Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN OSTEOPOROSIS GENE
RELATED APPLICATION
This application is a continuation of and claims priority to U.S. Application
No. 10/346,723, filed January 16, 2003, which is a continuation-in-part of
U.S.
Application No. 09/952,360, filed September 13, 2001, and which is also a
continuation-in-part and claims priority to International Application No.
PCT/IBO1/01667, which designated the United States and was filed on September
12, 2001, published in English, which is a continuation-in-part of U.S.
Application
No. 09/661,~g7, filed September 14, 2000. The entire teachings of the above
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Osteoporosis is a debilitating disease characterized by low bone mass and
deterioration of bone tissue, as defined by decreased bone mineral density
(BMD).
A direct result of the experienced microarchitectural deterioration is
susceptibility to
fractures and skeletal fragility, ultimately causing high mortality, morbidity
and
medical expenses worldwide. Postmenopausal woman are at greater risk than
others
because the estrogen deficiency and coiTesponding decrease in bone mass
experienced during menopause increase both the probability of osteoporotic
fracture
and the number of potential fracture sites. 51 et aging women are not the only
demographic group at risk. young woman who are malnourished, ammenorrheic, or
insufficiently active are at risk of inhibiting bone mass development at an
early age.
Furthermore, androgens play a role in the gain of bone mass during pubez-ty,
so
elderly or hypogonadal men face the risk of osteoporosis if their bones were
insufficiently developed.
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The need to fmd a cure for this disease is complicated by the fact that there
are many contributing factors that cause osteoporosis. Nutrition (particularly
calcium, vitamin I? and vitamin K intake), hormone levels, age, sex, race,
body
weight, activity level, and genetic factors all account for the variance seen
in bone
mineral density among individuals. Currently, the drugs approved to treat
osteoporosis act as inhibitors of bone reabsorption, and include methods such
as
hormone replacement therapy (HRT), selective estrogen receptor modulators,
calcitonin, and biophosphonates. However, these treatments may not
individually
reduce risk with consistent results and while some therapies improve BMI~ when
co-
administered, others show no improvement or even lose there efficacy when used
in
combination. Clearly, as life expectancy increases and health and economic
concerns of osteoporosis grow, a solution for the risks associated with this
late-onset
disease is in great demand. Early diagnosis of the disease or predisposition
to the
disease would be desirable.
SUMMARY OF THE INVENTI~N
As described herein, it has been discovered that polymorphisms in the
nucleic acid sequence for human bone morphogenetic protein 2 (BMP2) have been
correlated through human linkage studies to a number of osteoporosis
phenotypes.
In particular, it has been discovered that one or more single nucleotide
polymorphisms within the nucleotide sequence encoding the BMP2 gene product is
correlated to osteoporosis. Accordingly, this invention pertains to an
isolated
nucleic acid m~lecule containing the EI~J~2 nucleic acid of SEQ III N~:1
having at
least one altered nucleotide as shov~n in Table 2 and to gene products encoded
thereby (referred to herein as a "variant EMh2 nucleic acid or gene" or
"variant
Ell 2 gene product")
A number of polymorphisms have been observed in the EMP2 nucleic acid
as repouted in Table 2. Thus, in particular embodiments, the isolated nucleic
acid
molecule of the invention can have one or a combination of these nucleotide
polymorphisms. These polymorphisms can be part of a group of other
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polymorphisms in the BMP2 nucleic acid that contributes to the presence,
absence or
severity of osteoporosis. In one embodiment, the nucleic acid molecule will
comprise at least the polymorphism at nucleotide position 3747, and the gene
products will comprise the polymorphism at nucleotide position 3747 (amino
acid
change serine to alanine).
The invention further provides a method for assaying a sample for the
presence of a nucleic acid molecule comprising all or a portion of BMP2 in a
sample, comprising contacting said sample with a second nucleic acid molecule
comprising a nucleotide sequence encoding a BMP2 polypeptide (e.g-., SEQ ID
N~:1 or the complement of SEQ ID N~:1 and which comprises at least one
polymorphism as shown in Table 2); a nucleotide sequence encoding SEQ II7 N~:
2
which comprises at least one polymorphism as shown in Table 2, or a fragment
or
derivative thereof, under conditions appropriate for selective hybridisation.
In one
embodiment, the nucleic acid molecule will comprise at least the polymorphism
at
nucleotide position 3747, and the gene products will comprise the polymorphism
at
nucleotide position 3747 (amino acid change serine to alanine). The invention
additionally provides a method for assaying a sample for the level of
expression of a
BMP2 polypeptide, or fragment or derivative thereof, comprising detecting
(directly
or indirectly) the level of expression of the BMP2 polypeptide, fragment or
derivative thereof.
The invention also relates to a vector comprising an isolated nucleic acid
molecule of the invention operatively linked to a regulatory sequence, as well
as to a
recombinant host cell comprising the vector. The invention also provides a
method
for preparing a polypeptide encoded by an isolated nucleic acid molecule
described
herein (a BMP2 polypeptide), comprising culturing a recombinant host cell of
the
invention under conditions suitable for expression of said nucleic acid
molecule.
The invention further provides an isolated polypeptide encoded by isolated
nucleic acid molecules of the invention (e.g., BMP2 polypeptide), as well as
fragments or derivatives thereof. In a particular embodiment, the polypeptide
comprises the amino acid sequence of SEQ II? N~:2 and comprising at least one
polymorphism as shown in Table 2, and in another embodiment comprising at
least
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the polymorphism at 3747 resulting in an amino acid change from serine to
alanine.
The invention also relates to an isolated polypeptide comprising an amino acid
sequence that is greater than about 90 percent identical to the amino acid
sequence
of SEQ ID N0:2 and comprising at least one polymorphism described herein; in
another embodiment, the amino acid sequence is about 95 percent identical to
the
amino acid sequence of SEQ ID N0:2.
The invention also relates to an antibody, or an antigen-binding fragment
thereof, which selectively binds to a polypeptide of the invention, as well as
to a
method for assaying the presence of a polypeptide encoded by an isolated
nucleic
acid molecule of the invention in a sample, comprising contacting said sample
with
an antibody which specifically binds to the encoded polypeptide.
The invention fLU-ther relates to methods of diagnosing osteoporosis or a
predisposition to osteoporosis. The methods of diagnosing a predisposition to
osteoporosis in an individual include detecting the presence of a mutation in
BMP2,
as well as detecting alterations in expression of an EMP2 polypeptide, such as
the
presence of different splicing variants of BIl~IP2 polypeptides. The
alterations in
expression can be quantitative, qualitative, or both quantitative and
qualitative. The
methods of the invention alone or in combination with other assays, e.g., bone
turnover marker assays (e.g., bone scans), allow for the accurate diagnosis of
osteoporosis at or before disease onset, thus reducing or minimizing the
debilitating
effects of osteoporosis.
The invention additionally relates to an assay for identifying agents that
alter
(e.g., enhance or inhibit) the activity or expression of one or more EIe~IP2
polypeptides. For example, a cell, cellular fraction, or solution containing
an E1~IP2
polypeptide or a fragment or derivative thereof, can be contacted with an
agent to be
tested, and the level of EhlIF2 polypeptide e~~pression or activity can be
assessed.
The activity or expression of more than one EI~11~2 polypeptides can be
assessed
concurrently (e.g., the cell, cellular fraction, or solution can contain more
than one
type of BIYIP2 polypeptide, such as different splicing variants, and the
levels of the
different polypeptides or splicing variants can be assessed).
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In another embodiment, the invention relates to assays to identify
polypeptides which interact with one or more BMP2 polypeptides. In a yeast two-
hybrid system, for example, a first vector is used which includes a nucleic
acid
encoding a DNA binding domain and also an BMP2 polypeptide, splicing variant,
or
fragment or derivative thereof, and a second vector is used that includes a
nucleic
acid encoding a transcription activation domain and also a nucleic acid
encoding a
polypeptide that potentially can interact with the BMP2 polypeptide, splicing
variant, or fragment or derivative thereof (e.~., a BMP2 polypeptide binding
agent or
receptor). Incubation of yeast containing both the first vector and the second
vector
Lender appropriate conditions allows identification of polypeptides which
interact
with the BMP2 polypeptide or fragment or derivative thereof, and thus can be
agents
which alter the activity of expression of an BMP2 polypeptide.
Agents that enhance or inhibit BMP2 polypeptide expression or activity are
also included in the current invention, as are methods of altering (enhancing
or
inhibiting) BMP2 polypeptide expression or activity by contacting a cell
containing
BMP2 and/or polypeptide, or by contacting the BMP2 polypeptide, with an agent
that enhances or inhibits expression or activity of BMP2 or polypeptide.
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 BMP2 polypeptide. The invention
further
pertains to methods of treating osteoporosis, by administering BMP2
therapeutic
agents, such as nucleic acids of the invention, polypeptides of the invention,
the
agents that alter activity of BMP2 polypeptide, or compositions comprising the
nucleic acids, polypeptides, and/or the agents that alter activity of BMP2
polypeptide. In another embodiment, the BI~~P2 therapeutic agent is the human
BMP2 nucleic acid or its gene product from a healthy individual who does not
have
osteoporosis.
In a further embodiment, the invention is directed to a method of diagnosing
a susceptibility to osteoporosis in an individual, comprising detecting a
polymorphism in a human BMP2 gene of SEA ~ NO: l, wherein the presence of a
'6('!79 at nucleotide position 122247 of AL03566~; the presence of a "G"
allele at
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position 118920 of AL035668; the presence of a "T" allele at position 167584
of
AL035668; the presence of a "G" allele at position 138476 of AL035668; the
presence of a "T" allele at position 167584 of AL035668; the presence of a "T"
allele at TSC0428253; or the presence of the "G" allele of TSC0293456 is
indicative
of a susceptibility to osteoporosis, compared with an individual having a "T"
at
nucleotide position 122247 of AL035668; an "A" allele at position 118920 of
AL035668; a "C" allele at position 167584 of AL035668; an "A" allele at
position
138476 of AL035668; a "C" allele at position 167584 of AL035668; a "C" allele
at
TSC04~28253; or the presence of the "A" allele of TSC0293456. In a particular
embodiment, the polymorphism is detected in a sample from a source selected
from
the group consisting of: blood, serum, cells and tissue.
In another embodiment, the invention is directed to a method of diagnosing a
susceptibility to osteoporosis in an individual, comprising detecting a
polymorphism
in a hulnan EMI'2 gene of SEQ III N~: 1, wherein the polymorphism is selected
from the group consisting of T to G at position 122247 of AL035668; A to G at
position 118920 of AL035668; C to T at position 167584 of AL035668; A to G at
position 138476 of AL035668; C to T at position 167584 of AL035668; C to T at
TSC0428253; A to G at TSC0293456 and combinations thereof. In a particular
embodiment, the polymorphism is detected in a sample from a source selected
from
the group consisting of: blood, serum, cells and tissue.
In another embodiment, the invention is directed to an isolated nucleic acid
molecule comprising the nucleic acid having SEQ I17 N~:1 with one or more of
the
nucleic acid changes selected from the group consisting of: T to G at position
122247 of AL035668; C to T at position 121366 of AL035668; A to G at position
118920 of AL035668; C to T at position 167584 of AL035668; A to G at position
138476 of AL035668 a_nd combinations thereof.
In another embodiment, the invention is directed to an is~lated polypeptide
comprising an amino acid sequence selected from the group consisting of: an
alanine
at amino acid position 37, a serine at amino acid position 94, a serine at
amino acid
position 189, or combinations thereof.
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In yet another embodiment, the invention is directed to a method of
diagnosing a susceptibility to osteoporosis, comprising detecting a
polypeptide
described herein that is indicative of a susceptibility to osteoporosis. In a
particular
embodiment, the determination of an amino acid at a position selected from the
group consisting of: position 37, position 94, position 1891 and combinations
thereof, comprises contacting the sample with an antibody specific for either
the
reference amino acid or the variant amino acid. In another embodiment, the
invention is directed to a pharmaceutical composition comprising a polypeptide
described herein. In another embodiment, the invention is directed to a method
of
treating osteoporosis in an individual, comprising administering to the
individual,
isolated polypeptide described herein, in a therapeutically effective amo~.mt.
In another embodiment, the invention is directed to a kit comprising: a) at
least one antibody selected from the group consisting of: an antibody specific
for the
BMP2 protein, comprising a serine at amino acid position 37, an alanine at
amino
acid position 37, an alanine at amino acid position 94, a serine at amino acid
position
94~, an arginine at amino acid position 189, a serine at amino acid position
189, or
combinations thereof; and b) a reference BMP2 protein sample.
DETAILED DESCRIPTION OF TFIE 1NVENTION
As described herein, Applicants have completed a genome wide scan on
patients with various forms of osteoporosis and identified a region on
chromosome
20 linked to osteoporosis. Until now there have been no known linkage studies
of
osteoporosis in humans showing any connection to this region of the
chromosome.
Based on the linkage studies conducted, Applicants have discovered a direct
relationship between BI~~2 and osteoporosis. R~lthough the 812 nucleic acid
from normal individuals is knov~n, there have been no studies directly
investigating
the link between BI 1~2 and osteoporosis. Moreover, there have been no variant
forms reported that have been associated with osteoporosis. The linkage
studies are
based on genome wide scans encompassing affected persons having different
osteoporosis phenotypes; i.e., hip, spine, combined and combined severe
(e.g..,
patients having vertebral compression fracture, hip fracture, other
osteoporosis
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related low impact fracture). From the data obtained in the linkage study, a
region
on chromosome 20, specifically the BMP2 gene, was identified. The variant BMP2
nucleic acid has previously unreported nucleotide changes that were observed
in the
patient population (see Tables 2, 3 and 4 for the polymorphic changes).
All nucleotide positions are relative to SEQ ID NO:1 or to GenBank number
AL035668, where indicated. The polymorphism at nucleotide position 3747
appears
statistically more frequent in the osteoporosis test population than in the
control
population.
NUCLEIC ACIDS OF TIIE IN'~ENTION
~llIP2 hluele~tides, 1'~rti~ns aid T~crr~iat~ts
Accordingly, the invention pertains to an isolated nucleic acid molecule
comprising the human BMP2 nucleic acid having at least one nucleotide
alteration
and correlated with incidence of osteoporosis. The term, "variant BMP2", as
used
herein, refers to an isolated nucleic acid molecule in chromosome 20 having at
least
one altered nucleotide. The variants of BMP2 of the present invention are
associated with a susceptibility to a number of osteoporosis phenotypes. The
invention includes a portion or fragment of the isolated nucleic acid molecule
containing the alteration (e.g.., cDNA, the gene, or a fragment thereof),
including
fragments encoding a variant BMP2 polypeptide (e.~., the polypeptide having
SEQ
ID NO:2). In one embodiment, the isolated nucleic acid molecules comprises a
polymorphism selected from the group consisting of any one or combination of
those
shown in Tables 2, 3 and 4 of the BMP2 gene, including comprising at least the
polymorphism at nucleotide 3747. In certain embodiments for therapeutic
purposes
for example, the nucleic acid comprises the sequence of SEA ID NO:1 that
represents the human BMh2 nucleic acid of a healthy individual.
