Note: Descriptions are shown in the official language in which they were submitted.
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1
SUSCEPTTBILITY GENE FOR HUMAN STROKE;
METHODS OF TREATMENT
RELATED APPLICATIONS
S This application is a continuation of and claims priority to U.S.
Application
No. 10/650,120, filed August 27, 2003, which is a continuation-in-part of U.S.
Application No. 10/419,723 filed April 18, 2003, which is a continuation-in-
part of
U.S. Application No. 101255,120, Bled September 25, 2002, which is a
continuation-
in-part of U.S. Application No. 10/067,514, filed February 4, 2002, which is a
continuation-in-part of U.S. Application No. 09/811,352, filed March 19, 2001.
The
entire teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
Stroke is a common and serious disease. Each year in the United States more
than 600,000 individuals suffer a strolce and more than 160,000 die from
stroke-
related causes (Sacco, R.L. et al., Sti°oke 28, 1507-17 (1997)). In
western countries
stroke is the leading cause of severe disability and the third leading cause
of death
(Bonita, R., Lancet 339, 342-4 (1992)). The lifetime risk of those who reach
the age
of 40 exceeds 10%.
The clinical phenotype of stroke is complex but is broadly divided into
ischemic (accounting for 80-90%) and hemorrhagic stroke (10-20%) (Caplan, L.R.
Caplafz's Strolze; A Clinical Appooaclz, 1-556 (Buttenvorth-Iieinemann,
2000)).
Ischemic stroke is further subdivided into large vessel occlusive disease
(referred to
here as carotid stroke), usually due to atherosclerotic involvement of the
common
and internal carotid arteries, small vessel occlusive disease, thought to be a
non-
atherosclerotic narrowing of small end-arteries within the brain, and
cardiogenic
stroke due to blood clots arising from the heart usually on the background of
atrial
fibrillation or ischemic (atherosclerotic) heart disease (Adams, H.P., Jr. et
al., Sts°oke
24, 35-41 (1993)). Therefore, it appears that stroke is not one disease but a
heterogeneous group of disorders reflecting differences in the pathogenic
mechanisms (Alberts, M.J. Genetics of Cef~ebrovascula~ Disease, 3 86 (Future
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Publishing Company, Tnc., New York, 1999); Hassan, A. & Markus, H.S. Bruin
123,
1784-812 (2000)). However, all forms of stroke share rislc factors such as
hypertension, diabetes, hyperlipidemia, and smoking (Sacco, R.L. et al.,
Stroke 28,
1507-17 (1997); Leys, D. et al., J. Neurol. 249, 507-17 (2002)). Family
history of
stroke is also an independent risk factor suggesting the existence of genetic
factors
that may interact with environmental factors (Hassan, A. & Markus, H.S. Brain
123,
1784-812 (2000); Brass, L.M. & Alberts, M.J. Baillieres Clira. Neurol. 4, 221-
45
(1995)). .
The genetic determinants of the common forms of stroke are still largely
unknown. There are examples of mutations in specific genes that cause rare
Mendelian forms of stroke such as the Notch3 gene in CADASIL (cerebral
autosomal dominant arteriopathy with subcortical infarctions and
leukoencephalopathy) (Tournier-Lasserve, E. et al., Nat. Genet. 3, 256-9
(1993);
Joutel, A. et al., Nature 383, 707-10 (1996)), Cystatira C in the Icelandic
type of
hereditary cerebral hemorrhage with amyloidosis (Palsdottir, A. et al., Lancet
2, 603-
4 (1988)), APP in the Dutch type of hereditary cerebral hemorrhage (Levy, E.
et al.,
Science 248, 1124-6 (1990)) and the KRIT1 gene in patients with hereditary
cavernous angioma (Gunel, M. et al., Proc. Natl. Acad. Sci. USA 92, 6620-4
(1995);
Sahoo, T. et al., Hufn. Mol. Genet. 8, 2325-33 (1999)). None of these raze
forms of
stroke occur on the background of atherosclerosis, and therefore, the
corresponding
genes are not likely to play roles in the common forms of stroke which most
often
occur with atherosclerosis.
It is very important for the health care system to develop strategies to
prevent
stroke. Once a stroke happens, irreversible cell death occurs in a significant
poition
of the brain supplied by the blood vessel affected by the stroke.
Unfortunately, the
neurons that die cannot be revived or replaced from a stem cell population.
Therefore, there is a need to prevent strokes from happening in the first
place.
Although we already know of certain clinical risk factors that increase stroke
risk
(listed above), there is an uiunet medical need to define the genetic factors
involved
in stroke to more precisely define stroke risk. Further, if predisposing
alleles are
common in the general population and the specificity of predicting a disease
based
on their presence is low, additional loci such as protective Ioci are needed
for
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meaningful prediction of disposition of the disease state. There is also a
great need
for therapeutic agents for preventing the first stroke or further strokes in
individuals
who have suffered a previous stroke or transient ischemic attack.
SUMMARY OF THE INVENTION
A locus conferring susceptibility to ischemic stroke to chromosome Sql2 in the
Icelandic~population has been mapped and the identification of
phosphodiesterase 4D
(PDE4D) as the gene at Sql2 contributing to the risk of ischemic stroke has
been
reported. This locus was extensively fine mapped and tested for association to
stroke.
Most striking is that haplotypes can be classified into three distinct groups:
wild type, at-
risk and protective. Additionally, a significant disregulation of multiple
PDE4D
isoforms in stroke patients was observed. The strongest association was within
the
PDE4D, especially to the two major subtypes of ischemic stroke, carotid and
cardiogenic
stroke. We have found variation in PDE4D that more than doubles the risk for
cardiogenic and carotid stroke, two of the most common forms of ischemic
stroke. We
have shown that there are at least 9 isoforms of PDE4D at the mRNA level and
the
protein level. The basis for these isoforms is the use of alternative 5 prime
exons that are
alternatively spliced into a common set of exons defining the catalytic domain
as well as,
in the case of the long forms, a set of exons def ring a common core in the
regulatory
domain. The PDE4D gene is involved in the pathogenesis of stroke. The PDE4D
gene
may be involved through artherosclerosis, the major pathological process
underlying
ischemic stroke. Our results indicate that atherosclerosis is a cAMP disease
resulting
from disregulation of its levels within the vasculature.
In one aspect, the invention relates to methods of diagnosing a predisposition
to stroke. The methods of diagnosing a predisposition to stroke in an
individual
include detecting the presence of a polymorphism in PDE4D, as well as
detecting
alterations in expression of a PDE4D polypeptide or isoform, such as the
presence of,
or relative expression of different splicing variants of PDE4D polypeptides.
For
example, it may be that the ratio of certain splice variants could be used as
a
diagnostic marker for stroke predisposition. Also an abnormal splice form can
be
detected (that is one that is not normally expressed but is created from a DNA
sequence mutation that leads to an abnormal splice form to be created from the
primary transcript) may be created from mutations in the PDE4D gene. For
example,
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new splice sites might be created from a single base substitution within an
intron that
is inappropriately used as a splice acceptor or donor site, resulting in an
abnormal
message which is likely to have a premature stop codon leading to a truncated
form
of PDE4D protein. The alterations in expression can be quantitative,
qualitative, or
both quantitative and qualitative. The methods of the invention allow the
accurate
diagnosis of stroke at or before disease onset, thus reducing or minimizing
the
debilitating effects of stroke. The methods of the invention also diagnose
those
individuals who are protected against developing stroke even in the face of
other risk
factors including but not restricted to hypertension, diabetes,
hyperlipidemia,
smoking history, previous stroke, TIA, MI or PAOD, or caxxiers of stroke
associated
gene variants. In one embodiment, predisposition to stroke or susceptibility
to stroke
can be assessed by determining PDE4D isoform levels in the individual compared
to
control levels, wherein a difference in isoform expression is indicative of
predisposition or susceptibility to stroke. Preferably, the level of
expression of
I S PDE4D7 and/or PDE4D9 is assessed.
The invention additionally relates to an assay for identifying agents that
alter
(e.g., enhance or inhibit) the activity or expression or transcription of one
or more
PDE4D polypeptides or isoforms. Such an assay may also identify agents that
alter
the relative expression of one or more PDE4D isoforms with respect to other
isoforms at either the mRNA level or polypeptide level. For example, a cell,
cellular
fraction, or solution containing a PDE4D polypeptide or a fragment or
derivative
thereof, can be contacted with an agent to be tested, and the level of PDE4D
polypeptide expression or activity can be assessed. Alternatively, a cell, or
cell with
artificial DNA construct with part or all of the PDE4D gene with or without a
reporter gene can be used to identify agents that may directly affect
transcription at
one or more of the many alternative PDE4D promoters upstream of the
alternative 5
prime exons or splicing efficiency of the primary transcript to one or more
mRNA
isoforms. The activity or expression of more than one PDE4D polypeptides can
be
assessed concurrently (or the corresponding reporter gene activity) (e.g., the
cell,
cellular fraction, or solution can contain more than one type of PDE4D
polypeptide,
such as different splicing variants, and the levels of the different
polypeptides or
splicing mRNA variants can be assessed).
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Agents that enhance or inhibit PDE4D mlZNA or polypeptide expression or
activity are also included in the current invention, as are methods of
altering
(enhancing or inhibiting) PDE4D mRNA or poLypeptide expression or activity by
contacting a cell containing PDE4D gene, mRNA, and/or polypeptide, or by
contacting the PDE4D gene, mRNA, and/or polypeptide, with an agent that
enhances
or inhibits expression or activity of PDE4D mlZNA or polypeptide. In another
embodiment, isoform mLZNA and/or protein levels can be altered, compared to
control Levels, using the agents of the invention.
Additionally, the invention pertains to pharmaceutical compositions
comprising the nucleic acids of the invention, the polypeptides of the
invention,
and/or the agents that alter activity of PDE4D polypeptide. The invention
further
pertains to methods of treating stroke, by administering PDE4D therapeutic
agents,
such as nucleic acids of the invention, poLypeptides of the invention, the
agents that
alter activity of PDE4D polypeptide, or compositions comprising the nucleic
acids,
polypeptides, and/or the agents that alter activity of PDE4D polypeptide.
The invention further relates to methods for preventing the occurrence of
stroke in an individual in need thereof by regulating a PDE4D mLRNA and/or
polypeptide isoform Level compared to control levels, whereby the regulated
isoform
level mimics the level of a healthy individual. Isoform expression at the mRNA
and/or polypeptide level can be regulated using the agents and pharmaceutical
compositions of the invention, by genetic alteration, by altering the ratio of
isoforms
and/or their absolute expression. In one embodiment, isoforms PDE4D7 and/or
PDE4D9 can be regulated.
The invention further provides a method of diagnosing susceptibility to stroke
in an individual. This method comprises screening for one of the at-risk
haplotypes
in the phosphodiesterase 4D gene that is more frequently present in an
individual
susceptible to stroke, compared to the frequency of its presence in the
general
population, wherein the presence of an at-risk haplotype is indicative of a
susceptibility to stroke. An "at-risk haplotype" is intended to embrace one or
a
combination ofhaplotypes described herein over the PDE4D gene that show high
correlation to stroke. In one embodiment, the at-risk haplotype is
characterized by
the presence of at least one single nucleotide polymorphism at nucleic acid
positions
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at risk haplotype 1 is G at nucleic acid position 142780 respectively,
relative to SEQ
ID NO: 1 and allele 0 of microsatellite marker AC0088181-1. In another
embodiment, the at-risk haplotype 2 is characterized by the presence of at
least one
single nucleotide polymorphism and microsatellite marker at nucleic acid
positions
142780, 135112, 132562, 131865, 129361, 129360, 125304, 123426, 123312,
120628, 118914, 111781, 111252, 109301, 107849, 105225, 104552, 102977,
100795, 99035, 88614, 88456, 83119, 82244, 80127, 78552, relative to SEQ ID
NO:
1 and allele 0 microsatellite marker AC0088181-1.
In yet another embodiment, the at-risk haplotype 3 is characterized by the
presence of at least one polymorphism at nucleic acid positions 138806,
131865,
129361, 120628, 91470 relative to SEQ ID NO: 1.
Also described are methods for diagnosing susceptibility to stroke in an
individual comprising screening for an at-risk haplotype in the
phosphodiesterase 4D
gene that is more frequently present in an individual susceptible to stroke
(affected),
compared to the frequency of its presence in a healthy individual (control)
wherein
the screening for the presence of an at-risk haplotype within or near PDE4D
that
significantly correlates with at least one of the haplotypes described herein
or stroke
susceptibility. As an example of a simple test for correlation would be a
Fisher-exact
test on a two by two table. Given a cohort of chromosomes the two by two table
is
constructed out of the number of chromosomes that include both of the
haplotypes,
one of the haplotype but not the other and neither of the haplotypes.
A protective haplotype is intended to embrace one or a combination of
haplotypes described herein over the PDE4D gene that show a protective
characteristic or property of a reduced risk of stroke. The particular
combination of
genetic markers (haplotypes) are present at a higher than expected frequency
in
controls than patients. Individuals with a protective allele or haplotype are
about
30% less likely to have a stroke compared to the general population. In one
embodiment, a protective haplotype is characterized by the presence of at
least one
single nucleotide polymorphism, such as the allele A at nucleotide position
142780
relative to SEQ ID NO: 1. The presence of the polymorphisms that comprise the
at-
risk haplotype or protective haplotype can be determined by electrophoretic
analysis,
restriction length polymorphism analysis, fluorescence energy transfer
detection,
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kinetic PCR, allele specific PCR, sequence analysis, hybridization analysis or
other
known techniques.
Kits for diagnosing susceptibility to stroke in an individual are also
disclosed
and comprise primers for nucleic acid amplification of a region of PDE4D
comprising the at-risk haplotype and/or protective haplotype.
The first major application of the current invention involves prediction of
those at higher risk of developing a stroke. Diagnostic tests that define
genetic
factors contributing to stroke might be used together with or independent of
the
known clinical risk factors to define an individual's risk relative to the
general
population. Better means for identifying those individuals at risk for stroke
should
lead to better prophylactic and treatment regimens, including more aggressive
management of the current clinical risk factors such as hypertension,
diabetes,
hypercholesterolemia, hypertriglyceridemia, obesity, and inflammatory
components
as reflected by increased C-reactive protein levels or other inflammatory
markers.
Information on genetic risk may be used by physicians to help convince
particular
patients to adjust life style and quit smoking. This invention provides the
means to
define a genetic component that doubles an individual's risk for stroke. Also
described are means to define the genetic components that protect an
individual from
stroke.
The second major application of the current invention is the specific
identification of a rate-limiting pathway involved in stroke. While many have
attempted to find genes that are over-expressed or under-expressed in
atherosclerosis
plaques in the carotid arteries, the vast majority of the changes seen in
diseased blood
vessels compared to normal blood vessels are simply a reaction to the
underlying
process of atherosclerosis and stroke predisposition and are not the
underlying cause.
A disease gene with genetic variation that is significantly more common in
stroke
patients as compared to controls represents a specifically validated causative
step in
the pathogenesis of stroke. That is, the uncertainty about whether a gene is
causative
or simply reactive to the disease process is eliminated. The protein encoded
by the
disease gene defines a rate-limiting molecular pathway involved in the
biological
process of stroke predisposition. The proteins encoded by such stroke genes or
its
interacting proteins in its molecular pathway may represent drug targets that
may be
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selectively modulated by small molecule, protein, antibody, or nucleic acid
therapies.
Such specific information is greatly needed since strolce prevention and
treatment is a
major unmet medical need that affects over a half million Americans each year.
Also useful is determining the gene that is protective against stroke. The
proteins
encoded by the protective gene and the biological pathway that it is a member
may
represent another target selectively modulated by small molecule, protein
antibody or
nucleic acid therapies.
A third application of the current invention is its use to predict an
individual's
response to a particular drug, even drugs that do not act on PDE4D or its
pathway. It
is a well-known phenomenon that in general, patients do not respond equally to
the
same drug. Much of the differences in drug response to a given drug is thought
to be
based on genetic and protein differences among individuals in certain genes
and their
corresponding pathways. Our invention defines the PDE4D pathway and its effect
on cAMP levels in cells where it is expressed as one key molecular pathway
involved in stTOke risk. Some current or future therapeutic agents may be able
to
affect this pathway directly or indirectly and therefore, be effective in
those patients
whose stroke risk is in part determined by PDE4D pathway genetic variation. On
the
other hand, those same drugs may be less effective or ineffective in those
patients
who do not have at risk variation in the PDE4D gene or pathway. Therefore,
PDE4D
variation or haplotypes may be used as a pharmacogenomic diagnostic to predict
drug response and guide choice of therapeutic agent in a given individual.
The invention helps meet the unmet medical needs in at least two major
ways: 1) it provides a means to define patients at higher risk for stroke than
the
general population who can be more aggressively managed by their physicians in
an
effort to prevent stroke; and 2) it defines a drug target that can be used to
screen and
develop therapeutic agents that can be used to prevent stroke before it
happens or
prevent a second stroke in those who have already suffered a stroke or
transient
ischemic attack.
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9
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 certain
embodiments
of the invention, as illustrated in the accompanying drawings.
FIGS. 1.1 and 1.2 show two family pedigrees each affected by several of the
strolce subtypes, including hemorrhagic strolce.
FIGS. 2.1, 2.2 and 2.3 show the genetic, combined and physical maps for
locating the PDE4D gene using 30 polymorphic markers. For the combined map,
all
markers have been assigned in the genetic and physical map unless otherwise
indicated (* indicates marker only assigned in the physical map; ** indicates
markers
only assigned in genetic map).
FIG. 3 shows the schematic representations of PDE4D splice variants. Splice
variants PDE4D9 are novel, as well as exons D7A-1, D7A-2, D7A-3, D8 and D9.
Splice variants 4DN1, 4DN2 and 4DN3 (Miro, et al., Bioclzena. Bioplays. Res.
Cor~afn., 274: 415-421 (2002), and 4D1, 4D2, 4D3, 4D4 and 4D5 are known
(Bolger
et al., Biochena. J. pt. 2: 539-548 (1997).
FIG. 4 is a graphic representation showing PDE4D isoform expression in
EBV transformed cells (expression of PDE4D3 and PDE4D9 below detection
limits).
FIG. 5 is a graphic representation showing expression of PDE4D isoforms in
EBV transformed cells from patients with or without the stroke-associated
haplotype.
FIG. 6 is a graphic representation showing expression of PDE4D isoforms in
EBV cells from controls with or without the stroke-associated haplotype.
FIGS. 7.1 to 7.10 show the amino acid sequences for the isofonns of the
PDE4D gene. SEQ ID NO: 2 is D4; SEQ ID NO: 3 is N2; SEQ ID NO: 4 is D5;
SEQ ID NO: 5 is N3; SEQ ID NO: 6 is D3; SEQ ID NO: 7 is N1; SEQ TD NO: 8 is
D8; SEQ TD NO: 9 is Dl; and SEQ ID NO: 10 is D2.
FIGS. 8.1 and 8.2 list all publicly available PDE4D mRNAs and novel cDNA
segments identified by deCODE genetics.
FIGS. 9.1 to 9.351 show the genomic sequence of the human PDE4D gene.
FIGS. 10.1 to 10.3 show a graphic representation showing the single marker
allelic association within the PDE4D gene. FIG. 10.1 is a schematic showing
the
gene structures. FIG. 10.2 shows graphic representation of the microsatellite
and
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SNP distribution within thePDE4D gene. FTG. 10.3 shows graphic representation
of
the single marker allelic association across the PDE4D gene for both
microsatellites
(filled circles) and SNPs (open circles); negative log p-valve versus the
physical
location in kilobases.
5 FIGS. 11.1 to 11.3 graphically depict the haplotype association for carotid
and cardiogenic stroke combined. Estimated haplotype frequencies for patients
and
controls respectively, are indicated within parentheses. FIG. 11.1 is a
comparison of
groups of haplotypes constructed from SNP45 and AC008818-1, two markers
separated by 6kb. Note that X is a co~aposite allele that denotes jointly all
alleles of
10 AC0088 t 8-1 except allele 0. Apart from haplotype AO that is not found in
our
samples, other haplotypes can be grouped into three groups with distinct
risks. Each
arrow corresponds to a comparison between two groups and RR is the estimated
risk
of the group the arrow is pointing at relative to the other group. The
difference
between 1 and the information (Info) is a measure of the fraction of
information that
is lost due to uncertainty in phase and missing genotypes. FIG. 11.2 shows
intermediate results when the investigation is extended from SNP45 and
AC008818-
1, which are both in LD block B, to include 25 SNPs in LD block C. He is the
at-
risk haplotype, identified in FIG. 13 and L~ is a composite haplotype that
denotes
jointly all haplotypes of the 25 SNPs except H~. Together with AC008818-1 and
SNP45, the haplotypes here span 64kb. Haplotype GO in A is split info extended
haplotypes GOH~ and GOLD. GOH~ has significantly higher risk than GOLD, and
the
risk of GOLD is not distinguishable from the wild type GX. FIG. 11.3 shows a
refinement of the groupings in A- GOLD is moved from the at-risk group to the
wild type group. Also noted is that the extended haplotype AXH~ does not exist
indicating that blocks B and C are in LD.
FIG. 12 is a schematic representation of the physical map of STRKl interval
showing all genes and mRNAs in region. Markers identified with an asterisk (*)
indicate those with significant single marker association.
FIGS 13.1 to 13.3 show a graphical depiction of the linkage disequilibrium
(LD) and haplotypes in the 5'end of PDE4D gene. FIG. 13.1 shows pairwise
linkage
disequilibrium between SNPs in a 600 kb region in the 5' end of PDE4D. The
markers are plotted equidistant. Two measures of LD are shown: D' in the upper
left
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11
triangle and p-values in the lower right triangle. This region can be divided
into
three blocks of strong LD, each with limited haplotype diversity, block A,
block B
and block C. The lines indicate the position of the three exons D7-1, D7-2 and
D7-3
and the microsatellite marker AC008818-1. FIG. 13.2 show all common haplotypes
identified within each of the three blocks. Association results for all the
haplotypes
are presented in Table 2C. FIG. 13.3 depicts the percentage of chromosomes
within
each block that match one of the common haplotypes.
DETAILED DESCRIPTION OF THE INVENTION
The first major stroke locus, STRKl, was mapped to Sql2 using a genome-
wide search for susceptibility genes in the common forms of stroke. A broad
but
rigorous definition of the phenotype Was used including patients with ischemic
stroke, transient ischemic attack (TIA.), and hemorrhagic stroke. The lod
score after
adding a higher density of markers (one marker every 1 cM) was 4.40 (P=3.9x10-
6)
I S at marker DSS2080. The lod score increased to 4.9 after the hemorrhagic
stroke
patients were removed, suggesting that the gene at the locus is primarily
important '
for ischemic stroke. The most promising region harboring a stroke
susceptibility
gene was narrowed down to a segment less than 6 cM (approximately 3.8 Mb),
from
DSS 14'4 to DSS398, as defined by a decrease of one in LOD scare (will be
referred
to as the "one-LOD interval" hereafter).
We describe here the positional cloning of a stroke susceptibility gene
located
in the STRKI locus. This region was extensively fine-mapped and tested for
association to stroke. The strongest association found in the one-LOD interval
was
within the phosphodiesterase 4D gene (PDE4D), a member of the Iarge
superfamily
of cyclic nucleotide phosphodiesterases. The strongest signal observed at
PDE4D
was to the two major subtypes of ischemic stroke, carotid and cardiogenic
stroke.
Relative expression of PDE4D isoforms correlated with stroke and with the
genetic ,
variation within PDE4D which is associated to stroke. Our results suggest that
this
gene is involved in pathogenesis of stroke through atherosclerosis, the major
pathological process underlying stroke.
Our results also indicate that genetic variation in the PDE4D gene is
associated
with ischemic stroke. The direct involvement of PDE4D is strongly supported by
both
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12
linkage and haplotype association. Multiple markers and haplotypes within the
PDE4D
gene show strong association to stroke. The haplotypes can be classified into
three
distinct groups, wild type, at-risk and protective. We first identified the
association
using microsatellite marlcers, and supplementing the microsatellite data with
a denser set
of SNPs further supported this. The strongest association was to the two
ischemic
subtypes, carotid and cardiogenic stroke. This gene shows no association to
small vessel
occlusive disease, the form of stroke thought to be independent of
atherosclerosis.
Haplotype analyses show that the most significant haplotype extends over an
area of 260
kb covering the first exon of the PDE4D gene. The haplotype is significantly
associated
I O to carotid and cardiogenic stroke with a relative risk of 2.3 and
approximately 4'~ % of
carotid/cardiogenic stroke patients carry at least one copy of this haplotype.
This same
haplotype has a relative risk of 1.~ for stroke in general. This haplotype
extends over the
5'exon unique to the PDE4D7 isoform and the presumed promoter region of this
isoform
suggesting that the functional variation rnay be involved in transcriptional
regulation.
This hypothesis is also supported by our PDE4D expression analysis that shows
that
there is significant correlation between the disease associated haplotype and
the level of
PDE4D7 message.
The strongest association found for this PDE4D haplotype was to the two
major subtypes of ischemic stroke, carotid and cardiogenic stroke suggesting a
role
for this gene in the vascular biology of atherosclerosis. While there are
multiple
etiologies for ischemic stroke, atherosclerosis remains the most important
one.
Atherosclerosis is a chronic progressive disease characterized by accumulation
of
lipids, fibrous, and cellular elements within the large arteries. These
lesions can
grow sufficiently large to impede blood flow and, more importantly, their
surfaces
can rupture leading to local thrombus formation occluding the blood vessel and
causing a stroke or myocardial infarction. The major pathological process for
the
two ischemic subtypes, carotid and cardiogenic stroke is atherosclerosis.
First, it is
the major cause of stenotic and occlusive lesions of the internal and common
carotids
that lead to carotid strokes. Second, cardiac thrombi which shed emboli to the
brain
most commonly occur on the background of coronary artery disease, such as
following acute myocardial infarction or ischemic cardiomyopathy, and/or due
to
atrial fibrillation on the basis of poor compliance of ischemic ventricles
(diastolic
dysfunction/stiffening). Although atrial fibrillation may occur on the
background of
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13
other diseases such as valvular disease, hyperthyroidism, and hypertension, in
the
age group that tends to suffer from stroke, ischemic heart disease remains one
of the
most important causes. Ischemic stroke resulting from occlusion of small
penetrating arteries within the brain (small vessel occlusive disease or
lacunar stroke)
is generally thought to result from local endothelial proliferation since
atherosclerosis only occurs in larger arteries. PDE4D does not show
association to
small vessel stroke, consistent with it role in atherosclerosis. In summary,
atherosclerosis accounts for the majority of all strokes, particularly carotid
and
cardiogenic stroke, two subphenotypes that show the strongest association to
the
PDE4D gene.
REPRESENTATIVE TARGET POPULATION
An individual at risk for stroke is an individual who has at least one risk
factor, such as previous stroke or TIA, an at-risk haplotype in one or more
stroke risk
genes, an at-risk haplotype for the PDE4D gene; a polymorphism in a PDE4D
gene;
disregulation of PDE4D isoform expression; diabetes; hypertension;
hypercholesterolemia; elevated Ip(a); obesity; a past or current smoker; an
elevated
inflammatory marker (e.g., a marker such as C-reactive protein (CRP), serum
amyloid A, fibrinogen, tissue necrosis factor-alpha, a soluble vascular cell
adhesion
molecule (sVCAM), a soluble intervascular adhesion molecule (sICAM), E-
selectin,
matrix metalloprotease type-l, matrix metalloprotease type-2, matrix
metalloprotease
type-3, and matrix metalloprotease type-9); increased LDL cholesterol andlor
decreased HDL cholesterol; and/or at least one previous myocardial infarction,
concurrent MI, acute coronary syndrome, stable angina, atherosclerosis,
carotid
stenosis, peripheral vascular occlusive disease, or requires treatment for
restoration
of coronary artery blood flow (e.g., angioplasty, stmt, coronary artery bypass
graft).
An individual who has a protective haplotype is one who is less likely to have
a stroke. In another embodiment of the invention, an individual who is at risk
for
stroke is an individual who has a polymorphism in a PDE4D gene, in which the
presence of the polymorphism is indicative of a susceptibility to stroke. An
individual who has a protective haplotype and less likely to have a stroke is
an
individual who has a polymorphism in a PDE4D gene such as the A allele at
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14
nucleotide position 142780 relative to SEQ ID NO: 1, in which the presence of
the
polymorphism is indicative of a protection from stroke. The term "gene," as
used
herein, refers to not only the sequence of nucleic acids encoding a
polypeptide, but
also the promoter regions, transcription enhancement elements, splice
donor/acceptor
sites, splice enhancer and silencer sequences and other regulators of
splicing, and
other non-transcribed nucleic acid elements. Representative polymorphisms
include
those presented in Table 11, below.
In one embodiment of the invention, an individual who is at risk for stroke is
an individual Who has an at-risk haplotype in PDE4D, as described herein,
particularly but not limited to ischemic stroke. Increased risk for the two
major
subtypes of ischemic stroke, carotid and cardiogenic stroke, can be assessed
by
screening for at-risk haplotype that comprises SNPSPDM361194,
SNPSPDM368135, SNPSPDM370640, SNPSPDM379372 and SNPSPDM408531 at
the 5' UTR of PDE4D7. Results reported herein indicate that PDE4D is involved
in
pathogenesis of stroke through atherosclerosis. The major pathological process
for
carotid stroke and cardiogenic stroke is atherosclerosis. Thus, an individual
who is
at-risk for atherosclerosis, peripheral arterial occlusive disease, or
myocardial
infarction can also benefit from the teachings of the invention.
ASSESSMENT FOR AT-RISK. AND PROTECTIVE HAPLOTYPES
A "haplotype," as described herein, refers to a combination of genetic
markers ("alleles"), such as those set forth in Tables 1, 2C, 4A and 4B. In a
certain
embodiment, the haplotype can comprise one or more alleles, two or more
alleles,
three or more alleles, four or more alleles, or five or more alleles. The
genetic
markers are particular "alleles" at "polymorphic sites" associated with PDE4D.
A
nucleotide position at which more than one sequence is possible in a
population
(either a natural population or a synthetic population, e.g., a library of
synthetic
molecules), is referred to herein as a "polymorphic site". Where a polymorphic
site
is a single nucleotide in length, the site is referred to as a single
nucleotide
polymorphism ("SNP"). For example, if at a particular chromosomal location,
one
member of a population has an adenine and another member of the population has
a
thymine at the same position, then this position is a polymorphic site, and,
more
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specifically, the polymorphic site is a SNP. Polymorphic sites can allow for
differences in sequences based on substitutions, insertions or deletions. Each
version
of the sequence with respect to the polymorphic site is referred to herein as
an
"allele" of the polymorphic site. Thus, in the previous example, the SNP
allows for
S both an adenine allele and a thymine allele.
