCCACAGAGCACTTTGTTAGA GCCTCCCTTAAGCTGTTATGC
5718DG13S1927SEQ ID NO: 296 SEQ ID NO: 297 401
33.50 CACTCTTTACTGCCAATCACTCCGCCGTGTGGGTGTATGAAT
3440DG13S1503SEQ ID N0:298 SEQ ID NO: 299 226
33.56 TTGTACCAGGAACCAAAGACAA CACAGACAGAGGCACATTGA
8100DG13S332SEQ 1D NO: 300 SEQ ID NO: 301 176
33.67 GCTCTGGTCACTCCTGCTGT CATGCCTGGCTGATTGTTT
5841DG13S333SEQ ID NO: 302) SEQ ID NO: 303 446
33.77 CCAACATCGGGAACTG (SEQ TGCATTCTTTAAGTCCATGTC
ID
1389D13S220 NO: 304 SEQ ID NO: 305 ~ 191
33.81 CAGCAACTGACAACTCATCCA CCTCAATCCTCAGCTCCAAC
8041DG13S1919SEQ ID NO: 306 SEQ ID N0.307 255
33.87 TCCTTCACAGCTTCAAACTCA AGTGAGAAGCTTCCATACTGG
3614DG13S1439SEQ ID NO: 308 ) T (SEQ ID NO: 309) 239
33.90DG13S335GCCAACCGTTAGACAAATGA CTACATGTGCACCACAACACC~
~ 201
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_87_
6065 (SEQ ID NO: 310) (SEQ ID NO: 311)
33.92 AGTTTATTGCCGCCGAGAG ACCCACCACATTCACAAGC
(SEQ
8653DG13S340ID NO. 312 SEQ ID NO: 313 373
34.01 CGATTGCCATGTCTCTTTGA GAGATCTGGCCTGGATI-fGT
9455DG13S1496SEO ID NO: 314 SEQ ID NO: 315 155
34.03 TGAGGCCAGCCTTACCTCTAT CCAGACATGGTGGCTTGT
4089DG13S342SEO ID NO: 316 SEQ ID NO: 317 366
34.06 GAAGGAAGGAAGGGAAGGAA AAGGATGAGAAGAGTCCATGC
1777DG13S344SEQ ID NOv 318 SEQ ID NO: 319 292
34.06 AAATACCCTTTGAACAGACACACTAGCTGAGCATGGTGGTACG
7239DG13S345SEQ ID NO: 320 SEQ ID NO: 321 201
34.07 AAAGACAAGACAGCAATCCAAA GCAGAACCCAGGCTACAGAT
7874DG13S346SEQ ID NO: 322 SEQ ID NO. 323 152
34.08 TCATTGTCAGCACAGAATGAACT(GGAGGGAGGGAAGAAAGAGA
4138DG13S347SEQ ID NO: 324 SEQ ID NO: 325 ) 338
34.08 GCAACACAGTGAAAGCCCA(SEQACAGGAGCATGCCACCATG(SE
4326D13S624 ID NO: 326 Q ID NO: 327 191
34.15 GGGAAGAGGAGATTGACTTGTT(GGAACACCATCATTCCAACC(S
6075DG13S339SEQ ID NO: 328 EQ ID NO: 329 232
34.19 TACAAGCTCCACCGTCCTTC(SETGAGTTGCTGCCTCTTCAAA(S
2478DG13S1926Q ID NO: 330 EQ ID NO: 331 ) 261
34.22 TGCTAATGGGCCAAGGAATA(SEGCTAAATGTCCTCATGAATAGC
0227DG13S1469Q ID NO: 332) C(SEQ ID NO: 333 ) 382
34.30 TGTCCTGCAGACAGATGGTC(SECCTCCGGAGTAGCTGGATTA(S
1448DG13S351Q ID NO: 334) EQ ID NO: 335 ) 294
34.38 GAGACTGGCCCTCATTCTTG(SEAAGAAGCCAGAGACAAAGAAA
7883DG13S26 Q ID NO: 336 TACA SEQ ID NO: 337 330
)
34.53 CATCTATCTTTGGATTCAGTGGTTGCTCCCAACATCTTACCAG(S
5441DG13S30 G SEQ ID NO: 338 EQ ID NO: 339 388
34.56 TGTCCTCTGGTCATTTCTATGGT(CATGAATGAGAAGTGATGAAT
5594DG13S1435SEQ ID NO: 340 GG SEQ ID NO: 341 235
)
34.65 AACACGGGAAATTCCAACAG(SETGAAGAACTGAAATTGCCAGTA
9858DG13S1446Q ID NO: 342 A SEQ ID NO: 343 379
34.71 CAGACACTGTAAACTGGCTTCG(GCCACATTGCTATCAGCGTA(S
2260DG13S356SEQ ID NO: 344 EQ ID NO: 345 212
34.73 TGTCATAGGCTTGCGGTATTT(SETTGGTAGGGTCCTTTCCTTT(S
8756DG13S357Q ID NO: 346 EQ ID NO: 347 202
34.77 GCCTGCTCACTGTTGTTTGA(SECGGTTATCAGAGACTGGTGGT(
0571DG13S1032Q ID NO: 348 SEQ ID NO: 349 211
34.79 GGCTTATTTCATGTACGGCTA(SEGGTTAAACTCTACTTAGTCCTG
9679DG13S1557Q ID NO: 350 ATGC SEQ ID NO: 351 158
34.88 GAACTCTGCAGGCACCTCTT(SECCTGAAGCGCTTGTACTGAA(S
2934DG13S1925Q ID NO: 352 EQ ID NO: 353 456
34.93 TGTTGCGTACTCAGCCCATA GACAGGTGTCAAACGGGTCT(S
2690DG13S1484SEQ ID N0:354 EQ ID NO: 355 246
34.94 TTGGCTTCTCGCTCTTTCTT(SEQAGCCATCAGTCACATGCAAA
2547DG13S360ID NO: 356 SEQ ID NO: 357 350
34.99 AGATCTCCAGGGCAGAGGAC(SECCTTCCTCCCTCCTTCTCTC(S