The isolated nucleic acid molecules of the present invention can be FgNA, for
example, mI~NA, or DNA, such as cDNA and genomic DNA. DNA molecules can
be double-stranded or single-stranded; single-stranded I~NA or DNA can be
either
the coding, or "sense" strand or the non-coding, or "antiasnsa" strand. The
nucleic
acid molecule can include all or a portion of the coding sequence of the gene
and can
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further comprise additional non-coding sequences such as introns and non-
coding 3'
and 5' sequences (including regulatory sequences and other flanking sequences,
for
example). Additionally, the nucleic acid molecule can be fused to a marker
sequence, affinity tag, or other gene or 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
hemagglutin
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 I~NA library). For example, an isolated
nucleic
acid of the invention can 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 fomn part of a
composition
(for example, a crude extract containing other substances), buffer system or
reagent
mix. In other circumstances, the material can be purified to essential
homogeneity,
for example as determined by polyacrylamide gel electrophoresis (FAGS) or
column
chromatography such as HPLC. An isolated nucleic acid molecule of the
invention
can comprise at least about 50, ~0 or 90% (on a molax basis) of all
macromolecular
species present. With regard to genomic DNA, the term "isolated" also can
refer to
nucleic acid molecules that are separated from the chromosome with which the
genomic DNA is naturally associated. For example, the isolated nucleic acid
molecule can contain less than about 5 kb, 4~ kb, 3 kb, 2 kb, 1 kb, 0.5 kb or
0.1 kb of
the nucleotides that flank the nucleic acid molecule in the genomic I~1~TA 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 DhTA contained
in a
vector is included in the definition of "isolated" as used herein. Also,
isolated
nucleic acid molecules include recombinant DIVA molecules in heterologous host
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cells or heterologous organisms, as well as partially or substantially
purified DNA
molecules in solution. "Isolated" nucleic acid molecules also encompass in
vivo and
in vitro RNA transcripts of the DNA molecules of the present invention. An
isolated
nucleic acid 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 are included in the
definition of
"isolated" as used herein. Such isolated nucleotide sequences are useful, for
example, in the manufacture of the encoded polypeptide, as probes for
isolating
homologous sequences (e.~., from other mammalian species), for gene mapping
(e.~., by its situ hybridization with chromosomes), or for detecting
expression of the
gene in tissue (e.~.9 human tissue), such as by Northern blot analysis or
other
hybridization techniques.
The present invention also pertains to nucleic acid molecules that are not
necessarily found in nature but, nonetheless, encode a BMP2 polypeptide (e.~.,
a
polypeptide having the amino acid sequence of SEQ ID N~:2 and comprising at
least one polymorphism as shown in Tables 2, 3 and 4). Thus, for example, DNA
molecules that comprise a sequence that is different from the naturally-
occurring
nucleotide sequence but that, due to the degeneracy of the genetic code,
encode an
BMF2 polypeptide of the present invention are also the subject of this
invention.
The invention also encompasses variants of the nucleotide sequences of the
invention, such as those encoding portions, analogues or derivatives of the
BMP2
polypeptide. Such variants can be naturally-occurring, such as in the case of
allelic
variation, 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 nucleotides9 which can result in
conservative or non-conser~rative amino acid changes, in eluding additions and
deletions. The nucleotide (and/or resultant amino acid) changes can be silent
or
conserved9 that is, they do not necessarily have to alter the characteristics
or activity
of the BMP2 polypeptide. In one embodiment, the nucleotide sequences are
fragments that comprise one or more polymorphic microsatellite markers. In
another
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embodiment, the nucleotide sequences are fragments that comprise one or more
single nucleotide polymorphisms in the B1VIP2 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 sequences 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 that hybridize under high stringency
hybridization
and wash conditions (e.g., for selective hybridization) to a nucleotide
sequence
comprising a nucleotide sequence selected from SEQ III N~:1 comprising at
least
one polymorphism as shown in Tables 2, 3 and 4 or the complement thereof, or a
nucleotide sequence encoding an amino acid sequence of SEQ ID N~:2 comprising
at least one polymorphism as shown in Tables 29 3 and 4~. In one embodiment,
the
variant that hybridizes under high stringency hybridizations has an activity
of BI~?.
Such nucleic acid molecules can be detected and/or isolated by allele- or
sequence-specific hybridization (e.g., under high stringency conditions).
"Specific
hybridization,'9 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 complementarity to the second nucleic acid
than to any
other nucleic acid in a sample wherein the hybridization is to be performed).
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"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, that permit hybridization of a particular nucleic acid to a
second
nucleic acid; the first nucleic acid can be perfectly (i.e., 100%)
complementary to the
second, or the first and second can share some degree of complementarity that
is less
than perfect (e.g., 70%, 75%, 85%, 90%, 95%). For example, certain high
stringency conditions can be used to 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
Cu~f°eazt 1'~~toc~ls i~ 1Vl~lecula~ viol~gy (t~usubel, F.Ie~I. et al.,
"Cu~t~ev~t Pr~t~c~ls i~z
ll~l~lecul~zr~ viol~gy", John Wiley ~ Sons, (1990, 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, 6~°C) and
the concentration
of destabilizing agents such as formamide or denaturing agents such as SIBS,
but
also on factors such as the length of the nucleic acid sequence, base
composition,
percent mismatch between hybridizing sequences and the fiequency 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 least about
60%, at
least about 70%, at least about ~0°/~, at least about 90°/~ or
at least about 95°/~ or
more identical to each other remain hybridized to on a another. Ey varying
hybridization conditions from a level of stringency at which no hybridization
occurs
to a level at which hybridization is first obser~red9 conditions that will
allow a given
sequence to hybridize (e.g., selectively) with the most complementary
sequences in
the sample can be determined.
Exemplary conditions are described in I~rause, M.H. and S.A. ~-laronson,
Methods iyz E~zymol~gy, 200:546-556 (1991). Also, in, Ausubel, et al.,
"Cur°rent
F~otocols i~ Molecular Bi~logy", John Wiley ~z Sons, (1990, that describe the
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determination of wash 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 mismatch percentage among the
sequences that hybridize. Generally, doubling the concentration of SSC results
in an
increase in Tm of about 17°C. Using these guidelines, the wash
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 0.2~SSC/0.1% SIBS for 10 min at room temperature; a moderate
stringency wash can comprise washing in a pre-warmed 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 pre-warmed (6~°C) solution containing
O.1XSSC/0.1%SI~S for
15 min at 6~°C. Furthermore, washes can be performed repeatedly or
sequentially to
obtain a desired result as known in the art. Equivalent conditions can be
determined
by varying one or more of the parameters given as an example, as known in the
art,
while maintaining a similar degree of complementarity between the target
nucleic
acid molecule and the primer or probe used (e.g., the sequence to be
hybridized).
The percent identity of two nucleotide or amino acid sequences can be
determined by aligning the sequences for optimal comparison purposes (e.~.,
gaps
can be introduced in the sequence of a first sequence). The nucleotides or
amino
acids at corresponding positions are then compared, and the percent identity
between
2~ the t~vo sequences is a function of the number of identical positions
shared by the
sequences (i.e., °/~ identity = # of identical positions/total # of
positions x 100). In
certain embodiments, the length of a sequence aligned for comparison purposes
is at
least 30%, at least 4~0%, at least 60%, at least 70°/~, at least
SO°/~ or at least 90% of
the length of the reference sequence. The actual comparison of the two
sequences
can be accomplished by well-known methods, for example, using a mathematical
algorithm. A non-limiting example of such a mathematical algorithm is
described in
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Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an
algorithm
is incorporated into the NBLAST and XBLAST programs (version 2.0) as described
in Altschul et al., Nucleic Acids Res., 25:389-3402 (1997). When utilizing
BLAST
and Gapped BLAST programs, the default parameters of the respective programs
(e.g., NBLAST) can be used. See the website on the world wide web at
ncbi.nlm.nih.gov. 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 non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller, CABI~S (1989).
Such an algorithm is incorporated into the ALIG1V 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 FAM120 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 AI~VAIVCE and ADAM as
described in Torellis and Robotti (1994) Coyrr~aut. Aplal. Piosci., 10:3-5;
and FASTA
described in Pearson and Lipman (1988) PNAS, 55:2444-8.
In another embodiment, the percent identity between two amino acid
sequences can be accomplished using the GAP program in the GCG software
package (Accelrys, Cambridge, UK) 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.
The present invention also provides isolated nucleic acid molecules that
contain a fragment or pol-tion that hybridizes under highly stringent
conditions to a
nucleotide sequence comprising a nucleotide sequence selected from SE(~ III
N~:1
and comprising at least one polymorphism as shown 111 Tables 2, 3 and 4~ and
the
complement thereof and also provides isolated nucleic acid molecules that
contain a
fragment or portion that hybridizes under highly stringent conditions to a
nucleotide
sequence encoding an amino acid sequence selected from SEQ II) N~:2, a
polymorphic variant thereof, or a fragment or portion thereof. The nucleic
acid
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fragments of the invention are at least about 15, at least about 18, 20, 23 or
25
nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length.
Longer
fragments, for example, 30 or more nucleotides in length, which encode
antigenic
polypeptides described herein, are particularly useful, such as for the
generation of
antibodies as described below.
Probes aid P~~i~aers
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 hybridise in a base-specific manner to a
complementary
strand of nucleic acid molecules. In addition to I~Nh and I~NA, such probes
and
primers include polypeptide nucleic acids (PNA), as described in Nielsen et
al.,
Science, 254, 1497-1500 (1991).
t~ probe or primer comprises a region of nucleotide sequence that hybridises
to at least about 15, typically about 20-25, and in certain embodiments about
40, 50
or 75, consecutive nucleotides of a nucleic acid molecule comprising a
contiguous
nucleotide sequence selected from: SEQ III N~:1 and comprising at least one
polymorphism as shown in Tables 2, 3 and 4 and the complement thereof, or a
sequence encoding an amino acid sequence selected from SEQ ID N~:2 or
polymorphic variant thereof. In particular embodiments, a probe or primer can
comprise 100 or fewer nucleotides; for example, in certain embodiments from 6
to
50 nucleotides, or for example from 12 to 30 nucleotides. In other
embodiments, the
probe or primer is at least 70°/~ identical to the contiguous
nucleotide sequence or to
the complement of the contiguous nucleotide sequence, for example at least
30°f°
identical in certain embodiments at least ~5°/~ identical in other
embodiments at
least 90% identical, and in other embodiments at least 95°~o identical,
or even
capable of selectively hybridising to the contiguous nucleotide sequence or to
the
complement of the contiguous nucleotide sequence. ~ften, the probe or primer
further comprises a label, e.g., radioisotope, fluorescent compound, er~yme,
or
enzyme co-factor.
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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 in SEQ III NO:l. 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 17~ NO:1 (and optionally comprising at least one polymorphism
as
shoran in Tables 2, 3 and 4) and/or the complement thereof. See generally PCR
Techv~ology: Principles a~cd Applications fog DNA Amplifrcation (ed. H.A.
Erlich,
Freeman Press, NY, NY, 1992); PCR P~~toc~ls: A Guide t~ Methods ayZd
Applications (Eds. Innis, et al., Academic Press, San Diego, CA, 1990);
lVlattila et
al., Nucleic Acids Res., 1~:4~967 (1991); Eckert et al., PCR Nletlz~ds a~cd
Applicati~~cs, 1:17 (1991); PCI~ (eds. ll~IcPherson et al., I1~L Press,
Oxford); and
U.S. Patent 4,683,202. The nucleic acid molecules can be amplified using cDNA,
mI~NA or genomic I~NA as a template, cloned into an appropriate vector and
characterized by l~NA sequence analysis.
Other suitable amplification methods include the ligase chain reaction (LCIZ)
(see Wu and Wallace, Gehomics, 4:560 (1989), Landegren et al., Science,
241:1077
(1988), transcription amplification (Kwoh et al., P~oc. 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 I~NA (ssI~NA)
and
double-stranded IaNA (dsI~I~TA) as the amplification products in a ratio of
about 30
and 100 to 1, respectively.
The amplified I~NA can be labeled9 for e~~ample radiolabeled, and used as a
probe for screening a cT~NA library derived from human cells. The cDI~JA can
be
derived from mI~lVA and contained in zap express (Stratagene, La Jolla, CA),
~IPLO~ (Gibco B1~L, Gaithesburg, l~) or other suitable vector. Corresponding
clones can be isolated, DIVA can obtained following i~c viv~ 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
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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 Cloning, A Laboratofy Manual (2nd Ed., CSHP, New York 1989);
Zyskind et al., Recombinant DNA Laborato~ y Manual, (Aced. Press, 19~g)).
Additionally, fluorescence methods are also available for analyzing nucleic
acids
(Chen, et al., Genome Res. 9, 492 (1999)) and polypeptides. Using these or
similar
methods, the polypeptide and the DNA encoding the polypeptide can be isolated,
sequenced and further characterized.
Antisense nucleic acid molecules of the invention can be designed using the
nucleotide sequences of SECT 1T) N~:1 and comprising at least one polymorphism
as
shown in Tables 2, 3 and 4 and/or the complement thereof, and/or a portion of
SEQ
III N~:1 and comprising at least one polymorphism as shown in Tables 2, 3 and
4 or
the complement thereof, and constructed using chemical synthesis and enzymatic
ligation reactions using procedures kno~m in the art. For example, an
antisense
nucleic acid molecule (e.g., an antisense oligonucleotide) can be chemically
synthesized using naturally occurring nucleotides or variously modified
nucleotides
designed to increase the biological stability of the molecules or to increase
the
physical stability of the duplex formed between the antisense and sense
nucleic
acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides
can be
used. Alternatively, the antisense nucleic acid molecule can be produced
biologically using an expressi~n vector into which a nucleic acid molecule has
been
subcloned in an antisense orientation (i.~.,1NA transcribed from the inserted
nucleic acid molecule will be of an antisense orient~.tion 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 I?NA sequences in patients to identify genetic
disorders
(e.g., a predisposition for or susceptibility to osteoporosis), and as probes,
such as to
hybridize and discover related I~NA sequences or to subtract out known
sequences
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from a sample. The nucleic acid sequences can further be used to derive
primers for
genetic fingerprinting, to raise anti-polypeptide antibodies using
immunization
techniques, and as an antigen to raise anti-DNA antibodies or elicit immune
responses. Fortions 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 identification of a biological sample.