Typically, a reference sequence is referred to for a particular sequence.
Alleles that differ from the reference are referred to as "variant" alleles.
For
example, the reference PDE4D sequence is described herein by SEQ >D NO: 1. The
term, "variant PDE4D", as used herein, refers to a sequence that differs from
SEQ m
10 NO: l, but is otherwise substantially similar. The genetic markers that
make up the
haplotypes described herein are PDE4D variants.
Additional variants can include changes that affect a polypeptide, e.g., the
PDE4D polypeptide. These sequence differences, when compared to a reference
nucleotide sequence, can include the insertion or deletion of a single
nucleotide, or of
1 S more than one nucleotide, resulting in a frame shift; the change of at
least one
nucleotide, resulting in a change in the encoded amino acid; the change of at
least
one nucleotide, resulting in the generation of a premature stop codon; the
deletion of
several nucleotides, resulting in a deletion of one or more amino acids
encoded by
the nucleotides; the insertion of one or several nucleotides, such as by
unequal
recombination or gene conversion, resulting in an interruption of the coding
sequence of a reading frame; duplication of all or a part of a sequence;
transposition;
or a rearrangement of a nucleotide sequence, as described in detail above.
Such
sequence changes alter the polypeptide encoded by a PDE4D nucleic acid. For
example, if the change in the nucleic acid sequence causes a frame shift, the
frame
2S 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 stroke or a susceptibility to
stroke can
be a synonymous change in one or more nucleotides (i.e., a change that does
not
result in a change in the amino acid sequence). Such a polymorphism can, for
example, alter splice sites, affect the stability or transport of mRNA, or
otherwise
affect the transcription or translation of the polypeptide. The polypeptide
encoded by
the reference nucleotide sequence is the "reference" polypeptide with a
particular
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16
reference amino acid sequence, and polypeptides encoded by variant alleles are
referred to as "variant" polypeptides with variant amino acid sequences.
Haplotypes are a combination of genetic markers, e.g., particular alleles at
polymorphic sites. The haplotypes described herein, e.g., having markers such
as
those shown in Table 3, Table 4A and 4B, are found more frequently in
individuals
with stroke than in individuals without stroke. Therefore, these haplotypes
have
predictive value for detecting stroke or a susceptibility to stroke in an
individual.
The haplotypes described herein are a combination of various genetic markers,
e.g.,
SNPs and microsatellites. Therefore, detecting haplotypes can be accomplished
by
methods known in the art for detecting sequences at polymorphic sites, such as
the
methods described above.
In certain methods described herein, an individual who is at risk for stroke
is
an individual in whom an at-risk haplotype is identified. In one embodiment,
the at
' risk haplotype is one that confers a significant risk of stroke. In one
embodiment,
significance 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 least about 1.2, including
but 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 not limited to about 25%, 30%, 35%, 40%,
45%,
SO%, SS%, 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.
An at-risk haplotype in, or comprising portions of, the PDE4D gene, is one
where the haplotype is more frequently present in an individual at risk for
stroke
(affected), compared to the frequency of its presence in a healthy individual
(control), and wherein the presence of the haplotype is indicative of stroke
or
susceptibility to stroke. A protective haplotype in or comprising portions of
the
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17
PDE4D gene is one where the haplotype is more frequently present in an
individual
where the haplotype is protective against being affected by stroke compared to
the
frequency of its presence in an individual with stroke. The presence of the
haplotype
is indicative of a protection from strolce or protection from susceptibility
to stroke as
described above.
Standard techniques for genotyping for the presence of SNPs and/or
microsatellite markers can be used, such as fluorescent-based techniques
(Chen, et
al., C~efaome Res. 9, 492 (1999)), PCR, LCR, Nested PCR and other techniques
for
nucleic acid amplification. In one embodiment, the method comprises assessing
in
an individual the presence or frequency of SNPs and/or microsatellites in,
comprising portions of, the PDE4Dgene, wherein an excess or higher frequency
of
the SNPs and/or microsatellites compared to a healthy control individual is
indicative
that the individual has stroke, or is susceptible to stroke. See, for example,
Table l,
Table 2C, Table 2D, Table 3, Table 4A and 4B (below) for SNPs and markers that
can form haplotypes that can be used as screening tools. These markers and
SNPs
can be identified in at-risk haploptypes. For example, an at-risk haplotype
can
include microsatellite markers and/or SNPs such as those set forth in Table
2C, Table
4B and 4B. The presence of the haplotype is indicative of stroke, or a
susceptibility
to stroke, and therefore is indicative of an individual who falls within a
target
population for the treatment methods described herein.
Haplotype analysis first involves defining a candidate susceptibility locus
using LOD scores. The defined regions are then ultra-fine mapped with
microsatellite markers with an average spacing between markers of less than
100kb.
All usable microsatellite markers that found in public databases and mapped
within
that region can be used. In addition, microsatellite markers identified within
the
deCQDE genetics sequence assembly of the human genome can be used. The
frequencies of haplotypes in the patient and the control groups using an
expectation-
maximization algorithm can be estimated (Dempster A. et al., 1977. J. R. Stat.
Soc.
B, 39:1-3~9). An implementation of this algorithm that can handle missing
genotypes and uncertainty with the phase can be used. Under the null
hypothesis, the
patients and the controls are assumed to have identical frequencies. Using a
likelihood approach, an alternative hypothesis where a candidate at-risk-
haplotype,
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18
which can include the markers described herein, 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 is tested. Likelihoods
are
maximized separately under both hypotheses 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 or protective haplotypes, for
example, association of all possible combinations of genotyped markers is
studied,
provided those maxkers span a practical region. The combined patient and
control
groups can be randomly divided into two sets, equal in size to the original
group of
patients and controls. The haplotype analysis is then repeated and the most
significant p-value registered is determined. This randomization scheme can be
repeated, for example, over 100 times to construct an empirical distribution
of p-
values.
In one embodiment, the at-risk haplotype is characterized by the presence of
the polymorphism(s) represented by one or a combination of single nucleotide
polymorphisms at nucleic acid positions 1425923, 1415979, 1414804, 1371388,
1307403 and 1257206, relative to SEQ ID NO: 1. In another embodiment, a
diagnostic method for susceptibility to stroke can comprise determining the
presence
of at-risk haplotype represented by one or a combination of single nucleotide
polymorphisms and microsatellite markers at nucleic acid positions 263539,
252772,
189780, 175259, 171240, 136550 and 120628, relative to SEQ 1D NO: 1. In
another
embodiment, the at-risk haplotype is characterized by the following SNPs:
SNP5PDM361194, SNP5PDM368135, SNP5PDM370640, SNP5PDM379372, and
SNPSPDM408531. In one embodiment, the protective haplotype comprises the A
allele of SNP45 at position 142780 relative to SEQ I~ NO: 1. This haplotype is
particularly useful for assessing susceptibility to the two major subtypes of
ischemic
stroke, carotid and cardiogenic stroke. In another embodiment, an at-risk
haplotype,
particularly for carotid and cardiogenic stroke, is characterized by use of
microsatellite marker AC008818-1 to define the presence of an at-risk allele.
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NUCLEIC ACID THERAPEUTIC AGENTS
In another embodiment, 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); or a nucleic acid encoding a
PDE4D
polypeptide, can be used in "antisense" therapy, in which a nucleic acid
(e.g., an
oligonucleotide) which specifically hybridizes to the mRNA and/or genomic DNA
of
a nucleic acid is administered or generated in situ. The antisense nucleic
acid that
specifically hybridizes to the mRNA andlor DNA inhibits expression of the
polypeptide encoded by that mRNA and/or DNA, e.g., by inhibiting translation
and/or transcription. Binding of the antisense nucleic acid can be by
conventional
base pair complementarity, or, for example, in the case of binding to DNA
duplexes,
through specific intexaction in the major groove of the double helix.
An antisense construct can be delivered, for example, as an expression
plasmid as described above. When the plasmid is transcribed in the cell, it
produces
RNA that is complementary to a portion of the mRNA andlor DNA that encodes a
PDE4D 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 polypeptide. In one
embodiment, the oligonucleotide probes are modified oligonucleotides that are
resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases,
thereby
rendering them stable ifZ vivo. Exemplary nucleic acid molecules for use as
antisense
oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate
analogs of DNA (see also U.S. Patent 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. (Cancer 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 polypeptide. The
antisense
oligonucleotides bind to mRNA transcripts and prevent translation. Absolute
complementarity, although preferred, is not required. A sequence
"complementary"
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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
5 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 skilled in
the art can
ascertain a tolerable degree of mismatch by use of standard procedures.
10 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
15 peptides (e.g. for targeting host cell receptors in vivo), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger et al., P~oc. Natl. Acad. Sci.
USA
86:6553-6556 (1989); Lemaitre et al., Pf-oc. Natl. Acad. Sci. USA 84:648-652
(1987);
PCT International Publication No. WO 88/09810) or the blood-brain barrier
(see,
e.g., PCT International Publication No. WO 89/10134), or hybridization-
triggered
20 cleavage agents (see, e.g., Krol et al., BioTeclaniques 6:958-976 (1988))
or
intercalating agents. (See, e.g., Zon, Pharm.Res. 5: 539-549 (1988)). To this
end, 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 a PDE4D
polypeptide ifs vivo. A number of methods can be used for delivering antisense
DNA
or RNA to cells; e.g., antisense molecules can be injected directly into the
tissue site,
or modified antisense molecules, designed to target the desired cells (e.g.,
antisense
linked to peptides or antibodies that specifically bind receptors or antigens
expressed
on the target cell surface) can be administered systematically. Alternatively,
in a
another 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
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21
117. The use of such a construct to transfect target cells in the patient
results in the
transcription of sufficient amounts of single stranded RNAs that will form
complementary base pairs with the endogenous transcripts and thereby prevent
translation of the mRNA. For example, a vector can be introduced ira 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 by recombinant DNA technology methods standard in the art and
described above. For example, a plasmid, cosmid, YAC or viral vector can be
used
to prepare the recombinant DNA construct that can be introduced directly into
the
tissue site. Alternatively, viral vectors can be used which selectively infect
the
desired tissue, in which case administration may be accomplished by another
route
(e.g., systemically).
In another embodiment of the invention, small double-stranded interfering
RNA (RNA interference (RNAi)) can be used. RNAi is a post-transcription
process,
in which double-stranded RNA is introduced, and sequence-specific gene
silencing
results, though catalytic degradation of the targeted mRNA. See, e.g.,
Elbashir,
S.M. et al., Nature 411:494-498 (2001); Lee, N.S., Nature Biotech. 19:500-505
(2002); Lee, S-K. et al., Nature Medicisze ~(7): 681-686 (2002); the entire
teachings
of these references are incorporated herein by reference.
Endogenous expression of a gene product can also be reduced by inactivating
or "knocking out" the gene or its promoter using targeted homologous
recombination
(e.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell
51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989)). For example, an
altered, non-functional gene (or a completely unrelated DNA sequence) flanked
by
DNA homologous to the endogenous gene (either the coding regions or regulatory
regions of the gene) can be used, with or without a selectable marker and/or a
negative selectable marker, to transfect cells that express the gene ih vivo.
Insertion
of the DNA construct, via targeted homologous recombination, results in
inactivation
of the gene. The recombinant DNA constructs can be directly administered or
targeted to the required site i~a vivo using appropriate vectors, as described
above.
Alternatively, expression of non-altered genes can be increased using a
similar
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22
method: targeted homologous recombination can be used to insert a DNA
construct
comprising a non-altered functional gene, or the complement thereof, or a
portion
thereof, in place of an gene in the cell, as described above. In another
embodiment,
targeted homologous recombination can be used to insert a DNA construct
comprising a nucleic acid that encodes a polypeptide variant that differs from
that
present in the cell.
Alternatively, endogenous expression of a gene product can be reduced by
targeting deoxyribonucleotide sequences complementary to the regulatory region
(i.e., the promoter and/or enhancers) to form triple helical structures that
prevent
transcription of the gene in target cells in the body. (See generally, Helene,
C.,
Anticancer Drug Des., 6(6):569-84 (1991); Helene, C. et al., Ah~.. 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 the gene product, can be used in the manipulation of tissue, e.g., tissue
differentiation, both ifa vivo and for 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 a nucleic acid RNA or
nucleic acid sequence) can be used to investigate the role of one or more
members of
the PDE4D pathway in the development of disease-related conditions. Such
techniques can be utilized in cell culture, but can also be used in the
creation of
transgenic animals.
The therapeutic agents as described herein can be delivered in a composition,
as described above, or alone. They can be administered systemically, or can be
targeted to a particular tissue. The therapeutic agents can be produced by a
variety of
means, including chemical synthesis; recombinant production; in vivo
production
(e.g., a transgenic animal, such as U.S. Patent No. 4,873,316 to Meade et
al.), for
example, and can be isolated using standard means such as those described
herein.
In addition, a combination of any of the above methods of treatment (e.g.,
administration of non-altered polypeptide in conjunction with antisense
therapy
targeting altered mRNA; administration of a first splicing variant in
conjunction with
antisense therapy targeting a second splicing variant) can also be used.
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The invention additionally pertains to use of such therapeutic agents, as
described herein, for the manufacture of a medicament for the treatment of
stroke,
TIA., MI, and/or atherosclerosis, e.g., using the methods described herein.
MONITORING PROGRESS OF TREATMENT
The current invention also pertains to methods of monitoring the
effectiveness of treatment on the regulation of expression (e.g., relative or
absolute
expression) of one or more PDE4D isoforms at the RNA or protein level or its
enzymatic activity. PDE4D message or protein or enzymatic activity can be
measured in a sample of peripheral blood or cells derived therefrom. An
assessment
of the levels of expression or activity can be made before and during
treatment with
PDE4D therapeutic agents.
For example, in one embodiment of the invention, an individual who is a
member of the target population can be assessed for response to treatment with
a
PDE4D inhibitor, by examining cAMP levels or PDE4D enz~nnatic activity or
absolute and/or relative levels of PDE4D protein or mRNA isofonns in
peripheral
blood iii general or specific cell subfractions or combination of cell
subfractions. In
addition, variation such as haplotypes or mutations within or near (within 100
to
200kb) of the PDE4D gene may be used to identify individuals who are at higher
risk
for stroke or TIA to increase the power and efficiency of clinical trials for
pharmaceutical agents to prevent or treat first or subsequent stroke. The
haplotypes
and other variations may be used to exclude or fractionate patients in a
clinical trial
who are likely to have non-CAMP or non-PDE4D pathway involvement in their
stroke risk in order to enrich patients who have other pathways involved and
boost
the power and sensitivity of the clinical trial. Such variation may be used as
a
pharmacogenomic test to guide selection of pharmaceutical agents for
individuals.
NUCLEIC ACIDS OF THE INVENTION
Nucleic Acids, Po3~tioras and Variants
All nucleotide positions are relative to SEQ ID NO: 1. The nucleic acids,
polypeptides and antibodies described herein can be used in methods of
diagnosis of
susceptibility to stroke, as well as in kits useful for diagnosis of a
susceptibility to
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stroke. In addition, the invention pertains to isolated nucleic acid molecules
comprising a human PDE4D nucleic acid. The term, "PDE4D nucleic acid," as used
.herein, refers to an isolated nucleic acid molecule encoding PDE4D
polypeptide.
The PDE4D 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 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 further comprise additional non-coding sequences such as introns
and
non-coding 3' and 5' sequences (including regulatory sequences, for example,
as well
as promoters, transcription enhancement elements, splice donor/acceptor sites,
etc.).
For example, a PDE4D nucleic acid can comprise the nucleic acid of SEQ m NO: 1
which may optionally comprise at least one polymorphism as shown in Tables 11
and 12, 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 PDE4D
polypeptide.
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 nucleotide sequence (as in
genomic sequences) and/or has been completely or partially purified from other
transcribed sequences (e.g., as in an RNA library). For example, an isolated
nucleic
acid of the invention may be substantially isolated with respect to the
complex
cellular milieu in which it naturally occurs, or culture medium when produced
by
recombinant techniques, or chemical precursors 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.
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Preferably, an isolated nucleic acid molecule comprises at least about S0, 80
or 90%
(on a molar basis) of all macromolecular species present. With regard to
genomic
DNA, the term "isolated" also can refer to nucleic acid molecules that are
separated
from the chromosome with which the genomic DNA is naturally associated. For
5 example, the isolated nucleic acid molecule can contain less than about 5
kb, 4 kb, 3
kb; 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which flank the nucleic acid
molecule
in the genomic DNA of the cell from which the nucleic acid molecule is
derived.
The nucleic acid molecule can be fused to other coding or regulatory
sequences and still be considered isolated. Thus, recombinant DNA contained in
a
10 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 ih vivo-and isa vitf~o RNA
transcripts of the DNA molecules of the present invention. An isolated nucleic
acid
15 molecule or nucleotide sequence can include a nucleic acid molecule or
nucleotide
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
20 DNA molecules in solution. Ifa 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 ifa situ hybridization with
25 chromosomes), or for detecting expression of the gene in tissue (e.g.,
human tissue),
such as by Northern blot analysis.
The present invention also pertains to variant nucleic acid molecules which
are not necessarily found in nature but which encode a PDE4D polypeptide
(e.g., a
polypeptide having the amino acid sequence of SEQ 1D NO: 2, 3, 4, 5, 6, 7, 8,
9, 10,
12 or 14), or another splicing variant of PDE4D polypeptide or polymorphic
variant
thereof. Thus, for example, DNA molecules which comprise a sequence that is
different from the naturally-occurring nucleotide sequence but which, due to
the
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26
degeneracy of the genetic code, encode a PDE4D polypeptide of the present
invention are also the subject of this invention. The invention also
encompasses
nucleotide sequences encoding portions (fragments), or encoding variant
polypeptides such as analogues or derivatives of the PDE4D polypeptide. Such
variants can be naturally-occurring, such as in the case of allelic variation
or single
nucleotide polymorphisms, or non-naturally-occurring, such as those induced by
various mutagens and mutagenic processes. Intended variations include, but are
not
limited to, addition, deletion and 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 the
PDE4D polypeptide. In one embodiment, the nucleotide sequences are fragments
that comprise one or more polymorphic microsatellite markers. In another
embodiment, the nucleotide sequences are fragments that comprise one or more
single nucleotide polymorphisms in the PDE4D gene.
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 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
nucleotide sequence described herein (e.g., nucleic acid molecules which
specifically
hybridize to a nucleotide sequence encoding polypeptides described herein,
and,
optionally, have an activity of the polypeptide). In one embodiment, the
invention
includes variants described herein which hybridize under high stringency
hybridization conditions (e.g., for selective hybridization) to a nucleotide
sequence
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27
comprising a nucleotide sequence selected from SEQ m NO: 1 which may
optionally comprise at least one polymorphism as shown in Tables 11 and 12 or
the
complement thereof. In another embodiment, the invention includes variants
described herein which hybridize under high stringency hybridization
conditions
(e.g., for selective hybridization) to a nucleotide sequence encoding an amino
acid
sequence selected from SEQ m NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14 or
polymorphic variant thereof. In another embodiment, the protein product of the
variant that hybridizes under high stringency conditions has an activity of
PDE4D.
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 similarity to the second nucleic acid than to any other nucleic
acid in a
sample wherein the hybridization is to be performed). "Stringency conditions"
for
hybridization is a term of art which refers to the incubation and wash
conditions, e.g., ,
conditions of temperature and buffer concentration, which permit hybridization
of a
particular nucleic acid to a second nucleic acid; the first nucleic acid may
be
perfectly (i. e.,100%) complementary to the second, or the first and second
may share
some degree of complementarity which 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 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., "Current Protocols ih 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., 0.2XSSC, O.1XSSC), 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
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28
hybridizing sequences and the frequency of occurrence of subsets of that
sequence
within other non-identical sequences. Thus, equivalent conditions can be
determined
by varying one or more of these parameters while maintaining a similar degree
of
identity or similarity between the two nucleic acid molecules. Typically,
conditions
are used such that sequences at Ieast 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 I~rause, M.H. and S.A. Aaronson,
Methods in Enzyrnology, 200:546-556 (1991). Also, in, Ausubel, et aZ.,
"Cut°rent
Protocols in 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 sought.
For example, a low stringency wash can comprise washing in a solution
containing 0.2XSSC/0.1% SDS for 10 min at room temperature; a moderate
stringency Wash can comprise washing in a prewarmed solution (42°C)
solution
containing 0.2XSSC/0.1 % SDS for 15 min at 42°C; and a high stringency
wash can
comprise washing in prewarmed (68°C) solution containing
O.1XSSC/0.1%SDS for
15 min 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,
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29
while maintaining a similar degree of identity or similarity between the
target nucleic
acid molecule and the primer or probe used.
The percent homology or identity of two nucleotide or amino acid sequences
can be determined by aligning the sequences for optimal comparison purposes
(e.g.,
gaps can be introduced in the sequence of a first sequence for optimal
alignment).
The nucleotides or amino acids at corresponding positions are then compared,
and
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
positionsltotal # of positions x I00). When a position in one sequence is
occupied by
the same nucleotide or amino acid residue as the correspondingeposition in the
other
sequence, then the molecules are homologous at that position. As used herein,
nucleic acid or amino 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 3.0%, 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.
One, non-limiting example of such a mathematical algorithm is described in
Marlin et
al., Ps°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 algoritlnn utilized
for the comparison of sequences is the algorithm of Myers and Miller, CABIOS
(1989). Such an algoritlun is incorporated into the ALIGN program (version
2.0)
which is part of the GCG sequence alignment software package. When utilizing
the
ALIGN program for comparing amino acid sequences, a PAMI20 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
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ADAM as described in Torellis and Robotti (1994) Corraput. Appl. Biosci., 10:3-
5;
and FASTA described in Pearson and Lipman (1988) PNAS, X5:2444-8.
In another embodiment, the percent identity between two amino acid
sequences can be accomplished using the GAP program in the GCG software
5 package (Accelrys, Cambridge, UI~) using either a Blossom 63 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.
10 The present invention also provides isolated nucleic acid molecules that
contain a fragment or portion that hybridizes under highly stringent
conditions to a
nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO:
1 .
which may optionally comprise at least one polymorphism as shown in Tables 11
and 12 and the complement thereof, and also provides isolated nucleic acid
15 molecules that contain a fragment or portion that hybridizes under highly
stringent
conditions to a nucleotide sequence encoding an amino acid sequence selected
from
SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or polymorphic variant
thereof. The
nucleic acid fragments of the invention are at least about 15, preferably at
least about
18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more
nucleotides in
20 length. Longer fragments, for example, 30 or more nucleotides in length,
which
encode antigenic polypeptides described herein are particularly useful, such
as for
the generation of antibodies as described below.
Probes and Py°iiyaers
25 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. By "base specific manner" is meant that the
two
sequences must have a degree of nucleotide complementarity sufficient for the
30 primer or probe to hybridize. Accordingly, the primer or probe sequence is
not
required to be perfectly complementary to the sequence of the template. Non-
complementary bases or modified bases can be interspersed into the primer or
probe,
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31
provided that base substitutions do not inhibit hybridization. The nucleic
acid
template may also include "non-specific priming sequences" or "nonspecific
sequences" to which the primer or probe has varying degrees of
complementarities.
Such probes and primers include polypeptide nucleic acids, as described in
Nielsen et
al., Science, 254, 1497-1500 (I991).
A probe or primer comprises a region of nucleic acid that hybridizes to at
least about 15, fox example about 20-25, and in certain embodiments about 40,
50 or
75, consecutive nucleotides of a nucleic acid of the invention, such as a
nucleic acid
comprising a contiguous nucleic acid sequence of SEQ m NO: 1 or the complement
of SEQ ID NO: 1, or a nucleic acid sequence encoding an amino acid sequence of
SEQ 1D NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, I2 or 14, or polymozphic variant
thereof. In
certain 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 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 by the polymerase chain reaction using synthetic
oligonucleotide primers designed based on one or more of the sequences
provided in
SEQ ID NO: 1 which may optionally comprise at least one polymorphism shown in
Tables 1 l and 12, and/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 Teclanology: PYiyaciples and Applications for DNA
Ayraplificatio~a (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR
Protocols: A
Guide to Methods afad Applications (Eds. Innis, et al., Academic Press, San
Diego,
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32
CA, 1990); Mattila et al., Nucleic Acids Res., 19:4967 (1991); Eckert et al.,
PGR
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
(I988), transcription amplification (Kwon et al., Proc. Natl. Acad. Sci. USA,
86:1173
(1989)), and self sustained sequence replication (Guatelli et al., Pf-oc. Nat.
Acad. Sci.
USA, 87:1874 (1990)) and nucleic acid based sequence amplification (NASBA).
The
latter two amplification methods involve isothermal reactions based on
isothermal
transcription, which produce both single stranded RNA (ssRNA) and double
stranded
DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to l,
respectively.
The amplified DNA can be labeled (e.g., with radiolabel or other reporter
molecule) and used as a probe for screening a eDNA 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 nucleotide sequence of nucleic
acid
molecules of the present invention can be accomplished using well-known
methods
that are commercially available. See, for example, Sambrook et al., Molecular
Clo~zing, A Laboratory Mafzual (2nd Ed., CSHP, New York 1989); Zyslcind et
al.,
Recombinant DNA Laboy~atofy 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 ZD NO: 1 and/or the complement of SEQ lD NO: l,
and/or a portion of SEQ ID NO: 1 or the complement of SEQ ID NO: I and/or a
sequence encoding the amino acid sequences or SEQ ID NO: 2, 3, 4, 5, 6, 7, 8,
9, 10,
12 and/or 14, or encoding a portion of SEQ )D NO: 2, 3, 4, 5, 6, 7, 8, 9, 10,
I2 and/or
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33
14, (wherein any one of these may optionally comprise at least one
polymorphism as
shown in Tables 11 and 12) and 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 occurnng nucleotides or variously modified
nucleotides
designed to increase the biological stability of the molecules or to increase
the
physical stability of the duplex formed between the antisense and sense
nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted nucleotides can be
used.
Alternatively, the antisense nucleic acid molecule can be produced
biologically using
an expression vector into which a nucleic acid molecule has been subcloned in
an
antisense orientation (i. e., RNA transcribed from the inserted nucleic acid
molecule
will be of an antisense orientation to a target nucleic acid of interest).
In general, the isolated nucleic acid sequences of the invention can be used
as
molecular weight markers on Southern gels, and as chromosome markers that are
labeled to map related gene positions. The nucleic acid sequences can also be
used
to compare with endogenous DNA sequences in patients to identify genetic
disorders
(e.g., a predisposition for or susceptibility to stroke), and as probes, such
as to
hybridize and discover related DNA sequences or to subtract out known
sequences
from a sample. The nucleic acid sequences can further be used to derive
primers for
genetic fingerprinting, to raise anti-polypeptide antibodies using DNA
immunization
techniques, and as an antigen to raise anti-DNA antibodies or elicit immune
responses. Portions or fragments of the nucleotide sequences identified herein
(and
the corresponding complete gene sequences) can be 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 associated
with
genetic disease; (ii) identify an individual from a minute biological sample
(tissue
typing); and (iii) aid in forensic identif canon of a biological sample.
Additionally,
the nucleotide sequences of the invention can be used to identify and express
recombinant polypeptides for analysis, characterization or therapeutic use, or
as
markers for tissues in which the corresponding polypeptide is expressed,
either
constitutively, during tissue differentiation, or in diseased states. The
nucleic acid
sequences can additionally be used as reagents in the screening and/or
diagnostic
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34
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.
hectors
Another aspect of the invention pertains to nucleic acid constructs containing
a nucleic acid molecule selected from the group consisting of SEQ m NO: 1
which
may optionally comprise at Ieast one polymorphism shown in Tables 11 and 12
and
the complement thereof (or a portion thereof). Yet another aspect of the
invention
pertains to nucleic acid constructs containing a nucleic acid molecule
encoding the
amino acid sequence of SEQ DJ NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14 or
polyrnorphic variant thereof. The constructs comprise a vector (e.g., an
expression
vector) into which a sequence of the invention has been inserted in a sense or
antisense orientation. As used herein, the term "vector" refers to a nucleic
acid
molecule capable of transporting another nucleic acid to which it has been
linked.
One type of vector is a "plasmid", which r efers 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
vectors, expression vectors, are capable of directing the expression of genes
to which
they are operably linked. In general, expression vectors of utility in
recombinant
DNA techniques are often in the form of plasmids. However, the invention is
intended to include such other forms of expression vectors, such as viral
vectors
(e.g., replication defective retroviruses, adenoviruses and adeno-associated
viruses)
that serve equivalent functions.
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
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for expression, which is operably linked to the nucleic acid sequence to be
expressed.
Within a recombinant expression vector, "operably or operatively linked" is
intended
to mean that the nucleotide sequence of interest is linked to the regulatory
sequences) in a manner which allows for expression of the nucleotide sequence
5 (e.g., in an in vita°o transcription/translation system or in a host
cell when the vector is
introduced into the host cell). The term "regulatory sequence" is intended to
include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation
signals). Such regulatory sequences are described, for example, in Goeddel,
Gehe
ExpYessioh T'eclanology: Methods in Ehzytnology 185, Academic Press, San
Diego,
10 CA (1990). Regulatory sequences include those which direct constitutive
expression
of a nucleotide sequence in many types of host cell and those which direct
expression
of the nucleotide sequence.only in certain host cells (e.g., tissue-specific
regulatory
sequences). It will be appreciated by those 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
I S 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
20 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),
yeast cells or mammalian cells. Suitable host cells are discussed further in
Goeddel,
supra. Alternatively, the recombinant expression vector can be transcribed and
translated in vitro, for example using T7 promoter regulatory sequences and T7
25 polymerase.
Another aspect of the invention pertains to host cells into which a
recombinant expression vector of the invention has been introduced. The terms
"host
cell" and "recombinant host cell" are used interchangeably herein. It is
understood
that such terms refer not only to the particular subject cell but also to the
progeny or
30 potential progeny of such a cell. Because certain modifications may occur
in
succeeding generations due to either mutation or environmental influences,
such
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36
progeny may not, in fact, be identical to the parent cell, but are still
included within
the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic
acid molecule of the invention can be expressed in bacterial cells (e.g., E.
coli),
insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells
(CHO) or
COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized techniques for introducing a foreign nucleic acid molecule (e.g.,
DNA)
into a host cell, including calcium phosphate or calcium chloride co-
precipitation,
DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable
methods for transforming or transfecting host cells can be found in Sambrook,
et al.,
(supf-a), and other laboratory manuals.