8979DG13S1522Q ID NO: 358 EQ ID NO: 359 355
35.07 CGTCATTGATCCCAATCATCT(SEGGCTGATAGCCTCCCTTGTA
4962DG13S1517Q ID NO: 360 ) (SEQ ID N0:361) 235
35.07DG13S1521GAGAGAGAGCAGCTTGCATGT(SGGCTGATAGCCTCCCTTGTA(S172
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_88_
4962 EQ ID N0:362) EQ ID N0:363)
35.12 ACCTTTCAAGCTTCCGGTTT(SEQTTCCATCCGTCCATCTATCC(S
6882DG13S364ID NO: 364 EQ ID NO: 365 172
35.32 TTAAAGTCACTTGTCTGTGGTCA(TTTGTAGGAATCAAGTCAAATA
8663DG13S1036SEQ ID NO: 366 ATGTA SEQ ID NO: 367 216
35.33 CAAACATCACACTGGGCAAA(SETGCTTTGGAATCTTTCTTGCT(S
5364DG13S367Q ID NO: 368) EQ ID NO: 369 301
35.37 CTGCCAGGATGTCAGCATT(SEQTCCACACTTTCTCATCACCTAA
1957DG13S1901ID NO: 370 A SEQ ID NO: 371 440
35.42 CTTTCGGAAGCTTGAGCCTA(SECCCAAGACCACTGCCATATT(S
0295DG13S1037Q ID NO: 372 EQ ID NO: 373) 269
35.42 TGACAGGTTTGGGTATATTGGA(TGCTTAATGTAGTGGCAGCA(S
5841DG13S1854SEQ ID NO: 374) EQ ID NO: 375 124
35.50 TCCTGCCTTTGTGAATTCCT(SEQGTTGAATGAGGTGGGCATTA(S
6053DG13S1038ID NO: 376 EQ ID NO: 377 334
35.54 CCATTTAATCCTCCAGCCATT(SEGCTCCACCTTGTTACCCTGA(S
7210DG13S1039Q ID NO: 378 EQ ID NO: 379 167
35.60 ACAACCCTGGAATCTGGACT(SEGAAGGAAAGGAAAGGAAAGAA
9252DG13S1840Q ID NO: 380 A SEQ ID NO: 381 217
35.61 TGACAAGACTGAAACTTCATCAG(GATGCTTGCTTTGGGAGGTA(S
9286DG13S369SEQ ID NO: 382) EQ ID NO: 383) 257
35.62 TTGAGGACCTGTCGTTACG TTATAGAGCAGTTAAGGCACA
(SEQ
7911D13S305 D NO: 384) (SEQ ID NO: 385) 394
I
35.65 TGAGGGTGGTAAGCCCTTATT(SGGAGTTGTGGCCTCTCTCTCT(
6659DG13S375EQ ID NO: 386) SEQ ID NO: 387) 192
35.76 AAGCAAATATGCAAAATTGC(SEQTCCTTCTGTTTCTTGACTTAAC
0368D13S219 D NO: 388 A SEQ ID NO: 389 125
I
35.82 TGCTAAGAGGGCAGATCTCA(SEGGCTCATAGCCAATl-fCTCC
5852DG13S378Q ID NO: 390 SEQ ID NO: 391 324
35.83 CGGCATTCTCAATAACCTCAA TCTTTGATGAGGATCAATTAGT
2127DG13S32 SEQ ID NO: 392 GG SEQ ID NO: 393 214
35.87 ACGCACACACACACACACAC TGCCTCTGTAATCCTGTGTAGC
2936DG13S1549SEQ ID NO: 394 SEQ ID N0:395 260
35.91 GCTCTAAGGTGGGTCCCAATA GGGAATGACAAGATCAGTTTA
2321DG13S1473SEQ ID N0:396 CC SEQ ID NO: 397 163
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-~9-
Table 7.
The selected SNP haplotypes and the corresponding p-values, relative rislc
(RR), number of patients (#aff), allelic frequency in patients (aff.frq.),
carrier
frequency in patients (carr.frq.),number of controls (#con), allelic frequency
in
controls (con.frq.), population attributable risk (PAR). The patients used for
this
analysis were all unrelated within 4 meioses.
_ a
ci~ ~ a n c n
M ~ ~ ~ O M M n M
M
O _ QOO oO_ _ _ _ _
C~ M C~C~C~C~
-val RR #affaff.frcarr.fr#concon.frPAR
. . .
B44.8E-052.089030.1060.20 619 0.0540.11 2 2 2 0
B52.4E-052.209100.1010.19 623 0.0490.113 2 2 2 0
B61.8E-062.229130.1310.24 623 0.0630.143 2 2 2 0 2
A45.1 1.819190.1590.29 623 0.0950.14 2 3 2 0
E-06
A52.6E-061.919200.1500.28 624 0.0850.143 2 3 2 0
to All references cited herein are incorporated by reference in their
entirety.
While this invention has been particularly shown and described with references
to
preferred embodiments thereof, it will be understood by those skilled in the
art that
various changes in form and details may be made therein without departing from
the
scope of the invention encompassed by the appended claims.