Additionally,
the nucleotide sequences of the invention can be used to identify and express
recombinant polypeptides for analysis, characterization or therapeutic use, or
as
markers for tissues in which the corresponding polypeptide is expressed,
either
constitutively, during tissue differentiation, or in diseased states. The
nucleic acid
sequences can additionally be used as reagents in the screening and/or
diagnostic
assays described herein, and can also be included as components of kits (e.g.,
reagent kits) for use in the screening and/or diagnostic assays described
herein.
hectors
Another aspect of the invention pertains to nucleic acid constructs containing
a nucleic acid molecule selected from the group consisting of SEQ ID N~:1 and
comprising at least one polymorphism as shown in Tables 2, 3 and 4 and the
complement 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 ~EQ ID N~:2 or polymorphic variant thereof. The constructs
comprise
a vector (e.~., an expression vector) into which a sequence of the invention
has been
inserted in a sense or antiasnsa 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. ~ne type of vector is a "plasmid", which refers to a circular
double-stranded DNA loop into which additional DNA segments can be ligated.
Another type of vector is a viral vector, wherein additional DNA segments can
be
ligated into the viral genome. Certain vectors are capable of autonomous
replication
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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, called 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.
Recombinant expression vectors of the invention can comprise a nucleic acid
molecule of the invention in a form suitable for expression of the nucleic
acid
molecule in a host cell. This means that the recombinant expression vectors
include
one or more regulatory sequences, selected on the basis of the host cells to
be used
for 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 that allows for expression of the nucleotide
sequence (e.g., in an i~ vitro transcriptioutranslation system or in a host
cell where
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 Caoeddel, Gene Exp~~,ssi~n Techvc~l~gy: l~rleth~ds ih Ev~zyrh~l~gy
1 ~5,
Academic Press, San Diego9 CA (1~~0). Regulatory sequences include those that
direct constitutive expression of a nucleotide sequence in many types of host
cell and
those that 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 transformed and the level of expression of
polypeptide
desired. The expression vectors of the invention can be introduced into host
cells to
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thereby produce polypeptides, including fusion polypeptides, encoded by
nucleic
acid molecules as described herein.
The recombinant expression vectors of the invention can be designed for
expression of a polypeptide of the invention in prokaryotic or eukaryotic
cells, e.g.,
bacterial cells such as E. coli, insect cells (e.g., using baculovirus
expression
vectors), yeast cells or mammalian cells. Suitable host cells are discussed
further in
Goeddel, supf°a. Alternatively, the recombinant expression vector can
be transcribed
and translated in vitro, for example using T7 promoter regulatory sequences
and T7
polymerise.
Another aspect of the invention pertains to host cells into which a
recombinant expression vector of the invention has been introduced. The terms
"host cell" and "recombinant host cell" are used interchangeably herein. It is
understood that such terms refer not only to the particular subject cell but
also to the
progeny or potential progeny of such a cell. because certain modifications can
occur
in succeeding generations due to either mutation or environmental influences,
such
progeny might 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
(CH~)
or C~S cells). ~ther suitable host cells are known to those skilled in the
art.
Vector I?IVA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
'"transf~rmati~n" and ~'transfection99 are intended to refer to a variety of
aurt-recognised techniques for introducing a foreign nucleic acid molecule
(~.g.,
I~1~TA) into a host cell, including calcium phosphate or calcium chloride
co-precipitation, L~EAE-dextrin mediated trinsfection, lipofection, or
electroporition. Suitable methods for transforming or transfecting host cells
can be
found in Sambrook, et al. (supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the expression vector and transfection technique used, only a small fraction
of cells
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can 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.
Selectable markers can include those that confer resistance to drugs such as
G41 S,
hygromycin and methotrexate. Nucleic acid molecules encoding a selectable
marker
can be introduced into a host cell on the same vector as the nucleic acid
molecule of
the invention or can be introduced on a separate vector. 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
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 BMf2 gene, or an
exogenous
nucleic acid encoding BI~IP2 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, e.g., a
mammal, e.g., a rodent such as a rat or mouse, in which one or more of the
cells of
the animal includes a transgene. ~ther examples of transgenic animals include
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non-human primates, sheep, dogs, cows, pigs, goats, chickens and amphibians. A
transgene is exogenous DNA that is integrated into the genome of a cell from
wluch
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, e.8., a mammal, e.8., 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.8., 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,~66 and
4,870,009,
U.S. Patent No. 49873,191 and in Hogan, ~Vfaszipulati~g the ll~f~use Errabr~r~
(Cold
Spring Harbor Laboratory Press, Cold spring Harbor, N.~'., 1986). Methods for
constuucting homologous recombination vectors and homologous recombinant
animals are described further in Bradley (1991) Curr~e~t ~pii~io~c afz
~iolTeeha~~logy,
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)
lVatuf°e, 385:810-813 and PCT Publication Nos. WO 97/07668 and WO
97/07669.
POL~PEPTIDES OF THE 1NVENTION
The present invention also pertains to isolated BMP2 polypeptides, e.8.,
proteins, and variants thereof as well as polypeptides encoded by nucleotide
sequences described herein (e.8., other splicing variants). The term
"polypeptide"
refers to a polymer of amino acids9 and not to a specific length9 thus,
peptides,
oligopeptides and proteins are included within the definition of a
polypeptide. As
used herein, a polypeptide is said to be 'nsolated" 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
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polypeptide with which it is not normally associated in a cell 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
"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 comprises an amino acid sequence
encoded by a nucleic acid molecule comprising a nucleotide sequence selected
from
the group consisting of SEA Ih hJ~:l and comprising at least on a polymorphism
as
shown in Tables 2, 3 and 4~ and complements and portions thereof. However, the
invention also encompasses sequence variants. variants include a substantially
homol~gous polypeptide encoded at 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
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nucleotide sequence selected from the group consisting of SEQ ID NO:1 and
comprising at least one polymorphism as shown in Tables 2, 3 and 4 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 III N0:2 or
polymorphic variants thereof. Variants also include polypeptides substantially
homologous or identical to these polypeptides but derived from another
organism,
i.e., an oi-tholog. 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%, in other
embodiments
at least about ~0-~5%, and in other embodiments greater than about 90% or more
homologous or identical. A substantially homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic acid molecule
hybridizing to SEQ II? N0:1 and comprising at least one polymorphism as shown
in
Tables 2, 3 and 4, or portion thereof, under stringent conditions as more
particularly
described above or will be encoded by a nucleic acid molecule hybridizing to a
nucleic acid sequence encoding SEQ III NO:2 portion thereof or polymorphic
variant thereof, under stringent conditions as more particularly described
thereof.
The invention also encompasses polypeptides having a lower degree of
identity but having sufficient similarity so as to perform one or more of the
same
functions performed by a polypeptide en coded by a nucleic acid bnolecule of
the
invention. Similarity is determined by conserved amino acid substitution or by
structural similarity. Such conservative substitutions are those that
substitute a given
amino acid in a polypeptide by another amino acid of life characteristics.
Conservative substitutions are likely to be phenotypically silent. Typically
seen as
conservative substitutions are the replacements, one for another, among the
aliphatic
amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser
and Thr,
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exchange of the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg and
replacements
among the aromatic residues Phe and Tyr. Guidance concerning which amino acid
changes are likely to be phenotypically silent are found in Bowie et al.,
Science
247:1306-1310 (1990).
A variant polypeptide can differ in amino acid sequence by one or more
substitutions, deletions, insertions, inversions, fusions, and truncations or
a
combination of any of these. Further, variant polypeptides can be fully
functional or
can lack function in one or more activities. Fully functional variants
typically
contain only conservative variation or variation in non-critical residues or
in
non-critical regions. Functional vaaiants can also contain substitution of
similar
amino acids that result in no change or an insignificant change in function.
Alternatively, such substitutions can positively or negatively affect function
to some
degree. IVon-functional variants typically contain one or more non-
conservative
amino acid substitutions, deletions, insertions, inversions, or truncation or
a
substitution, insertion, inversion, or deletion in a critical residue or
critical region.
Amino acids that are essential for function can be identified by methods
known in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis
(Cunningham et al., Scievcce, 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 irZ vita~. Sites that
are
critical for polypeptide activity can also be determined by structural
analysis, for
example, by crystallisation, nuclear magnetic resonance or photoaffmity
labeling
(smith ~t al., J ll~f~l. ~i~l., 224:899-904 (1992); de 5~os et al. Scievrce,
255:306-312
(1992)).
The invention also in eludes polypeptide fragments of the polypeptides of the
lllventloll. Fragments can be derived from a polypeptide encoded by a nucleic
acid
molecule comprising SEQ l~ I~T~:l and comprising at least one polymorphism as
shown in Tables 2, 3 and 4~ or a portion thereof and the complements thereof.
However, the invention also encompasses fragments of the variants of the
polypeptides described herein. As used herein, a fragment comprises at least 6
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contiguous amino acids. Useful fragments include those that retain one or more
of
the biological activities of the polypeptide as well as fragments that can be
used as
an immunogen to generate polypeptide-specific antibodies.
Biologically active fragments (peptides which are, for example, 6, 9, 12, 15,
16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) can
comprise a domain, segment, or motif that has been identified by analysis of
the
polypeptide sequence using well-known methods, e.g., signal peptides,
extracellular
domains, one or more transmembrane segments or loops, ligand binding regions,
zinc finger domains, I~NA binding domains, acylation sites, glycosylation
sites, or
phosphorylation sites.
Fragments can be discrete (not fused to other amino acids or polypeptides) or
can be within a larger polypeptide. Further, several fragments can be
comprised
within a single larger polypeptide. In one embodiment a fragment designed for
expression in a host can have heterologous pre- and pro-polypeptide regions
fused to
the amino terminus of the polypeptide fragment and an additional region fused
to the
carboxyl terminus of the fragment.
The invention thus provides chimeric or fusion polypeptides. These
comprise 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 pey° se. For
example, the
fusion polypeptide can be a SST-fusion polypeptide in which the polypeptide
sequences are fused to the ~-terminus of the ~~T sequences. ~ther types of
fusion
polypeptides include, but are not limited to, en ~ymatic fission polypeptides,
for
example (~-galactosidase fusions, yeast two-hybrid C~I~L fusions, poly-FIis
fusions
and Ig fusions. Such fusion polypeptides, particularly poly-Ibis fusions, can
facilitate the purification of the recombinant polypeptide. In certain host
cells (e.g.,
mammalian host cells), expression and/or secretion of a polypeptide can be
increased by using a heterologous signal sequence. Therefore, in another
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embodiment, the fusion polypeptide contains a heterologous signal sequence at
its
N-terminus.
EP-A-O 464 533 discloses fusion proteins comprising various portions of
immunoglobulin constant regions. The Fc is useful in therapy and diagnosis and
thus results, for example, in improved pharmacokinetic properties (EP-A 0232
262).
In drug discovery, for example, human proteins have been fused with Fc
portions for
the purpose of high-throughput screening assays to identify antagonists.
Bennett et
al., J. Mol. Rec., x:52-SS (1995) and Johanson et al., J. Bi~l. Clzenz.,
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
subclass
(IgG, IgIe~I, IgA, IgE).
A chimeric or fission polypeptide can be produced by standard recombinant
DNA techniques. For example, DNA fragments coding for the different
polypeptide
sequences are ligated together in-frame in accordance with conventional
techniques.
In another embodiment, the fusion gene can be synthesized by conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of nucleic acid fragments can be carried out using anchor
primers that
give rise to complementary overhangs between two consecutive nucleic acid
fragments that can be subsequently annealed and re-amplified to generate a
chimeric
nucleic acid sequence (see Ausubel et al., Cuf°rent Pr'~t~c~ls ih
Molecula~° Biology,
1992). Moreover, many expression vectors are commercially available that
already
encode a fusion moiety (e.g., a CaST protein). A nucleic acid molecule
encoding a
polypeptide of the invention can be clop ed 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 techuques. 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
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polypeptide can then be isolated from the cells by an appropriate purification
scheme
using standard protein purification techniques.
In 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
constitutively9 during tissue differentiation, or in a diseased state. The
polypeptides
can be used to isolate a corresponding binding partner, e.g., receptor or
ligand, such
as, for example, in an interaction trap assay, and to screen for peptide or
small
molecule antagonists or agonists of the binding interaction.
ANTIB~DIES ~F THE 1NVENTI~N
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
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 N~:2 and comprising at least
one polymorphism as shown in Tables 2, 3 and 4~, or a portion thereof, or
having an
amino acid sequence encoded by a nucleic acid molecule comprising all or a poz-
tion
of SEQ TD N~:1 and comprising at least one polymorphism as shown in Tables 2,
3
and 4~. The tm-m "antibody9' as used herein refers to immw~oglobulin 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 that naturally contains the polypeptide.
Examples
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of immunologically active portions of immunoglobulin molecules include Flab)
and
F(ab')2 fragments that can be generated by treating the antibody with an
enzyme such
as pepsin. The invention provides polyclonal and monoclonal antibodies that
bind to
a polypeptide of the invention. The term "monoclonal antibody" or "monoclonal
antibody composition", as used herein, refers to a population of antibody
molecules
that contain only one species of an antigen binding site capable of
immunoreacting
with a particular epitope of a polypeptide of the invention. A monoclonal
antibody
composition thus typically displays a single binding affinity for a particular
polypeptide of the invention with which it immunoreacts.
folyclonal antibodies can be prepared as described above by immunizing a
suitable subject with a desired immunogen, e.~., 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 an immobilized polypeptide. If desired, the antibody
molecules
directed against the polypeptide can be isolated from the mammal (e.~., 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 fiom the subject and used to prepare monoclonal antibodies by
standard techniques, such as the hybridoma technique originally described by
Kohler
and Milstein (1975) Nature, 256:495-497, the human B cell hybridoma technique
(I~ozbor et al. (193) Immu~ol. T~day, 4:72), the EEC-hybridoma technique (Cole
~t al. (195), Ill~az~cloyzal Afatzb~dies arid C'aazce~ Therapy, Alan 1Z. hiss,
Inc., pp.
77-96) or trioma techniques. The technology for producing hybridomas is well
knov~n (see generally C'a~a°b°ev~t Pr~t~~~l,~ ire Ia~aaaaua~~h~y
(1994) Coligan et al. (eds.)
John iTliley ~c Sons, Inc., i~le~rr Fork, N~. briefly, an immortal cell
(typically a
myeloma) is fused to a lymphocyte (typically a splenocyte) from a mammal
immunized with an immunogen as described above, and the culture supernatants
of
the resulting hybridoma cells are screened to identify a hybridoma producing a
monoclonal antibody that binds a polypeptide of the invention.