I S For stable transfection of mammalian cells, it is known that, depending
upon
the expression vector and transfection technique used, only a small fraction
of cells
may integrate the foreign DNA into their genome. In order to identify and
select
these integrants, a gene that encodes a selectable marker (e.g., for
resistance to
antibiotics) is generally introduced into the host cells along with the gene
of interest.
Preferred selectable markers include those that confer resistance to drugs,
such as
G4I ~, hygromycin and methotrexate. Nucleic acid molecules encoding a
selectable
maxker 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 will
survive,
while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (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
encoding a polypeptide of the invention has been introduced) in a suitable
medium
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37
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 PDE4D gene, or an exogenous
nucleic acid encoding PDE4D 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 in which
endogenous nucleotide sequences have been altered. Such animals are useful for
studying the function and/or activity of the nucleotide sequence and
polypeptide
encoded by the sequence and for identifying 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 "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.
Patent No. 4,873,191 and in Hogan, Manipulating tlae Mouse Errabryo (Cold
Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Methods for
constructing homologous recombination vectors and homologous recombinant
animals are described further in Bradley (1991) Cur~refzt Opifzion in
BiolTechnology,
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38
2:823-829 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.
(1997)
Nature, 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.
POLYPEPTD~ES OF THE INVENTION
The present invention also pertains to isolated polypeptides encoded by
PDE4D ("PDE4D polypeptides") and fragments and variants thereof, as well as
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
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
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
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39
"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 nucleotide sequence
selected from the group consisting of SEQ DJ NO: 1 which may optionally
comprise
at least one polymorphism shown in Tables 11 and 12 and complements and
portions
thereof, e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or a portion
or
pol5nnorphic variant thereof. However, the polypeptides of the invention also
encompass fragment and sequence variants. Variants include a substantially
homologous polypeptide encoded by the same genetic locus in an organism, i,
e., an
allelic variant, as well as other splicing variants. Variants also encompass
polypeptides derived from other genetic loci in an organism, but having
substantial
homology to a polypeptide encoded by a nucleic acid molecule comprising a
nucleotide sequence selected from the group consisting of SEQ ~ NO: 1 which
may
optionally comprise at least one polymorphism shown in Tables 11 and 12 and
complements and portions thereof, or having substantial homology to a
polypeptide
encoded by a nucleic acid molecule comprising a nucleotide sequence selected
from
the group consisting of nucleotide sequences encoding SEQ ID NO: 2, 3, 4, 5,
6, 7, 8,
9, 10, 12 or 14, 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
homologous or identical. A substantially homologous amino acid sequence,
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according to the present invention, will be encoded by a nucleic acid molecule
hybridizing to SEQ ID NO: 1 which may optionally comprise at least one
polymorphism shown in Tables 11 and 12, or portion thereof, under stringent
conditions as more particularly described above, or will be encoded by a
nucleic acid
5 molecule hybridizing to a nucleic acid sequence encoding SEQ ID NO: 2, 3, 4,
5, 6,
7, ~, 9, 10, 12 or 14, 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
10 functions performed by a polypeptide encoded by a nucleic acid molecule of
the
invention. Similarity is determined by conserved amino acid substitution. Such
substitutions are those that substitute a given amino acid in a polypeptide by
another
amino acid of like characteristics. Conservative substitutions are likely to
be
phenotypically silent. Typically seen as conservative substitutions are the
l~ 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 GIu, 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
20 silent are found in Bowie et al., Scies2ce ~4?:1306-1310 (1990).
A variant polypeptide can differ in amino acid sequence by one or more
substitutions, deletions, insertions, inversions, fusions, and truncations or
a
combination of any of these. Further, variant polypeptides can be fully
functional or
can lack function in one or more activities. Fully functional variants
typically contain
25 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
30 substitutions, deletions, insertions, inversions, or truncation or a
substitution,
insertion, inversion, or deletion in a critical residue or critical region.
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41
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
(Cunningharn et al., Scie~zce, 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 ira vitro, or ifa
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, 2SS:306-3I2 (1992)).
The invention also includes polypeptide fragments of the polypeptides of the
invention. Fragments can be derived from a polypeptide encoded by a nucleic
acid
molecule comprising SEQ m NO: 1 which may optionally comprise at least one
polymorphism shown in Tables 11 and 12 or a portion thereof and the
complements
thereof (e.g., SEQ m NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or other
splicing
I S variants). However; the iilvention 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, I5,
16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) can
comprise a domain, segment, or motif that has been identified by analysis of
the
polypeptide sequence using well-known methods, e.g., signal peptides,
extracellular
domains, one or more transmembrane segments or loops, ligand binding regions,
zinc finger domains, DNA binding domains, acylation sites, glycosylation
sites, or
phosphorylation sites.
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.
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42
The invention thus provides chimeric or fusion polypeptides. These comprise
a polypeptide of the invention operatively linked to a heterologous protein or
polypeptide having an amino acid sequence not substantially homologous to the
polypeptide. "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 per 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 axe not limited to, enzymatic fusion polypeptides,
for
example (3-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His
fusions
and Ig fusions. Such fusion polypeptides, particularly poly-His fusions, can
facilitate
the purification of recombinant polypeptide. In certain host cells (e.g.,
mammalian '
host cells), expression and/or secretion of a polypeptide can be increased by
using a
I S heterologous signal sequence. Therefore, in another embodiment, the fusion
polypeptide contains a heterologous signal sequence at its N-terminus.
EP-A-0 464 533 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 of Molecular Recognition, x:52-58 (1995) and Johanson et al.,
The
.Iournal ofBiological Chemistry, 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 carned out using anchor primers which give
rise to
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43
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., Cuf°~eht PYOtocols ifa 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
produced by recombinant DNA techniques. For example, a nucleic acid molecule
encoding the polypeptide is cloned into an expression vector, the expression
vector
introduced into a host cell and the polypeptide expressed in the host cell.
The
polypeptide can then be isolated from the cells by an appropriate purification
scheme
I S using standard protein purification techniques.
W general, polypeptides of the present invention can be used as a molecular
weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns
using
art-recognized methods. 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 receptor or a ligand) in
biological
fluids. The polypeptides can also be used as markers for cells or tissues in
which the
corresponding polypeptide is preferentially expressed, either constitutively,
during
tissue differentiation, or in a diseased state. The polypeptides can be used
to isolate a
corresponding binding agent, e.g., receptor or Iigand, such as, for example,
in an
interaction trap assay, and to screen for peptide or small molecule
antagonists or
agonists of the binding interaction.
ANTIBODIES OF THE INVENTION
Polyclonal and/or monoclonal antibodies that specifically bind one form of
the gene product but not to the other form of the gene product are also
provided.
Antibodies are also provided that bind a portion of either the variant or the
reference
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44
gene product that contains the polymorphic site or sites. The invention
provides
antibodies to the polypeptides and polypeptide fragments of the invention,
e.g.,
having an amino acid sequence encoded by SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9,
10, 12
or 14, or a portion thereof, or having an amino acid sequence encoded by a
nucleic
acid molecule comprising all or a portion of SEQ ID NO: 1 which may optionally
comprise at least one polymorphism shown in Tables 11 and 12 (e.g., SEQ ID NO:
2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or another splicing variant or portion
thereof). The
term "antibody" as used herein refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e., molecules
that
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
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')Z 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 irmnunoreacting with a particular epitope of a polypeptide of
the
invention. A monoclonal antibody composition thus typically displays a single
binding affinity for a particular polypeptide of the invention with which it
immunoreacts.
Polyclonal antibodies can be prepared as described above by immunizing a
suitable subject with a desired immunogen, e.g., polypeptide of the invention
or
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
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and used to prepare monoclonal antibodies by standard techniques, such as the
hybridoma technique originally described by Kohler and Milstein (1975)
Natur°e,
256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983)
Irrarnunol.
Today, 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal
5 Antibodies arid Caracer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma
techniques.
The technology for producing hybridomas is well known (see generally Current
Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc.,
New
York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to
lymphocytes (typically splenocytes) from a mammal immunized with an immunogen
I O as described above, and the culture supernatants of the resulting
hybridoma cells are
screened to identify a hybridoma producing a monoclonal antibody that binds a
polypeptide of the invention.
Any of the many well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied fox the purpose of generating a
monoclonal .
I S antibody to a polypeptide of the invention (see, e.g., Current Protocols
in
Immunology, supra; Galfre et al. (1977) Nature, 266:55052; R.H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum
Publishing Corp., New York, New York (1980); and Lerner (1981) gale J. Biol.
Med., 54:387-402. Moreover, the ordinarily skilled worker will appreciate that
there
20 are many variations of such methods that also would be useful.
Altenzative 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
25 members that bind the polypeptide. Kits for generating and screening phage
display
libraries are commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods and
reagents
particularly amenable for use in generating and screening antibody display
library
30 can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication
No. WO
92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791;
PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; FCT
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46
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication
No. WO 90/02809; Fuchs et al. (1991) BiolTeclz.jaology, 9:1370-1372; Hay et
al.
(1992) Hutn. Antibod. Hybridomas, 3:81-85; Huse et al. (1989) Science,
246:1275-
1281; Griffiths et al. (1993) EMBO J., 12:725-734.
Additionally, recombinant antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made using standaxd 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.
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
example, determine the efficacy of a given treatment regimen. Coupling the
antibody to a detectable substance can facilitate detection. Examples of
detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase,
~3-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
materials include luciferase, luciferin, and aequorin, and examples of
suitable
radioactive material include 125I, 131I, 35S or 3H.
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DIAGNOSTIC ASSAYS
The nucleic acids, probes, primers, polypeptides and antibodies described
herein can be used in methods of diagnosis of stroke or diagnosis of a
susceptibility
to stroke or to a disease or condition associated with an stroke gene, such as
PDE4D,
as well as in kits useful for diagnosis of stroke or a susceptibility to
stroke or to a
disease or condition associated with PDE4D. In one embodiment, the kit useful
for
diagnosis of stroke or susceptibility to stxoke, or to a disease or condition
associated
with PDE4D comprises primers as described herein, wherein the primers contain
one
or more of the SNPs identified herein. In parallel, definition of stroke risk
associated with PDE4D/cAMP pathway is useful and novel to define subgroups of
individuals who would be best treated by pharmaceutical agents acting on PDE4D
and/ cAMP pathways (and vice versa).
In one embodiment of the invention, diagnosis of stroke or susceptibility to
stroke (or diagnosis of or susceptibility to a disease or condition associated
with
PDE4D) is made by detecting a polymorphism in a PDE4D nucleic acid as
described
herein. The polymorphism can be an alteration in a PDE4D nucleic acid, such as
the
insertion or 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 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 cause an alteration in the polypeptide encoded by a PDE4D
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 PDE4D nucleic acid or a susceptibility to a disease or condition associated
with a
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48
PDE4D 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
PDE4D
nucleic acid). For diagnostic applications, there may be polymorphisms
informative
for prediction of disease risk that are in linkage disequilibrium with the
functional
polymorphism. 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 PDE4D nucleic acid that has any of the alteration described above is
referred
to herein as an "altered nucleic acid."
In a first method of diagnosing stroke or a susceptibility to stroke,
hybridization methods, such as Southern analysis, Northern analysis, or in
situ
hybridizations, can be used (see Current Protocols in Molecular Biolog,~,
Ausubel, F.
et al., eds., Tohn 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 obtained from an individual suspected of having, being
susceptible to or predisposed for, or carrying a defect for, a susceptibility
to a disease
or condition associated with a PDE4D 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 in a stroke nucleic acid is
present, and/or to determine which splicing variants) encoded by the PDE4D 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 PDE4D
nucleic acid or contains a nucleic acid encoding a particular splicing variant
of a
PDE4D 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.).
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49
To diagnose a susceptibility to stroke, a hybridization sample is formed by
contacting the test sample containing PDE4D, with at least one nucleic acid
probe.
A preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid
probe capable of hybridizing to mRNA or genomic DNA sequences described
herein. The nucleic acid probe can be, for example, a full-length nucleic acid
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 SEQ ID NO: 1 which may
optionally
comprise at least one polymorphism shown in Tables 1 l and 12, or the
complement
thereof, or a portion thereof; or can be a nucleic acid encoding. a portion of
SEQ ID
NO: 2, 3, 4, 5, 6, 7, ~, 9, 10, 12 or 14. Other suitable probes for use in the
diagnostic
assays of the invention are described above (see e.g., probes and primers
discussed
under the heading, "Nucleic Acids of the Invention").
The hybridization sample is maintained under conditions that are sufficient to
allow specific hybridization of the nucleic acid probe to PDE4D. "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, fox example, as described above.
In a
particularly preferred embodiment, the hybridization conditions for specific
hybridization are high stringency.
Specific hybridization, if present, is then detected using standard methods.
If
specific hybridization occurs between the nucleic acid probe and PDE4D in the
test
sample, then PDE4D 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. In one embodiment, specific hybridization of at
least
one of the nucleic acid probes is indicative of a polymorphism in PDE4D, or of
the
presence of a particular splicing variant encoding PDE4D and is therefore
diagnostic
for a susceptibility to stroke.
In Northern analysis (see Current Protocols in Molecular Biology, Ausubel,
F. et al., eds., John Wiley & Sons, supYa) the hybridization methods described
above
are used to identify the presence of a polymorphism or a particular splicing
variant,
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associated with a susceptibility to stroke. For Northern analysis, a test
sample of
RNA is obtained from the individual by appropriate means. Specific
hybridization of
a nucleic acid probe, as described above, to RNA from the individual is
indicative of
a polymorphism in PDE4D, or of the presence of a particular splicing variant
5 encoded by PDE4D, and is therefore diagnostic for a susceptibility to
stroke.
For representative examples of use of nucleic acid probes, see, for example,
U.S. Patents No. 5,288,611 and 4,851,330.
Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a
nucleic acid probe in the hybridization methods described above. PNA is a DNA
10 mimic having a peptide-like, inorganic backbone, such as N-(2-
aminoethyl)glycine
units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen
via a
methylene carbonyl linker (see, for example, Nielsen, P.E, et al.,
Biocofzjugate
Chefraistry, 1994, 5, American Chemical Society, p. 1 (1994). The PNA probe
can be
designed to specifically hybridize to a gene having a polymorphism associated
with a
15 susceptibility to stroke. Hybridization of the PNA probe to PDE4D is
diagnostic for
a susceptibility to stroke.
In another method of the invention, mutation analysis by restriction digestion
can be used to detect a mutant gene, or genes containing a polymorphism(s), if
the
mutation or polymorphism in the gene results in the creation or elimination of
a
20 restriction site. If a restriction site is not naturally created, one can
be created by
PCR that depends on the polymorphism and allows genotyping. A test sample
containing genomic DNA is obtained from the individual. Nucleic acid
amplification methods, including but not limited to Polymerase chain reaction
(PCR), Transcription Mediated Amplifications (TMA), and Ligase Mediate
25 Amplification (LMA), can be used to amplify PDE4D. The digestion pattern of
the
relevant DNA fragment indicates the presence or absence of the mutation or
polymorphism in PDE4D, and therefore indicates the presence or absence of this
susceptibility to stroke. RFLP analysis is conducted as described (see Current
Protocols in Molecular Biology, supYa). Amplification techniques based upon
30 detection of sequence of interest using reverse dot blot technology (linear
array or
strips) can be used and are described, for example, in U.S. Patent No.
5,468,613.
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Sequence analysis can also be used to detect specific polymorphisms in
PDE4D. A test sample of DNA or RNA is obtained from the test individual. PCR
or
other appropriate methods can be used to amplify the gene, and/or its flanking
sequences, if desired. The sequence of PDE4D, or a fragment of the gene, or
cDNA,
or fragment of the cDNA, or rnRNA, or fragment of the mRNA, is determined,
using
standard methods. The sequence of the gene, gene fragment, cDNA, cDNA
fragment, mRNA, or mRNA fragment is compared with the known nucleic acid
sequence of the gene, cDNA (e.g., SEQ ID NO: 1 which may optionally comprise
at
least one polymorphism shown in Tables 11 and I2, or a nucleic acid sequence
I O encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or a fragment
thereof) or
mRNA, as appropriate. In one embodiment, the presence of at least one of the
polymorphisms in PDE4D indicates that the individual has a susceptibility to
stroke.
Allele-specific oligonucleotides can also be used to detect the presence of a
polymorphism in PDE4D, through the use of dot-blot hybridization of amplified
oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for
example,
Saiki, R. et al., (1986), NatuYe (London) 324:163-166). An "allele-specific
oligonucleotide" (also referred to herein as an "allele-specific
oligonucleotide
probe") is an oligonucleotide of approximately 10-50 base pairs, preferably
approximately I S-30 base pairs, that specifically hybridizes to PDE4D, and
that
contains a polymorphism associated with a susceptibility to stroke. An allele-
specific oligonucleotide probe that is specific for particular polymorphisms
in
PDE4D can be prepared, using standard methods (see Current Protocols in
Molecular
Biology, supf~a). To identify polymorphisms in the gene that are associated
with a
susceptibility to stroke, a test sample of DNA is obtained from the
individual. PCR
can be used to amplify all or a fragment of PDE4D, and its flanking sequences.
The
DNA containing the amplified PDE4D (or fragment of the gene) is dot-blotted,
using
standard methods (see Current Protocols in Molecular Biology, supra), and the
blot
is contacted with the oligonucleotide probe. The presence of specific
hybridization
of the probe to the amplified PDE4D is then detected. Specific hybridization
of an
allele-specific oligonucleotide probe to DNA from the individual is indicative
of a
polymorphism in PDE4D, and is therefore indicative of a susceptibility to
stroke.
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The invention further provides allele-specific oligonucleotides that hybridize
to the reference or variant allele of a nucleic acid comprising a single
nucleotide
polymorphism or to the complement thereof These oligonucleotides can be probes
or primers.
An allele-specific primer hybridizes to a site on target DNA overlapping a
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 that
hybridizes at a
distal site. Amplification proceeds from the two primers, resulting in a
detectable
product that 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 polymorphic site and the other of which exhibits perfect
complementarity to a
distal site. The single-base mismatch prevents amplification and no detectable
product is formed. The method works best when the mismatch is included in the
3'-
most position of the oligonucleotide aligned with the polymorphism because
this
position is most destabilizing to elongation from the primer (see, e.g., WO
93/22456).
With the addition of such analogs as locked nucleic acids (LNAs), the size of
primers and probes can be reduced to as few as 8 bases. LNAs are a novel class
of
bicyclic DNA analogs in which the 2' and 4' positions in the furanose ring are
joined
via an O-methylene (oxy-LNA), S-methylene (thin-LNA), or amino methylene
(amino-LNA) moiety. Common to all of these LNA variants is an affinity toward
complementary nucleic acids, which is by far the highest reported for a DNA
analog.
For example, particular all oxy-LNA nonamers have been shown to have melting
temperatures of 64 °C and 74 ° C when in complex with
complementary DNA or
RNA, respectively, as opposed to 28 °C for both DNA and RNA for
the
corresponding DNA nonamer. Substantial increases in Tm are also obtained when
LNA monomers are used in combination with standard DNA or RNA monomers.
For primers and probes, depending on where the LNA monomers are included
(e.g.,
the 3' end, the 5'end, or in the middle), the Tm could be increased
considerably.
In another embodiment, arrays of oligonucleotide probes that axe
complementary to target nucleic acid sequence segments from an individual, can
be
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used to identify polymorphisms in PDE4D. For example, in one embodiment, an
oligonucleotide linear 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 "Genechips.TM.," have been generally described in the art, for example,
U.S.
Patent No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092.
These arrays can generally be produced using mechanical synthesis methods or
light
directed synthesis methods that incorporate a combination of photolithographic
methods and solid phase oligonucleotide synthesis methods. See Fodor et al.,
Science, X51:767-777 (1991), Pirrung et al., U.S. Patent No. 5,143,854 (see
also PCT
Application No. WO 90/15070) and Fodor et al., PCT Publication No. WO 92/10092
and U.S. Patent No. 5,424,186, the entire teachings of each of which are
incorporated
by reference herein. Techniques for the synthesis of these arrays using
mechanical
synthesis methods are described in, e.g., U.S. Patent No. 5,384,261, the
entire
teachings of which are incorporated by reference herein. In another
embodiment,
linear arrays or microarrays can be utilized.
Once an oligonucleotide array is prepared, a nucleic acid of interest is
hybridized with the array and scanned for polyrnorphisms. 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. Patent
No. 5,424,186, the entire teachings of which are incorporated by reference
herein. In
brief, a target nucleic acid sequence that includes one or more previously
identified
polymorphic markers is amplified by well-known amplification techniques, e.g.,
PCR. Typically, this involves the use of primer sequences that are
complementary to
the two strands of the target sequence both upstream and 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.
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Although primarily described in terms of a single detection block, e.g., fox
detection of a single polymorphism, arrays can include multiple detection
blocks, and
thus be capable of analyzing multiple, specific polymorphisms. In alternate
arrangements, it will generally be understood that detection blocks may be
grouped
within a single array or in multiple, separate arrays so that varying, optimal
conditions may be used during the hybridization of the target to the array.
For
example, it may often be desirable to provide for the detection of those
polymorphisms that fall within G-C rich stretches of a genomic sequence,
separately
from those falling in A-T rich segments. This allows for the separate
optimization of
hybridization conditions for each situation.
Additional description of use of oligonucleotide arrays for detection of
polymorphisms can be found, for example, in U.S. Patents 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 PDE4D or splicing variants encoding by PDE4D. Representative methods
include
direct manual sequencing (Church and Gilbert, (1988), Pr~oc. Natl. Acad. Sci.
USA
81:1991-1995; Sanger, F. et al. (1977) Pf°oc. Natl. Acad. Sci. 74:5463-
5467; Beavis
et al., U.S. Patent No. 5,288,644); automated fluorescent sequencing; single-
stranded
conformation polymorphism assays (SSCP); clamped denaturing gel
electrophoresis
(CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V.C. et al.
(I9891) Proc. Natl. Acad. Sci. USA 86:232-236), mobility shift analysis
(Orita, M. et
al. (1989) Pf-oc. Natl. Acad. Sci. USA 86:2766-2770), restriction enzyme
analysis
(Flavell et al. (1978) Cell 15:25; Geever, et al. (1981) Pr~oc. Natl. Acad.
Sci. USA
78:5081); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et
al.
(I985) P~oc. Natl. Acad. S'ci. USA 85:4397-4401); RNase protection assays
(Myers,
R.M. et al. (1985) Science 230:1242); use of polypeptides which recognize
nucleotide mismatches, such as E. coli mutS protein, for example.
In one embodiment of the invention, diagnosis of a disease or condition
associated with PDE4D (e.g., stroke) or a susceptibility to a disease or
condition
associated with PDE4D (e.g., stroke) can also be made by expression analysis
by
quantitative PCR (kinetic thermal cycling). This technique utilizing TaqMan
° or
Lightcycler~ can be used to allow the identification of polymorphisms and
whether
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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
PDE4D nucleic acid or splicing variants encoded by a PDE4D nucleic acid.
Further,
the expression of the variants can be quantified as physically or functionally
5 different.
In another embodiment of the invention, diagnosis of a susceptibility to
stroke can also be made by examining expression and/or composition of an PDE4D
polypeptide, by a variety of methods, including enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. A
10 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
PDE4D, or for the presence of a particular variant (e.g., an isoform) encoded
by
PDE4D. An alteration in expression of a polypeptide encoded by PDE4D can be,
for
example, an alteration in the quantitative polypeptide expression (i.e., the
amount of
15 polypeptide produced); an alteration in the composition of a polypeptide
encoded by
PDE4D is an alteration in the qualitative polypeptide expression (e.g.,
expression of
a mutant PDE4D polypeptide or of a different splicing variant or isoform). In
one
embodiment, detecting a particular splicing variant encoded by that PDE4D, or
a
particular pattern of splicing variants makes diagnosis of the disease or
condition
20 associated with PDE4D or a susceptibility to a disease or condition
associated with
PDE4D.
Both such alterations (quantitative and qualitative) can also be present. An
"alteration" in the polypeptide expression or composition as used herein,
refers to an
alteration in expression or composition in a test sample, as compared with the
25 expression or composition of polypeptide by PDE4D 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 stroke. An alteration
in the
expression or composition of the polypeptide in the test sample, as compared
with
the control sample, is indicative of a susceptibility to stroke. Similarly,
the presence
30 of one or more different splicing variants or isoforms 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
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stroke. Various means of examining expression or composition of the
polypeptide
encoded by PDE4D can be used, including spectroscopy, colorimetry,
electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al.,
U.S.
Patent No. 4,376,110) such as immunoblotting (see also Current Protocols in
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')~) can be used. The term "labeled", with regard to the probe or
antibody, is
intended to encompass direct labeling of the probe or antibody by coupling (i.
e.,
physically linking) a detectable substance to the probe or antibody, as well
as indirect
labeling of the probe or antibody by reactivity with another reagent that is
directly
labeled. Examples of indirect labeling include detection of a primary antibody
using
a fluorescently labeled secondary antibody and end-labeling of a DNA probe
with
biotin such that it can be detected with fluorescently labeled streptavidin.
Western blotting analysis, using an antibody as described above that
specifically binds to a polypeptide encoded by a mutant PDE4D, or an antibody
that
specifically binds to a polypeptide encoded by a non-mutant gene, or an
antibody
that specifically binds to a particular splicing variant encoded by PDE4D, can
be
used to identify the presence in a test sample of a particular splicing
variant or
isoform, or of a polypeptide encoded by a polyrnorphic or mutant PDE4D, or the
absence in a test sample of a particular splicing variant or isoform, or of a
polypeptide encoded by a non-polymorphic or non-mutant gene. The presence of a
polypeptide encoded by a polymorphic or mutant gene, or the absence of a
polypeptide encoded by a non-polymorphic or non-mutant gene, is diagnostic for
a
susceptibility to stroke, as is the presence (or absence) of particular
splicing variants
encoded by the PDE4D gene.
In one embodiment of this method, the level or amount of polypeptide
encoded by PDE4D in a test sample is compared with the level or amount of the
polypeptide encoded by PDE4D 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,
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is indicative of an alteration in the expression of the polypeptide encoded by
PDE4D,
and is diagnostic for a susceptibility to stroke. Alternatively, the
composition of the
polypeptide encoded by PDE4D in a test sample is compared with the composition
of
the polypeptide encoded by PDE4D 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 fox a susceptibility to stroke. 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
polypeptide in the test sample, compared to the control sample; a difference
in
composition in the test sample, compared to the control sample; or both a
difference
in the amount or level, and a difference in the composition, is indicative of
a
susceptibility to stroke.
In another embodiment, assessment of the splicing variant or isoform(s) of a
polypeptide encoded by a polymorphic or mutant PDE4D, can be performed. .The
assessment can be performed directly (e.g., by examining the polypeptide
itself), or
indirectly (e.g., by examining the mRNA encoding the polypeptide, such as
through
mRNA profiling). For example, probes or primers as described herein can be
used
to determine which splicing variants or isoforms are encoded by PDE4D rnRNA,
using standard methods.
The presence in a test sample of a particular splicing variants) or isoform(s)
associated with stroke or risk of stroke, or the absence in a test sample of a
particular
splicing variants) or isoform(s) not associated with stroke or risk of stroke,
is
diagnostic for a disease or condition associated with a PDE4D gene or a
susceptibility to a disease or condition associated with a PDE4D gene.
Similarly, the
absence in a test sample of a particular splicing variants) or isoform(s)
associated
with stroke or risk of stroke, or the presence in a test sample of a
particular splicing
variants) or isoform(s) not associated with stroke or risk of stroke, is
diagnostic for
the absence of disease or condition associated with a PDE4D gene or a
susceptibility
to a disease or condition associated with a PDE4D gene.
In another embodiment, differential expression of isoforms PDE4D7,
PDE4D9 and combinations thereof can be assessed and compared to control
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individuals. Decreased expression of these isoforms is indicative of
susceptibility to
stroke, particularly carotid stroke and/or caxdiogenic stroke.
The invention further pertains to a method for the diagnosis and
identification
of susceptibility to stroke in an individual, by identifying an at-risk
haplotype in
PDE4D. lii one embodiment, the at-risk haplotype is a haplotype for which the
presence of the haplotype increases the risk of stroke significantly. Although
it is to
be understood that identifying whether a risk is significant may depend on a
variety
of factors, including the specific disease, the haplotype, and often,
environmental
factors, the significance may be measured by an odds ratio or a percentage. 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 least about
1.2,
including but 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 not limited
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 stroke or a
susceptibility to stroke in an individual, comprising screening for an at-risk
haplotype in the PDE4D nucleic acid that is more frequently present in an
individual
susceptible to stroke (affected), compared to the frequency of its presence in
a
healthy individual (control), wherein the presence of the haplotype is
indicative of
stroke or susceptibility to stroke. Standard techniques for genotyping for the
presence of SNPs and/or microsatellite markers that are associated with stroke
can be
used, such as fluorescent-based techniques (Chen, et al., Genome Res. 9, 492
(1999),
PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. In
one
embodiment, the method comprises assessing in an individual the presence or
frequency of SNPs and/or microsatellites in the PDE4D nucleic acid that are
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associated with stroke, wherein an excess or higher frequency of the SNPs
and/or
microsatellites compared to a healthy control individual is indicative that
the
individual has stroke or is susceptible to stroke.
See Table 2C, Table 3, Table 4A, and 4B for SNPs and markers that comprise
haplotypes that can be used as screening tools. See also, Table 5, Table 6,
Table 11
and Table 12 that set forth previously known SNP and novel rnicrosatellite
markers
and their counterpart sequence m reference numbers. SNPs and markers from
these
lists represent at-risk haplotypes and can be used to design diagnostic tests
for
determining a susceptibility to stroke.
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) PDE4D polypeptide, means fox amplification of nucleic acids
comprising PDE4D, or means for analyzing the nucleic acid sequence of PDE4D or
for analyzing the amino acid sequence of an PDE4D polypeptide, etc. In one
embodiment, a kit for diagnosing susceptibility to stroke can comprise primers
for
nucleic acid amplification of a region in the PDE4D gene comprising an at-risk
haplotype that is more frequently present in an individual susceptible to
stroke. The
primers can be designed using portions of the nucleic acids flasiking SNPs
that are
indicative of stroke. In a particularly preferred embodiment, the primers axe
designed to amplify regions of the PDE4D gene associated with an at-risk
haplotype
for stroke, shown in Tables 8A and 8B. In another embodiment of the invention,
a
kit for diagnosing susceptibility to stroke can further comprise probes
designed to
hybridize to regions of the PDE4D gene associated with an at-risk haplotype
for
stroke, shown in Table 5 and table 6 andlor generated from SEQ m Nos: 85-102.