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Any of the many well known protocols used for fusing lymphocytes and
immortalized cells can be applied for the purpose of generating a monoclonal
antibody to a polypeptide of the invention (see, e.g., Current Protocols in
Immunology, supra; Galfre et al. (1977) Natuy°e, 266:55052; R.H.
Kenneth, in
Movcoclonal A~rtibodies: A New I~in2ensi~n Ih Biological Analyses, Plemun
Publishing Corp., New York, New York (1980); and Lerner (1981) Yale J. Biol.
Med., 54:387-402). Moreover, the ordinarily skilled worker will appreciate
that
there are many variations of such methods that also would be useful.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal antibody to a polypeptide of the invention can be identified and
isolated
by screening a recombinant combinatorial immunoglobulin library (e.g., an
antibody
phage display library) with the polypeptide to thereby isolate immunoglobulin
library members that bind the polypeptide. Kits for generating and screening
phage
display libraries are commercially available (e.g., the Pharmacia
Recombiazafat Phage
AyZtabody System, Catalog No. 27-9400-O1; and the Stratagene Smf~APTM Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods and
reagents
particularly amenable for use in generating and screening antibody display
library
can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No.
WO
92/18619; PCT Publication No. W~ 91/17271; PCT Publication No. W~ 92/20791;
PCT Publication No. W~ 92/15679; PCT Publication No. W~ 93/01288; PCT
Publication No. W~ 92/01047; PCT Publication No. W~ 92/09690; PCT
Publication No. W~ 90/02809; Fuchs et al. (1991) Bi~lTechva~l~gy, 9:1370-1372;
Hay et al. (1992) ~Ium. Aa~tib~d I~yb~idcmas, 3:81-85; Huse et al. (1989)
S'cievcce,
26:1275-1281; Griffiths ct al. (1993) B'MB~.I, 12:725-734.
Additionally, recombinant antibodies9 such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made using standard recombinant DNA techniques, are within the scope of the
invention. Such chimeric and humanized monoclonal antibodies can be produced
by
recombinant DNA techniques k~zown 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
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chromatography or immunoprecipitation. A polypeptide-specific antibody can
facilitate the purification of natural polypeptides from cells and of
recombinantly
produced polypeptides 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. Detection can be
facilitated by coupling the antibody to a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish peroxidase,
alkaline
phosphatase, (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'ZSh 13'I, 3sS, 3zp~ 33P~ i4C or 3H.
DIAGNOSTIC AND SCREENING ASSAYS OF THE INVENTION
The present invention also pertains to a method of diagnosing or aiding in the
diagnosis of osteoporosis or a susceptibility to osteoporosis associated with
the
presence of the Eh lv/T'2 nucleic acid or gene product in an individual.
Diagnostic
assays can be designed for assessing EMP2 gene expression, or for assessizig
activity
of EMP2 polypeptides of the invention. Such assays can be used alone or in
combination with other assays, ~.g., bone turnover marker assays (e.g., bone
scans).
In one embodiment, the assays are used in the context of a biological sample
(e.g.,
blood, serum, cells, tissue, synovial fluid) to thereby determine whether an
individual is afflicted with osteoporosis, or is at risk for (has a
predisposition for or a
susceptibility to) developing osteoporosis. The invention also provides for
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prognostic (or predictive) assays for determining whether an individual is
susceptible to developing osteoporosis. For example, alterations in the gene
can be
assayed in a biological sample. Such assays can be used for prognostic or
predictive
purpose to thereby prophylactically treat an individual prior to the onset of
symptoms associated with osteoporosis. Another aspect of the invention
pertains to
assays for monitoring the influence of agents (e.g., cliwgs, compounds or
other
agents) on the gene expression or activity of polypeptides of the invention,
as well as
to assays for identifying agents that bind to BMP2 polypeptides. These and
other
assays and agents are described in further detail in the following sections.
I~IAGIV~STIC ASSAYS
The nucleic acids, polypeptides and antibodies described herein can be used
in methods of diagnosis of osteoporosis or a susceptibility to osteoporosis,
as well as
in kits useful for diagnosis of osteoporosis or a susceptibility to
osteoporosis.
In one embodiment of the invention, diagnosis of a susceptibility to
osteoporosis is made by detecting a polymorphism in BMP2 as described herein.
The polymorphism can be a change in BMP2, such as the insertion or deletion of
a
single nucleotide, or of more than one nucleotide, resulting in a frame shift;
the
change of at least one nucleotide, resulting in a change in the encoded amino
acid;
the change of at least one nucleotide, resulting in the generation of a
premature stop
codon; the deletion of several nucleotides, resulting in a deletion of one or
more
amino acids encoded by the nucleotides; the insertion of one or several
nucleotides,
such as by unequal recombination or gene conversion, resulting in an
interruption of
the coding sequence of the gene; duplication of all or a part of the gene;
transposition of all or ~ part of the gene; ox rearrangement of all or a part
of the gene.
More than one such change can be present in a single gene. such sequence
changes
alter the polypeptide encoded by a BMP2 nucleic acid. For example, if the
change in
the nucleic acid sequence causes a frame shift, the frame shift can result in
a change
in the encoded amino acids, and/or can result in the generation of a premature
stop
codon, causing generation of a truncated polypeptide. Alternatively, a
polymorphism associated with a susceptibility to osteoporosis can be a
synonymous
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change in one or more nucleotides (i.e., a change that does not result in a
change in
the polypeptide encoded by an BMP2 gene). Such a polymorphism can, for
example, alter splicing sites, affect the stability or transport of mRNA, or
otherwise
affect the transcription or translation of the gene. A BMP2 nucleic that has
any of
the alterations described above is referred to herein as a "variant nucleic
acid" or,
sometimes, "mutant gene."
In a first method of diagnosing a susceptibility to osteoporosis,
hybridization
methods, such as Southern analysis, Northern analysis, or in situ
hybridizations, can
be used (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds.,
John
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, osteoporosis (the "test individual"). The
individual can
be an adult, child, or fetus. The test sample can be from any source that
contains
genomic DNA, such as a blood sample, sample of amniotic fluid, sample of
cerebrospinal fluid, sample of synovial fluid, or tissue sample fiom 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 BMP2 is
present.
The presence of the polymorphism can be indicated by hybridization of the gene
in
the genomic DNA, RNA, or cDNA to a nucleic acid probe. A "nucleic acid probe",
as used herein, can be a DNA probe or an RNA probe9 the nucleic acid probe
contains at least one polymorphism in B1~~IP2. The probe can be any of the
nucleic
acid molecules described above (~.~., the gene or nucleic acid9 a fragment, a
vector
comprising the gene, etca).
To diagnose a susceptibility to osteoporosis, a hybridization sample is
formed by contacting the test sample containing BMP2, with at least one
nucleic
acid probe. A non-limiting example of a 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
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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
and optionally comprising at least one polymorphism as shown in Tables 2, 3
and 4,
or the complement or a portion thereof. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
The hybridization sample is maintained order conditions which are sufficient
to allow specific hybridization of the nucleic acid probe to BMP2. "Specific
hybridization", as used herein, indicates exact hybridization (e.~., with no
mismatches). Specific hybridization can be performed under high stringency
conditions or moderate stringency conditions, for example, as described above.
In
one 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 BMP2 in the
test
sample, then BMP2 has the polymorphism that is present in the nucleic acid
probe.
More than one nucleic acid probe can also be used concurrently in this method.
Specific hybridization of any one of the nucleic acid probes is indicative of
a
polymorphism in BMP2, and is therefore diagnostic for a susceptibility to
osteoporosis.
In another hybridization method, Northern analysis (see Current Protocols in
Molecular Biology, Ausubel, F. ~t czl., eds., John Whey ~; Sons, sai~a~cz) is
used to
identify the presence of a polymorphism associated with a susceptibility to
osteoporosis. For Northern analysis, ~ test sample of IOTA 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
111
BMP2, and is therefore diagnostic for a susceptibility to osteoporosis.
For representative examples of use of nucleic acid probes, see, for example,
ZJ.S. Patents No. 5,288,611 and 4,851,330.
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Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a
nucleic acid probe in the hybridization methods described above. PNA is a DNA
mimic having a peptide-like, inorganic backbone, such as N-(2-
aminoethyl)glycine
units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen
via a
methylene carbonyl linker (see, for example, Nielsen, P.E. et al.,
Biocohjugate
Chenaist~y, 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 susceptibility to osteoporosis. Hybridization of the PNA probe to BMP2
is
diagnostic for a susceptibility to osteoporosis.
In one embodiment of the invention, diagnosis of osteoporosis or a
susceptibility to osteoporosis associated with BMP2, can be made by expression
analysis using quantitative PCPv (kinetic thermal cycling). In one embodiment,
the
diagnosis of osteoporosis is made by detecting at least one BMP2-associated
allele
and in combination with a bone turnover marker assay (e.g., bone scans). This
technique can, for example, utilize commercially available technologies such
as
TaqMan~ (Applied Biosystems, Foster City, CA), to allow the identification of,
for
example, polymorphisms. The technique can assess the presence of an alteration
in
the expression or composition of the polypeptide encoded by BMP2 or splicing
variants. Further, the expression of the variants can be quantified as
physically or
functionally different.
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
restriction sites A test sample containing genomic DNA is obtained from the
individual. Polymerase chain reaction (PCI~) can be used to amplify B1~~P2
(ands if
necessary, the flanking sequences) in the test sample of genomic DNA from the
test
individual. I~FLP analysis is conducted as described (see C~.uTent Protocols
in
Molecular Biology, supf~a). The digestion pattern of the relevant DNA fragment
indicates the presence or absence of the mutation or polymorphism in BMP2, and
therefore indicates the presence or absence of this susceptibility to
osteoporosis.
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Sequence analysis can also be used to detect specific polymorphisms in
BMP2. 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 BMP2, or a fragment of the gene, or
cDNA,
or fragment of the cDNA, or mRNA, 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 and comprising at least one
polymorphism as shown in Tables 2, 3 and 4) or mRNA, as appropriate. The
presence of a specific polymorphism in BMP2 indicates that the individual has
a
susceptibility to osteoporosis.
Allele-specific oligonucleotides can also be used to detect the presence of a
polymorphism in BMP2, through the use of dot-blot hybridization of amplified
oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for
example, Saiki, R. et al., (1956), Nature (Lord~n) 34:163-166). An "allele-
specific
oligonucleotide" (also referred to herein as an "allele-specific
oligonucleotide
probe") is an oligonucleotide of approximately 10-50 base pairs or
approximately
15-30 base pairs, that specifically hybridizes to BMP2, and that contains a
polymorphism associated with a susceptibility to osteoporosis. An allele-
specific
oligonucleotide probe that is specific for particular polymorphisms in BMP2
can be
prepared, using standard methods (see Current Protocols in Molecular Biology,
supra). To identify polymorphisms in the gene that are associated with a
susceptibility to osteoporosis, a test sample of DNA is obtained from the
individual.
PCR can be used to amplify all or a fr~.gnaent of BMP2, amd its flanking
sequences.
The DNI~ containing the amplified Bl~~~ (or fragment of the gene) is dot-
blotted,
using standard methods (see Current Protocols in Molecular Biology,
su~aa°a), and the
blot is contacted with the oligonucleotide probe. The presence of specific
hybridization of the probe to the amplified BMP2 is then detected. Specific
hybridization of an allele-specific oligonucleotide probe to DNA from the
individual
is indicative of a specific polymorphism in BMP2, and is therefore indicative
of a
susceptibility to osteoporosis.
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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 (Gibbs, R et al., 199. Nucleic Acids Res.,
17:2437-244g). This primer is used in conjunction with a second primer, which
hybridizes at a distal site. Amplification proceeds from the two primers,
resulting in
a detectable product, which indicates the particular allelic form is present.
A control
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 fornled. 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.~., W~ 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 ~ 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 ~-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 oposed 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.~., the 3' end,
the
5'end, or in the middle), the Tm could be increased considerably.
In another embodiment, arrays of oligonucleotide probes that are
complementary to target nucleic acid sequence segments from an individual, can
be
used to identify polymorphisms in a ~1~2 nucleic acid. For example, in one
embodiment, an oligonucleotide array can be used. ~ligonucleotide arrays
typically
comprise a plurality of different oligonucleotide probes that are coupled to a
surface
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of a substrate in different known locations. These oligonucleotide arrays,
also
described as "GenechipsTM," have been generally described in the art, for
example,
U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and
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
(Fodor, S. et al., 1991. Seie~rce, 251:767-773; Pirrung et al., U.S. Pat. No.
5,143,854
(see also PCT Application No. WO 90/15070); and Fodor. S. et al., PCT
Publication
No. WO 92/10092 and U.S. Pat. No. 5,424,186, the entire teachings of each of
which are incorporated by reference herein). Techniques for the synthesis of
these
arrays using mechanical synthesis methods are described in, e.g., U.S. Pat.
No.
5,384,261; the entire teachings of which are incorporated by reference herein.
In
another example, linear arrays can be utilized.
Once an oligonucleotide array is prepared, a nucleic acid of interest is
hybridized with the array and scanned for polymorphisms. Ilybridi~ation and
scanning are generally carried out by methods described herein and also in,
e.g.,
published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat.
No. 5,424,186, the entire teachings of which are incorporated by reference
herein. In
brief, a target nucleic acid sequence, which 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
downstre~~n, from the polymorphism. Asymmetric PCI~ techniques can also be
used. Amplified target, generally in corporating 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.
Although primarily described in terms of a single detection block, e.g., for
detection of a single polymorphism, arrays can include multiple detection
blocks,
and thus be capable of analyzing multiple, specific polymorphisms. In
alternate
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arrangements, it will generally be understood that detection blocks can be
grouped
within a single array or in multiple, separate arrays so that varying, optimal
conditions can be used during the hybridization of the target to the array.
For
example, it will 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 descriptions of use of oligonucleotide arrays for detection of
polymorphisms can be found, for example, in LT.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 EMP2. representative methods include, for example, direct manual sequencing
(Church and Gilbert, (1988), Pr~c. Natl. Acad. ~'ci. LIS'A 81:1991-1995;
Sanger, F. et
al. (1977) P~~c. Natl. Acad. Sci. 74:5463-5467; Beavis et al. LT.S. Pat. No.
5,288,644); automated fluorescent sequencing; single-stranded conformation
polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE);
denaturing gradient gel electrophoresis (DGGE) (Sheffield, V.C. et al. (19891)
P~~c.
Natl. Acad. Sci. ZI8'A 86:232-236), mobility shift analysis (~rita, M. et al.
(1989)
Proc. Natl. Acad. Sci. USA 86:2766-2770), restriction enzyme analysis (Flavell
et al.
(1978) Cell 15:25; Geever, et al. (1981) F~oc. Natl. Acad. Sci. LISA 78:5081);
heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al. (1985)
Proc. Natl. Aeaa'. ~'~i. LISA 85:4397-4401); rNase protection assays (Myers9
1.M. et
al. (1985) S~ie~cce 230:1242); use of polypeptides that recognize nucleotide
mismatch es9 such as E. c°~li mutS protein; and allele-specific PCr.
In one embodiment of the invention, diagnosis of a disease or condition
associated with a EMP2 nucleic acid (~.g.9 osteoporosis) or a susceptibility
to a
disease or condition associated with a Eh/~1P2 nucleic acid (~.~.,
osteoporosis) can
also be made by expression analysis by quantitative PCr (kinetic themnal
cycling).
In one embodiment, the diagnosis of the disease itself, e.~., osteoporosis9 is
made by
detecting at least one EMP2-associated allele and in combination with a bone
turnover marker assay (e.~., bone scans). This technique, utilizing TaqMan~,
can be
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used to allow the identification of polymorphisms and whether a patient is
homozygous or heterozygous. The technique can assess the presence of an
alteration
in the expression or composition of the polypeptide encoded by a BMP2 nucleic
acid
or splicing variants encoded by a BMP2 nucleic acid. Further, the expression
of the
variants can be quantified as physically or functionally different.
In another embodiment of the invention, diagnosis of a susceptibility to
osteoporosis can also be made by examining expression and/or composition of an
BMP2 polypeptide, by a vaxiety of methods, including enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. A test sample from an individual is assessed for the
presence
of an alteration in the expression and/or an alteration in composition of the
polypeptide encoded by BMP2. An alteration in expression of a polypeptide
encoded by BMP2 can be, for example, an alteration in the quantitative
polypeptide
expression (i.e., the amount of polypeptide produced); an alteration in the
composition of a polypeptide encoded by BMP2 is an alteration in the
qualitative
polypeptide expression (e.g., expression of a mutant BMP2 polypeptide or of a
different splicing variant). In one embodiment, diagnosis of a susceptibility
to
osteoporosis is made by detecting a particular splicing variant encoded by
BMP2, or
a particular pattern of splicing variants.
Both such alterations (quantitative and qualitative) can also be present. An
"alteration" in the polypeptide expression or composition, as used herein,
refers to
an alteration in expression or composition in a test sample, as compared with
the
expression or composition of polypeptide by BMP2 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 v~ho is not affected by osteoporosis.
Similarly, the
presence of one or more different splicing variants in the test sample, or the
presence
of significantly different amounts of different splicing variants in the test
sample, as
compared with the control sample, is indicative of a susceptibility to
osteoporosis.
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
osteoporosis.
Various means of examining expression or composition of the polypeptide
encoded
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by BMP2 can be used, including spectroscopy, colorimetry, electrophoresis,
isoelectric focusing, and immunoassays (e.g., David et al., U.S. Pat. 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), e.g., an antibody with a detectable
label, can
be used. Antibodies can be polyclonal or monoclonal. An intact antibody, or a
fragment thereof (e.g., Fab or F(ab')2) 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 blot analysis, using an antibody as described above that specifically
binds to a polypeptide encoded by a mutant BMP2, or an antibody that
specifically
binds to a polypeptide encoded by a non-mutant gene, can be used to identify
the
presence in a test sample of a polypeptide encoded by a polymorphic or mutant
BMP2, or the absence in a test sample 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
osteoporosis.
In one embodiment of this method, the level or amount of polypeptide
encoded by Bh!!P2 in a test sample is coanpared with the level or amount of
the
polypeptide encoded by BMP2 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 sample9 such that the difference is statistically
significant,
is indicative of an alteration in the expression of the polypeptide encoded by
BMP2,
and is diagnostic for a susceptibility to osteoporosis. Alternatively, the
composition
of the polypeptide encoded by BMP2 in a test sample is compared with the
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composition of the polypeptide encoded by BMP2 in a control sample. A
difference
in the composition of the polypeptide in the test sample, as compared with the
composition of the polypeptide in the control sample, is diagnostic for a
susceptibility to osteoporosis. 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
osteoporosis.
Fits useful in the methods of diagnosis comprise components useful in any
of the methods described herein, including for example, hybridization probes,
restriction enzymes (e.g., for 1~FLP analysis), allele-specific
oligonucleotides,
antibodies which bind to altered or to non-altered (native) B1VIP2 polypeptide
(e.g.,
to SEQ III IV~:2 and comprising at least one polymorphism as shown in Tables
2, 3
and 4), means for amplification of nucleic acids comprising BIi~2, or means
for
analyzing the nucleic acid sequence of BMP2 or for analyzing the amino acid
sequence of an BMP2 polypeptide, etc. Additionally, kits can provide reagents
for
assays to be used in combination with the methods of the present invention,
e.g.,
bone turnover marker assays (e.g., bone scans).
Fits (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 moleculess restriction enzymes (e.g., for
I~LP
analysis), allele-specific oligonucleotides9 antibodies that bind to altered
or to
non-altered (native) Bh/1P2 polypeptide, means for amplification of nucleic
acids
comprising a BI~fPI 2, or means for analy~;ing the nucleic acid sequence of a
BI~~/IP2
nucleic acid or for analyzing the amino acid sequence of a BI~IP2 polypeptide
as
described herein, etc. In one embodiment, the kit for diagnosing osteoporosis
or a
susceptibility to osteoporosis can comprise primers for nucleic acid
amplification of
a region in the BMP2 nucleic acid comprising one or more polymorphic sites
that
are more fiequently present in an individual having osteoporosis or is
susceptible to
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osteoporosis. The primers can be designed using portions of the nucleic acids
flanking SNPs that are indicative of osteoporosis. Additionally, kits can
provide
reagents for assays to be used in combination with the methods of the present
invention, e.g., bone turnover marker assays (e.g., bone scans).
The invention further pertains to a method for the diagnosis and
identification of susceptibility to osteoporosis in an individual, by
identifying an
at-risk marker, e.g., an SNP, in BMP2. In one embodiment, the at-risk marker
is one
that confers a significant risk of osteoporosis. In one embodiment,
significance
associated with a marker 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 2.2, including by not limited to:
1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.g, 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%, g0%, g5%, 90%, 95% and 9~%. In a fiu-ther
embodiment, a significant increase in risk is at least about 50%. It is
understood
however, that identifying whether a risk is medically significant can also
depend on
a variety of factors, including the specific disease, the marker, and often,
environmental factors.
The invention also pertains to methods of diagnosing osteoporosis or a
susceptibility to osteoporosis in an individual, comprising screening for an
at-risk
marker associated with the BI~P2 nucleic acid that is more frequently present
in an
individual susceptible to osteoporosis (affected)9 compared to the frequency
of its
presence in a healthy indi~ridual (control)9 wherein the presence of the
marker is
indicative of osteoporosis or susceptibility to osteoporosis. Standard
techniques for
genotyping for the presence of GNPs and/or microsatellite markers that are
associated with osteoporosis can be used, such as fluorescent based techniques
(Chen, et a1.9 1999. Ge~orne Res., 9:492), PCR, LCR, Nested PCR and other,
techniques for nucleic acid amplification. In one embodiment, the method
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comprises assessing in an individual the presence or frequency of a specific
SNP
allele or microsatellite allele associated with the BMP2 nucleic acid that are
associated with osteoporosis, wherein an excess or higher frequency of the
marker
compared to a healthy control individual is indicative that the individual has
osteoporosis or is susceptible to osteoporosis. See Tables 2, 3 and 4 for SNPs
and
markers that can be used as screening tools.
SCREENING ASSAYS AND AGENTS IDENTIFIED THEREBY
The invention provides methods (also referred to herein as "screening
assays") for identifying the presence of a nucleotide that hybridizes to a
nucleic acid
of the invention, as well as for identifying the presence of a polypeptide
encoded by
a nucleic acid of the invention. In one embodiment, the presence (or absence)
of a
nucleic acid molecule of interest (e.g., a nucleic acid that has significant
homology
with a nucleic acid of the invention) in a sample can be assessed by
contacting the
sample with a nucleic acid comprising a nucleic acid of the invention (e.g..,
a nucleic
acid having the sequence of SEQ ID N~:1 and comprising at least one
polymorphism as shown in Tables 2, 3 and 4, or the complement thereof, or a
nucleic acid encoding an amino acid having the sequence of SEQ ID N0:2 or a
fragment or variant of such nucleic acids), under stringent conditions as
described
above, and then assessing the sample for the presence (or absence) of
hybridization.
In one embodiment, high stringency conditions are conditions appropriate for
selective hybridization. In another embodiment, a sample containing the
nucleic
acid molecule of interest is contacted with a nucleic acid containing a
contiguous
nucleotide sequence (e.~., a primer or a probe as described above) that is at
least
partially complementary to a part of the nucleic ~.cid molecule of interest
(~.~., a
variant Bl~iP2 nucleic acid), and the contacted sample is assessed for the
presence or
absence of hybridization. In one 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
can be subjected to amplification prior to performing the hybridization.
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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
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
(e.g., increase or decrease) the activity of the polypeptides described
herein, or that
otherwise interact with the polypeptides herein. For example, such agents can
be
agents that bind to polypeptides described herein (~.g., ~MP2 binding agents);
that
have a stimulatory or inhibitory effect on, for example, activity of
polypeptides of
the invention; that change (e.g., enhance or iWibit) the ability of the
polypeptides of
the invention to interact with ~MP2 binding agents (e.g., receptors or other
binding
agents); or that alter posttranslational processing of the BMf2 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)9 as well as agents identifiable by the
assays.
Test agents can be obtain ed using any of the numerous approaches in
combinatorial
library methods lmown in the art, including: biological libraries; spatially
addressable parallel solid phase or solution phase libraries; synthetic libray
methods
requiring deconvolution; the "one-bead one-compound" library method; snd
synthetic libral-y 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, I~.S. (1997) Anticaazce~ Drug Des., 12:145).
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In one embodiment, a cell, cell lysate, or solution containing or expressing
an
BMP2 polypeptide (e.g., SEQ III N~:2 and comprising at least one polymorphism
as shown in Tables 2, 3 or 4), or an active fragment or derivative thereof (as
described above), can be contacted with an agent to be tested to identify
agents
which alter the activity of an BMP2 polypeptide; alternatively, the
polypeptide can
be contacted directly with the agent to be tested. The level (amount) of BMP2
activity is assessed (e.g., the level (amount) of BMP2 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 BMP2 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 BMP2 polypeptide. An increase in the level of BI~1 2 activity relative to a
control, indicates that the agent is an agent that enhances (is an agonist of)
BMl'2
activity. Similarly, a decrease in the level of BIi~2 activity relative to a
control,
indicates that the agent is an agent that inhibits (is an antagonist of)
Bll~IP2 activity.
In another embodiment, the level of activity of an BMF2 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 BMF2 activity.
The present invention also relates to an assay for identifying agents that
alter
the expression of the BI~2 nucleic acid (e.g., antisense nucleic acids, fusion
proteins9 polypeptides, peptidomimetics, prodrugs~ receptors9 binding agents,
antibodies, small molecules or other drugs or ribo~ymes), that alter (e.g.,
increase or
decrease) expression (e.g., transcription or translation) of the gene, or that
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 BIenP2
polypeptide (e.g., BMP2 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
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comprises elements necessary for transcription/translation of the nucleic
acid. Cells
not suspended in solution can also be employed, if desired. The level and/or
pattern
of BMP2 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 BMP2 expression in the absence of the agent to be
tested). If
the level and/or pattern in the presence of the agent differs, by an amount or
in a
mamier 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 BMf2.
Enhancement of BMP2 expression indicates that the agent is an agonist of BMP2
activity. Similarly, inhibition of BMP2 expression indicates that the agent is
an
antagonist of BMP2 activity. In another embodiment, the level and/or pattern
of
BMP2 polypeptide(s)(e.g., different splicing variants) in the presence of the
agent to
be tested, is compared with a control level and/or pattern that has previously
been
established. A level and/or pattern in the presence of the agent that differs
from the
control level and/or pattern by an amount or in a manner that is statistically
significant indicates that the agent alters BMP2 expression.
In another embodiment of the invention, agents that alter the expression of
the BMP2 nucleic acid or that 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 BMP2 nucleic acid operably linked to a
reporter
gene. After contact with an agent to be tested, the level of expression of the
reporter
gene (e.g., the level of mI~TA or of protein expressed) is assessed, and is
compared
with the level of expression in a control (a. 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 mamier that is statistically
significant, from
the level in the absence of the agent, then the agent is an agent that alters
the
expression of BMP2, as indicated by its ability to alter expression of a gene
that is
operably linked to the BMP2 gene promoter. Enhancement of the expression of
the
reporter indicates that the agent is an agonist of BMF2 activity. Similarly,
inhibition
of the expression of the reporter indicates that the agent is an antagonist of
BMP2
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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 BMF2 expression.
Agents that alter the amounts of different splicing variants encoded by BMF2
(e.g., an agent that enhances the activity of a first splicing variant, and
that inhibits
activity of a second splicing variant), as well as agents that are agonists of
activity of
a first splicing variant and antagonists of activity of a second splicing
variant, can 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 BMP2
binding
agent. For example, a cell that expresses a compound that interacts with BMP2
(herein refereed to as a "BMP2 binding agent", which can be a polypeptide or
other
molecule that interacts with BMP2, such as a receptor) is contacted with BMP2
in
the presence of a test agent, and the ability of the test agent to alter the
interaction
between BMP2 and the BMP2 binding agent is determined. Alternatively, a cell
lysate or a solution containing the BMP2 binding agent, can be used. An agent
that
binds to BMP2 or the BMP2 binding agent can alter the interaction by
interfering
with, or enhancing the ability of BMP2 to bind to, associate with, or
otherwise
interact with the BMP2 binding agent. Determining the ability of the test
agent to
bind to BMP2 or an BMP2 binding agent can be accomplished, for example, by
coupling the test agent with a radioisotope or er~ymatic label such that
binding of
the test agent to the polypeptide can be determined by detecting the labeled
with''~I,
~~S, 3'P, 33h,'4C or 3H, either directly or indirectly, and the radioisotope
detected by
direct counting of radioemmission or by scintillation counting. Alternatively,
test
agents can be er~ymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to product. It is also
within
the scope of this invention to determine the ability of a test agent to
interact with the
polypeptide without the labeling of any of the interactants. For example, a
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microphysiometer can be used to detect the interaction of a test agent with
BMP2 or
a BMP2 binding agent without the labeling of either the test agent, BMP2, or
the
BMP2 binding agent (McConnell, H.M. et al. (1992) Science, 257:1906-1912). As
used herein, a "microphysiometer" (e.g., CytosensorTM) is an analytical
instrument
that measures the rate at wluch 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 know BMP2 binding partners. Thus,
these receptors can be used to screen for compounds that are BMP2 receptor
agonists for use in treating osteoporosis or BMP2 receptor antagonists for
studying
osteoporosis. The linkage data provided herein, for the first time, provides
such
correction to osteoporosis. Drugs could be designed to regulate BMP2 receptor
activation, and, in turn, could be used to regulate signaling pathways and
transcription events of genes downstream.