SCREENING ASSAYS AND AGENTS IDENTIFIED THEREBY
The invention provides methods (also referred to herein as "screening
assays") for identifying the presence of a nucleotide that hybridizes to a
nucleic acid
of the invention, as well as for identifying the presence of a polypeptide
encoded by
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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 signiEcant
homology
with a nucleic acid of the invention) in a sample can be assessed by
contacting the
sample with a nucleic acid comprising a nucleic acid of the invention (e.g., a
nucleic
5 acid having the sequence of SEQ JD NO: 1 which may optionally comprise at
least
one polymorphism shown in Tables 11 and 12, or the complement thereof, or a
nucleic acid encoding an amino acid having the sequence of SEQ ID NO: 2, 3, 4,
S,
6, 7, S, 9, 10, 12 or 14, or a fragment or variant of such nucleic acids),
under
stringent conditions as described above, and then assessing the sample for the
10 presence (or absence) of hybridization. Tn another embodiment, high
stringency
conditions are conditions appropriate for selective hybridization. In axzother
embodiment, a sample containing the nucleic acid molecule of interest is
contacted
with a nucleic acid containing a contiguous nucleotide sequence (e.g., a
primer or a
probe as described above) that is at least partially complementary to a part
of the
1 S nucleic acid molecule of interest (e.g., a PDE4D nucleic acid), and the
contacted
sample is assessed for the presence or absence of hybridization. In another
embodiment, the nucleic acid containing a contiguous nucleotide sequence is
completely complementary to a part of the nucleic acid molecule of interest.
In any of these embodiments, all or a portion of the nucleic acid of interest
20 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 to the polypeptide of interest (e.g., an antibody such as those
described
2S above), and 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) that
alter
30 (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., PDE4D binding
agents);
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which have a stimulatory 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 PDE4D binding agents
(e.g.,
receptors or other binding agents); or which alter posttranslational
processing of the
PDE4D 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
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. (1997) Araticahcey~ Df-ug Des., 12:145).
In one embodiment, to identify agents which alter the activity of a PDE4D
polypeptide, a cell, cell lysate, or solution containing or expressing a PDE4D
polypeptide (e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or I4, or another
splicing
variant encoded by PDE4D), 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 PDE4D
activity is assessed (e.g., the level (amount) of PDE4D 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 PDE4D 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 PDE4D polypeptide. An increase in the level of PDE4D activity relative to
level
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62
of the control, indicates that the agent is an agent that enhances (is an
agonist of)
PDE4D activity. Similarly, a decrease in the level of PDE4D activity relative
to
level of the control, indicates that the agent is an agent that inhibits (is
an antagonist
of) PDE4D activity. In another embodiment, the level of activity of a PDE4D
polypeptide or derivative or fragment thereof in the presence of the agent to
be
tested, is compared with a control level that has previously been established.
A level
of the activity in the presence of the agent that differs from the control
level by an
amount that is statistically significant indicates that the agent alters PDE4D
activity.
The present invention also relates to an assay for identifying agents which
alter the expression of the PDE4D gene (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 gene or which otherwise
interact
with the nucleic acids described herein, as well as agents identifiable by the
assays.
For example, a solution containing a nucleic acid encoding PDE4D polypeptide
{e.g.,
PDE4D gene) 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 transeription/translation of the nucleic acid. Cells
not
suspended in solution can also be employed, if desired. The level and/or
pattern of
PDE4D expression (e.g., the level and/or pattern 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 PDE4D 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 PDE4D.
Enhancement of PDE4D expression indicates that the agent is an agonist of
PDE~.D
activity. Similarly, inhibition of PDE4D expression indicates that the agent
is an
antagonist of PDE4D activity. In another embodiment, the Level and/or pattern
of
PDE4D polypeptide(s) (e.g., different splicing variants) in the presence of
the agent
to be tested, is compared with a control level andlor pattern that have
previously been
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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 PDE4D expression. In one
embodiment,
agents that can alter expression levels of isoforms PDE4D7 and/or PDE4D9 can
be
assessed, preferably to complement the expression levels to approximate the
ratios of
a healthy individual.
In another embodiment of the invention, agents which alter the expression of
the PDE4D gene or which otherwise interact with the nucleic acids described
herein,
can be identified using a cell, cell lysate, or solution containing a nucleic
acid
encoding the promoter region of the PDE4D gene operably linked to a reporter
gene.
After contact with an agent to be tested, the level of expression of the
reporter gene
(e.g., the level of mRNA or of protein expressed) is assessed, and is compared
with
the level of expression in a control (i. e., the level of the expression of
the reporter
gene in the absence of the agent to be tested). If the level in the presence
of the agent
differs, by an amount or in a manner that is statistically significant, from
the level in
the absence of the agent, then the agent is an agent that alters the
expression of
PDE4D, as indicated by its ability to alter expression of a gene that is
operably
linked to the PDE4D gene promoter. Enhancement of the expression of the
reporter
indicates that the agent is an agonist of PDE4D activity. Similarly,
inhibition of the
expression of the reporter indicates that the agent is an antagonist of PDE4D
activity.
In another embodiment, the level of expression of the reporter in the presence
of the
agent to be tested, is compared with a control level that has previously been
established. A level in the presence of the agent that differs from the
control level by
an amount or in a manner that is statistically significant indicates that the
agent alters
PDE4D expression.
Agents which alter the amounts of different splicing variants encoded by
PDE4D (e.g., an agent which enhances activity of a first splicing variant, and
which
inhibits activity of a second splicing variant), as well as agents which are
agonists of
activity of a first splicing variant and antagonists of activity of a second
splicing
variant, can easily be identified using these methods described above.
In other embodiments of the invention, assays can be used to assess the
impact of a test agent on the activity of a polypeptide in relation to a PDE4D
binding
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agent. For example, a cell that expresses a compound that interacts with PDE4D
(herein referred to as a "PDE4D binding agent", which can be a polypeptide or
other
molecule that interacts with PDE4D, such as a receptor) is contacted with
PDE4D in
the presence of a test agent, and the ability of the test agent to alter the
interaction
between PDE4D and the PDE4D binding agent is determined. Alternatively, a cell
lysate or a solution containing the PDE4D binding agent, can be used. An agent
which binds to PDE4D or the PDE4D binding agent can alter the interaction by
interfering with, or enhancing the ability of PDE4D to bind to, associate
with, or
otherwise interact with the PDE4D binding agent. Determining the ability of
the test
agent to bind to PDE4D or an PDE4D 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 lash 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 Iabel
detected by
determination of conversion of an appropriate substrate to product. It is also
within
the scope of this invention to determine the ability of a test agent to
interact with the
polypeptide without the labeling of any of the interactants. For example, a
microphysiometer can be used to detect the interaction of a test agent with
PDE4D or
a PDE4D binding agent without the labeling of either the test agent, PDE4D, or
the
PDE4D binding agent. McConnell, H.M. et al. (1992) Science, 257:I906-I9I2. 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. See
the
Examples Section for a discussion of known PDE4D binding partners. Thus, these
receptors can be used to screen for compounds that are PDE4D receptor agonists
for
use in treating stroke or PDE4D receptor antagonists for studying stroke. The
linkage data provided herein, for the first time, provides such connection to
stroke.
Drugs could be designed to regulate PDE4D receptor activation that in turn can
be
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used to regulate signaling pathways and transcription events of genes
downstream,
such as Cbfal.
In another embodiment of the invention, assays can be used to identify
polypeptides that interact with one or more PDE4D polypeptides, as described
5 herein. For example, a yeast two-hybrid system such as that described by
Fields and
Song (Fields, S. and Song, O., Nature 340:245-246 (1989)) can be used to
identify
polypeptides that interact with one or more PDE4D 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
10 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 maxkers 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
15 vector is used which includes a nucleic acid encoding a DNA binding domain
arid
also an PDE4D polypeptide, splicing variant, fragment or derivative thereof,
and a
second vector is used which includes a nucleic acid encoding a transcription
activation domain and also a nucleic acid encoding a polypeptide which
potentially
may interact with the PDE4D polypeptide, splicing variant, or fragment or
derivative
20 thereof (e.g., a PDE4D 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)
allows
identification of colonies which express the markers of interest. These
colonies can
be examined to identify the polypeptide(s) that interact with the PDE4D
polypeptide
25 or fragment or derivative thereof. Such polypeptides may be useful as
agents that
alter the activity of expression of a PDE4D polypeptide, as described above.
In more than one embodiment of the above assay methods of the present
invention, it may be desirable to immobilize either PDE4D, the PDE4D binding
agent, or other components of the assay on a solid support, in order to
facilitate
30 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
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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 PDE4D or a PDE4D binding agent to be bound to a matrix or other solid
support.
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 PDE4D 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 is identified as a stimulator or enhancer of the mRNA or polypeptide
expression. Alternatively, when expression of the mRNA or polypeptide is less
(statistically significantly less) in the presence of the test agent than in
its absence,
the test agent is identified as an inhibitor of the mRNA or polypeptide
expression.
The level of mRNA or polypeptide expression in the cells can be determined by
methods described herein for detecting mRNA or polypeptide.
This invention further pertains to novel agents identified by the above-
described screening assays. Accordingly, it is within the scope of this
invention to
further use an agent identified as described herein in an appropriate animal
model.
For example, an agent identified as described herein (e.g., a test agent that
is a
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 above-described screening assays for
treatments as
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described herein. In addition, an agent identified as described herein can be
used to
alter activity of a polypeptide encoded by PDE4D, or to alter expression of
PDE4D,
by contacting the polypeptide or the gene (or contacting a cell comprising the
polypeptide or the gene) with the agent identified as described herein.
PHARMACEUTICAL COMPOSITIONS
The present invention also pertains to pharmaceutical compositions
comprising agents described herein, particularly nucleotides encoding the
polypeptides described herein; comprising polypeptides described herein (e.g.,
one or
more of SEQ ID NO: 2, 3, 4, 5, 6, 7, ~, 9, 10, 12 or 14); and/or comprising
other
splicing variants encoded by PDE4D; and/or an agent that alters (e.g.,
enhances or
inhibits) PDE4D gene expression or PDE4D polypeptide activity as described
herein.
For instance, a polypeptide, protein (e.g., an PDE4D receptor), an agent that
alters
PDE4D gene expression, or a PDE4D binding agent or binding partner, fragment,
fusion protein or prodrug thereof, or a nucleotide or nucleic acid construct
(vector)
comprising a nucleotide of the present invention, or an agent that alters
PDE4D
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.
Suitable pharmaceutically acceptable carriers include but are not limited to
water, salt solutions (e.g., NaCI), saline, buffered saline, alcohols,
glycerol, ethanol,
gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,
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.
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
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powder. The composition can be formulated as a suppository, with traditional
binders and Garners such as triglycerides. Oral formulation can include
standard
carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, polyvinyl pyrolidone, sodium saccharine, cellulose, magnesium
carbonate,
etc.
Methods of introduction of these compositions include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intraocular, intravenous,
subcutaneous,
topical, oral and intranasal. Other suitable methods of introduction can also
include
gene therapy (as described below), rechargeable or biodegradable devices,
particle
acceleration devises ("gene guns") and slow release polymeric 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.
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 Garner compatible with topical application and having a
dynamic
viscosity preferably greater than water, can be employed. Suitable
formulations
include but are not limited to solutions, suspensions, emulsions, creams,
ointments,
powders, enemas, lotions, sols, 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
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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 Garner material, is packaged in a
squeeze
bottle or in admixture with a pressurized volatile, normally gaseous
propellant, e.g.,
pressurized air.
Agents described herein can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free amino groups
such
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. W addition,
in
vita°o or in vivo assays may optionally be employed to help identify
optimal dosage
ranges. The precise dose to be employed in the formulation will also depend on
the
route of administration, and the seriousness of the symptoms of stroke, 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 ira
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
by the agency of manufacture, use of sale for human administration. The pack
or kit
can be labeled with information regarding mode of administration, sequence of
drug
administration (e.g., separately, sequentially or concurrently), or the like.
The pack
or kit may also include means for reminding the patient to take the therapy.
The
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
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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
5 standard time courses.
METHODS OF THERAPY
The present invention encompasses methods of treatment (prophylactic
andlor therapeutic) for stroke or a susceptibility to stroke, such as
individuals in the
10 target populations described herein particularly ischemic (e.g., carotid
and
cardiogenic strokes) and TIA, using a PDE4D therapeutic agent. A "PDE4D
therapeutic agent" is an agent that alters (e.g., enhances or inhibits) PDE4D
polypeptide (enzymatic activity) and/or PDE4D gene expression, as described
herein
(e.g., a PDE4D agonist or antagonist). PDE4D therapeutic agents can alter
PDE4D
15 polypeptide activity or nucleic acid expression by a variety of means, such
as, for
example, by providing additional PDE4D polypeptide or by upregulating the
transcription or translation of the PDE4D gene; by altering posttranslational
processing of the PDE~.D polypeptide; by altering transcription of PDE4D
splicing
variants; or by interfering with PDE4D polypeptide activity (e.g., by binding
to a
20 PDE4D polypeptide), or by downregulating the transcription or translation
of the
PDE4D gene.
In particular, the invention relates to methods of treatment for stroke or
susceptibility to stroke (for example, for individuals in an at-risk
population such as
those described herein); as well as to methods of treatment for myocardial
infarction,
25 atherosclerosis, acute coronary syndrome (e.g., unstable angina, non-ST-
elevation
myocardial infarction (NSTEM~ or ST-elevation myocardial infarction (STEMI));
for decreasing risk of a second myocardial infarction; for atherosclerosis,
such as for
patients requiring treatment (e.g., angioplasty, stems, coronary artery bypass
graft) to
restore blood flow in arteries (e.g., coronary arteries) and peripheral
arterial
30 occlusive disease.
Representative PDE4D therapeutic agents include the following:
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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, cDNA, and/or mRNA, double-
stranded
interfering RNA, a nucleic acid encoding a PDE4D polypeptide or active
fragment or
derivative thereof, or an oligonucleotide; for example, SEQ m NO: 1 which may
optionally comprise at least one polymorphism shown in Tables 11 and 12 or a
nucleic acid encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14, or
fragments or
derivatives thereof), antisense nucleic acids or small double-stranded
interfering
RNA;
polypeptides described herein (e.g., one or more of SEQ m NO: 2, 3, 4, 5, 6,
7, ~, 9, I0, 12 or I4, and/or other splicing variants encoded by PDE4D, or
fragments
or derivatives thereof);
other polypeptides (e.g., PDE4D receptors); PDE4D binding agents;
peptidomimetics; fusion pxoteins or prodrugs thereof; antibodies (e.g., an
antibody to
a mutant PDE4D polypeptide, or an antibody to a non-mutant PDE4D polypeptide,
or an antibody to a particular splicing variant encoded by PDE4D, as described
above); ribozymes; other small molecules;
and other agents that alter (e.g., inhibit or antagonize) PDE4D gene
expression or polypeptide activity, or that regulate transcription of PDE4D
splicing
variants (e.g., agents that affect which splicing variants are expressed, or
that affect
the amount of each splicing variant that is expressed).
More than one PDE4D therapeutic agent can be used concurrently, if desired.
The PDE4D therapeutic agent that is a nucleic acid is used in the treatment of
stroke. The term, "treatment" as used herein, refers not only to ameliorating
symptoms associated with the disease, but also preventing or delaying the
onset of
the disease, and also lessening the severity or frequency of symptoms of the
disease,
preventing or delaying the occurrence of a second episode of the disease or
condition; andlor also lessening the severity or frequency of symptoms of the
disease
or condition. In the case of atherosclerosis, "treatment" also refers to a
minimization
or reversal of the development of plaques. The therapy is designed to alter
(e.g.,
inhibit or enhance), replace or supplement activity of a PDE4D polypeptide in
an
individual. For example, a PDE4D therapeutic agent can be administered in
order to
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upregulate or increase the expression or availability of the PDE4D gene or of
specific
splicing variants of PDE4D, or, conversely, to downregulate or decrease the
expression or availability of the PDE4D gene or specific splicing variants of
PDE4D.
Upregulation or increasing expression or availability of a native PDE4D gene
or of a
particular splicing variant could interfere with or compensate for the
expression or
activity of a defective gene or another splicing variant; downregulation or
decreasing
expression or availability of a native PDE4D gene or of a particular splicing
variant
could minimize the expression or activity of a defective gene or the
particular
splicing variant and thereby minimize the impact of the defective gene or the
particular splicing variant.
The PDE4D therapeutic agents) are administered in a therapeutically
effective amount (i. e., an amount that is sufficient to treat the disease,
such as by
ameliorating 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, in vitro or ira vivo assays may optionally be employed to help
identify
optimal dosage ranges. The precise dose to be employed in the formulation will
also
depend on the route of administration, and the seriousness of the disease or
disorder,
and should be decided according to the judgment of a practitioner and each
patient's
circumstances. Effective doses may be extrapolated from dose-response curves
derived from in vitf~o or animal model test systems.
In one embodiment, a nucleic acid of the invention (e.g., a nucleic acid
encoding a PDE4D polypeptide, such as SEQ ID NO: 1 which may optionally
comprise at least one polymorphism shown in Tables 1 l and 12; or another
nucleic
acid that encodes a PDE4D polypeptide or a splicing variant, derivative or
fragment
thereof, such as a nucleic acid encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9,
10, 12 or
14) can be used, either alone or in a pharmaceutical composition as described
above.
For example, PDE4D or a cDNA encoding the PDE4D polypeptide, either by itself
or included within a vector, can be introduced into cells (either ira vitro or
ifa vivo)
such that the cells produce native PDE4D polypeptide. If necessary, cells that
have
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been transformed with the gene or cDNA or a vector comprising the gene or cDNA
can be introduced (or re-introduced) into an individual affected with the
disease.
Thus, cells which, in nature, lack native PDE4D expression and activity, or
have
mutant PDE4D expression and activity, or have expression of a disease-
associated
PDE4D splicing variant, can be engineered to express PDE4D polypeptide or an
active fragment of the PDE4D polypeptide (or a different variant of PDE4D
polypeptide). In another embodiment, nucleic acid encoding the PDE4D
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 gene transfer systems, including viral
and
nonviral transfer systems, can be used. Alternatively, nonviral gene transfer
methods, such as calcium phosphate coprecipitation, mechanical techniques
(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; ar
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 PDE4D is
administered or generated in situ. The antisense nucleic acid that
specifically
hybridizes to the mRNA andlor DNA inhibits expression of the PDE4D
polypeptide,
e.g., by inhibiting translation and/or transcription. Binding of the antisense
nucleic
acid can be by conventional base pair complementarity, or, for example, in the
case
of binding to DNA duplexes, through specif c 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 PDE4D 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 PDE4D.
In one embodiment, the oligonucleotide probes are modif ed oligonucleotides
that
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74
are resistant to endogenous nucleases, e.g., exonucleases andlor
endonucleases,
thereby rendering them stable iri vivo. Exemplary nucleic acid molecules for
use as
antisense oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate analogs of DNA (see also U.S. Patent 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. ((1988) Biotechhiques 6:958-976); and Stein et al. ((1988) Cancer
Res
48:2659-2668). With respect to antisense DNA, oligodeoxyribonucleotides
derived
from the translation initiation site, e.g., between the -10 and +10 regions of
PDE4D
sequence, are preferred.
To perform antisense therapy, oligonucleotides (mRNA, cDNA or DNA) are
designed that are complementary to mRNA encoding PDE4D. The antisense
oligonucleotides bind to PDE4D 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 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. (1989) Proc. Natl. Acad.
Sci.
USA 86:6553-6556; Lemaitre et al., (1987), Proc. Natl. Acad. Sci. USA 84:648-
652;
PCT International Publication No. W088/09810) or the blood-brain barrier (see,
e.g.,
CA 02499320 2005-03-23
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PCT International Publication No. W089/10134), or hybridization-triggered
cleavage
agents (see, e.g., Krol et al. (1988) BioTechniques 6:958-976) or
intercalating agents.
(See, e.g., Zon, (1988), P7zafnrc. Res. 5:539-549). To this end, the
oligonucleotide
may be conjugated to another molecule (e.g., a peptide, hybridization
triggered cross-
5 linking agent, transport agent, hybridization-triggered cleavage agent). '
The antisense molecules are delivered to cells that express PDE4D in 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
10 antibodies that specifically bind receptors or antigens expressed on the
target cell
surface) can be administered systematically. Alternatively, in another
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
15 sufficient amounts of single stranded RNAs that will form complementary
base pairs
with the endogenous PDE4D transcripts and thereby prevent translation of the
PDE4D mRNA. For example, a vector can be introduced in vivo such that it is
taken
up by a cell and directs the transcription of an antisense RNA. Such a vector
can
remain episomal or become chromosomally integrated, as long as it can be
20 transcribed to produce the desired antisense RNA. Such vectors can be
constructed
by recombinant DNA technology methods standard in the art and described above.
For example, a plasmid, cosmid, YAC or viral vector can be used to prepare the
recombinant DNA construct that can be introduced directly into the tissue
site.
Alternatively, viral vectors can be used which selectively infect the desired
tissue, in
25 which case administration may be accomplished by another route (e.g.,
systemically).
Methods of modulating PDE4D expression by administering an RNA
inhibitor of the activity of the target protein are also possible. The term
"RNA
inhibitor" refers to an inhibitory RNA that silences expression of the target
protein
30 by RNA interference (McManus, M.T. and Sharp, P.A., 2002. Nat. Rev. Genet.
3:737-47; Hannon, G.J., 2002. Nature 418:244-51; Paddison, P.J. and Hannon,
G.J.,
2002. Cancer Cell 2:I7-23). RNA interference is conserved throughout
evolution,
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76
from C. elegafzs to humans, and is believed to function in protecting cells
from
invasion by RNA viruses. When a cell is infected by a dsRNA virus, the dsRNA
is
recognized and targeted for cleavage by an RNaseIII-type enzyme termed Dicer.
The Dicer enzyme "dices" the RNA into short duplexes of 2I nucleotides, termed
S short-interfering RNAs or siRNAs, composed of I9 nucleotides of perfectly
paired
ribonucleotides with two unpaired nucleotides on the 3' end of each strand.
These
short duplexes associate with a multiprotein complex termed RISC, and direct
this
complex to mRNA transcripts with sequence similarity to the siRNA. As a
result,
nucleases present in the RISC complex cleave the mRNA transcript, thereby
abolishing expression of the gene product. In the case of viral infection,
this
mechanism would result in destruction of viral transcripts, thus preventing
viral
synthesis. Since the siRNAs are double-stranded, either strand has the
potential to
associate with RISC and direct silencing of transcripts with sequence
similarity.
Recently, it was determined that gene silencing could be induced by
presenting the cell with the siRNA, mimicking the product of Dicer cleavage
(Elbashir, S.M., et al., 2001. NatuYe 411:494-BElbashir, S.M., et al., 2001.
Gefzes
Dev. 15:188-200). Synthetic siRNA duplexes maintain the ability to associate
with-
RISC and direct silencing of mRNA transcripts, thus providing researchers with
a
powerful tool for gene silencing in mammalian cells. Yet another method to
introduce the dsRNA for gene silencing is shRNA, for short hairpin RNA
(Paddison,
P.T., et al., 2002. Genes Dev. 16:948-58; Brummelkamp, T.R., et al., 2002
Science
296:550-3; Sui, G., et al., 2002. P~oc. Natl. Acad. Sci. U.S.A. 99:5515-20).
In this
case, a desired siRNA sequence is expressed from a plasmid (or virus)
containing an
"shRNA" gene having an inverted repeat with an intervening loop sequence to
form
a hairpin structure. The resulting shRNA transcript containing the hairpin is
subsequently processed by Dicer to produce siRNAs for silencing. Plasmid-based
shRNAs can be expressed stably in cells, allowing long-term gene silencing in
cells,
or even in animals (McCaffrey,A.P., et al., 2002. Natzzy~e 4I8:38-9; Xia, H.,
et al.,
2002. Nat. Biotech. 20:1006-10; Lewis, D.L., et al., 2002. Nat. Geyzetics 32:
I07-8;
Rubinson, D.A., et al., 2003. Nat. Genetics 33:401-6; Tiscornia, G., et al.,
(2003)
P~oc. Natl. Acad. Sci. U.S.A. IOO:I844-8). RNA interference has been
successfully
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77
used therapeutically to protect mice from fulminant hepatitis (Song, E., et
al., 2003.
Nat. Medicine 9:347-51).
Endogenous PDE4D expression can be also reduced by inactivating or
"knocking out" PDE4D or its promoter using targeted homologous recombination
(e.g., see Smithies et al. (1985) Nature 327:230-234; Thomas & Capecchi (1987)
Cell 51:503-512; Thompson et al. (1989) Cell 5:313-321). For example, a
mutant,
non-functional PDE4D (or a completely unrelated DNA sequence) flanked by DNA
homologous to the endogenous PDE4D (either the coding regions or regulatory
regions of PDE4D) can be used, with or without a selectable marker and/or a
negative selectable marker, to transfect cells that express PDE4D ira vivo.
Insertion
of the DNA construct, via targeted homologous recombination, results in
inactivation
of PDE4D. 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-mutant PDE4D can be increased using a similar
I S method: targeted homologous recombination can be used to insert a DNA
construct
comprising a non-mutant, functional PDE4D (e.g., a gene having SECT ID NO: 1
which may optionally comprise at least one polymorphism shown in Tables 11 and
12), or a portion thereof, in place of a mutant PDE4D in the cell, as
described above.
In another embodiment, targeted homologous recombination can be used to insert
a
DNA construct comprising a nucleic acid that encodes a PDE4D polypeptide
variant
that differs from that present in the cell.
Alternatively, endogenous PDE4D expression can be reduced by targeting
deoxyribonucleotide sequences complementary to the regulatory region of PDE4D
(i.e., the PDE4D promoter and/or enhancers) to form triple helical structures
that
prevent transcription of PDE4D in target cells in the body. (See generally,
Helene, C.
(1991) Anticancer Drug Des., 6(6):569-84; Helene, C., et al. (1992) Ahh, N. Y.
Acad.
Sci., 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-25). Likewise,
the
antisense constructs described herein, by antagonizing the normal biological
activity
of one of the PDE4D proteins, can be used in the manipulation of tissue, e.g.,
tissue
differentiation, both isi vivo and for 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 a PDE4D mRNA or gene
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78
sequence) can be used to investigate role of PDE4D in developmental events, as
well
as the normal cellular function of PDE4D in adult tissue. 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 PDE4D therapeutic agents
as described herein can also be used in the treatment or prevention of stroke.
The
therapeutic agents can be delivered in a composition, as described above, or
by
themselves. They can be administered systemically, or can be targeted to a
particular
tissue. The therapeutic agents can be produced by a variety of means,
including
chemical synthesis; recombinant production; ifa vivo production (e.g., a
transgenic
animal, such as U.S. Patent 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-mutant PDE4D polypeptide in conjunction with antisense therapy
targeting
mutant PDE4D mRNA; administration of a first splicing variant encoded by PDE4D
in conjunction with antisense;therapy targeting a second splicing encoded by
PDE4D), can also be used.
The invention will be further described by the following non-limiting
examples. The teachings of all publications cited herein are incorporated
herein by
reference in their entirety.
EXAMPLES
EXAMPLE 1: PDE4D VARIATIONS AND HAPLOTYPES INCREASE RISK
FOR STROKE
Icelandic Stroke Patients and Phenotype Characterization
A population-based list containing 2543 Icelandic stroke patients, diagnosed
from 1993 through 1997, was derived from two major hospitals in Iceland and
the
Icelandic Heart Association (the study was approved by the Icelandic Data
Protection
Commission of Iceland and the National Bioethics Committee). Patients with
hemorrhagic stroke represented 6% of all patients (patients with the Icelandic
type of
hereditary cerebral hemorrhage with amyloidosis and patients with subarachnoid
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hemorrhage were excluded). Ischemic stroke accounted for 67% of the total
patients
and TIAs 27%. The distribution of stroke suptypes in this study is similar to
that
reported in other Caucasian populations (Mohr, J.P., et al., Neurology, 2:754-
762
(1978); L. R. Caplan, In Sts°oke, A Clinical Apps°oach
(Butterworth-Heinemann,
Stoneham, MA, ed 3, (1993)).
The list of approximately 2000 living patients was run through our
computerized genealogy database. A comprehensive genealogy database that has
been established at deCODE genetics was used to cluster the patients in
pedigrees.
Each version of the computerized genealogy database was reversibly encrypted
by
the Data Protection Commission of Iceland before arriving at the laboratory
(Gulcher, J.R., et al., EuY. J. Hum. Genet. 8:739 (2000)). The database uses a
patient.
list, with encrypted personal identifiers, as input, and recursive algorithms
to find all
ancestors in the database who are related to any member on the input list
within a
given number of generations back (Gulcher, J.R., and Stefansson, K., Clin.
Chem.
Lab. Med. 36:523 (2998)) covering the whole Icelandic nation. The cluster
function
then searches for ancestors who are common to any two or more members of the
input list. One hundred and seventy-nine families with two or more living
patients
were chosen for the study with a total of 476 patients connected within 6
meioses (6
meioses connect second cousins). Informed consent was obtained from all
patients
and their relatives whose DNA samples were used in the linkage scan. The mean
separation between affected pairs is 4.8 meioses. Of the patients selected for
the
study 73% had ischemic strokes, 23% TIAs and 4% hemorrhagic strokes.
In the selected families, hemorrhagic stroke patients clustered with ischemic
stroke and TIA. patients, and there were no families with a striking
preponderance of
hemorrhagic stroke or of the subtypes of ischemic stroke. Patients with
ischemic
stroke were reclassified according to the TOAST (Trial of Org 10172 in Acute
Stroke Treatment) sub-classification system for stroke (Adams, H.P., Jr., et
al.,
Stroke, 24:34-4I (1993)). This system includes five categories: (1) large-
artery
atherosclerosis, (2) cardioembolism, (3) small-artery occlusion (lacune), (4)
stroke of
other determined etiology and (5) stroke of undetermined etiology. The
diagnoses
were based on clinical features and on data from ancillary diagnostic studies.