In another embodiment of the invention, assays can be used to identify
polypeptides that interact with one or more BMP2 polypeptides, as described
herein.
For example, a yeast two-hybrid system (Fields, S. and Song, ~., Nature
340:245-
246 (1989)) can be used to identify polypeptides that interact with one or
more
BMP2 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
I~NA binding domain and a transcription activation domain). If the two domains
are
separated but fused to two different proteins that interact with one another,
transcriptional activation can be achieved, and transcription of specific
markers (e.g.,
nutritional markers such as His and Ade, or color markers such as lack) can be
used
to identify the presence of interaction and transcriptional activation. for
a«ample, in
the methods of the invention, a first vector is used that includes a nucleic
acid
encoding a D1VA binding domain and also an BMP2 polypeptide, splicing variant,
or
fragment or derivative thereof, and a second vector is used that includes a
nucleic
acid encoding a transcription activation domain and also a nucleic acid
encoding a
polypeptide that potentially can interact with the BMP2 polypeptide, splicing
variant, or fragment or derivative thereof (e.g., a BMP2 polypeptide binding
agent or
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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 that express the
markers of
interest. These colonies can be examined to identify the polypeptide(s) that
interact
with the BMP2 polypeptide or fragment or derivative thereof. Such polypeptides
can be useful as agents that alter the activity of expression of an BMP2
polypeptide,
as described above.
In more than one embodiment of the above assay methods of the present
invention, it could be desirable to immobilize either BMP2, the BMP2 binding
agent, or other components of the assay on a solid support in order to
facilitate
separation of complexed from uncomplexed forms of one or both of the
polypeptides, as well as to accommodate automation of the assay. Binding of a
test
agent to the polypeptide, or interaction of the polypeptide with a binding
agent in the
presence and absence of a test agent, can be accomplished in any vessel
suitable for
containing the reactants. Examples of such vessels include microtitre plates,
test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein (e.g.,
a CaST
fusion protein) can be provided, thus adding a domain that allows BMP2 or a
BMP2
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 BMP2 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 mI~TA
or polypeptide(s) in the presence of the test agent is compared to the level
of
expression of mI~NI~ 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.
F'or example9 when expression of ml~Tl~ 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 mRNt~ or polypeptide
expression. Alternatively, when expression of the mI~NA or polypeptide is less
(statistically significantly less) in the presence of the test agent than in
its absence,
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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 fiu-ther 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 described herein. In addition, an agent identified as
described
herein can be used to alter activity of a polypeptide encoded by EMP2, or to
alter
expression of BMP2, 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 nucleic acids described herein, particularly nucleotides encoding
the
polypeptides described herein; comprising polypeptides described herein (e.g.,
SEQ
III N0:2); and/or comprising the agent that alters (e.g., enhances or
inhibits) EMP2
polypeptide activity described herein. For instance, a polypeptide, protein9
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 EMP2
polypeptide activity, can be formulated with a physiologically acceptable
carrier or
excipient to prepare a pharnlaceutical composition. The carrier and
composition can
be sterile. The formulation should suit the mode of administration.
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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 pI~ buffering agents. The composition can be a liquid
solution, suspension, emulsion, tablet, pill, capsule, sustained release
formulation, or
powder. The composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. ~ral 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. ~ther suitable methods of
introduction
can also include gene therapy (as described below), rechargeable or
biodegradable
devices, particle acceleration devises (6'gene guns") and slow release
polymeric
devices. The phal-maceutical compositions of this invention can also be
administered as pert of a combinatorial therapy with other agents.
The composition can be formulated 111 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
can
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
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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 can be mixed prior to administration.
For topical application, nonsprayable forms, viscous to semi-solid or solid
forms comprising a carrier compatible with topical application and having a
dynamic
viscosity greater than water, can be employed. Suitable formulations include
but are
not limited to solutions, suspensions, emulsions, creams, ointments, powders,
enemas, lotions, sole, 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 can be
incorporated
into a cosmetic formulation. For topical application, also suitable are
sprayabhe
aerosol preparations wherein the active ingredient, optionally in combination
with a
solid or liquid inert carrier material, is packaged in a squeeze bottle or in
admixture
with a pressurized volatile, normally gaseous propellant, e.g., pressurized
air.
Agents described herein can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free amino groups
such
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 the agent that will be therapeutically effective in the treatment of
a
pat~ticular disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques. In addition,
iiz
vita°~ or i~r viv~ assays can optionally be emphoyed 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 osteoporosis,
and
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should be decided according to the judgment of a practitioner and each
patient's
circumstances. Effective doses can be extrapolated from dose-response curves
derived from i~c 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. ~ptionally 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.~., separately, sequentially or concurrently), or the like.
The pack
or kit can also include means for reminding the patient to take the therapy.
The pack
or kit can be a single unit dosage of the combination therapy or it can be a
plurality
of unit dosages. In particular, the agents can be separated, mixed together in
any
combination, present in a single vial or tablet. Agents assembled in a blister
pack or
other dispensing means is envisioned by the present invention. For the purpose
of
this invention, unit dosage is intended to mean a dosage that is dependent on
the
individual pharmacodynamics of each agent and administered in FDA approved
dosages in standard time courses.
METH~DS OF THERAFY
The present invention also pertains to methods of treatment (prophylactic
and/or therapeutic) for osteoporosis or a susceptibility to osteoporosis,
using an
EMP2 therapeutic agent. A '6EI~~2 therapeutic agent" is an agent that alters
(~.g.9
enhances or inhibits) EMh2 polyrpeptide activity and/or EMP2 expression, as
described herein (e.~., an EMP2 agonist or antagonist).
EMP2 therapeutic agents can alter ~l~lth2 polypeptide activity or gene
expression by a variety of means, such as, for example, by providing
additional
EMP2 polypeptide or by upregulating the transcription or translation of the
EMP2
gene; by altering posttranslational processing of the BMP2 polypeptide; by
altering
transcription of BMP2 splicing variants; or by interfering with BMP2
polypeptide
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activity (e.g., by binding to a BMP2 polypeptide), or by downregulating the
transcription or translation of the BMP2 gene. Representative BMP2 therapeutic
agents include the following: nucleic acids or fragments or derivatives
thereof
described herein, particularly nucleotides encoding the polypeptides described
herein
and vectors comprising such nucleic acids (e.g., a gene, cDNA, and/or mRNA,
such
as a nucleic acid encoding a BMP2 polypeptide or active fragment or derivative
thereof, or an oligonucleotide; for example, SEQ ID NO:1 and optionally
comprising
at least one polymorphism as shown in Tables 2, 3 and 4 or a nucleic acid
encoding
SEQ ID NO:2 and optionally comprising at least one polymorphism as shown in
Tables 2, 3 and 4~, or fiagments or derivatives thereof); polypeptides
described herein
(e.g., SEQ ID NO:2 comprising at least one polymorphism as shown in Tables 2,
3
and 4, andlor other splicing variants encoded by BMP2, or fragments or
derivatives
thereof); other polypeptides (e.g., BMP2 receptors); BMP2 binding agents,
including
but not limited to BMP2R2, BMPR1A and BMPR1B; peptidomimetics; fusion
proteins or prodrugs thereof; antibodies (e.g., an antibody to a mutant BMP2
polypeptide, or an antibody to a non-mutant BMP2 polypeptide, or an antibody
to a
particular splicing variant encoded by BMP2, as described above); ribozymes;
other
small molecules; and other agents that alter (e.g., enhance or inhibit) BMP2
gene
expression or polypeptide activity, or that regulate transcription of BMP2
splicing
variants (e.g., agents that affect which splicing variants are expressed, or
that affect
the amount of each splicing variant that is expressed.
The BMP2R2, BMPR1A and BMPR1B are cell-surface receptors binding the
BMP2 protein; the BMP2 protein binds to these receptors, and, in turn,
stimulates
certain intracellular responses (Fujii,1VI., et al.~, l~~l. ~i~l. Cell,
1~(11):3501-13
(1999); Massague, J.9 d9~a7zd. Reo. I~i~elzetrr., ~a7:753-91 (199g); '.hen,
D.g et al., ,a:
Cell hi~l., 12(1):295-305 (199g); and Kirsch, T., et. al., l~at~ra~e
Sta°ac~t~ca°al.~i~~~gy,
70):492-496 (2000)). There are also known direct inhibitors of BMP2, e.g.,
noggin
and chordin (at least noggin; chordin is a known inhibitor of other BMF's)
(~immerman, L.B., et. al., Cell, 86:599- 606 (1996); Aspenberg, P., et. al.,
J. ~~ne
ll~Iitzer. Res., 16(3):497-500 (2001); and Dale., L. et.al., ~i~essays,
21(9):751-60
(1999)).
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More than one BMP2 therapeutic agent can be used concurrently with
another, if desired.
The BMP2 therapeutic agent that is a nucleic acid is used in the treatment of
osteoporosis or in the treatment for a susceptibility to osteoporosis. 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. The therapy is
designed to alter (e.g., inhibit or enhance), replace or supplement activity
of a BMP2
polypeptide in an individual. For example, a BMP2 therapeutic agent can be
administered in order to upregulate or increase the expression or availability
of the
BMP2 gene or of specific splicing variants of BMP2, or, conversely, to
downregulate or decrease the expression or availability of the BMP2 gene or
specific
splicing variants of BMP2. LJpregulation or increasing expression or
availability of
a native BMP2 nucleic acid 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
BMF2
nucleic acid 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. In one
embodiment, the BMP2 therapeutic agent is the healthy gene or gene product
(SEQ
ID N~:2), e.g., a BMP2 nucleic acid and gene product that is not associated
with
osteoporosis.
The BMP2 therapeutic agents) are administered in a therapeutically
effective amount (e.g., 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 that 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, i~ vitro or i~c vivo assays can optionally be employed to help
identify
optimal dosage ranges. The precise dose to be employed in the formulation will
also
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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 can be extrapolated from dose-response curves
derived from ivy vitro or animal model test systems.
In one embodiment, a nucleic acid of the invention (e.g., a nucleic acid
encoding a BMP2 polypeptide, such as SEQ ID NO:1 optionally comprising at
least
one polymorphism as shown in Tables 2, 3 and 4 or another nucleic acid that
encodes a BMP2 polypeptide or a splicing vaxiant, derivative or fragment
thereof,
such as a nucleic acid encoding SEQ lI~ N0:2 and comprising at least one
polymorphism as shown in Tables 2, 3 and 4 can be used, either alone or in a
pharmaceutical composition as described above. For example, B1VIP2 or a cI~Nt~
encoding the BIvIP2 polypeptide, either by itself or included within a vector,
can be
introduced into cells (either ih vital~ or ih viv~) such that the cells
produce native
B1~IP2 polypeptide. If necessary, cells that have been transformed with the
gene or
cDNA or a vector comprising the gene or cI~NA can be introduced (or re-
introduced) into an individual affected With the disease. Thus, cells that, in
nature,
lack native BMP2 expression and activity, or have mutant BMP2 expression and
activity, or have expression of a disease-associated BMP2 splicing variant,
can be
engineered to express BMP2 polypeptide or an active fragment of the BMP2
polypeptide (or a different variant of BMP2 polypeptide). In one embodiment, a
nucleic acid encoding the BMP2 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 usedo
Alternatively,
nonviral gene transfer methods, such as calcium phosphate coprecipitation,
mechanical techniques (e.g., microinjection); membrane fusion-mediated
transfer via
liposomes; or direct I~NA uptake, can also be used.
Alternatively, in another embodiment of the invention, a nucleic acid of the
invention; a nucleic acid complementary to a nucleic acid of the invention; or
a
portion of such a nucleic acid (e.g., an oligonucleotide as described below),
can be
used in "antisense" therapy, in wluch a nucleic acid (e.g., an
oligonucleotide) that
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specifically hybridizes to the mRNA and/or genomic DNA of BMP2 is administered
or generated i~c situ. The antisense nucleic acid that specifically hybridizes
to the
mRNA and/or DNA inhibits expression of the BMP2 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 specific interaction in the major groove of the double
helix.
An antisense construct of the present invention can be delivered, for
example, as an expression plasmid as described above. When the plasmid is
transcribed in the cell, it produces I~NA that is complementary to a portion
of the
mIZNA and/or DNA that encodes BMP2 polypeptide. Alternatively, the antisense
constmct can be an oligonucleotide probe that is generated ex viv~ and
introduced
into cells; it then inhibits expression by hybridizing with the mIZNA and/or
genomic
DNA of BMP2. 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 ire viv~. Exemplary nucleic acid
molecules for use as antisense oligonucleotides are phosphoramidate,
phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to
constructing oligomers useful in antisense therapy are also described, for
example,
by Van der I~rol et al. ((1988) ~ioteclziziques 6:958-976); and stein et al. (
(1988)
Cav~ce~ 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 BMP2 sequence, can be utilized.
To perform antisense therapy, oligonucleotides (mI~NA9 cDNA or DNA) are
designed that are complementary to mI~NA encoding BI~/11~2e The antisense
oligonucleotides bind to BI~1P2 mI~NA transcripts and prevent translation.
Absolute
complementarity is not required. A sequence "complementary" to a portion of an
I~NA, as referred to herein, indicates that a sequence has sufficient
complementarity
to be able to hybridize with the I~NA, forming a stable duplex; in the case of
double-stranded antisense nucleic acids, a single strand of the duplex DNA can
thus
be tested, or triplex formation can be assayed. The ability to hybridize will
depend
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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 can contain and still form a stable duplex
(or
triplex, as the case may be). ~ne 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 ivc viv~), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger et al. (1989) Pr~c. Natl. Acad.
S'ci.