Patients defined with large-artery atherosclerosis had clinical and brain
imaging
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findings of cerebral cortical dysfunction and either significant (>70%)
stenosis (this
is a stricter criteria than used in TOAST where 50% stenosis is the cut-off)
or
occlusion of a major brain artery or branch cortical artery. Potential sources
of
cardiogenic embolism were excluded. The category caxdioembolism included
5 patients with at least one cardiac source for an embolus and potential large-
artery
sources of thromobosis and embolism was eliminated. Patients with small-artery
occlusion had one of the traditional clinical lacunar syndromes and no
evidence of
cerebral cortical dysfunction. Potential cardiac source of embolus and
stenosis >70%
in an ipsilateral extracranial artery was excluded. The category, acute stroke
of other
10 determined etiology, included patients with rare causes of stroke and
patients with
two or more potential causes of stroke. If the causes of stroke could not be
determined despite extensive evaluation patients were included in the category
stroke
of undeterniined etiology. FIG. 1 displays two pedigrees each affected by
several of
the stroke subtypes, including hemorrhagic stroke. Apparently what is
inherited in
IS stroke is the broadly defined phenotype.
Genonae-wide scab
A genome-wide scan was performed using a framework map of about 1000
microsatellite markers. The DNA samples were genotyped using approximately
20 1000 fluorescently labelled primers. A microsatellite screening set based
in part on
the ABI Linkage Marker (v2) screening set and the ABI Linkage Marker (v2)
intercalating set in combination with S00 custom-made markers were developed.
All
markers were extensively tested for robustness, ease of scoring, and
efficiency in 4X
multiplex PCR reactions. In the framework marker set, the average spacing
between
25 markers was approximately 4 cM With no gaps larger than 10 cM. Marker
positions
were obtained from the Marshfield map, except for a three-marker putative
inversion
on chromosome 8 (Jonsdottir, G.M., et al., Am. J. Hum. Genet., 67 (Suppl.
2):332
(2000); Yu, A., et al., Am. J. Hutn. Genet. 67 (Suppl. 2):10 (2000). The PCR
amplifications were set up, run and pooled on Perkin Elmer/Applied Biosystems
877
30 Integrated Catalyst Thermocyclers with a similar protocol for each marker.
The
reaction volume used was 5 ~,l and for each PCR reaction 20 ng of genomic DNA
was amplified in the presence of 2 pmol of each primer, 0.25 U AMPLITAQ GOLD
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(DNA polymerise; trademark of Roche Molecular Systems), 0.2 mM dNTPs and 2.5
rnM MgCl2 (buffer was supplied by manufacturer). The PCR conditions used were
95°C for 10 minutes, then 37 cycles of 15 s at 94°C, 30s at
55°C and 1 min at 72°C.
The PCR products were supplemented with the internal size standard and the
pools
were separated and detected on Applied Biosystems model 377 Sequencer using
v3.0
GENESCAN (peak calling software; trademark of Applied Biosystems). Alleles
were called automatically with the TRUEALLELE (computer program for alleles
identification; trademark of Cybergenetics, Inc.) program, and the program,
DECODE-GT (computer editing program that works downstream of the
TRUEALLELE program; trademark of deCODE genetics), was used to fractionate
according to quality and edit the called genotypes (Palsson, B., et al.,
Genome Res.
9:1002 (1999)). At least 180 Icelandic controls were genotyped to derive
allelic
frequencies.
A total of 476 patients and 438 relatives were genotyped. The data was
analyzed and the statistical significance determined by applying affecteds-
only
allele-sharing methods (which does not specify any particular inheritance
model)
implemented in the ALLEGRO (computer program for multipoint linkage analysis;
trademark of deCODE genetics) program that calculates lod scores based on
multipoint calculations. Our baseline linkage analysis uses the Spars scoring
function
(Kruglyak, L., et al., Am. J. Hum. Genet., 58:1347 (1996)), the exponential
allele-
sharing model (Kong, A. and Cox, N.J., Am. J. Hum. Genet., 61:1179 (1997)),
and a
family weighting scheme which is halfway, on the log scale, between weighting
each
affected pair equally and weighting each family equally. In the analysis we
treat all
genotyped individuals who are not affected as "unknown". All linkage analyses
in
this paper were performed using multipoint calculation with the program
ALLEGRO
(deCODE genetics) (Gudbjartsson, D.F., et al., Nat. Genet. 25:12 (2000)).
The allele sharing lod scores for the genome scan using the framework map
showed three regions that achieved a lod score above 1Ø Two of these regions
are
on chromosome Sq. The first peak is at approximately 69 cM with a lod score of
2.00. The second peak is at 99 cM with a lod score of 1.14. The third region
is on
chromosome 14q at 55 cM with a lod score of 1.24.
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The information for linkage at the Sq locus was increased by genotyping an
additional 45 markers over a 45 cM segment which spanned both peals. The
information used here is defined by Nicolae (D. L. Nicolae, Thesis, University
of
Chicago (1999)) and has been demonstrated to be asymptotically equivalent to a
classical measure of the fraction of missing information (Dempster, A.P., et
al., J. R.
Statist. Soc. B, 39:1 (19?7)). While the lod score at the second peak dropped
slightly
to around 1.05, the lod score at the first peak increased to 3.39. However,
close
inspection of our results suggested that not only does the Marshfield genetic
map
lack resolution (many markers assigned the same map location), but also there
may
be some errors in their order. As a result, the genetic length of the region
estimated
using our material was substantially greater than what is reported. By
modifying the
ALLEGRO (deCODE genetics) program, we applied the EM algorithm to our data to
estimate the genetic distances between markers. We found that our estimate of
the
genetic length of the region was substantially longer than that given in the
Marshfield.
map. Tbis indicates a problem with marker order because, in general, incorrect
marker order leads to an increased number of apparent crossovers and increases
the
apparent genetic length.
Playsical and genetic mapping
The marker order and inter-marker distances were improved by constructing
high density physical and genetic maps over a 20 cM region between maxkers
DSS474 and DSS2046. A combination of data from coincident hybridizations of
BAC membranes using a high density of STSs and the Fingerprinting Contig
database was used to build laxge contigs of BACs from the RPCI -11 library.
The
order of the linkage maxkers was also confirmed by high-resolution genetic
mapping
using the stroke families supplemented with over 112 other large nuclear
families.
High resolution genetic mapping was used both to anchor and place in order
contigs
found by physical mapping as well as to obtain accurate inter-marker distances
for
the correctly ordered markers. Data from 112 Icelandic nuclear families
(sibships
with their parents, containing from two to seven siblings) were analyzed
together
with the nuclear families available within the stroke pedigrees. For the
purpose of
genetic mapping the 112 nuclear families alone provide 588 meioses, and the
total
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83
number of meioses available for mapping was over 2000. By comparison, the
Marshfield genetic map was constructed based on I82 meioses. The large number
of
meiotic events within our families provides the ability to map markers to the
resolution of 0.5 to 1.0 cM. Combining this information with the physical map
resulted in a highly reliable order of markers and inter-marker distances
within this
20 cM region. Linkage markers common to the genetic and physical maps were
used
to anchor and place in order four of the physically mapped contigs. By
integrating
the genetic and physical maps a most likely order of 30 polymorphic markers
was
derived.
BAC contigs were generated by a method that combines coincident primer
hybridization with data mining. The RPCI-11 human male BAC library segments 1
.
& 2 (Dieter de Jong; Children's Hospital Oakland Research Institute)
containing
about 200,000 clones with a 12X coverage, were gridded using a 6x6 double
offset
pattern in 23 cm x 23 cm membranes with a BioGrid robot (Biorobotics Ltd., , .
Cambridge, UI~). Initially, hybridizations were performed with markers in the
region of interest according to their location in the Weizmann Institute
Unified
Database. Primer sequences were analyzed and discarded according to their
content
of known repeats, E. coli and vector sequences (the analysis was performed
using
software developed at deCODE genetics). One hundred and fifty markers in the
region (30 polymorphic markers used in linkage and I20 generated from STSs)
separated by an average of 130 kb were used. The selected markers were used to
generate two 32P labelled probes, F that contained the pooled forward primers
and R
that contained the pooled reverse primers. Reading of positive signals was
performed automatically from digitized images of resulting autoradiograms by
informatics tools developed at deCODE genetics. The coincident signals in both
hybridizations were selected as positive clones. A set of overlapping clones
was
assembled through a combination of hybridization and BAC fingerprint walking.
Fingerprints of positive clones were analyzed using the FPC database developed
at
the Sanger Center. Data from FPC contigs prebuilt with a cutoff of 3e-12 and
from
sequence datamining was integrated with the hybridization results. BACs in the
region detected by data mining and hybridization were re-arrayed using a
Multiprobe
IIex robot (Packard, Meriden, CT). SmaII membranes (8 cm x 12 cm) were gridded
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84
in 6x6 double offset pattern and individually hybridized with the markers of
interest.
Positive patterns were transferred using transparencies to an Excel file
containing
macros to provide BAC to marker associations. A visual map was generated by
combining the hybridization, fingerprinting and sequence data. New markers
were
generated from BAC end sequences to close the gap. After several rounds of
hybridization positive BACs were assembled into 7 contigs covering
approximately
20 Mb. Thirty of the polymorphic markers used in linkage were assigned to four
of
the contigs. Estimation of contig lengths and distance between markers
assigned to
them was based on the FPC program.
Twenty-seven of our 30 linkage markers mapped to three contigs in the
October 2000 release from UCSC, the UC Santa Cruz (LJCSC) draft assembly. The
marker order within the contigs is in agreement with our order with the
exception of
two markers. Although the UCSC assemblies are improving, some contigs have
incorrect order, orientation, or contig assembly. We believe that high
resolution
genetic mapping and perhaps focused hybridization experiments are still
necessary o
confirm accuracy of sequence assemblies. In addition, high resolution genetic
mapping provides better estimates of inter-marker genetic distances that are
also
important for linkage analysis (Halpern, J. and Whittennore, A.S., Hum. Hered.
49:194 (1999); Daw, E.W., et al., Genet. Epidemiol. 19:366 (2000)).
Statistical Methods for Linkage Analysis
lVIuItipoint, affected-only allele-sharing methods were used in the analyses
to
assess evidence for linkage. All results, both the LOD-score and the non-
pararnetric
linkage (NPL) score, were obtained using the program Allegro (Gudbjartsson et
al.,
Nat. Genet. 25:12-3, 2000). Our baseline linkage analysis, as previously
described
(Gretarsdottir et al., Am JHom Genet, 70:593-603, 2002), uses the Spars
scoring
function (Whittemore, A.S., Halpern, J. (1994), Biometrics 50:118-27; Kruglyak
L,
et al. (1996), Am JHum Genet 5:1347-63), the exponential allele-sharing model
(Kong, A. and Cox, N.J. (1997), Ana JHuna Genet 61:1179-88) and a family
weighting scheme that is halfway, on the log-scale, between weighting each
affected
pair equally and weighting each family equally. The information measure we use
is
part of the Allegro program output and the information value equals zero if
the
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marker genotypes are completely uninformative and equals one if the genotypes
determine the exact amount of allele sharing by decent among the affected
relatives
(Gretarsdottir et al., Am. J. Horn. Ge3aet, 70:593-603, (2002)). We computed
the P-
values two different ways and here report the less significant result. The
first P-value
5 was computed on the basis of large sample theory; the distribution of Zlr =
~(2[loge(10)LOD]) approximates a standard normal variable under the null
hypothesis of no linkage (Kong, A. and Cox, N.J. (1997), Ana JHum Genet
61:1179-
88). The second P-value was calculated by comparing the observed LOD-score
with
its complete data sampling distribution under the null hypothesis
(Gudbjartsson et
10 al., Nat. Gefzet. 25:12-3, 2000). When the data consist of more than a few
families,
as is the case here, these two P-values tend to be very similar.
Final liyakage Yesults a~td localization
Linkage analysis including genotypes from the higher density markers using
IS the deCODE marker order resulted in a lod score of 4.40 (P = 3.9 X 10-6) on
chromosome Sql2 at the marker DSS2080. The reported P value is part of the
output
of the ALLEGRO (deCODE genetics) program which was developed at deCODE
and has become a standard linkage program worldwide over the last 3 years
(Gudbjartsson et al., Nat.~Geyaet. 25:12-3, 2000). We have given it to over
200
20 academic departments around the world free of charge and it is widely used.
The
locus has been designated as STRKI. With the addition of these extra markers,
it was
possible to narrow down the region to a segment less than 6 cM, from DSS1474
to
DSS398, as defined by one drop in lod.
To further investigate the contribution of this susceptibility locus to
stroke, a
25 range of parametric models were fitted to the data. However, all analyses
were still
affeeteds oyr.ly in the sense that individuals were either classified as
affecteds or
having unknown disease status. A lod score of 4.08 was obtained with a
dominant
model where the allele frequency of the susceptibility gene was assumed to be
5%
and Garners of the alteration were assumed to have seven-fold the risk of a
non-
30 carrier. By inspecting the individual families, no obvious correlation was
seen
between families that contribute positively to the linkage results with the
prevalence
of hypertension, diabetes or hyperlipidemias. When the data were reanalyzed
with
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86
the hemorrhagic stroke patients removed, the allele sharing lod score
increased to
4.86 at DSS2080. Although this 0.46 increase in log score suggests that STRI~1
is
involved primarily in ischemic stroke and TTAs, it is not statistically
significant
based on simulations (one sided P equals 0.09). In order to assess whether
such a
S change in lod score would be likely to occur by chance we selected 1000
random sets
of 22 patients whose status we then changed to "unknown" in an analysis. The P
value we present is the fraction of the 1000 simulations which produce a lod
score
increase at the peak locus equal to or greater than that which we observed by
changing the affection status of the 22 hemorrhagic stroke patients to
"unknown".
Idef2tificatioyz ofAllelicAssociatiofa
All micxosatellite markers in the approx. 6 cM interval (markers from
DSS398 to DSS1474) were analyzed with respect to allelic association.
1 S MicYOSatellite allelic association
We initially genotyped 864 Icelandic stroke patients and 908 controls using a
total of 98 microsatellite markers. These markers axe distributed over a
region of
approximately 11 Mb. The region is centered on our linkage peak and
corresponds
to the 2 LOD drop. The density of markers is greater in the central 3.7 Mb
portion of
the region, which includes the 1 LOD drop, with an average spacing of one
marker
every S3 kb. We have designated this central region, which is flanked by
maxkers
DSS1474 and DSS398, as the STRKI interval. Three markers, AC027322-S;
DSS2121 and AC008818-1, showed a difference in allelic frequency between
patients and controls with p-values less than 0.01 (Table 1). Correcting for
the
2S relatedness of the Icelandic patients had little impact on the p-values,
but after
correcting for the number of markers and alleles tested none of these p-values
were
significant (Table 1).
We had previously observed that our linkage peak increased, albeit not
significantly, when excluding the hemorrhagic stroke patients. We therefore
tested
only those patients with ischemic stroke or TIA for association to the
markers. In
addition, our ischemic stroke and TIA. patients have been sub-classified
according to
the TOAST research criteria and we also repeated the association analysis
separately
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for patients with the three TOAST subcategories: cardiogenic, carotid (greater
than
70% stenosis) and small vessel occlusive disease. Lastly, we tested the
combination
of patients with cardiogenic and carotid stroke, since these categories of
stroke are
most clearly related to atherosclerosis. The results for each of these
association
studies are presented in Table 1. Three of the tests, one for cardiogenic
stroke
(AC008818-1), one for carotid stroke (DGSS397), and one for the combination of
carotid and cardiogenic stroke (AC008818-1) were significant even after
correcting .
for multiple testing (Table 1). The marker DGSS397 is located within the PDE4D
gene and AC008818-1 is in the 5' end of PDE4D and in the overlapping gene
Prostate androgen-regulated transcript (PARTI) whose transcript is on the
other
strand going in the opposite direction. PDE4D is an important regulator of
intracellular levels of cAMP and is expressed widely. PARTI encodes a putative
protein with unknown function predominantly expressed in the prostate gland
and in
several cancer cell lines. Physical locations of all genotyped markers and
PDE4D
and PARTl exons are available in Table 2C. The association results for the
combination of carotid and cardiogenic stroke ware particularly striking with
an
allele frequency of 35.5% in patients for allele 0 (the CEPH reference allele)
of
marker AC008818-1 versus 25.5% in controls. The unadjusted p-value for this
marker is 0.0000015, and after adjusting fox multiple testing of markers is
0.00025
(Table 1). This remains significant even after adjusting for the several
phenotypes
sW died. The risk of this allele to the other alleles of this marker, assuming
the
multiplicative model Terwilliger, J.D. & Ott, J. A haplotype-based 'haplotype
relative
risk' approach to detecting allelic associations. Hmn Hef~ed 42, 337-46 (1992)
and
Falk, C.T. & Rubinstein, P. Haplotype relative risks: an easy reliable way to
construct a proper control sample for risk calculations. Ann Hutn Genet 51 (
Pt 3),
227-33 (1987), was estimated to be 1.60, and the corresponding population
attributable risk was 25%.
Thus, the strong association signals from our initial microsatellite
association
studies helped to focus our attention on the STRKI interval and, in
particular, to the
PDE4D gene region.
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Table 1.
Microsatellite allelic association analysis of the two-lod drop of the STRKl
locus.
All microsatellites that show association with a p-value less than 0.09 for
all stroke, all stroke
excluding hemorrhagic stroke, cardiogenic stroke, carotid stroke, small vessel
disease and
Phenotype Marker Allele p-value RR # Aff. '4~' # Cfirl
All
AC027322-5 10 0.001 3.34 787 1.90 779 0.6
D5S2121 -2 0.0027 2.19 824 2.7 870 1.3
AC008818-1 0 0.0045 1.25 815 29.9 891 25.5
All patients -
excluding
hemorrhagic
stroke
AC027322-5 10 0.00052 3.56 740 20 779 0.6
D5S2121 -2 0.0023 2.23 774 2.8 870 1.3
AC008818-1 0 0.0062 1.24 764 29.9 891 25.5
Cardiogenic
stroke
AC008818-1 0 0.000054*1.60216 35.4 891 25.5
D5S1990 20 0.00053 2.18223 7.9 879 3.8
D5S2089 -10 0.0027 2.22219 5.9 813 2.8
D5S1359 2 0.0044 1.39214 36.0 777 28.8
AC016604-2 0 0.0048 1.44170 51.8 446 42.7
AC008804-1 0 0.0068 1.52128 36.3 367 27.3
AC022125-3 0 0.0077 1.36223 36.8 775 30.0
DG5S2066 0 0.0095 1.80166 92.5 501 87.2
DG5S2039 9 0.0084 2.00167 8.7 491 4.6
D5S647 -6 0.0091 2.43199 3.8 789 1.6
Carotid stroke
DG5S397 4 0.00024* 1.70124 65.7 577 53.0
DG5S2056 12 0.0009 3.3380 8.8 464 2.8
AC008818-1 0 0.001 1.61125 35.6 891 25.5
DG5S2039 -3 0.003 1.6296 45.8 491 34.3
DG5S2045 0 0.0051 1.8055 57.3 339 42.6
DG5S818 6 0.0079 1.50111 63.1 563 53.3
AC016604-3 4 0.0072 1.5399 40.9 645 31.2
Small vessel
disease
D5S1359 2 0.0085 1.41157 36.3 777 28.8
D5S2080 2 0.0092 1.38153 54.6 885 46.5
D5S2121 -2 0.0059 2.93152 3.6 870 1.3
Combined
cardiogenic &
carotid stroke
AC008818-1 0 0.0000015*1.60341 35.5 891 25.5
AC008833-6 0 0.0026 1.35335 70.3 868 63.8
DG5S2066 0 0.0032 1.74258 92.3 501 87.2
DG5S397 4 0.009 1.29345 59.3 577 53.0
D5S2121 -2 0.0081 2.39336 3.0 870 1.3
the combination
of cardiogenic
and carotid
stroke
*significant for
after adjusting multiple
testing
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Alleles #'s: For SNP alleles A = 0, C = 1, G = 2, T = 3; for microsatellite
alleles: the
CEPH sample 1347-02 (CEPH genomics repository) is used as a reference, the
lower allele
of each microsatellite in this sample is set at 0 and all other alleles in
other samples are
numbered accordingly in relation to this reference. Thus allelel is 1 by
longer than the lower
allele in the CEPH sample 1347-02, allele 2 is 2 by longer than the lower
allele in the CEPH
sample 1347-02, allele 3 is 3 by longer than the lower allele in the CEPH
sample 1347-02,
allele 4 is 4 by longer than the lower allele in the CEPH sample 1347-02,
allele -I is 1 by
shorter than the lower allele in the CEPH sample 1347-02, allele -2 is 2 by
shorter than the
lower allele in the CEPH sample 1347-02, and so on. Note that this same CEPH
sample is a
standard that is widely used throughout the world for calibration and
comparison of alleles.
AC008818-1 amplimer:
TGCTTGGTGAAGGAATAGCCACCCCAGAGAAGGAGTATGGACTTC
TATACACAATCATTCATTCATTCATTCATTCATTCATTCATTCATTCATTC
ACTACTCATGCATGATCTTTGTCCTTATCTTCCTCCACTGTCACATGAATA
CCCACCCACTGCACCTACCTGCTTCCTATTCCTGAGAACCCAGGCTC(SEQ
ID NO: 86)
AC008818-1, allele 0 is the same allele as the minimum allele observed in
CEPH 1347-02, family 137, individual 02.
Swedish patients have also been genotyped and microsatellite single and
multimarker association has been analyzed using the E-M algorithm. A total
number
of 943 Swedish patients (stroke patients and patients with carotid stenosis)
and 322 '
Swedish controls were analyzed (results shown in Table 2A). At least three
haplotypes were more common in patients compared to controls, confirming in a
second population that PDE4D shows association to stroke.
Table 2A
Swedish Patient Association
Markers Alleles pAllelicAll Frq All Frq # #
Aff Ctrl aff ctrl
Swedish patients
(n=943)
DSS2000 2 0.0024 912 318
(Sw 2) AC022125-3 0 0 Z 0.006 0.035 0.01 717 284
0
AC008833-6 DSS2000
DSS2091
(Sw-1) AC008804-2 -2 4 0.00280.057 0.05 672 I
D17-H -2 10 13
DI7-G DSS2080
AC008804-2 D17-H -4 0 0.00370.056 0.03 700 123
D17-G -2
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Scf°eefaing for polyn2orplaisms in PDE4D
We next considered whether a functional variant in the PDE4D gene might
be the cause of our observed microsatellite association. We matched public
domain
ESTs and our own RT-PCR and RACE transcripts to our sequence of the STRKI
5 interval. We defined new alternative PDE4D transcripts, which together with
previously known transcripts indicated that the PDE4D gene contains 22 exons
over
at least 1.5 Mb and overlaps with PARTI. The PDE4D gene encodes eight protein
isoforms and has at least seven promoters. All isoforms identified have an
identical
C-terminal catalytic domain but differ at the N-terminal regulatory domain
(FIG. 2).
10 We then attempted to identify mutations by sequencing all known PDE4D
exons (including the overlapping PARTI exons) and, on average, 100 by of their
flanking introns in 188 patients and 94 controls. Forty-six polymorphisms were
identified; 44 SNPs and two intxonic deletions. Only two of the polymorphisms,
both SNPs, were found within the coding exons of the PDE4D gene, which is
15 consistent with the extraordinary lack of variation that others have
reported for all
four PDE4 classes Houslay, M.D. & Adams, D.R. PDE4 cAMP phosphodiesterases:
modular enzymes that orchestrate signalling cross-talk, desensitization and
compartmentalization. Biochern J370, 1-18 (2003). The two coding SNPs were
typed for additional patients and controls. However, these SNPs did not show
20 significant association to stroke (Table 2B). Therefore, if a functional
variant
conferring risk for stroke exists in the PDE4D gene, it may be within
regulatory
regions affecting transcription, splicing, message stability, or message
transport of
one ox more isoforms, or in exons that we have not yet identified.
25 Table 2B
Frequency of PDE4D coding mutations.
PDE4D
Markers AA change exon Allele p-val~re Aff. % Ctrl. % # Aff # Ctrl
SNP 250 Pro > Thr D1/D2 /a 0.163 2.0 1.5 604 369
SNP 257 Lys > Thr 4 C 0.381 0.2 0.0 474 294
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PDE4D isoform expression
Failing to find a functional mutation in the known coding exons of PDE4D,
we were interested to consider other possible evidence in favor of this gene
being a
source of the underlying association in this region. We conducted an
experiment to
study the expression levels of the various isoforms - with any significant
differences
between patients and controls potentially indicating that regulation of PDE4D
is a
key element in stroke susceptibility. We used EBV transformed B cell lines
from
randomly selected patients having ischemic stroke or TIA and from controls. We
carried out isoform-specific kinetic RT-PCR analysis to quantify each isoform
in 83
stroke patients and 84 controls. The patients were principally ischemic stroke
patients, with 32 of them having cardiogenic or carotid stroke. We observed
that the
total PDE4D message level, as assessed by amplification across exons present
in all
isoforms (PAID, was significantly lower in patients than in controls (p-value
=
0.0021). This decrease was due primarily to lower expression of the PDE4D 1,
PDE4D2 and PDE4D5 isoforms. This significant disregulation of the expression
of
multiple PDE4D isoforms greatly encouraged us to.continue our investigations
into
the association of the PDE4D gene to stroke.
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Table 2C: SNP icleh.tificatiort, sirtgle marker association grad LD trtapping
of the
PDE4D region
SNP marker Publicstart end in start end
or exon in NCBI NCBI in SEQ in
SEQ
code name build build ID NO: ID
31 33 1 NO:
1
AC016604-3 5754704557547304
AC016604-2 5762314857623287
exon 11 58241020582414321655335 1655747
y
exon 10 58242009582421911654576 1654758
exon 9 58242702582428241653943 1654065
exon 8 58243543582436971653070 1653224
exon 7 58254845582549441641818 1641917
exon 6 58256107582562711640491 1640655
exon 5 58257156582572541639508 1639606
exon 4 58258185582583561638406 1638578
exon 3 58259724582598171636944 1637037
exon D1/D2 58305211583055811591172 1591425
exon LF4 58446946584469951449835 1449884
exon LF3 58451540584516131445217 1445290
exon LF2 58459851584598871436943 1436979
exon LF1 58482128584823191414511 1414702
AC022125-3 58504109585042741392556 1392721
SNP SNP5PD890407 58506423585064231390407 1390407
204
AC008833-6 58507019585072221389608 1389811
exon D9 58541689585424701354347 1355128
D5S2000 5858546058585849
D5S2091 5859328458593634
exon D8 58623109586234141273404 1273709
D17- C 58645088586453861251432 1251730
AC008804-1 58784449587846411112181 1112373
AC008804-2 58817743588179311078881 1079069
exon D3 58852680588528191044051 1044190
D17- H 58860588588607251036142 1036279
D17- G 5894227058942541954298 954569
D5S2080 5899868558999021
exon D5 5903459859035009861791 862202
AC027322-5 5915922159159326737420 737519
exon D4 5915952059160492736254 737226
SNP SNP5PD166822rs7142915922989759229897
102
exon D7-3 5925484059255069641649 641878
SNP SNP5PD138604rs13474015925811359258113638605 638605
101
SNP SNP5PD121753rs15450705927496259274962
100
SNP SNP5PD118378rs15330195927833859278338
99
SNP SNP5PD117029rs9521105927968759279687
98
SNP SNP5PD104361rs19957805929235659292356
97
SNP SNP5PD97409 5929930859299308
96
SNP SNP5PD97281rs20163245929943759299437
95
SNP SNP5PD75406rs13964745932131359321313
94
SNP SNP5PD73383rs15088645932333659323336
93
SNP 5NP5PD72097rs15088595932462259324622
92
DG5S2045 5932531359325563571152 571406
DG5S2039 5933279959333077563636 563921
SNP SNP5PD46864rs15088635934985159349851
91
SNP SNP5PD43868rs21362035935284959352849
90
SNP SNP5PD29517rs13964765936716759367167
89
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DG5S2056 5938110259381367 515317 515582
DG5S818 5938477659384999 511685 511908
SNP88SNP5PDM14337rs15447885941102159411021
DG5S397 5943850659438784 457900 458178
SNP87SNP5PDM43741rs29108295944042459440424
exon D7 5945190959452039 444645 444775
-2
SNP86SNP5PDM57997rs29629725945468059454680
SNP85SNP5PDM65461rs29618975946214459462144
SNP84SNP5PDM67604rs7197025946428759464287
SNP83SNP5PDM76361rs9662215947304559473045
SNP82SNP5PDM83539rs29619035948022359480223
SNP81SNP5PDM89176 5948585959485859 410826 410826
SNP80SNP5PDM89683rs18626145948636859486368
DG5S2066 5952208559522346 374339 374600
SNP79SNP5PDM132154 5952883859528838 367847 367847
SNP78SNP5PDM153120 5954980459549804 346881 346881
SNP77SNP5PDM161561 5955824559558245 338440 338440
SNP76SNP5PDM166786 5956347059563470 333215 333215
SNP75SNP5PDM181173 5957785659577856 318829 318829
SNP74SNP5PDM182792 5957947559579475 317210 317210
SNP73SNP5PDM211974 5960865059608650 288027 288027
SNP72SNP5PDM217886 5961455759614557 282115 282115
SNP71SNP5PDM218639 5961531059615310 281362 281362
SNP70SNP5PDM224528 5962119059621190 275473 275473
SNP69SNP5PDM236461rs14232485963312459633124
SNP68SNP5PDM259844 5965650459656504 240157 240157
SNP67SNP5PDM261488 5965814859658148 238513 238513
SNP66SNP5PDM265669 5966232859662328 234332 234332
SNP65SNP5PDM271674rs9185905966833359668333
SNP64SNP5PDM275805rs14232475967246359672463
SNP63SNP5PDM280894rs7893895967755159677551
SNP62SNP5PDM285592 5968224759682247 214409 214409
SNP61SNP5PDM296955rs376915969361059693610
SNP60SNP5PDM299842 5969649759696497 200159 200159
SNP59SNP5PDM307243rs376845970389059703890
SNP58SNP5PDM308509rs28982785970515559705155
SNP57SNP5PDM310220rs4012075970686659706866
SNP56SNP5PDM310653rs7025535970729859707298
SNP55SNP5PDM324741rs2517265972138759721387
SNP54SNP5PDM326519rs272235972316559723165
SNP53SNP5PDM329913 5972655659726556 170088 170088
SNP52SNP5PDM332989 5972963259729632 166900 166900
SNP51SNP5PDM338487 5973512259735122 161514 161514
SNP50SNP5PDM345627rs1735915974224859742248
SNP49SNP5PDM349039rs272205974566159745661
SNP48SNP5PDM351840rs377605974846159748461
SNP47SNP5PDM356081 5975270159752701 143922 143922
SNP46SNP5PDM356447 5975306759753067 143555 143555
SNP45SNP5PDM357221 5975384259753842 142780 142780
SNP44SNP5PDM357245 5975386559753865 142757 142757
SNP43SNP5PDM357445 5975406659754066 142556 142556
PART1-exon 5975428459754775
1
exon D7-1 5975429459754415 142207 142328
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PART1-exon 5975601359757617
2
SNP SNP5PDM361194rs1530315975781659757816
42
SNP SNP5PDM361545 5975834159758341 138456 138456
41
AC008818 SEQ 5975988259760075 136740 136547
- 1 ID
NO:
86
SNP SNP5PDM363736 5976035759760357 136265 136265
40
SNP SNP5PDM364360rs38871755976098159760981
39
SNP SNP5PDM364848 5976146959761469 135152 135152
38
SNP SNP5PDM364888rs269565976151059761510 135112 135112
37
SNP SNP5PDM366629 5976325059763250 133371 133371
36
SNP SNP5PDM367438rs269555976406059764060 132562 132562
35
SNP SNP5PDM368135rs276535976475559764755 131865 131865
34
SNP SNP5PDM369610 5976622959766229 130391 130391
33
SNP SNP5PDM370640 5976725959767259 129361 129361
32
SNP SNP5PDM370641rs4570535976726059767261 129360 129360
31
SNP SNP5PDM374696rs272215977131659771316 125304 125304
30
SNP SNP5PDM376181rs29631105977280059772800
29
SNP SNP5PDM376575rs353875977319459773194 123426 123426
28
SNP SNP5PDM376688rs353865977330859773308 123312 123312
27
SNP SNP5PDM379372rs405125977599259775992 120628 120628
26
SNP SNP5PDM380376 5977699559776995
25
SNP SNP5PDM381086rs353855977770659777706 118914 118914
24
SNP SNP5PDM388220rs269535978483959784839 111781 111781
23
SNP SNP5PDM388748 5978536859785368 111252 111252
22
SNP SNP5PDM388749rs269545978536959785370
21
SNP SNP5PDM390700 5978731959787319 109301 109301
20
SNP SNP5PDM392152rs41334705978877159788771 107849 107849
19
SNP SNP5PDM392684 5978930259789302 107317 107317
18
SNP SNP5PDM394085 5979070459790704 105792 105792
17
SNP SNP5PDM394776rs353845979139559791395 105225 105225
16
SNP SNP5PDM395449rs353825979206859792068 104552 104552
15
SNP SNP5PDM397023rs269505979364359793643 102977 102977
14
SNP SNP5PDM399206rs269495979582559795825 100795 100795
13
SNP SNP5PDM400966rs1531535979758559797585 99035 99035
12
SNP SNP5PDM402736rs1523405979934959799349
11
SNP SNP5PDM407853 5980446859804468 92148 92148
i
SNP SNP5PDM408531 5980514559505145 91470 91470
9
SNP SNP5PDM408979 5980559359805593 91022 91022
8
SNP SNP5PDM409460 5980607459806074 90541 90541
7
SNP SNP5PDM411387 5980800159808001 88614 88614
6
SNP SNP5PDM411544rs275645980815959808159 88456 88456
5
SNP SNP5PDM416882rs1531525981349659813496 83119 83119
4
SNP SNP5PDM417756rs1874815981437159814371 82244 82244
3
SNP SNP5PDM419874rs1523415981648859816488 80127 80127
2
SNP SNP5PDM421449rs2489115981806359818063 78552 78552
1
D5S1990 6094559960945816
D5S1359 6354260363542894
D5S2089 6591431565914496
D5S647 6621767466218065
D5S2121 6658409166584385
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We next searched for SNPs in the intronic and flanking regions of PDE4D.
The SNPs were identified in the public NCBI SNP database or by sequencing
selected intronic and flanking regions in the gene in at least 94 patients and
94
controls. We initially identified 637 SNPS. Many of these SNPs were completely
5 correlated so we removed many redundant SNPs from further genotyping. Some
SNPs with very low minor allele frequencies were also ignored. This resulted
in a
set of 260 SNPs that were then genotyped for the entire patient and control
cohorts.
The preponderance of markers with significant associations was located at the
5' end
of the gene. One SNP (SNPSPDM76361;SNP83) for carotid stroke and five of the
10 SNPs (SNPSPDM357221=SNP45, SNPSPDM361545=SNP41,
SNPSPDM43741=SNP87, SNPSPDM29517=SNP89 and SNPSPDMSNP56) for the
combined cardiogenic and carotid stroke remained significant even after
adjusting
for all the SNPs .tested (Table 2D). Three of these significant SNPs flank
exon D7-l;
the other three are in a 100 kb region containing exon D7-2 (for physical
positions
15 see Table 2D). The two most significant SNPs, SNP45 and SNP41, are within 6
kb
of the microsatellite marker AC008818-1, and the at-risk alleles of all three
genetic
markers are in strong linkage disequilibrium with D' > 0.9 and p-value nearly
zero.
The square of the correlation (R2) is very high between the two SNPs (~ 0.93),
but is
substantially lower (~ 0.08) between each SNP and the at-risk allele of the
20 microsatellite. This is due to the fact that the frequency of the at-risk
alleles of the
two SNPs are similar, and much more frequent than that for the at-risk allele
of the
microsatellite. The LD block structure around the 5' end of PDE4D is displayed
in
FIG. 13.1. We delineate three blocks A, B and C encompassing the first three
exons
of PDE4D and its immediate downstream region. Exons D7-3 and D7-2 are both in
25 block A, while D7-1 (the first exon) is in block B, but close to its border
with block
C. Given this block structure we were prepared to investigate the haplotype
associated susceptibility to stroke in this region.
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Table 2D. All SNPs that show association with a p-value less than 0.01 for all
stroke
patients, all patients excluding hemozThagic stroke and the combined
cardiogenic and
carotid stroke.
Phenotype Marker Allele p-value RR # Affect Aff. % # Ctrl Ctrl.
All patients
All patients
excl.
hemorrhagic
stxoke
Combined
cardiogenic ~z
carotid
SNP 32 C 0.00024 1.46 400 37.9 475 29.5
SNP 56 T 0.0028 1.31 550 71.4 615 65.5
SNP 45 G 0.0065 1.33 723 82.4 492 78.0
SNP 48 T 0.0091 1.28 547 68.3 481 62.8
SNP 32 C 0.00034 1.45 377 37.8 475 29.5
SNP 56 T 0.0066 1.28 518 70.9 615 65.5
SNP 45 G 0.0095 1.31 679 82.3 492 78.0
SNP G 0.000034*1.77 309 86.3 492 78.0
45
SNP A 0.000078*1.86 236 86.0 368 76.8
41
SNP T 0.00019*1.49 263 58.2 583 48.4
87
SNP A 0.00025*1.84 232 88.8 450 8I.1
89
SNP T 0.00027*1.56 230 74.8 615 65.5
56
SNP T 0.00032 1.58 326 84.4 589 77.3
39
SNP G 0.00047 1.80 233 88.6 451 8I.3
91
SNP C 0.00069 1.61 144 40.3 475 29.5
32
SNP A 0.00089 I.73 153 83.0 556 73.8
62
SNP T 0.00080 1.51 229 71.8 481 62.8
48
SNP A 0.0018 1.49 259 72.0 403 63.6
42
SNP G 0.0025 1.68 252 90.7 570 85.3
184
SNP T 0.0042 1.54 234 85.3 569 79.0
58
SNP C 0.0041 1.58 146 36.0 269 26.2
53
SNP G 0.0046 1.40 225 54.2 450 45.9
97
SNP A 0.0049 1.32 334 63.9 651 57.3
204
SNP A 0.0054 1.59 228 89.0 612 83.7
8
SNP C 0.0074 1.39 223 60.1 349 52.0
83
SNP T 0.0093 1.48 243 85.4 550 79.8
43
* significant after adjusting for multiple testing
Haplotype analysis
Our general approach to haplotype analysis involves using likelihood-based
inference applied to NEsted MOdels. The method is implemented in our program
MEMO, which allows for many polymorphic markers, SNPs and microsatellites. The
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method and software axe specif cally designed for case-control studies where
the
purpose is to identify haplotype groups that confer different rislcs. It is
also a tool for
studying LD structures.
When investigating haplotypes constructed from many markers, apart from
looking at each haplotype individually, meaningful summaries often require
putting
haplotypes into groups. A particular partition of the haplotype space is a
model that
assumes haplotypes within a group have the same risk, while haplotypes in
different
groups can have different risks. Two models/partitions are nested when one,
the
alternative model, is a finer partition compared to the other, the null model,
i. e, the
alternative model allows some haplotypes assumed to have the same risk in the
null
model to have different risks. The models are nested in the classical sense
that the
null model is a special case of the alternative model. Hence traditional
generalized
likelihood ratio tests can be used to test the null model against the
alternative model.
Note that, with a multiplicative model, if haplotypes lal and h~ are assumed
to have
I S the same risk, it corresponds to assuming that flpl = flp~ where f and p
denote
haplotype frequencies in the affected population and the control population
respectively.
One common way to handle uncertainty in phase and missing genotypes is a
two-step method of first estimating haplotype counts and then treating the
estimated
counts as the exact counts, a method that can sometimes be problematic (e.g.,
see the
information measure section below) and may require randomization to properly
evaluate statistical significance. W NEMO, maximum likelihood estimates,
likelihood ratios and p-values are calculated directly, with the aid of the EM
algorithm, for the observed data treating it as a missing-data problem.
MEMO allows complete flexibility for partitions. Fox example, the first
haplotype problem described in the Methods section on Statistical analysis
considers
testing whether h~ has the same risk as the other haplotypes h2, ..., hk. Here
the
alternative grouping is [lal), [1a2, ..., lak] and the null grouping is [hl,
..., lak]. The
second haplotype problem in the same section involves three haplotypes hl =
G0, la2
= GX and 7z3 = AX, and the focus is on comparing hl and h2. The alternative
grouping is [hl], [la2), [h3] and the null grouping is [h~, h2], [h3]. The
actual problem
we faced in FIG. I I .I is actually slightly more complicated because allele X
is a
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composite allele that includes fve alleles other than allele 0, and hence GX
and AX
each correspond to five haplotypes. One could have collapsed these alleles
into one
at the data processing stage, and performed the test as described. This is a
perfectly
valid approach, and indeed, whether we collapse or not makes no difference if
there
were no missing information regarding phase. But, with the actual data, each
of the
5 alleles making up X correlates differently with the SNP alleles and this
provides
some partial information on phase. Collapsing at the data processing stage
will
unnecessarily increase the amount of missing information. What was actually
done
is natural in the nested-models/partition framework. Let laa be split into
hZa,122b, . . ..,
hZe, and h3 be split into h3a, h3v, ..., h3e. Then the alternative grouping
is. [lal], [h2a,
hZb, . . .., hae ], [h3~, h3b, .. ., h3e] and the null grouping is [hl, h2a,
lZ2b, . . .., h2~], [h3a,
h3b, ..., 723e]. The same method is used to handle the composite haplotypes in
FIG.
11.2 and 11.3 where collapsing at the data processing stage is not even an
option
since L~ represents multiple haplotypes constructed from 25 SNPs. Here, we
also
want to mention that, apart from the pair-wise comparisons presented in FIG.
11.1, a
3-way test with the alternative grouping of [lal], [la2a, h2b, ...., hZe ],
[h3Q, h3b, ..., h3e]
versus the null grouping of [7aI, h2a, hZb, ...., h2e, h3a, h3b, ..., h3e]
could also be
performed. Note that the generalized likelihood ratio test-statistic would
have two
degrees of freedom instead of one. We actually have performed this test and it
gave
a p-value of 2.4 x 10~~.
Measuring information
Even though likelihood ratio tests based on likelihoods computed directly for
the observed data, which have captured the information Ioss due to uncertainty
in
phase and missing genotypes, can be relied on to give valid p-values, it would
still be
of interest to know how much information had been lost due to the information
being
incomplete. Interestingly, one can measure information loss by considering a
two-
step procedure to evaluating statistical significance that appears natural but
happens
to be systematically anti-conservative. Suppose we calculate the maximum
likelihood estimates for the population haplotype frequencies calculated under
the
alternative hypothesis that there are differences between the affected
population and
control population, and use these frequency estimates as estimates of the
observed
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frequencies of haplotype counts in the affected sample and in the control
sample.
Suppose we then perform a likelihood ratio test treating these estimated
haplotype
counts as though they are the actual counts. We could also perform a Fisher's
exact
test, but we would then need to round off these estimated counts since they
are in
general non-integers. This test will in general be anti-conservative because
treating
the estimated counts as if they were exact counts ignores the uncertainty with
the
counts, overestimates the effective sample size and underestimates the
sampling
variation. It means that the chi-square likelihood-ratio test statistic
calculated this
way, denoted by A*, will in general be bigger than 11, the likelihood-ratio
test-
statistic calculated directly from the observed data as described in methods.
But ~1.*
is useful because the ratio t1/A* happens to be a good measure of information,
or 1 -
(!1/.~l*) is a measure of the fraction of information lost due to missing
information.
This information measure for haplotype analysis is described in Nicolae and
Kong,
Technical Report 537, Department of Statistics, University of Statistics,
University
I S of Chicago, Revised for BionaetYics (2003} as a natural extension of
information
measures defined for linkage analysis, and is implemented in NEMO.
Haplotype associatiofz
We first considered haplotypes based on the most significantly associated
SNPs and microsatellite, SNP45, SNP41 and AC008818-I, which are all in block B
and are separated by only 6 kb. Not surprisingly given the high degree of
correlation
between SNP45 and SNP41, we found that it was sufficient to consider only the
two
marker haplotypes consisting of the microsatellite and SNP45 - the SNP with
the
higher genotype yield. The results of this association study for the
combination of
carotid and cardiogenic stroke are displayed in FIG. 11.1. Note that, for
convenience, we have designated by the letter X the joint set of alleles that
are not
the at-risk allele, 0, of microsatellite AC008818-1. Thus, GX should be
understood
as the composite of all haplotypes including the G nucleotide of SNP45 except
for
the GO haplotype. For our samples, the AO haplotype does not exist. This
suggests
that allele 0 originated in a haplotype background with allele G of SNP45, and
since
then no recombination has occurred between those two markers for chromosomes
that carried allele 0. AX, GO and GX have significantly distinct risks for the
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combined carotid and cardiogenic stroke phenotype. We refer to GX as the wild
type
because it is the most common (53.4% in controls) and also because it has the
intermediate level risk that is not too different from the population risk.
The
haplotype GO has increased risk and AX is protective, with risks of 1.46 and
0.70
relative to the wild type, respectively. The GO risk is 2.07 times that of the
protective
haplotype AX. Each of the three pairwise comparisons is highly significant,
with p-
values ranging from 0.006 to 7.2x 10-g. It is interesting to observe that even
though
both AX and GX are composite haplotypes, the AX haplotype can be simply
summarized by the allele A of SNP45, since the AO haplotype does not exist.
For a
I O similar reason, the GO haplotype is completely determined by the 0 allele
of
AC008818-1. Also displayed in FIG. 11.1 is the information content (Info) of
each
test. The difference between Info and 1 is a measure of the information that
is Lost
due to the uncertainty with phase and missing genotypes. Note that Info is
very close
to 1 for each of the three tests in FIG. 1 I .1. That is a result of SNP45 and
AC008818-I being in very strong LD. Note that tests presented later in FIG. I
1.2
and I I .3, involving longer haplotypes have lower information content.
We next identified and estimated the risks for the common SNP haplotypes
within each block. For this portion of the analysis only those SNPs with minor
allele
frequency greater than 20% were considered. Block A (300 kb) contained I9 such
SNFs, block B (200 kb) 22 SNPs, and block C (60 kb) 25 SNPs. All haplotypes
within each block with an estimated frequency in the population of 2% or
greater
have been identified. Within each block there were fewer than ten such
haplotypes,
and they accounted for approximately 80% of the total haplotype frequency for
that
block. A brief schematic of the identified haplotypes are displayed in FIG.
13.2
and the risks and frequencies of these haplotypes are available in Table 3.
Within
block A no common haplotype has greater risk than SNP87 alone. The strongest
signals were for haplotypes in block B and C. Each block contained a haplotype
significantly associated with the combination of carotid and cardiogenic
stroke and
having relative risk around 1.5. The common at-risk haplotype in block B is
the SNP
background of the GO haplotype previously identified.
While there were no significant single marker associations in block C, a
common haplotype with 15.4% frequency in controls was observed. We designate
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this haplotype H~. Investigation of the contribution of H~ in conjunction with
the
SNP45 and AC008818-1 haplotypes leads to another interesting observation. For
notation, all haplotypes defined by the 25 SNPs in block C that are not H~ are
jointly
denoted by the composite haplotype L~. First, it is noted that AX and H~ do
not exist
together on the same chromosome (see FIG. 11.3), at least in these samples,
and thus
blocks B and C are far from being independent. As a consequence, the extended
composite haplotype AXLE is the same as AX. The haplotype GO can be split into
the two extended haplotypes GOHo and the composite GOLo, which, as indicated
in
FIG. 11.2, have significantly different risks (p value = 0.0067). Moreover, it
appears
that the elevated risk of GO is totally accounted for by GOI~'o as GOLC has
risk that is
not significantly different from GX = GXH~ + GXL~ (see FIG. 11.2). This
observation allows us to refine the haplotype groupings of FIG. 13.I into the
groupings indicated by FIG. 13.3. The extended at-risk haplotype GOH~ (8.8% in
controls) and protective composite haplotype AXLE (21.1% in controls), have,
respectively, relative risks of 1.98 and 0.68 compared to the wild type (70.1%
in
controls). Based on these risk estimates, if everybody's risk can be made to
correspond to that of a homozygote carrier of the protective variant, the
number of
cases would be reduced by 55%, which can be interpreted as the population
attributed risk of the at-risk haplotype and the wild type combined.
The at-risk haplotype GOH~ spans a region of about 64kb. While it is
possible that the increased risk is due to multiple polymorphisms over that
region,
the results are also consistent with a relatively recent mutation, as yet to
be
identified, which occurred in that haplotype background, and since then no
recombination has occurred in that extended region for chromosomes carrying
the
mutation. By contrast, the protective composite haplotype AXLE can be simply
represented by allele A of SNP45. Hence, it is possible that allele A of SNP45
is the
functional protective variant, although it is possible that the functional
variant is
simply in strong LD with allele A of SNP45 and has yet to be identified.
Indeed,
statistically, the effects of SNP45 and SNP41 are indistinguishable from each
other.
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Statistical analysis.
For single marker association to the disease, the Fisher exact test was used
to
calculate two-sided p-values for each individual allele. All p-values were
presented
unadjusted for multiple comparisons unless specifically indicated. The
presented
frequencies (for microsatellites, SNPs and haplotypes) were allelic
frequencies as
opposed to carrier frequencies. To minimize any bias due the relatedness of
the
patients who were recruited as families for the linkage analysis, we
eliminated first
and second-degree relatives from the patient list. Furthermore, we have
repeated the
test for association correcting for any remaining relatedness among the
patients, by
extending a variance adjustment procedure described in Risch, N. & Teng, J.
(Genome Res., 8:I278-1288 (1998)). The relative power of family-based and case-
control designs for linkage disequilibrium studies of complex human diseases
I.
DNA pooling. (ibis for sibships so that it can be applied to general familial
relationships, and present both adjusted and unadjusted p-values for
comparison. The
IS differences are in general very small as expected. To assess the
significance of
single-marker association corrected for multiple testing we carned out a
randomisation test using the same genotype data. We randomised the cohorts of
patients and controls and redid the association analysis. This procedure was
repeated
up to 500,400 times and the p-value we presented is the fraction of
replications that
produced a p-value for some marker allele that is lower than or equal to the p-
value
we observed using the original patient and control cohorts.
For both single-marker and haplotype analyses, relative risk (RR) and the
population attributable risk (PAR) were calculated assuming a multiplicative
model
(haplotype relative risk model), (Terwilliger, J.D. & Ott, J., Huyfa He~ecl,
42, 337-46
(1992) and Falk, C.T. & Rubinstein, P, Aran Hurn Gefzet 51 ( Pt 3), 227-33
(1987)),
i.e., that the risks of the two alleles/haplotypes a person carries multiply.
For
example, if RR is the risk of A relative to a, then the risk of a person
homozygote
AA will be RR times that of a heterozygote Aa and RRZ times that of a
homozygote
aa. The multiplicative model has a nice property that simplifies analysis and
computations-haplotypes are independent, i.e., in Hardy-Weinberg equilibrium,
within the affected population as well as within the control population. As a
consequence, haplotype counts of the affecteds and controls each have
multinomial
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distributions, but with different haplotype frequencies under the alternative
hypothesis. Specifically, for two haplotypes la; and h~, risk(hl)/risk(h~) _
(flpl)l~lp~),
where f and p denote respectively frequencies in the affected population and
in the
control population. While there is some power loss if the true model is not
multiplicative, the loss tends to be mild except for extreme cases. Most
importantly,
p-values are always valid since they are computed with respect to null
hypothesis.
In general, haplotype frequencies are estimated by maximum likelihood and
tests of differences between cases and controls are performed using a
generalized
likelihood ratio test (Rice, J.A. Mathematical Statistics anel Data Analysis,
602
(International Thomson Publishing, (1995)). deCODE's haplotype analysis
program
called MEMO, which stands for NEsted MOdels, was used to calculate all the
haplotype results presented. To handle uncertainties with phase and missing
genotypes, it is emphasized that we do not use a common two-step approach to
association tests, where haplotype counts are first estimated, possibly with
the use of
I S the EM algorithm, Dempster, (A.P., Laird, N.M. & Rubin, D.B., Journal of
the Royal
Statistical Society B, 39, 1-38 (1971)) and then tests are performed treating
the
estimated counts as though they are true counts, a method that can sometimes
be
problematic and may require randomisation to properly evaluate statistical
significance. Instead, with NEMO, maximum likelihood estimates, likelihood
ratios
and p-values are computed with the aid of the EM-algoritlun directly for the
observed data, and hence the loss of information due to uncertainty with phase
and
missing genotypes is automatically captured by the likelihood ratios. Even so,
it is of
interest to know how much information is retained, or lost, due to incomplete
information. Described herein is such a measure that is natural under the
likelihood
framework. For a fixed set of markers, the simplest tests we performed, with
results
presented in Table 3, compare one selected haplotype against all the others.
Call the
selected haplotype hl and the others 1a2, ..., hk. Letpl, ..., pk denote the
population
frequencies of the haplotypes in the controls, and f , . . ., fk denote the
population
frequencies of the haplotypes in the affecteds. Under the null hypothesis, f =
p1 for
all i. The alternative model we use for the test assumes laz, ..., I~k to have
the same
risk while lal is allowed to have a different risk. This implies that whilepl
can be
different from fl, f l(f'2+. . .+fk) = p~/(p2+, , ,+pk) _ ~i1 for i = 2, . .
., k. Denoting f lpl by
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~, and noting that (3a+.. .+~3k =1, the test statistic based on generalized
likelihood
ratios is
2 [~~rrpl; ~2~ ...~ PJk-I) -'~~l~~l ~2~ ~,.~ ~k-1),
where .E denotes logelikelihood and '" and ~ denote maximum likelihood
estimates
under the null hypothesis and alternative hypothesis respectively. A has
asymptotically a chi-square distribution with 1-df, under the null hypothesis
and it
was used to compute p-values presented in Table 3. The tests presented in FIG.
11
have slightly more complicated null and alternative hypotheses. For the
results in
FIG. 11, let hl be G0, h2 be GX and h3 be AX. When comparing GO against GX,
i.e.,
this is the test which gives estimated RR of 1.46 and p-value = 0.0002, the
null
assumes GO and GX have the same risk but AX is allowed to have a different
risk.
The alternative hypothesis allows all three haplotype groups to have different
risks.
This implies that, under the null hypothesis, there is a constraint that f lpt
= filp2, or
w = ~lpi)l [filp2] =1. The test statistic based on generalized likelihood
ratios is
~ - 2 ~ ~ ~pl ~ .fn ~~ ~ 'w) - ~~Pn fu ~a > 1 ),
that again has asymptotically a chi-square distribution with 1-df under the
null
hypothesis. There is actually an extra complication to the test due to h2 and
h3 being
composite haplotypes. That is handled in a natural manner under the nested
models
framework. Other tests presented in FIG. 11.2 and 11.3 were similarly
performed.
LD between pairs of SNPs was calculated using the standard definition of D'
and R2 (Lewontin, R., GefZetics 49, 49-67 (1964) and Hill, W.G. & Robertson,
A.
Theo~. Appl. Genet. 22, 226-231 (1968)). Using MEMO, frequencies of the two
marker allele combinations are estimated by maximum likelihood and deviation
from
linkage equilibrium is evaluated by a likelihood ratio test. The definitions
of D' and
RZ were extended to include microsatellites by averaging over the values for
alI
possible allele combination of the two markers weighted by the marginal allele
probabilities. When plotting all marker combination to elucidate the LD
structure in
a particular region, we plot D' in the upper left corner and the p-value in
the lower
right corner. In the LD plots we present the markers are plotted equidistant
rather
than according to their physical location.
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Table 3 Haplotype diversity at the 5'end of the PDE4D gene.
All haplotypes shown that have > 2% population frequency within each of the 3
blocks of strong LD together with the haplotype association results comparing
the
combination of cardiogenic and carotid stroke versus controls.
Block A:
O O O °~ h CO 4y M N O h CC 'd' M N r O 01
r r O 1~ O O O O 1~ O O) ~ OD OD GO 00 CO CO 00 h
z z ~, z z z z z z z z ~ z z z z z z z z p-value Aff % Ctrl % RR
v~ ~n ~n N ~n ~n cn
T G T A T G A G AG A ~',T A G C G G T C 0.030226.722.21.28
C G C A C C G A GA G ,!~C G G C G G T G 0.3662.1 2.9 0.73
C G C A C C G A GA G ,4 C G A T G G A T 0.3032.6 3.6 0.71
C G C A C C G A GA G p, C G A T A G A T 0.33522.524.50.89
C G C G C G A G AG A C, T A G C G A A T 0.21612.610.61.23
C A C A C C A G AG A ~',T A G C G A A T 0.8762.2 2.4 0.94
C A C A C C A G AG A G C A A T G G T C 0.0016.5 11.20.55
C A C G C G A G AG A (',T A G C G A A T 0.4017.9 6.6 1.20
Block B:
1~COM r O1h f0d'M N r 07 h et O 00InN r O
1 1 C C f t G C Cf1 7 M C~'~'~'e~'
I 7 N 7 l
l
V1
~ ~ ~ C O 0 D Q O ~ ~ ~ ~ a a a a RR
a ~ a a a a a a a a a a w a a p-value Aff
a ,~; a % Ctr
a, /
a ~ ~ ~ ~ ~ ~ ~ ~ z ~ ~ ~ ~ z N
~ ~ ~ z
z
c c c n c
n n n n
c
A A T G T A A G A A C A G T A C C T G A A T 0.0004 29.2 21.4 1.52
A T G T AA G A CT A A A A T T C A G G A 0.0074.37.6 0.55
A
G C A T AA G A AC A G T A C C T G A A T 0.9582.02.1 D.98
A
G C A T GG A G AT A A A A T T C G G A T 0.6106.25.6 1.13
A
G C A T GG A G CT A A A A T T C A G G A 0.01043.46.3 0.52
A
G C A T GA G A AC C G T G T C T G A A T 0.80314.614.11.04
G
Block C:
h ~f! et N r O O h. ID V' M N O 07 CC In d' M N
M M M M M M N N N N N N N r r r r r r ~' ~ ~' M N r
a a a a a a a a a a a a a a a a a a a a o- a a a a p-value Aff % Ctrl RR
z z z z z z z z z z z z z z z z z z z z z z z z z
N ~
T A A C C A C G A A C T T A T T G A A T T T G A A 0.0006 22.2 15.4 9.58
GA A C C A CG A A T C C G C CG A G C A TC A A 0.5332.22.80.78
GA A C C A CG A A T C C G C CG A G T T TG A A 0.24 3.02.01.52
GA A C C A CG A T T C T A C CA G G C A CC T G 0.3022.61.81.46
~
GG C T T C CG A A C T T A T TG A A T T TG A A 0.2582.03.00.68
GG C T T C CG A A T C C G C TG A G C A TC T G 0.00787.812.00.62
GG C T T C GA G T T C T A C CA G G C A CC T G 0.78132.933,60.97
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Allelic definitions and polymorphisms for SNPs in the two most significant
haplotypes (in block B and C).
The analysis presented above represents a conservative analysis of the data
since it restricted the analysis to SNPs with minor allelic frequencies of
greater than
20%. To further understand the magnitude of the contribution of PDE4D to
stroke in
this 5 prime region, we repeated the analysis without such restrictions,
including all
SNPs selected for genotyping. We found a SNP haplotype for the two major
subtypes of ischemic stroke, carotid and cardiogenic stroke (Table 3, Block
C). This
is a 5 SNP haplotype that covers an area of 48 kb and is just upstream of the
5'exon
covering the presumed promoter region of isoform PDE4D7. It captures the same
information as the 0 allele for marker AC008818-1. However, the SNP haplotype
is
more specific in the sense that it has a higher relative risk, i. e., 2.3.
This haplotype is
carried by 47% of the patients and has the same population attributable risk
(PAR) of
0.25. The polymorphisms and alleles for the SNPs are presented in Table 4A.
Table 4A
SNP
Pol mor hismPositionAllele
Public Y P
name
Name (nucleotide)
if available
SNP42 rs153031 A/G 138806 0 (A)
SNPSPDM361194
SNP34 rs27653 C/A 131865 0 (A)
SNP5PDM368135
SNP32 rs456009 C/T 129361 1 (C)
SNP5PDM370640
SNP26 rs40512 GlA 120628 0 (A)
SNPSPDM379372
SNP9 fneW) G/A 91470 0(A)
SNPSPDM408531
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Table 4B
N Wit' N ~O
a n n n ~- # Aff. # Ctrl. R-risk PAR info
Phenotype ~ N N N ~ p-value Affect Freq.* Ctrl Freq*
All stroke A A C A A 2.17E-05 988 0.19 652 0.12 1.8 0.16 0.604
Cardiogenic/ A A C A A 3.37E-07 313 0.236 652 0.119 2.3 0.25 0.616
carotid
~ allelic frequency
The sequences for the microsatellite markers are as follows:
AC008879-2 amplimer:
ACAAAGAGCACCTTTCCAGTGGACAACTAACTAAAGTGGTGTGATTTTGG
TATAAGTTTGTGTGTGTGTGTGTGTGTGTGTTGTGTGTGTGTGTATGTGTA.