ZIS'A 56:6553-6556; Lemaitre et al., (1937), Pr~oc. Natl. Read. ~'ci. U~'A
~4:64~8-652;
PCT International Publication No. W088/09810) or the blood-brain barrier (see,
e.g.,
PCT International Publication No. W089/10134), or hybridization-triggered
cleavage agents (see, e.g., Krol et al. (1988) BioTechv~iques 6:955-976) or
intercalating agents. (See, e.g., Zon, (1985), Pharm. Res. 5:539-549). To this
end,
the oligonucleotide can 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 ~MP2 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 (~.g.q antisense
linked to
peptidee or 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 sufficient amounts of single-stranded RNAs that will form
complementary base pairs with the endogenous ~MP2 transcripts and thereby
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prevent translation of the BMP2 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 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 to selectively infect
the desired
tissue, in which case administration can be accomplished by another route
(e.~.,
systemically).
Endogenous BMP2 expression can also be reduced by inactivating or
"knocking out" BMP2 or its promoter using targeted homologous recombination
(e.~., see Smithies et al. (1955) Natz~re 317:230-234; Thomas ~ Capecchi (197)
Cell 51:503-512; Thompson et al. (199) Cell 5:313-321). For example, a mutant,
non-functional BMP2 (or a completely unrelated DNA sequence) flanked by DNA
homologous to the endogenous BMP2 (either the coding regions or regulatory
regions of BMP2) can be used, with or without a selectable marker and/or a
negative
selectable marker, to transfect cells that express BMP2 in vivo. Insertion of
the
DNA construct, via targeted homologous recombination, results in inactivation
of
BMP2. The recombinant DNA constructs can be directly administered or targeted
to
the required site i~c vivo using appropriate vectors, as described above.
Alternatively,
expression of non-mutant BMP2 can be increased using a similar method:
targeted
homologous recombination can be used to insert a DNA construct comprising a
non-
mutant, functional B1~~1P2 (e.~., a nucleic acid having SEA ID N~:1), or a
portion
thereof, in place of a mutant BMP2 in the celh as described above. In another
embodiment, targeted homologous recombination can be used to insert a DNA
construct comprising a nucleic acid that encodes a BMP2 polypeptide variant
that
differs from that present in the cell.
Alternatively, endogenous BMP2 expression can be reduced by targeting
deoxyribonucleotide sequences complementary to the regulatory region of BMP2
(i.e., the BMP2 promoter and/or enhancers) to form triple helical structures
that
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prevent transcription of BMP2 in target cells in the body. (See generally,
Helene, C.
(1991) Anticancer Drug Des., 6(6):569-84; Helene, C., et al. (1992) Ann, N. Y.
Acad.
Sci., 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15). Likewise,
the
antisense constructs described herein, by antagonizing the normal biological
activity
of one of the BMP2 proteins, can be used in the manipulation of tissue, e.g.
tissue
differentiation, both i~r 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 BMP2 mI~NA or
gene sequence) can be used to investigate role of BMP2 in developmental
events, as
well as the normal cellular function of BMP2 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 BMP2 therapeutic agents
as described herein can also be used in the treatment or prevention of
osteoporosis.
The therapeutic agents can be delivered in a composition, as described above,
or by
themselves. They ca.n 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; ifZ vivo production
(e.g., a
transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade et al.), for
example,
and can be isolated using standard means such as those described herein.
t~ combination of any of the above methods of treatment (e.g., administration
of non-mutant BMP2 polypeptide in conjunction with antisense therapy targeting
mutant BMP2 mI~NA; administration of a variant encoded by BMP2 in conjunction
with antisense therapy targeting a second variant encoded by BMP2), 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.
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EXAMPLES
Example 1. Identification of the BMP2 Nucleic Acid With Linkage to
Osteoporosis
Phenotype and Fauzily Cohstructio~c
Patients who have low impact fractures and/or take bisphosphonates for
treating osteoporosis are automatically treated as affecteds. People with low
bone
mass density (BMD) measurements are considered-to be osteoporotic, and have
been
shown to have substantially increased risk of fractures. BMD measurements are
taken for both the hip and the spine. For each person with BMD measurements, a
standardized BMD score is computed (mean 0, standard deviation 1 for the
population), which is adjusted for sex, age, body weight and hormone
replacement
therapy (I~T). For the combined analysis, the two measurements are summed.
Population Bl~ data from Iceland and the United States are used for
standardization and adjustment. For example, a person with a positive BMD
score is
above average and one with a negative score is below average for his/her age,
body
weight and possibly HRT. Assuming approximate normality, a score of -1
corresponds approximately to the lower 16th percentile, etc.
For analysis, we start with a current list of primary people, people who have
BMD measurements and/or are severely affected, and for whom we have genotypes.
We then use the genealogy database to create family clusters linking these
primary
people using a threshold distance of 5 meiotic events. This procedure produced
190
potentially informative clusters with a total of 1215 primary people.
Li~2l~ez~e data
Four genome wide scans (OWS) were performed using osteoporotic
phenotypes at different skeletal sites; the hip, the spin e, aaZd combined
phenotypes.
All GWS analysis located at 20 cl~I region on Chr20, between 10 cM and 30 cM
based on the Marshfield map.
All of the analyses were performed using the Allegro linkage program
developed at deCODE (Gudbjartsson et al., lVatu~e Geyzetics, 25: 12-13, May
2000).
The allele sharing analysis uses the Spairs scoring function of GENEI~LTN'TEI~
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(Kruglyak et al., Am. J. Hum. Gehet., 46: 1347-1363, 1996), but families were
weighted using a scheme that is a compromise between weighting families
equally
and weighting affected pairs equally. The allele-sharing LOD scores were
computed
using the 'exponential model' described in Kong and Cox, Am. J. Hum. Gevcet.,
61:
1179-llgS (1997).
Hip
The phenotype used was age, sex, weight and HRT corrected BMD < -1 SIB
at the hip (total hip). Hip fracture cases and bisphosphonate users are also
considered affected even if values are above -1 SIB. A total of 346 affected
were
used in this analysis. The GWS resulted in a L~I~ score of 3.1 using our
standard
set of markers. I-adding 10 extra markers at the region on interest, between
11 cM
and 39 cM, resulted in a L~I~ score of 3.3.
Spine
The phenotype was age, sex, weight and HRT corrected )3MD<-1 SD at
lumbar spine (L2-L4). Vertebral compression fracture cases and bisphosponate
users are also considered affected even if values are above -1 SD. A total of
402
affected people were used in this analysis. The GWS resulted in a LOD score of
2.4
at the same location as in the hip analysis using the standard set of markers,
but a
L~I~ score of 2.9 with the extra marker set.
C~fnbi'zed
The phenotype used was the sum of corrected I~MI~ < -1.5 SIB. Vertebral
compression fracture9 hip fracture, other osteoporosis related low impact
fracture (at
least two fractures) and bisphosphonate users (~MI~ measurements before
treatment
start are used if available) are all considered affected. A total of 522
affected were
used in this analysis. The CaWS resulted in a L~I~ score of 2.5 with the
standard
marker set, but a L~I~ score of 3.9 using the extra markers in the region.
Combined severe
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The phenotype used was the sum of the age, sex, weight and HRT corrected
BMD < -2.3 SD. Vertebral compression fracture, hip fracture, other
osteoporosis
related low impact fracture (at least two fractures) and bisphosphonate users
affected. The number of affected in this analysis was 290. The GWS resulted in
a
LOD score of 3.8 with the standard set but a LQD score of 4.7 was reached
using the
extra 10 markers in addition.
Corticosteroid users and women with early menopause were excluded as
affected in all analysis.
T'he ~NIP2 gea~e
The BMP2 nucleic acid is located in this region. ~nly 5 kb are between the
marker D20S846, which gives the highest L~D score, and the 3' end of the gene.
The gene has been sequenced and characterised in terms of exon/intron
structures,
promoter region and transcriptions! start sites. This information are publicly
available.
A number of nucleotide changes are observed in the Icelandic population.
These changes have not to our knowledge been described before (See Table 2).
BMP2 binds to the receptors BMFR-IA or BMPR-IB, and BMPR-II, leading
to formation of receptor complex heterodimer and phosphorylation of the BMPR-
IA
or BMPR-IB receptors. ~nce activated, these receptors subsequently
phosphorylate
SMADl, SMADS or SMADB, which in turn form complexes with SMAD4 and
translocate to the nucleus where the transcription of specific genes is
affected
(Massague, J., Atzrza~. Rev. ~i~chera-a., 67:753-791 (1998); Chen, D. et al.,
,~ Gel!
~i~l., 12(1):295-305 (1998)). SMADs 6 and 7 block signals by preventing the
activation of SMAD1, S~~ADS or Sl'~.tADB by the BMP2 receptors and have been
shown to inhibit osteoblast differentiation (lvliya~ono, I~.9 ~~r~e, 25(1):91-
93 (1999);
Fujii, M., et al.s 1!101. ~i~l. Cell, 1~(11):3801-3813 (1999)). BI~~IP2
stimulates Cbfal9
alkaline phosphatase and Collagen type I (osteoblast specific proteins)
expression
through BMPR-IB (Chen, D. et al., J: Cell ~i~l.,1~2(1):295-305 (1998). Cbfal
regulates the expression of osteoprotegerin (~PG), which is an osteoblast-
secreted
glycoprotein that functions as a potent inhibitor of osteoblast
differentiation and thus
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of bone resorption (Thirunavukkarasu, I~., et al., J. Biol. Chem., (2000)).
Cbfal
controls osteoblast differentiation and bone formation. During cellular aging
of
human osteoblasts, there is a significant reduction (up to 50%) of Cbfal mRNA
(Christiansen, M., et al., J. C~erohtol. A Biol. Sci. Med. Sci., 55(4):B194-
200 (2000)).
Results ahd Discussiov~
As a result of the linkage studies, the analysis shows that this locus is
involved in multiple osteoporosis phenotypes. Fm-thermore, mutation within the
human BMP2 nucleic acid is likely to explain the phenotypes in these families.
Sporadic occmTence of osteoporosis, i.e., occurrence without familial
connection,
can also be determined using the information contained herein.
~steoporosis could be caused by a defect in the BMP2 nucleic acid as
follows: An alteration in the BMP2 nucleic acid (transcription, splice,
protein variant
etc.) could lead to a reduction of its action on Cbfal through BMPR-IB and the
subsequent signaling pathway. This would lead to less bone formation because
of
fewer and less active osteoblasts and more bone resorption because of less OPG
and
more osteoclasts. This would lead to bone loss. Since a significant reduction
of
Cbfal levels is associated with aging osteoblasts, this effect could become
more
important with older age.
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Table 1
LOCUS 14759 by DNA
DEFINITION Human bone morphogenetic protein 2 (BMP2) gene, complete
cds,
complete sequence.
ACCESSION
VERSION
KEYWORDS
SOURCE human.
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;
Mammalia; Eutheria; Primates; Catarrhini; Hominidae;
Homo.
REFERENCE 1 (bases 1-14759)
AUTHORS Blakey,S.
TITLE Direct Submission
JOURNAL Submitted (04-APR-2000) Sanger Centre, Hinxton,
Cambridgeshire, CB10 1SA, UK. E-mail enquiries:
humquery@sanger.ac.uk Clone requests:
clonereauest(aOsan~er.ac.uk
COMMENT This sequence was taken from GenBank sequence AL035668
(VERSION AL035668.15, GI:4995292), by 118501..133259.
FEATURES Location/Qualifiers
source l.. 14759
/organism="Homo sapiens"
/db xref="taxon : 9606"
/chromosome="20"
/map="20p12"
/clone="RP5-859D4"
/clone lib="RPCI-5"
gene 2072..12634
/gene="BMP2"
/note="BMP2A"
/db xref="LocusID:650"
/db xref="MIM:112261"
exon 2072..2387
/gene="BMP2"
/number=1
exon 3632..3984
/gene="BMP2"
/number=2
CDS /join(3639..3984, 11757..12601)
/gene="BMP2"
/note="BMP2 exons defined by comparison to mRNA
sequence (NM 001200)'°
/codon start=1
/product="bone morphogenetic protein 2
precursor"
/protein id="NP 001191.1'°
/db xref="GI:4557369"
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Table 2
nucleotide nucleotide
position position
nucleotide relative relative to position in
to
change SEQ. ID NO SEQ. AL035668gene amino acid change
1
A to G -2047 116454 promoter
T to C -1136 117365 promoter
(ATTT)n -901 117600 promoter
C to T -638 117863 promoter
C to T -568 117933 promoter
T to C -72 118429 promoter
G to A 70 118570 promoter
A 368 118868 promoter
insertion
A to G 420 118920 promoter
A to G 472 118972 promoter
G to C 1464 119964 5'utr
G to A 1722 120222 5'utr
C to G 1914 120414 5'utr
A to C 2536 121036 intron 1
C to T 2866 121366 intron 1
G to T 3145 121645 intron 1
T to G 3747 122247 axon 2 serine to alanine
A to G 3899'~~ 122399 axon 2
G to T 3918 122418 axon 2 ~.lanine to
serina
A to G 4181 122681 intron 2
G to A 4244 12274~4~ intron 2
A to T 4359 122859 intron 2
G to A 4435 122935 intron 2
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Table 2 Cont'd.
nucleotide nucleotide
position position
nucleotidrelative relative to position in
to
a changeSEQ. ID NO:1SEQ. AL035668gene amino acid change
T 4712 123212 intron 2
insertion
T to 5041 123541 intron 2
A
C to 5048 123548 intron 2
T
G to 5787 124287 intron 2
A
G to 6217 124717 intron 2
A
G to 7111' 125611 intron 2
A
A to 7162 125662 intron 2
T
T to 7781' 126281 intron 2
C
A to 7828 126328 intron 2
G
C to 7874 126374 intron 2
T
G to 8035* 126535 intron 2
C
A to 8083 126583 intron 2
C
T to 8463 126963 intron 2
G
G to 9013 * 127513 intron 2
A
G to 9082 127582 intron 2
A
G to 10631 129131 intron 2
T
A to 10841 129341 intron 2
G
A to 11980~~ 130480 eon 2 arginina to serine
T
C to 12571 131071 exon 2
T
A to 1284~5~ 131345 3' utr
C
T to 13066 131566 3' utr
C
A to 13209~~ 131709 3' utr
G
C to 13296 131796 3' utr
A
4 by 13533-13536 132033-1320363' utr
deletion
*known in SNP databases
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Example 2. Direct Sequencing of the BMP2 Nucleic Acid Sequence Reveals Other
Polymorphisms.
Additional genetic markers were identified in the BMP2 nucleic acid by
direct sequencing of the region in different populations. These are listed in
Table 3
with nucleotide position relative to SEQ. AL035668 as in earlier SNP update
file.