TACATTTAGTTTTATTGTAA.CAAAGCAACTTGTACTTTTCACGTTTAAAA
(SEQ lD NO: 85)
* AC008879-2, allele 0 is the same allele as the minimwn allele observed in
CEPH 1347-02, family 137, individual 02.
In summary, this single SNP haplotype (which is only one haplotype of the
several found above but is probably the most tightly associated to stroke)
more than
doubles an individual's risk for cardiogenic and carotid stroke and accounts
for 25%
of such strokes in Iceland. The other haplotypes described above provide
additional
risk for stroke. The magnitude of this risk haplotype is comparable or higher
than
the well-known clinical risk factors for stroke such as hypertension,
diabetes,
hyperlipidemia, and smoking.
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b
b
d
W
w w o 00o N ~rvo000
V1 ~ .-nN N NN N M
~
N
U ~
d U E
d H H -
U
E.~d c
., 7
~ ~H U ~ H
H ~H ~ CH7~ E~-~C7
~ dd d ~~ ~ U
~ ~ d E E U
-~ -~
7 d E'~ E'.'C
C U U 7
U C7C7
w ~ U HH dd ~ H
~ ~ ~d d H~ C7.U
U
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a. U ~Ed-~~ d.t7d C7
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Table 7 a
Correlation between at-risk alleles for markers AC008818-1, SNP45 and SNP41.
Estimates of LD (correlation) between the at-risk alleles, allele 0 for marker
AC008818-1, allele
G for SNP45 and allele A for SNP41, the three most significant disease
associated genetic
markers.
A. Combined cardiogenic and carotid patients
,n r-
dwd-
a a a
.
z z
a cn v~
ACOO881s-~ 0.355 0.090 0.076
SNP 45 0.863 1 0.943
SNP 41 0.860 0.906 0.981
B. Controls
R2
T
U Z Z
u. Q Cn U)
AC008818-1 0.255 0.091 0.078
SNP 45 0.780 1 0.920
SNP 4'I 0.768 0.924 0.968
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Table 8
Association of risk factors.
Association of microsatellite AC008818-1 at-rislc allele 0, SNP45 allele A and
haplotype
GOH~ respectively with various risk factors.
Cases are stroke patients with risk factors and controls are stroke patients
without the
risk factors. P-values are two-sided.
AC008818-1 : Allele 0 SNP45 : Allele A Haplotype GOHc
Cases
Cases with Cases with Cases without Cases with Cases without
risk factor Without risk risk factor risk factor risk factor risk factor
factor
I N Frq. N Frq. __P__" N Frq. N Frq. P-value N Frq. N Frq. " p"sI
Hypertension 477 0.303 203 0.303 1.000 416 0.172 181 0.188 O.S10 503 0.134 216
0.123 0.634
Hyper- , 274 0.336 312 0.271 0.025 242 0.216 277 0.186 0.516 287 0.153 329
0.104 0.026
cholesterolemi
Diabetes [ 93 0.274 424 0.310 0.379 [ 79 0.196 398 0,176 0.422 [ 100 0.127 455
0.133 0.857
Peripheral
artery ~ 133 0.297 357 0.305 0.815 ~ 116 0.181 340 0.176 0.921 ~ 138 0.121 388
0.132 0.697
occlusive
Coronary ~ 179 0.302 429 0.318 0.588 ~ 153 0.170 406 0.182 0.662 ~ 181 0.122
467 0.141 0.444
artery disease
Early onset ~ 349 0.294 462 0.304 0.137 ~ 314 0.186 430 0.173 0.538 ~ 380
0.128 S06 0.125 0.876
(<68)
Males vs ~ 457 0.291 358 0.310 0.414 I 420 0.181 303 0.168 0.575 I 489 0.122
370 0.141 0.315
females
Discussion of Stoke Geyze Ide»tificatiofz
Genealogy, a comprehensive population-based list of broadly defined stroke
patients and non-parametric allele sharing methods have been combined to
successfully
map a major gene to chromosome 5 for one of the most complex diseases known.
We
then used a large case-control association study that showed that PDE4D is the
gene in
this location that is the gene conferring substantial risk for stroke. This is
the first gene
ever mapped and isolated for the common forms of stroke. There was no
correlation
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between the contribution of the families to this gene location and
hypertension, diabetes
or hyperlipidemias and this gene does not match any known gene contributing to
these
risk factors. The types of stroke studied in this work do not reflect a rare
or Icelandic-
specific form of stroke; rather, the diversity of the stroke phenotypes in
Icelanders as
well as risk factors are similar to those of most other Caucasian populations
(Agnarsson,
U., et al., Aura. Ifaterh. Med., 130:987 (1999); Eliasson, J.H., et al.,
Lc~knabla~i~, 85:517-
25 (1999); Sveinbjornsdottir, S., et al., Systematic registration of patients
with Stroke
and TIA admitted to The National University Hospital, Reykjavik, Iceland, in
1997,
XIII. Meeting of the Icelandic Association in Internal Medicine, Akureyri,
Tceland
(Valdimarsson, E.M., et al., Lreknabladid 84:921 (1998)).
The magnitude of the risk and the frequency of the disease haplotypes in the
general population confirm that we have mapped a gene for the common forms of
stroke
and not some rare form of stroke. This gene almost doubles one's risk for
stroke in
general, and more than doubles one's risk for the two most common subtypes of
stroke,
carotid and cardiogenic stroke. In addition, the most common disease haplotype
has. a
population attributed risk of 25% (which means it accounts for 25% of the
patients) and
there are other haplotypes that we describe herein that are less common that
accounts for
other patients. Thus PDE4D is a major cause of stroke and its relative risk
rivals those
of hypertension, smoking, diabetes, and hyperlipidemia. PDE4D shows tighter
correlation to the forms of stroke dependent on atherosclerosis (carotid and
cardiogenic
stroke) and it is expressed in cell types known to be important for
atherosclerosis such as
vascular smooth muscle cells, macrophages, and endothelial cells. This
suggests that the
strong effect that PDE4D variation has on stroke risk is through its role in
the vascular
biology of atherosclerosis (see discussion at the end of the examples).
Example 2 details
our sequencing of the entire PDE4D gene and the definition of its exon-intron
structure
based on new and old cDNAs, and Example 3 shows that the expression pattern of
PDE4D isoforms correlates with a stroke associated haplotype.
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EXAMPLE 2: SEQUENCING AND CHARACTERIZATION OF THE HUMAN
GENE AND ITS RNA/PROTEIN ISOFORMS
Sequence of the St>~oke Gene Regio~a
At the start of our work, there was little genomic sequence available in the
public
domain covering the stroke gene region. Therefore, we sequenced approximately
3 Mb
of the area defined by one drop in lod. The locus on Sql2 indicated in the
genome wide
scan was physically mapped using bacterial artificial chromosomes (BACs). A
set of
overlapping clones for a 20 cM region was assembled through a combination of
hybridization and BAC-fingerprint walking. Eighteen BACs (bacterial artificial
clones
(RP11-164A5, RPl I-188I15, RPl I-313P15, RP11-631M6, RP11-103A15, RP11-
489L13, RP11-621CI9, RP11-I13C1, RP11-567M18, RP11-412M9, RP11-ISIG2,
RP11-151F7, RP11-281M3, RP11- 42IL6, RP11-1A7, RP11-68E13, RP11-379P8, and
RP 11-422K3) covering the minimum tiling path of the one LOD interval were
analysed
using shotgun cloning and sequencing. Dye terminator (ABI PRISM BigDye)
chemistry
was used for fluorescent automated DNA sequencing. ABI prism 377 sequences
were
used to collect data and the Phred/Phrap/Consed software package in
combination with
the Polyphred software were used to assemble sequences (See Table 9A and 9B)
Publicly available sequences (AC008836, AC073546, AC021603, AC008498,
AC016435, AC02I60I, AC016591, AG008818, AC008879, AC008934, AC011929,
AC027322, AC008111, AC020924, AC026693, AC012315, AG08804, AC008791,
AC020975, AC008833, AC008829, AC022125, AC008790, AC026095, AC066693,
AC008852, AC016642, AC034250, AC025179, AC08814, AC008926, AC01039I,
AC016635 and AC016604) from this region were assembled with the obtained
sequence
and a 3.7 Mb sequence (with 22 gaps) was generated. Comparison of the current
public
human assembly (NCBI BUILD 33) to our sequence of the STRKl locus only showed
a
minor discrepance.
The BAC clones we sequenced are from the RCPI-11 Human BAC libraxy
(Pieter deJong, Roswell Park). The vector used was pBACe3.6. The clones were
picked
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into a 94 well microtiter plate containing LB/chloramphenicol (25
~,g/ml)/glycerol
(7.5%) and stored at -80°C after a single colony has been positively
identifted through
sequencing. The clones can then be streaked out on a LB agar plate with the
appropriate
antibiotic, chloramphenicol (25 ~g/ml)/sucrose (5%).
Table 9A
~ Sequenced at Decode
(BAC name) Comment Accession number
Rl'11-621C19 1 AC020733
RP11-113C1 2
RP11-412M9 2
RP11-15IG2 2
RPl1-151F7 2
RP11-281M3 2 '
RP 11-421L6 2 '
RPl 1-68E13 2
RP11-379P8 2
RP11-IA7 1 AC008111
Rl' 11-422K3 I 2
Key to "Comment" column:
1= This BAC has a publicly available sequence,
it was sequenced at Decode to make sure the sequence was correct
2= Only BAC end-sequence available for this BAC publicly,
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Table 9B
Sequences available
from Accession numberStatus of sequence
GenBank (SAC name)
RP 11-621 C 19 AC020733 17 unordered pieces
CTD-2003DS AC01659I complete sequence
CTD-2210C1 AC008879 7 unordered pieces
CTD-2124H11 AC008818 complete sequence
CTD-2301A11 AC008934 complete sequence
RP11-16B11 AC011929 7 unordered pieces
CTC-261E10 AC026693 complete sequence
CTD-2027610 AC027322 complete sequence
RP11-lA7 AC008111 8 unordered pieces
CTD-2122K7 AC012315 complete sequence
CTD-2085F10 AC008804 complete sequence
CTD-2040J22 AC008791 complete sequence
ltPl 1-235NI6 AC020975 16 ordered pieces
CTD-2146016 AC008833 complete sequence
CTD-2084I4 AC022125 1 T ordered pieces
CTD-2140K22 AC008829 26 ordered pieces
CTD-2I24D 11 AC020924 7 ordered pieces
RP11-731H6 AC026095 21 unordered pieces
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PDE4D Gene; Identification of New Exoras and Splice Variants
The gene, human cAMP specific phosphodiesterase 4D (HPDE4D) was
identified in the sequenced region by BLAST of our novel genomic sequence with
the
cDNAs/EST databases from GenBank. In addition, we ran RT-PCR reactions and 5
prime and 3 prime RACE reactions using cDNA libraries generated from a variety
of
tissues including human aorta. The primer sites used corresponded to known or
exons
predicted from our genomic sequence using Genscan, and Fgene. We found several
novel cDNAs and matched them to the 3Mb sequence in and around PDE4D. The
genomic sequence covering all known and novel exons in PDE4D so far is
approximately 1,SSO,OOQ bases in length.
We defined new alternative transcripts which together with previously known
transcripts showed that the PDE4D gene contains 22 exons over at least 1.5 Mb
and
overlaps with the PARTI gene whose transcript is on the other strand at the 5'
end. The
PDE4D gene has at least 7 promoters and encodes 8 protein isoforms. All
isoforms
have an identical C-terminal catalytic domain but differ at the N-terminal
regulatory
domain. Six of the 8 forms are so called long isoforms. Each of them have
unique N-
terminal regulatory domains but they are all characterized by two highly
conserved
regions found in all PDE4 subfamilies, i.e. upstream conserved regions 1 and 2
(UCR 1
and 2). The six long forms differ from each other by unique alternative 5
prime exons
which predicts six alternative promoters that are each upstream of the
corresponding 5
prime exon. The remaining two are the so-called short forms, variants that
lack the UCR
1 (Houslay, M.D. & Adarns, D.R., Biochem J, 370, 1-18 (2003)). The five
previously
known isoforms are encoded by I7 exons distributed over a segment of 0.9 Mb.
The three new exons D7A-1, D7A-2 and D7A-3 are spliced to one another and
together splice onto exon LF1 forming the splice variant we named PDE4D7 (FIG.
3).
Exon D7-1 is non-coding. Exons D8 and D9 are spliced by themselves onto exon
LF1
forming two splice variants we named PDE4D8 and PDE4D9, respectively (FIG. 3).
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In terms of genomic structure, the D7A exon extends the 5' end of PDE4D by
590,000 bp, and the D8 and D9 exons lie between exons D3 and LF1(physical
position
of exons presented in Table 2C). The new PDE4D7 isoform has an open reading
frame
extending into LF l, resulting in additional 91 amino acids at the N-terminus
of the
predicted protein. The D8 and D9 5' exons contain a long 5' UTR, followed by
an ATG
near the end of the exons that extends an ORF into LFl resulting in a novel N-
terminal
segments of 22 and 30 amino acids in the PDE4D8 and PDE4D9 predicted proteins,
respectively. The new splice variants were verified by RT-PCR on different
cDNA
tissue panels and subsequent cloning and sequencing of the products.
The PDE4D gene encodes at least eight different isoforms. Six of the eight
forms are the so-called long isoforms. Each of them has an unique N-terminal
regulatory domain but they are all characterized by two highly conserved
xegions found
in all PDE4 subfamilies, i. e., upstream conserved regions 1 and 2 (UCR 1 and
2). The
remaining two isoforms are the short forms, variants which lack the UCR 1.
Three PDE4D isoforms have been submitted to GenBank by Memory
Pharmaceuticals on September 16, 2002 and December 17, 2002, under accession
numbers AF536975 (isoform named PDE4D6), AF536976 (named PDE4D7) and
AF536977 (named PDE4D8). See also PCT WO 01/00851, published January 4, 2001.
The sequence AF536977 corresponds to our earlier reported PDE4D6 isoform and
AF536976 corresponds partly to our earlier reported PDE4D7 isoform, however
the first
untranslated exon we named D7-1 is missing from this sequence. The sequence
AF536975 is a new short PDE4D isoform. We have therefore changed the isoform
names accordingly herein as follows: PDE4D6 is now called PDE4D8, PDE4D7 is
now
called PDE4D7 and PDE4D8 is now called PDE4D9. We have submitted the new
PDE4D splice variants, PDE4D7 and PDE4D9 to GenBank (Accession numbers
AY245866 and AY245867, respectively).
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We have in addition identified 17 putative exons upstream of LF1, based on
ESTs, mouse homologies and GeneMiner exon predictions. Primers designed from
these exons were used in conjunction with primers from LF1 and exon3 for RT
PCR in
the hope of identifying novel exons. Novel exons were in turn used to design
primers
for various RT-PCR reactions. We also used 5'RACE primers, designed from the
known exons upstream of LFl. We have to date identified 14 new exons,
including
exons belonging to UniGene Cluster Hs.343602 that have now been connected to
LF1.
For the S' RACE reactions we used cDNA made from heart, SkNAS
(neuroblastoma cell line) and HVAEnd 5050 (endothelial cell line). For RT-PCR
reactions
a number of cDNAs made of total RNA were used (see below)
Novel exons in Table 1 OA are in italics; previously know PDE4D exons in
white.
Exon 3 of EST AW272330 is included on the table as a representative of the 3'
of ESTs
from UniGene cluster Hs 343602.The positions given are from SEQ ID NO: 1. Note
the
different splicing of 4D9-3.2, 4D9-3.1, and AW272330exon3.
Total RNA was isolated from HeLa, SkNAs and Jurkat 77 cell cultures according,
to
manual, using the TRIZOL° reagent provided by GibcoBRL. We used the
GeneRacerTM, ThermoZymeTM and TOPO TA cloning~ (containing pCR~2.1-
TOPO°)
kits from Tnvitrogen following the manufacturer's protocol.
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Table l0A
supported
exon EXON SEQ SEQ by
# tD ID EST(s)
1 1
1 4D7 A 108127108217 Yes
2 PDE4D7-1 142207142328 PDE4D
3 4D7-4 257650257705 Yes
4 4D7'-8 288224288393 No
4D7-B 295203295251 No
6 4D7-5 352169352317 No
7 4D7-6 441914442036 No
'
8 PDE4D7-2 444645444775 PDE4D
9 4D7 C 482438482719 No
4D7 7 597399597534 Yes
11 4D~ 9 626020626092 No
12 PDE4D7-3 641649641878 PDE4D
13 PDE4D4 736254737226 PDE4D
14 PDE4D5 861791862202 PDE4D
PDE4D3 10440511044190PDE4D
16 4D9-1 1Q695441069629Yes
17 4D9-2 10699361069993No
4D9-3.2 10716611071795
18 4D9-3.1 10716681071795Yes
AVV272330exon310716681071901
19 4D9-4 11218211121892No
4D9-5 12476211247696No
21 PDE4D8 12734041273709PDE4D
22 PDE4D9 13543471355128PDE4D
LF1exon 14145111414702PDE4D
5 Sequence of New Exons:
>4D7-A
GGCCTCGAGCAGAACTTCCCATTTGAGTGGGACCAAGAAGAGCATACAAAG
CTGAAA.TGTTCTCCAGAAGTTGATTTCCAATGGGGATAAA (SEQ ID NO: 88)
10 >4D7-4 From Forwaxd Primex
TGATTACAGGTTTTAGAGAAGAGGAACAATGCTTCCTCTGAGCCTGAAGAA
AAGAA (SEQ ID NO: 89)
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>4D7-8
AGTTCTGACCATGTCCTGTGTCACTCTCAAGCAGAGATTGAAAATGACATTC
GTCCTTTACTTGTTCCAAGGAAGCAAACATTTTATAGTTTGAAACTGTTTCTC
TTGCATTTGCTTTGCAAGAGGTTTGCAGAAGTTAAGCCTCATGGAGTCTTCTC
TGCTTAACTTAA (SEQ ID NO: 90)
>4D7-B
TGTGAAGAATTTGGAAATTGCAAGGAGCATGGGAAGGAGATGATTTGGG
(SEQ ID NO: 91)
>4D7-5
GAATGAAGAGGAAATGAAGACATACTTAGATAAAA.ACAGATTATCACCAGG
AGATCTGCTGTAAAAGAATGGCTAAAGGAAGTTAGCTAAGCAGAAAGGAAG
TAAGATAAAAAGGAACCTTGGAACATCAGGGAGGACAAAAGAAGATG(SEQ
ID NO: 92)
>4D7-C
TTTCTCTTTCTCCAATCACTCACTCTGGAGGCAGCTAGCTGTCAACTCACAAA
GACACTCAAGCAGCCTATGGAAGAAGGCCACATGGTAAAA.TATGGAGGCCT
CCAGCCAACAGTCAGC.AAGGAACTGAGACAAGTCAACAACCATGTGAGTGA
CTCGAGAAGTGCTTCTCTAGCTCCAGTTGAGACTTGCAGTAGCAGCAGCCTC
AGCTGGCGGCTTGACTGCAATCTCTTGAGAGACCCTAAGCTCTCCTGAATTC
TTGATCCTTAGAAACTGTGTGAG (SEQ ID NO: 93) .
>4D7-6
GGTCTAGCTGTGTCCCAGAGAGCAACTTCCCTTTTCAAGGGAGCCCACTCTG
TGTGATGCTTTTTCCTAGGTATGGGCAACCCATCCCTCCTAGGGTGAAAACT
TCGCTGTTGCTAGTTCGAG (SEQ ID NO: 94)
>4D7-7
AATGATGCCGTATTATTCTGCTGACCTAACTTCAAAGAAATAAAGAGTTTGC
AAGAAGAACTGCAGTTCTTCAAAGTACGCAATATGGATTTCCAAGATGAAT
GTAGTTTCTCTCTCTGAGGAATTCTGAACAGTG (SEQ ID NO: 95)
>4D7-9
GACTTGAGCATCTGAAGATTTTGGTTTCTGCAGAGGGTGGGAAAGGTTGAAC
CAATCCCCCATGGATACCAAG (SEQ ID NO: 96)
>4D9-1
GGCTTTCCAGATCCCTGAAGATAAAATACAAACTCTCCAACAAGACCTTT
TGGCCATCAGGAACGCAGCACCTGGCTCTCTCACTA (SEQ ID NO: 97)
>4D9-2
AAAGTCGCAGAGATAGCGGAGAACAAGAACCAGATCTCACAGTCATGGTGC
CAAAAGA (SEQ ID NO: 98)
>4D9-3.1
CTGTTACCCTAGCATGACTGCTTCAGCGAAGAGATAAGAGCTTGTTTGACTT
TTTCCACTGGAATTTTTCATGCCAGAAGAAATTGAACATGTGAGCCTGGTGT
CTGGAAGAGTAGCCTGGATTTATG (SEQ ID NO: 99)
>4D9-3.2
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AATTCAGCTGTTACCCTAGCATGACTGCTTCAGCGAAGAGATAAGAGCTTCT
TTGACTTTTTCCACTGGAATTTTTCATGCCAGAAGAAATTGAACATGTGAGC
CTGGTGTCTGGAAGAGTAGCCTGGATTTATG (SEQ ID NO: 100)
>4D9-4
TTCGTTGATAGTTCCAATATCTGTAATCTTGTTGGTCTACCTGTGCAGTTTAT
TCCACTGATTGTCTCTCAG (SEQ ID NO: 101)
>4D9-5
GCGAA.AATACTGAGGCTCAACAGACATA.AAATGGCTTGAGTTACCAGGCTA
CAGTAGAACTAGGATTTCAGTCCAG (SEQ ID NO: 102)
Spliciyag of the Exons as identified by RT PCRlRACE
New exons are in italics.
RT4D7: 4D?-1 + 4D?-2 + 4D?-3 + LF 1
RTI: 4D?-1 + 4D7-8 + 4D?-2 + Q.D?-3 + LF1
RT2: 4D7-4 + 4D?-2 + 4D7 9 + 4D?-3 + LF1
RT3: 4D?-2 + 4D7-3 + LF 1
RT4: 4D?-1 + 4D?-2 + 4D7 7 + 4D?-3 + 4D9-1 + 4D9-3.1
RTS: 4D?-1 + 4D?-2 + 4D?-3 + 4D9-2 + 4D9-3. I
Race6: 4D7-A + 4D7-B + 4D?-2 + 4D7-C + 4D?-3
RT7: 4D?-1 + 4D7-4
RTB: 4D?-1 + 4D7-5 + 4D?-2
RT9: 4D?-1 + 4D7-6 + 4D?-2
RT10: 4D9-1 + 4D9-3.1 + LF1
RTl l: 4D9-2 + 4D9-3.2 + LF1
RT12: 4D9-3 + 4D9-4 + LF1
RTI3: 4D9-3 + 4D9-4 + 4D9-5 + LF1
RT14: 4D9-3 + LF 1
Detection of variants in cDNA from various tissues:
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TablelOB
RT4D7RT1RT2RT3RT4RT5 RT6RT7RT8RT9RT10RT11RT12RT13RT14
Bone Marrow- - - * - - n n n n n - - -
Brain + - - * - - n n n n n n - + -
fetal + - - * - - n n n n n n - - -
Brain
colon + + _ * _ - n n n n n n - - -
Heart _ _ _ _ _ _ _ _ _ _ _ _ _ +
HVAEend - - n n n n + - - - + + n n +
5050
Kidne + - + + - - n n n n n n - - -
Liver - - - * - - n n n n n - - - -
Placenta - - - * - - n n n n n - - - -
Prostate + - - * + - n n n n n n - -
Saliva + - _ * - - n n n n n n - - -
gland
Skeletal - - - - - - n n n n n n + - +
Muscle ,
SkNAS + _ n * n n - + + + _ n n
cell
line
Spinal - - - - - - n n n n n n - -
Cord
Spleen - - * - - n n n n n n - - +
.
Testis - - - - - - n n n n n n - -
Th mus + - _ * - _ n n n n n n - - -
Thyroid + - - * - + n n n n n n - - +
Trachea + - - * - - n n n n n n - - -
~
Uterus - - - - - - n n n n n n - - +
other# - - - - - - n n n n n n - - -
+ = present and verified by sequencing
* = product of the correct size present; not yet verified by sequencing
- = not detected
n = not checked
# These are: Adrenal Gland, Fetal Liver, Cerebellum, Lung, Small Intestine.
Two of the variants that axe more widely expressed appear to be mutually
exclusive: 4D7 [with 4D7-1 as first exon] was detected in 10 cDNAs while RT14
is
found in 9cDNAs. Of these thyroid and prostate are the only tissues common to
both
variants.
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The 13 new RT and RACE variants presented above (we had previously described
the 4D7 variant), do not add any new translated sequence. The RT1 product is
expected
o be the same as the 4D7 putative protein. In variants RTZ and Race6 the exons
between 4D7-2 and 4D7~3 interfere with the ORF with the first AUG and ORF
being
just inside LFl. Similarly Exon 4D9-3 contains stop codons in all 3 reading
frames and
Variants RT10, 11,12,13, and 14 having their ATG initiation codon inside LF1.
It is not
clear whether variants RT4 and RTS, which contain exon 4D9-3 extend to LF 1 or
have
their 3' at 4D9-3 (the latter possibility is supported by the EST data).
It is noteworthy that all variants except 4D7, RT3 and RT14 have been observed
only in one of the cDNAs. Although all the new exons (except 4D9-3.1) have an
AG/GT splice signal, it is plausible that these variants represent rare or
aberrant events
with little physiological significance.
The following exons contain Alu repetitive element sequences: 4D7-5. and 4D7-
C. The gene specific reverse (3 °) primer was designed for PDE4D exon
LFl (5'
GGCAATGGAGGAGTTCCGGGACA TA-3'; SEQ ID NO: 87 origin from Homo
Sapiens).
A contig for the incomplete genomic sequence of the PDE4D gene was
submitted by others in November 2000 (GenBank entry NT 023193 by International
Human Genome Project collaborators). The size of the contig is 614 481 by
(including
gaps) whereas our novel genomic sequence for the whole PDE4D region (i. e.,
from the
first exon for PDE4D variant) is close to 1,690,000 by and contains no gaps.
The contig
NT 023193 comprises only 11 exons of the PDE4D gene (in FIG. 3, exons 4D1/D2 -
11) and the 5' differently spliced exons are missing in the contig (in FIG. 3,
exons D4,
D5, D3, D8, D9, D7A-l, D7A-2, D7A-3, LF1, LF2, LF3 and LF4).
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Table 13: New Isoforms
Isoform
Name Cell
line
Exon Size
PDE4D7 D7-1 5' 122 SKNAS
by
PDE4D7 D7-2 Internal131bp SKNAS
PDE4D7 D7-3 Internal230 SKNAS
by
PDE4D91 D9 5' 7~2 HeLa
by
1 Formerly referred to in previous applications as PDE4D~
The sequences are as follows:
D7A-1:
ATAGTTGGCGTACCCTGAGGCCTGCCAGTTCCTGCCTTAATGCATATGTAGT
CGTAATTGAGTTCTGACACGGCCTTGGATGTTTCTGTCCTAAATAGCTGACA
TTGCATCTTCAAGACTGT
D7A-2:
CATTCCAGTTGGCTTTTGAGTGGATACGTGCAGTGAGATCATTGACACTGGA
AACACTAGTTCCCATTTTAATTACTTA.AA.ACACCACGATGAA.AAGAAATACC
TGTGATTTGCTTTCTCGGAGCAAAAGT
D7A-3:
GCCTGTGAGGAAACACTACATTCCAGTAATGAAGAGGAAGACCCTTTCCGC
GGAATGGAACCGTATCTTGTCCGGAGACTTTCATGTCGCAATATTCAGCTTC
CCCCTCTCGCCTTCAGACAGTTGGAACAAGCTGACTTGAAAAGTGAATCAGA
GAACATTCAACGACCAACCAGCCTCCCCCTGAAGATTCTGCCGCTGATTGGT
ATCACTTCTGCAGAATCCAGTGG (SEQ ID NO: 11; includes D7A-1, D7A-2 and
D7A-3)
New predicted amino-terminal protein sequence from above (PDE4D7):
MKRNTCDLLSRSKSASEETLHSSNEEEDPFRGMEPYLVRRLSCRNIQLPPLAFRQ
LEQADLKSESENIQRPTSLPLKILPLIAITSAESS (90 amino acids) (SEQ ID NO: 1,2)
D9:
TTCTCACTGCCCTGCGGTGTTTTGAACTGCCTTCTTACAGACGTCATACAGCC
CTTGAGGAATAGTTTCTGCCTGGTGAGATTGAATGATAGTTCTCATTCACAA
AACCCTGGATTCTAAGCAGGGACACACAGAAATTACTTTCGCAGGTAAATC
AGCCCACCCAGCCAAAGTGTGGAGAGATTTGTTCCTTGGCTGACTTCTTTGC
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TCCACGGAGAGGAGTGTTTTCCTGTGCTTGCCCTGAAATGGAACTTCCTTGA
CAGCTCTCCCGTGTTACAGTACCTCCCGGTCATTTTCTTTTTCTCTCTCTCTAC
CTGCGCTCTTCGAGTGTCAGAAACCTTTAAAGCTGTTACTATGGAATTGCAA
AAAAGAGATCAAGTGACTCTTTCACTATGCTGGTTTCCCTTGTGACCCAGAT
GAAGAATCAATTCAGAATTCAGTTCCTCCCTTGGCATTGCAAGACACAGAAG
AAACTGTCACTTCCTAACAGCCTAGTACTGGAGTAAATTCAGTATGAAGGA.A
GAA.AGCGCTCCTGCGTGTTAGAACCTTGCCCATGAGCTGGACCGAGGACAG
GAGATGGACTCCAGGAAAATTGGATTTCTTCAAGCAGCCTCCCTTGGAAATG
GAATATCTTTAAAATCTTCTTTGCAGAAA.GACAGTTAGAATGTATTAATCAG
AATAGTTGAAGACTTATTTTCCTTTTTATTTTTTTTCAAAATGAGGATTATTAT
GAAGCCAAGATCCCGATCTACAAGTTCCCTAAGGACTGCAGAGGCAGTTTG
(SEQ ID NO: 13)
IS
New predicted amino-terminal protein sequence from above (PDE4D9):
MSIIMKPRSRSTSSLRTAEAV (21 amino acids) (SEQ ID NO: 14).
Table
11
Publically
Available
SNPS; SNP
117 No.
from NCBI
Database
rs286155 rs27960 rs27221 rs149079 rs789615 rs37708
rs286156 rs27564 rs27653 rs149324 rs401207 rs37709
rs2061250 rs27565 rs26955 rs153067 rs364917 rs789389
rs286150 rs26948 rs26956 rs40354 rs404202 rs1423247
rs206789 rs40131 rs153031 rs26951 rs440607 rs874768
rs1823062 rs26949 rs185190 rs153029 rs411255 rs2042315
rs1823063 rs26950 rs37762 rs27223 rs615429 rs918590
rs1445852 rs26954 rs37761 rs27222 rs789396 rs918591
rs766119 rs26953 rs1423471rs251726 rs37684 rs918592
rs956721 rs152324 rs27224 rs1862589 rs1445893 rs1115372
rs248910 rs35385 rsI645013rs702556 rs37685 rs1345782
rs248912 rs40512 rs1423472rs702554 rs1086121 rs1363862
rs187481 rs35386 rs27220 rs441391 rs42222 rs1423248
rs153152 rs35387 rs1423473rs446883 rs37707 rsI423246
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rs1862614rs1995780 rs1435077rs159624 rs1008709 rs298088
rs2194256rs1508865 rs1369287rs1159470 rs1027747 rs298087
rs889305rs952110 rs1017410rs159622 rs869685 rs1421401
rs2113071rs1533019 rs1017409rs256349 rs869686 rs298086
rs2113072rs2117552 rs1435076rs256348 rs924880 rs298085
rs966220rs1545069 rs1435075rs1501640 rs1504983 rs298084
rs966221rs1545070 rs1435074rs600611 rs1504982 rs298083
rs719702rs973700 rs978455 rs159621 rs877745 rs298073
rs2113073rs1583434 rs1827340rs159625 rs877744 rs298072
rs21130T4rs1347401 rs1393083rs1435072 rs2164661 rs298471
rs2113075rs1949017 rs988364 rs173945 rs981230 rs1421400
rs1035512rs723962 rs101T408rs256356 rs1437124 rs402874
rs1559277rs1355099 rs2053155rs185351 rs746477 rs434368
rs1981848rs1396473 rs181923 rs256355 rs893I91 rs37I0I1
rs1544788rs1369285 rs1546364rs2067024 rs1992112 rs298063
,rs1544790rs1435071 rs173942 rs256354 rs298102 rs298062
rs1544791rs1435070 rs159616 rs173944 rs298101 rs298061
rs851284rs1435083 rs159620 rs256353 rs2164660 rs298060
rs1396476rs991551 rs1501641rs986400 rs298100 rs298057
rs1508860rs1154790 rs159619 rs1504981 rs298098 rs298056
rs1974850rs1154789 rs159614 rs1120533 rs298096 rs1370230
rs2136203rs714291 rs159613 rs256351 rs298095 rs297975
rs2174994rs981760 rs159612 rs190458 rs298094 rs297974
rs1508863rs1369288 rs159611 rs256352 rs298093 rs379578
rs1508859rs977418 rs194368 rs171745 rs1362942 rs920190
rs1508864rs977417 rs661576 rs1157709 rs1362941 rs1865962
rs1396474rs977416 rs299627 rs1910790 rs298091 rs298018
rs1543951rs1529843 rs159608 rs1910789 rs298090 rs298021
rs20I6324rs1529842 rs159609 rs1504985 rs298089 rs298022
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rs298023rs20S3229 rs296406 rs697076 rs3757S rs1824154
rs298024rs295974 rs296405 rs29447$ rs37576 rs2112911
rs298025rs295973 rs295948 rs953302 rs1876209 rs1551564
rs298026rs295972 rs295947 rs294479 rs190486 rs2034895
rs298027rs295971 rs295946 rs697075 rs447261 rs2081092
rs298028rs295970 rs295945 rs294481 rs1506558 rs2112910
rs298029rs295969 rs295944 rs294482 rs1108916 rs918583
rs298030rs295968 rs1395334 rs294483 rs921942 rs1840838
rs169868rs295966 rs295943 rs702545 rs924998 rs13.50298
rs177077rs726652 rs1035321 rs294484 rs176705 rs1990985
rs298032rs295965 rs294494 rs294485 rs1156029 rs1379297
rs298033rs1307218 rs722923 rs294486 rsI156028 rs1817248
rs298034rs1307217 rs294495. rs702544 rs931857 rs244569
rs298035rs893190 rs294496 rs702543 rs931856 rs244568
rs298042rs1111495 rs294497 rs159194 rs931855 rs244567
rs298044rs295961 rs294498 rs402I5 rs 1506557rs244565
rs298045rs295960 rs294499 rs291118 rs462930 rs185417
rs298046rs295959 rs294500 rs1506560 rs458953 rs258128
rs298048rs295958 rs294501 rs37569 rs174039 rs258127
rs298049rs296410 rs294503 rs291119 rs2174624 rs258125
rs298050rs29595? rs295936 rs37571 rs2135480 rsI3.48710
rs298051rs295956 rs1395336 rs1870077 rs992726 rs1348709
rs298052rs295955 rs1395337 rs159195 rs294474 rs197I061
rs298053rs295954 rs294492 rs37572 rs294475 rs1S41673
rs190936rs295949 rs159196 rs37573 rs988827 rs1541672
rs298017rs29S980 rs159197 rs167161 rs988828 rs258112
rs298016rs295979 rs172362 rs37574 rs1350297 rs258111
rs298015rs295978 rs37579 rs1506562 rs1457110 rs171800
rs298014rsI154587 rs722784 rs291122 rs1457111 rs187716
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rs258110rs424839 rs1118965 rs35266 rs2556S2 rs26709
rs258109rs370891 rs154028 rs39672 rs255650 rs26710
rs258108rs434183 rs151802 rs958851 rs255649 rs280S5
rs258107rs444552 rs244580 rs244576 rs2194210 rs26711
rs665836rs433565 rs1457145 rs244575 rs255648 rs27723
rs392901rs1445918 rs244579 rs244573 rs255647 rs27185
rs383444rs441817 rs2S5812 rs35258 rs154221 rs2769S
rs662643rs433161 rs154029 rs352S9 rs256752 rs1445954
rs670I69rs428059 rs185333 rs40121 rs2S6120 rs27549
rs525099rs434422 rs35289 rs35261 rs255635 rs45S969
rs669240rs427433 rs35288 rs35264 rs185325 rs26712
rs381755rs391377 rs35287 rs40I22 rs26686 rs1867711
rs454702rs414746 rs35286 rs35265 rs1031197 rs1867712
~
rs443191rs187368 rs35285 rs3525S rs1031198 rs26713
rs380118rs244593 rs35284 rs721826 rs27183 rs26714
rs2168649rs244592 rs35283 rs244570 rs28044 rs27547
rs371775rs244591 rs35282 rs27171 rs27182 rs26715
rs378970rs244S90 rs35281 rs1824159 rs545611 rs27949
rs401013rs181736 rs35280 rs27170 rs649476 rs26700
rs427748rs193447 rs35279 rs27169 rs1664896 rs1306348
rs427740rs2028842 rs352?8 rs27168 rsI49106 rs35309
rs378869rs2028841 rs40126 rs2013979 rs1374028 rs27691
rs1902609rs1823068 rs35277 rs889231 rs531105 rs35310
rs389324rs1823067 rs35276 rs2014012 rs27184 rs26689
rs387647rs1823066 rs35275 rs37353 rs1445951 rs27187
rs377451rs244588 rs40125 rs187645 rs1947090 rs1445948
rs403695rsI68641 rs35274 rs1809012 rs26708 rs26687
rs403672rs2059175 rs244577 rs187644 rs2112959 rs166260
rs372309rs2059174 rs35267 rs153981 rs1445953 rs149506
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rs27722 rs1664886 rs1559251 rs15531I3
rs26695 rs1867724 rs1345791 rs1353748
rs27773 rs1445947 rs1345792 rs1498606
rs1471429rs42470 rs1345793 rs1353747
rs1471430rs1423308 rs1105577 rs1006431
rs26705 rs27174 rs1960 rs1948651
rs28054 rsI68834 rs1824788 rs1498605
rs26703 rs27727 rs1862563 rs1498604
rs27898 rs27I72 rs1551939 rs1498603
rs722010rs676449 rs1038080 rs1995166
rs27957 rs27186 rs997421 rs1498602
rs26702 rs2112957 rs1014317 rs1077183
rs27548 rs1023814 rs2059191 rs1078368
rs26701 rs27175 rs1551938 rs1874857
rs27188 rs1445950 rs1186170 rs1874858
rs27189 rs202I384 rs986067 rs1909294
rs149084rs736736 rs954740 rs1546221
rs153968rs745813 rs1363882 rs2055295
rs464787rs889229 rs1353749 rs1391648
rs153978rsl0?7978 rs1391651 rs2055298
rs464311rs2081106 rs1391650 rs1472456
rs149108rs1559252 rs1391649 rs1553114
rs153980rs2054443 rs1391652 rs1542842
rs153961rs922437 rs950446 rs1498611
rs1867725rs922436 rs950447 rsI532520
~
rs153965rs922435 rs1498599
rs153966rs922434 rs1498601
rs1988803rs716908 rs1498609
rs467300rs1971940 rs1498608
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Table 12
New SNPs identified by
deCODE
PositionVariation
AA Change
Exon
135641T/A 1268187 C/T
142780A/G 1268553 A!G
732790G/T 1272669 G/A
735966C/A 1272910 A/G
736226A/G 1273023 G/A
736516C/T 1273220 A/G
850001G/A 1273240 A/G
852776A/C 1273543 CfT
853079G/T 1288439 G/A
853575C/A 1289730 T/A
856468A/G 1290176 G/A
860845~A/G 1293745 T/C
870924A/G 1344605 A/G
1027267T/C 1344864 G/A
1027643T/G 1345135 C/G
1027757T/C 1345286 A/G
1028146T/A 1346112 C/T
1037657A/C 1352976 A/T
1044016G/A 1354291 T/C
1044045C/T 1354377 C/T
1254737T/C 1354554 C/A
1254849T/C 1354675 T/C
1255763G/T 1355114 T/C
1257206A/G 1355693 A/G
1258161T/C 1357081 A/G
1268007A/G 1362985 T/G
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1363021C/T 1580088 G/A
1363827C/T 1581078 G/A
1363911G/A 1582418 T/A
1364061C/T 1584580 A/C
1364066T/A 1585955 G/T
1367904A/G 1590608 T/C
1368193T/C 1590672 A/G
1368217G/C 1590673 G/T
1373349C/T 1590837 G/A
1373384A/G 1590936 C/A
1373415T/C 1591011 G/A
1373979T/G 1591047 C/T
1376149G/A 1591306 C/A Pro->Thr D1
1384931A/C 1591583 TlC
1385093A/T 1594788 C/A
1385107G/A 1594994 G/A
1385445T/C 1601831 C/T
1391418G/C 1636902 T/C
140921QC/A 1638550 A/C Lys->Thr exon
4
1414804C/T 1640663 T/C
1428284TlC 1641954 C/T
1431800A/T 1641960 C/T
1449904A/T 1653881 G/A
1574301C/G 1655748 G/A
1574615C/T 91470 G/A
1575634A/T
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Discussion of Example 2:
Here we present the first complete genomic sequence of human PDE4D, two
novel mRNAlprotein isoforms of PDE4D and their corresponding exons, and the
intron-
exon structure of known and novel isoforms. The basis for phosphodiesterases
is the
mammalian homolog of the "dunce" gene in Drosoplaila melanogastef°,
implicated in
learning and memory (Davis, R.L. and B. Dauwalder, Trends Genet., 7(7):224-229
(1991)). PDEs are members of a large superfamily of isoenzymes subdivided into
9 and
possibly 10 distinct families (Conti, M. and S.L. Jin, Prog. Nucleic Acid Res.
Mol. Biol.,
63:1-38 (1999)), with several genes in each family and more than one isoform
for each
gene. The significance of the diversity of PDEs is not known but many of the
isoforms
differ in their biochemical properties, phosphorylation, intracellular
targeting, protein-
protein interactions and patterns of expression in tissues, which suggests
that each of the
various isoforms might have distinct functions (Bolger, G.B., Cell Signal,
6(8):851-859
(1994); Conti, M., et al., Enclocr. Rev., 16(3):370-378 (1995)).
There are four genes that encode the type 5 PDEs (PDE4A, PDE4B, PDE4C and
PDE4D), which is a group of enzymes characterized by high affinity for cAMP.
The
gene for PDE4D was assigned to human chromosome Sql2 (Milatovich, A., et al.,
Somat. Cell Mol. Genet., 20(2):75-86 (1994); Szpirer, C., et al., Cytogenet.
Cell Genet.,
69(1-2):22-14 (1995)} and 5 distinct splice variants have been characterized
(the short
forms PDE4D1, PDE4D2 and the long forms PDE4D3, PDE4D4, and PDE4D5)
(Bolger, G.B., et al., Bioclaena. J., 328(Pt.2):539-548 (1997)) (FIG. 3). The
sequence of
the human PDE4D variants show a high degree of homology to the PDE4Ds
expressed
in mouse and rat. The pattern of splicing and different promoter usage is
highly
conserved during evolution indicating an important physiological role (Nemoz,
G., et
al., FEBSLett., 384(1):97-I02 (1996)). The PDE4D variants are generated at two
major
boundaries present in the gene. The first boundary corresponds to the junction
of exon
2. Differential splicing in this region generates the 2 short variants PDE4D1
(586 a.a.)
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and PDE4D2 (508 a.a.) (FIG. 3). This splicing boundary is conserved in mouse,
rat and
between different human PDE4 genes. The splicing variant PDE4D2 is generated
by the
removal of 256 by from the PDE4D1 sequence. The initiation codon in the PDE4D2
variant lies within exon D1/D2. Data demonstrates that the expression of the
short
PDE4D variants is under the control of an internal promoter regulated by cAMP
(Vicini,
E. and M. Conti, Mol. Ezzdocz°irzol., 11 (7):839-850 (1997)). The
second major splicing
boundary is also conserved during evolution and is identical to that described
in the
Drosophila dunce gene. Splicing occurs at the intron/exon boundary at the LFI
exon
(FIG. 3).
PDE furactiotz
The PDEs serve at least four major functions in the cell. They can (I) act as.
effector of signal transduction by interacting with receptors and G-proteins;
(2) integrate
the cyclic nucleotide-dependent pathway with othex signal transduction
pathways; (3)
IS function as homeostatic regulators, playing a role in feedback mechanisms
controlling
cyclic nucleotide levels during hormone and neurotransmitter stimulation; (4)
play an
important role in controlling the diffusion of cyclic nucleotides and in
cxeating
subcellular domains or channeling cyclic nucleotide signaling (Conti, M. and
S.L. Jin,
P>~og. Nucleic Acid Res. Mol Biol., 63:1-38.(1999)). Inhibition of PDE has
Long been
recognized as an effective pharmacological strategy to alter intracellular
cyclic
nucleotide levels (Flamm, E.S., et al., Arclz. Neurol., 32(8):569-71 (1975)).
It has been reported that PDE4 is the predominant isozyme regulating vascular
tone mediated by cAMP hydrolysis in cerebral vessels (Willette, R.N., et al.,
J. Ce~eb.
Blood Flow Metab., 17(2):210-9 (1997)).
A recent study on mice with targeted disruption of PDE4D gene (Hansen, G., et
al., Proc. Natl. Acad. Sci. USA, 97(12):6751-6 (2000)) has demonstrated a
cxucial role
of PDE4D in the control of smooth muscle contraction and muscarinic
cholinergic
receptor signaling but not in the control of airway inflammation. The Iung
phenotype of
the PDE4D-/- mice demonstrates that this gene plays a nonredundant role in
cAMP
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homeostasis. There is a significant reduction in PDE activity and an increase
in resting
and stimulated cAMP levels in the lung, indicating that other PDE4s (or other
PDEs) are
not up-regulated and cannot compensate for the loss of PDE4D. These findings
support
that PDE4D serves a unique, nonoverlapping functions in cell signalling.
No clear link between an established inherited disorder and known PDE loci has
emerged, with the exception of PDE6. Inhibitors of PDEs have been shown to
affect
airway responsiveness and pulmonary allergic inflammation (Schudt, C., et al.,
Pulm.
Pharmacol. Ther., 12(2):123-9 (1999)). There axe reports suggesting that
altered PDE4
function may be linked to nephrogenic diabetes insipidus (Takeda, S., et al.,
EradocrirZOlogy, 129(1):287-94 (1991)) or atopic dermatitis (Chan, S.C., et
al., J. Allergy
Clin. Immuhol., 91 (6):1179-88 (1993)), however no mutations have been
identified. It
has also been reported that vasorelaxation modulated by PDE4 (not mentioned
whether
it is A, B, C or D gene family) is compromised in chronic cerebral vasospasm
associated
with subarachnoid hemorrhage (Willette, R.N., et al., J. Cereb. Blood Flow
Metab.,
17(2):210-9 (1997)). PDE4D itself has not been linked to stroke before.
PDE4D expression arad cellular localization
PDE4Ds are expressed in human peripheral mononuclear cells (Nemoz, G., et
al., FEBSLett, 334(1):97-102 (1996)), brain (Bolger, G., et al., Mol. Cell
Biol.,
13(10):6558-71 (1993)), heart (I~ostic, M.M., et al., J. Mol. Cell
Caf°diol., 29(11):3135-
46 (1997)) and vascular smooth muscle cells (Liu, H. and D.H. Maurice, J.
Biol. Chem.,
274(15):10557-65 (1999)).
Immunoblotting of rat brain has shown that the PDE4D3, PDE4D4 and PDE4D5
proteins are present in brain (Bolger, G.B., et al., Biochem. J., 323(Pt
2):539-48 (1997))
and are expressed in cortex and cerebellum from rat (Iona, S., et al., Mol.
Pharmacol.,
53(1):23-32 (1998)). These proteins were recovered mostly or exclusively in
the
particulate fraction suggesting that these forms may be targeted to insoluble
cellular
structures. In addition a 68 kDa protein was detected which could represent
PDE4D1,
PDE4D2 or both. To verify this RT-PCR was performed on mRNA from rat brain and
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the results showed that transcripts for PDE4D1 and 2 were present. Their data
also
suggests that the N-terminal regions of the PDE4D3-5, derived from
alternatively
spliced regions of their mRNAs, are important in determining their subcellular
localization activity and differential sensitivity to inhibitors and there are
indications that
there is a propensity for the Iong PDE4D isoforms to interact with particulate
fraction of
the cell.
EXAMPLE 3: PDE4D ISOFORM EXPRESSION
Expression analysis in EB h tr~ansfor-med B cell limes
As a functional mutation in the known coding exons of PDE4D was not
identified, gene expression was next studied to determine if the genetic
association to
stroke relates to regulation of its expression levels. In order to test this,
we chose to use
cell lines instead of blood or tissues for these studies because expression
analysis of cell .
lines is not confounded by the presence of multiple cell types. Cell types may
express
PDE4D at different levels so it is generally more reliable to quantify
expression in cell
lines than tissues. Isoform-specific kinetic PCR analysis was carried out on
EBV
transformed B cell lines to quantify each isoform in 83 stroke patients and 84
controls.
These patients were not selected for this analysis based on any specific
subtype of
stroke. The majority of the patients had ischemic stroke and 38% of them had
carotid or
cardiogenic cause of stroke. Overall the total PDE4D message level as assessed
by
amplification across exons present in all isoforms (PAN), was significantly
lower in
patients than in controls (p value < 0.005). This decrease was due primarily
to lower
expression of the isoforms, PDE4D1, PDE4D2 and PDE4D5 (FIG. 4).
We selected individuals with a specific stroke associated haplotype and
compared the expression levels of carrier vs. non-carriers of this haplotype
and with
patients and controls examined separately (FIGS. 5 and 6). The haplotype was
constructed out of the at-risk allele for the microsatellite marker AC008818-1
and
SNP45 (SNPSPDM357221) and SNP41 (SNPSPDM361S45). This haplotype acts as a
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surrogate for the disease-associated haplotype we have identified in LD bloclc
B (Table
3). Patients with the haplotype had a significantly decreased expression of
the PDE4D7
and PDE4D9 isoforms (FIG. 5). Several other isoforms of PDE4D were expressed
but
did not show correlation to the disease haplotype. The PDE4D7 correlation was
also
present in controls but only marginally significant (FIG. 6). Of interest,
this at-risk
haplotype covers the 5' exon specific to PDE4D7 and presumably its promoter.
These results show that there is significant disregulation of the expression
of
multiple PDE4D isoforms in stroke patients.
Methodology for expression analysis using Quantitative Reverse Trahscriptase
PCR
Total RNA was isolated from EBV transformed B-cell cultures according to
manual, using the TRIZOL~ reagent provided by GibcoBRL. RNeasy mini Qiagen kit
with on column DNA digestion was used to clean RNA. Quality and quantity of
RNA
was assessed using 2100 Agilent Bioanalyser. cDNA was prepared from total RNA
using random hexamers with TaqlVIan Reverse Transcription Reagents kit from
Applied
Biosystems (N808-0234). Primer Express 2.0 and Oligo 6 software were used to
make
cDNA specific primers and probes for PDE4D and PDE4D isoforms. GAPDH "Assay-
On-Demand" was obtained from Applied Biosystems and used as a housekeeping
gene.
PDE assays were tested and optimized for 384 well high throughput expression
analysis
using ABI 7900 Instrument. A final concentration of 200 nM probes, 900 nM
primers
and 2 ng/mcl cDNA was used in a l Omcl reaction volume. Each plate was run
twice and
an average for each sample calculated. ABI7900 instrument was used to
calculate CT
(Threshold Cycle) values. Samples displaying a greater than 1 deltaCT between
duplicates were not used in our analysis. Quantity was obtained using the
formula 2-~CT
where ACT represents the difference of CT values between target and
housekeeping
assay.
Accession numbers AY245866 (PDE4D7) and AY245867 (PDE4D9).
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Discussion of the three examples arad conclusions:
Our results indicate that genetic variation in the PDE4D gene is associated
with
ischemic stroke. The direct involvement of PDE4D is strongly supported by
linkage in
conjunction with association and expression analysis. We first identified the
association
using microsatellite markers, and supplementing the microsatellite data with a
denser set
of SNPs further supported this. The strongest association is to the two
ischemic
subtypes, carotid arid cardiogenic stroke whereas we did not observe
association to small
vessel occlusive disease, the form of stroke thought to be independent of
atherosclerosis.
Although we have not identified a functional mutation in the PDE4D gene, we
have
identified a haplotype, that extends over the first exon of PDE4D that is
significantly
associated to carotid and cardiogenic stroke. This haplotype is present in 47%
of the
carotid/cardiogenic stroke patients, compared to 21% in the control group with
more
than two-fold stroke risk for the carriers of this haplotype. It has a
population attributed
risk of 25%. For the combined cardiogenic and carotid subtype of stroke, apart
from
finding individual SNP and microsatellite alleles that are significantly
associated with
the disease even after adjusting for multiple comparison, most interesting is
the
discovery that haplotypes covering the first exon of PDE4D can be classified
into three
groups with clearly distinct risks. Relative to the protective group, the
population
attributed risk of the at-risk and wild type groups combined is estimated to
be 55%.
Approximately 16% of the population carries one copy of the at-risk haplotype
in FIG.
12.3. They have about 1.8 times the risk of the general population for getting
cardiogenic or carotid stxoke. Approximately 0.8% of the population is
homozygous for
the at-risk haplotype and, assuming the multiplicative model, their risk is
estimated to be
about 3.8 times the risk of the general population. It is true that we have
not yet
identified or proved convincingly what is the functional variant, or variants,
which are
responsible for the observed effects of these haplotype groups. And, since
these
haplotype groups do not fully explain the linkage signal we observe in the
region for all
stroke patients, we certainly could not rule out, and indeed expect, that
there are other
variants/haplotypes within PDE4D not directly related to those we have
identified that
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confer risk to stroke. These are likely to be rare but could have very high
penetrance.
We also cannot rule out the possibility that some other genes in the linkage
region
independent of, or in conjunction With, PDE4D confer susceptibility to stroke.
We examined whether the disease associated alleles and haplotype are related
to
specific stroke risk factors such as hypertension, hypercholesterolemia,
diabetes,
peripheral artery occlusive disease and coronary artery disease in addition to
each onset
of stroke and gender (Table 8). A marginally significant association to
hypercholesterolemia was observed but it is clear that PDE4D's contribution to
stroke is
not strongly correlated with any of these known risk factors.
The PDE4D gene is a highly complex gene. By alternative splicing and use of
different promoters this gene generates at least 8 different isofoxms that
yield functional
proteins, differing from each other in their N-terminal regions. We have
identified four
new exons encoding the N-termini of two new isoforms PDE4D7 and PDE4D9. The
disease-associated haplotype extends over the 5'exon unique to the new PDE4D7
variant and the presumed promoter region of this isoform suggesting that the
functional
variation may be involved in transcriptional regulation. This hypothesis is
also
supported by our PDE4D expression analysis that shows significant correlation
between
the disease associated haplotype and the level of PDE4D7 message.
The strongest association found for this PD~'4D haplotype was to the two major
subtypes of ischemic stroke, carotid and cardiogenic stroke, suggesting a role
for this
gene in the vascular biology of atherosclerosis. While there are multiple
etiologies for
ischemic stroke, atherosclerosis remains the most important one and it is the
major
pathological process for the two ischemic subtypes, carotid and cardiogenic
strokes.
First, it is the major cause of stenotic and occlusive lesions of the internal
and common
carotids that lead to carotid strokes. Second, cardiac thrombi which shed
emboli to the
brain most commonly occur on the background of coronary artery disease, such
as
following acute myocardial infarction or ischemic cardiomyopathy, andlor due
to atrial
fibrillation on the basis of poor compliance of ischemic ventricles (diastolic
dysfunctionlstiffening). Although atrial fibrillation may occur on the
background of
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other diseases such as valvular disease, hyperthyroidism, and hypertension, in
the age
group that tends to suffer from stroke, ischemic heart disease remains one of
the most
important causes. Ischemic stroke resulting from occlusion of small
penetrating arteries
within the brain (small vessel occlusive disease or lacunar stroke) is
generally thought to
result from endothelial proliferation since atherosclerosis only occurs in
larger arteries.
PDE4D does not show association to small vessel stroke, consistent with its
role in
atherosclerosis. Carotid and cardiogenic stroke together account for the
majority of
ischemic stroke (note that our number for carotid is lower since we used a
more stringent
cutoff of stenosis).
PDE4D selectively degrades second messenger cAMP (Kong, A. et al., Nat
Genet 10, 10 (2002)), which plays a central role in signal transduction and
regulation of
physiological responses. It is expressed in most cell types important to the
pathogenesis
of atherosclerosis, including vascular smooth muscle cells (VSCM), endothelial
cells,
monocytes, macrophages and T-lymphocytes (Houslay, M.D. and Adams, D.R.,
Bioclaem J370, 1-18 (2003); Liu, H. and Maurice, D.H., JBiol Chena 274, 10557-
65.
(1999); Liu, H. et al., JBiol Chem 275, 26615-24. (2000); Baillie, G., et al.,
Mol
Phar°macol 60, 1100-11. (2001); Jin, S.L. and Conti, M., Proc Natl Acad
Sci LI S A 99,
7628-33. (2002)). Cyclic AMP is a key signalling-molecule in these cells
(Landells, L.J.
et al., Bf~ JPha~macol 133, 722-9 (2001); Fukumoto, S. et al., Cif°c
Res 85, 985-91.
(1999); Ogawa, S. et l., Am JPhysiol 262, C546-54 (1992)). In VSMC, low cAMP
levels lead to an increase in proliferation and migration that at least in
part is mediated
by PDE4 (Landells, L.J. et al., Br JPha~macol 133, 722-9 (2001); Stelzner,
T.J., et al., J
Cell Plzysiol 139, 157-66 (1989); Pan, X., et al., Biochem Pha~macol 48, 827-
35.
(1994)). Animal models have also shown that elevation of CAMP reduces
neointimal
lesion formation and inhibits proliferation of SMCs after arterial injury
(Palmer, D., et
al., Circ Res 82, 852-61. (1998); Indolfi, C. et al., Nat Med 3, 775-9.
(1997)). In
monocytes and T-lymphocytes, accumulation of cAMP is generally associated with
inhibition of immune functions such as proliferation and cytokine secretion
(Indolfi, C.
et al., JAm Coll Cardiol 36, 288-93. (2000)). It is attractive to postulate
that the
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regulation of cAMP through absolute or relative expression of one or more
PDE4D
isoforms may differ in individuals susceptible to stroke; some stroke patients
may have
increased PDE4D activity and, consequently lower cAMP levels in any of the
above cell
types, leading to development of the atherosclerotic plaque and/or its
instability.
However, contrary to what one might expect we see decreased expression in some
of the
PDE4D isoforms in EBV cell lines from stroke patients. It is of interest that
these
isoforms are all up regulated by CAMP (Liu, H. and Maurice, D.H., JBiol Cdaem
274,
10557-65. (1999); Tilley, S.L., et al., J Clip Invest 108, 15-23 (2001);
Vicini, E. and
Conti, M., Mol Eyadocf°ifzol 11, 839-50 (1997)) suggesting
disregulation at the level of
cAMP in patients. It is therefore possible that increased activity of one or
few splice
variants alters the effective PDE4D enzymatic activity of the cell decreasing
the cAMP
levels thus altering the expression of cAMP regulated isoforms as observed in
our
expression study. This relative expression of PDE4D isoforms may determine the
compartmental localization of PDE4D isoforms and thus the corresponding
gradients of
intracellular cAMP that have been recently observed (see Housley review).
In summary, we have presented association analyses (single marker and
haplotype analyses) that support the notion that the PDE4D gene confers risk
to
ischemic stroke. Furthermore, we have observed significant disregulation of
multiple
PDE4D isoforms in stroke patients. We propose that this gene is involved in
the
pathogenesis of stroke through atherosclerosis. PDE4D is expressed in cell
types
important in atherosclerosis and regulates a second messenger with a central
role to
processes important in the pathogenesis of atherosclerosis. Inhibition of
PDE4D in
general or specifically one or more isoforms, by a small molecule drug or
other
pharmacological agent might decrease the risk of stroke in general, and
especially those
who are predisposed to stroke through variation in the PDE4D gene.
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While this invention has been particularly shown and described with reference
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
spirit and scope of the invention as defined by the appended claims.