Table 3.
deG~~E type of nucleotidenucleotidelocationPublic
change name
numbering position position
relatide relative
to ~EC~. to 1~L035668
I~ f~~
1
P4019 C to T 112569
P4204 A to C 112754
P4337 T to G 112887
P4617 T to A 113167
P4730 A to G 113280
P4765 T to C 113315
P4822 A to G 113372
P5831 T to C 114381
P6121 A to C 114671 rs173106
P6136 A to T 114686
P6784 C to T 115334 rs969643
P6854 A to C 115404
P7420 G to A 115970
P7904 A to G -2047 116454 promoter
P8815 T to C -1136 117365 "
P9050 (ATTT)n -901 117600 "
P9313 C to T -638 117863 "
P9383 C to T -568 117933 "
P9879 T to C -72 118429
870 G to A 70 118570 "
B368 A insertion368 118868
B420 A to G 420 118920 "
8472 A to G 472 118972, "
81464 G to C 1464 119964 5'utr
81722 G toA 1722 120222 "
131914. C to G 1914 120414 ..
82636 A to C 2536 121036 intron
1
Et2866a C to T 2866 121366 "
Ei3145 G to T 3145 12164.5 ,
I~~7~Y T to G 374.7 122247 eon r~2273073
2
83809 A to G 3899 122399 " r~ ~
049007
83918 G to T 3918 1224.18 ,
841 fi~1A to G 4181 122681 intron
2
8244 G to A 4244 122744. "
B43~a9 A to T 4359 122859 "
844.35 G to A 4435 122935 ,
8.712 T insertion4712 123212
85041 T to A 5041 123541 "
8 x048 C to T 5048 123548 "
B578T G t~ A 5787 124287 "
86217 G to A 6217 124717 "
87111 G to A 7111 125611 " rS235764
87262 A to T 7162 125662 "
BP781* T to C 7781 126281 " rS1 X75274
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B7828* A to G 7828 126328 "
B7874* C to T 7874 126374 "
B8035* G to C 8035 126535 " rS235766
88083 A to C 8083 126583
88463 T to G 8463 126963 " rS235767
89013 G to A 9013 127513 " rS1005464
89082 G to A 9082 127582 "
B10631 G to T 10631 129131 "
810841 A to G 10841 129341 "
811980 A to T 11980 130480 exon 2 rS235763
812571 C to T 12571 131071 "
812845 A to C 12845 131345 3' utr rS15705
813066 T to C 13066 131566 " rS3178250
813209 A to G 13209 131709 " rS235769
813296 C to A 13296 131796 " rS170936
813533de144 by deletion13533-13536 132033 "
D841 C to T 132877
D873 T to C 132909
D1094 T to C 133130 rS235770
D1226 A to C 133262
D1354 G to A 133390
D1550 C to T 133586 TSC0078312/
rs28488
D1886 A to G 133922
D2o4s c to T 1340s4 rs235772
D2269 C to T 134305
D2319 T to A 134355
D2568 A to C 134604
D5348 C to T 137384
D5449 G to A 137485
D5498 C to T 137534
D5643 G to T 137679
D6220 A to G 138256 rS23151
D6440 A to G 138476
D6448 G to C 138484
D6683 C to T 138719
D6971 G to T 139007 TSC0191642/
rs910141
D7006 C to G 139042
D7355 C to G 139391
D7630 G to A 139666
Ds1 s3 c to T 140219 r~235750
D8629 T to C 140665
D8632 A to G 140668
D8862 G t~ A 140898
D9005 A to G 141041
D9036 C to T 141072
D9043 C to T 141079
D9126 G to A 14=1162
D9206 T to C 14.1242 r~235750
D9473 T to G 14.1509
D9617 C to T 141653
D9970 G to T 142006 r~235748
D10019 G to A 142055
D10402 T to C 142438
D10540 G to A 142576
D10554 T to C 142590
D10699 C to A 142735
D11023 T to C 143059
D11373 G to A 143409
D11395 A to G 143431
D11592 A to G 143628
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D12541 C to T 144577
D12645 A to T 144681
D12699 G to A 144735
D12908 C to A 144944
D13002 T to C 145038
D13071 T to A 145107
D13256 G to A 145292
D13259 G to T 145295
D13488 G to A 145524
D13749 A to G 145785
D14613 T to C 146649
D14664 C to T 146700
D14956 G to A 146992
D15562 C to T 147598
D15601 T to C 147637
D15827 C to T 147863
D16270 A to G 148306
D16345 C to T 148381
D16407 T to C 148443
D16595 C to G 148631
D17037 T to C 149073
D17242 G to A 149278
D17493 A to G . 149529 rs1116867
D17684 G to T 149720
D17794 G to A 149830
D18035 A to T 150071
D18292 C to A 150328
D18307 C to T 150343
D18513 C to G 150549
D18641 A to G 150677
D18855 A to T 150891
D19047 C to A 151083
D19354 G to A 151390
D19690 G to A 151726
D20383 A to G 152419
D20945 T to A 152981
D20958 C to T 152994
D20961 C fio T 152996
D21101 C to T 153137
D21190 C to A 153226
D21354 G to A 153390
D21382 T to C 153418
D22041 A to G 154077 TSC0278787
D22254. C to G 154290 TSC0278788
D22326 C to T 154362
D22530de16del6bp 154566
D22603 T to C 154.639
D22641 C to T 154677
D2264'i C to T 154677
D23348 C to T 155384
D24843 G to A 156879
D25216 A to C 157252
D25494 C to T 157530
D25528 T to C 157564 r~2876039
D25715 A to G 157751
D26836 A to C 158872
D28047 G to A 160083
D28047 G to A 160083
D28783 C to T 160819
D29019 G to A 161055
D29281 A to C 161317
D29461 T to C 161497
D29569 C to T 161605
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D30340C to T 162376
D30630G to A 162666
D31474G to T 163510
D31616T to A 163652
D32258T to C 164294
D32371A to C 164407
D33541T to C 165577
D34249T to G 166285
D34699A to G 166735
D35273C to A 167309
D35548C to T 167584
D35650G to T 167686 TSC032068
Additional linkage data of various SNPs to osteoporosis phenotypes is listed
in Table 4. Table 4 lists the p-value for the association of the SNP with the
phenotype (p-value), the relative risk (r), percentage of individuals affected
(Aff.
freq. caiTier), number of affected individuals (#aff), percentage of controls
(Ctrl.
freq. carrier), number of controls (#ctrl), identification of the allele, and
phenotype
screened.
Table 4. Association of SNPs with osteoporosis phenotypes.
p-valr #~afFAff.freq. allelebasemarker Phen~type
icon
Ctrl.freq.carrier
carrier
0.01712.1 648 6% 404 3% 2 G 83747 Linkage
phenotype
0.01741.4 832 13% 121110% 2 G 8420 Linkage
phenotype
0.04431.2 342 62% 428 56% 2 G D6440 Linkage
phenotype
0.04771.4 426 16% 577 12% 3 T D35548 Linkage
phenotype
0.04981.3 570 36% 401 30% 3 T TSC0428253Linkage
phenotype
0.02541.4 171 63% 356 54% 2 G TSC0293456firacture
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 can be made therein without
departing from the spirit and scope of the invention as defined by the
appended
claims.
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SEQUENCE LISTING
<110> deCODE genetics ehf.
Styrkarsdottir, Unnur
Johannsdottir, Vala Drofn
Gulcher, Jeffrey
<l20> Human Osteoporosis Gene
<130> 2345.2009009
<150> 10/346,723
<151> 2003-Ol-16
<150> 09/952,360
<151> 2001-09-13
<150> PCT/IBOl/01667
<151> 2001-09-12
<150> 09/661,887
<151> 2000-09-14
<160> 3
<170> FastSEQ for windows Version 4.0
<210> 1
<211> 14759
<212> DNA
<213> Homo Sapiens
<400> 1
ccttggtttt ggggatcatt tgggcaagcc cgaggtgctg tgcatggggg ctcctggaat 60
cctgggaagg gcagaaagcc ttggccccag actcatogtg cagcagctct gagcagtatt 120
tcggctgagg agtgacttca gtgaatattc agctgaggag tgacttggcc acgtgtcaca 180
gccctacttc ttgggggcct ggtggaagag ggtggcgtag aaggttccaa ggtcccaaac 240
tggaattgtc ctgtatgctt ggttcacaca gtgcgttatt ttaccttcct ctgagctgct 300
aatcgcctgc ctctgagctg ggtgagataa atatcacaag gcacaaagtg attgtacaat 360
aaaaaaatca aatccctccc atccatcctt cagtctgcca cacacgcagt ctacgttaca 420
cacatgtcac gtaaagcagg atgacatcca tgtcacatac atagacatat taaccgaaat 480
gtggcccttc ggttgcatat attctcatac atgaatatat ttatagaaat atatgcacat 540
atttttgtat attggatata tttatgtaac tataaattta catgcgtatg gatatgaaaa 600
taaatgcata cacatttatg taaaaaaatt tgtacacatg catttacata tgtaaataca 660
tacatctcta tgtattaatg tttaaaaaca ctcaatttcc agcctgctgt tttcttttaa 720
ttttcctcct attccgggga aacagaagcg tggatcccac gtctatgcta tgocaaaata 780
cgctgtaatt gaggtgtttt gttttgtttt gttttttgaa atcgtatatt acogaaaaac 840
ttcaaactga aagttgaata acgggcocag cggggaaata agaggccaga ccctgaccct 900
gcatttgtcc tggatttcgo ctccagagtc cccgcgaggg tccggcgcgc cagctgatct 960
ctectttgag agcagggagt ggaggcgcga gcgcccccct tggcggccgc gcgcccccgc 1020
cctccgcccc accccgccgc ggctgcccgg gcgcgccgtc cacacccctg cgcgcagctc 1080
ccgcccgctc ggggatcccc ggcgagccgc gccgcgaagg gggaggtgtt cggccgcggc 1140
cgggagggag ccggcaggcg gcgtcccctt taaaagccgc gagcgccgcg ccacggcgcc 1200
gccgccgccg tcgccgccgc cggagtcctc gccccgccgc gctgcgcccg gctcgcgctg 1260
cgctagtcgc tccgcttccc acaccccgcc ggggactggc agccgccgcc gcacatctgc 1320
cgccacagcc tccgccggct acccgaacgt tctcggggcc agcgccgagt ggatcaccgg 1380
ggaccgcgag gcacccgcgc gccgcagacc ccgcgcgggc tggagcaccc ggcagagcgc 1440
gccacagcgc cgtggcctct gctgcccggg ctgcgccaga gccgcggacg ggcgcgcaga 1500
gcgccgggga ctccggagcc gatccctagc gccgcgatgc ggagcaccta ctgcaggaga 1560
tcgggggcct gggacgcgct ggccgaggtg tgatcggacc ccaggctagc cacaaagggc 1620
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acttggcccc agggctagga gagcgagggg agagcacagc cacccgcctc ggcggcccgg 1680
gactcggctc gactcgccgg agaatgcgcc cgaggacgac ggggcgccag agccgcggtg 1740
ctttcaactg gcgagcgcga atgggggtgc actggagtaa ggcagagtga tgcggggggg 1800
caactcgcct ggcaccgaga tcgccgccgt gcccttccct ggacccggcg tcgcccagga 1860
tggctgcccc gagccatggg ccgcggcgga gctagcgcgg agcgcccgac cctcgacccc 1920
cgagtcccgg agccggcccc gcgcggggcc acgcgtccct cgggcgctgg ttcctaagga 1980
ggacgacagc accagcttct cctttctccc ttcccttccc tgccccgcac tcctccccct 2040
gctcgctgtt gttgtgtgtc agcacttggc tggggacttc ttgaacttgc agggagaata 2100
acttgcgcac cccactttgc gccggtgcct ttgccccagc ggagcctgct tcgccatctc 2160
cgagccccac cgcccctcca ctcctcggcc ttgcccgaca ctgagacgct gttcccagcg 2220
tgaaaagaga gactgcgcgg ccggcacccg ggagaaggag gaggcaaaga aaaggaacgg 2280
acattcggtc cttgcgccag gtcctttgac cagagttttt ccatgtggac gctctttcaa 2340
tggacgtgtc cccgcgtgct tcttagacgg actgcggtct cctaaaggta gaggacgcgg 2400
gccagggccc ggggtgggtg gtgggtggga gggggatttg ggcagccact gcggtagagc 2460
ccttccttac gtccaggcca gaagtaaaca gacccctctc cagtccacgt gcaacggagc 2520
cctgcagggg ctcccacttc cagctgcccc gggcgaccgt aagcctcacc ctcccggccc 2580
gcactcttcc acccctcttt cttcccctct ccctggaata cttttggagc tgttaacact 2640
tagatgaggt gttttattta tttatttatt tatttttaat ttttttaaaa acttttttgg 2700
gtcaaagaaa tccctttgag agggtagccc ctgggtttca cccgttagct gagaacctgt 2760
ccgctctgcc atggtgatct ccattcttca agtgtttccg ggagactt'gg tttctttgct 2820
cagagccgtg tcccatttag gaaagtacta ggagtttggg gttctcccta cttgtttcca 2880
gaaatgcgag gggtcagtac tgaaggatca cttggtactg tgtttttaac agctgacacg 2940
tgcattaata gatattcacc atttacgtaa tcccgggaag atacatgtgt atcttgactg 3000
cactgtgggg atgcgggatg gagctgcctt tcgagacacc cctgagggta ggggcctggg 3060
acacaagtca taagtggctt cagaagttgt ggccttgagc ttacagggtc tggaagctat 3120
aagggtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt caggaagttc tatacagtgc 3180
ctctaaggaa gtcacatgca ccatttatgt gtgtttatat gccagacagc gctcagcact 3240
ccgcatttgg gtttgtatag gggacgcagg gtgtcagatc aagcggtggt tttcccaggt 3300
tcccggcatt ggctgtcagc gctgtgtcac acacaaaaaa gtgacagtca ttggcgctgg 3360
tttggttggg ggggagggca aatcccaaat ctgatgtcag acgagctaag cgttggatgg 3420
gagcgataaa tcatctggtt caggaacttg ggacccttca ttatcccaaa cgtttgagct 3480
tcggtcggtc ttacctagac tcgtgagtgt gccaagccag gagggcatcc tggaggaggc 3540
acgccagcca aatgggagac cgggccgcgg gggcgcgagg ggggaggact gggcggggaa 3600
ctcgggtgac tcacgtcggt cctgtccgca ggtcgaccat ggtggccggg acccgctgtc 3660
ttctagcgtt gctgcttccc caggtcctcc tgggcggcgc ggctggcctc gttccggagc 3720
tgggccgcag gaagttcgcg gcggcgtcgt cgggccgccc ctcatcccag ccctctgacg 3780
aggtcctgag cgagttcgag ttgcggctgc tcagcatgtt cggcctgaaa cagagaccca 3840
cccccagcag ggacgccgtg gtgcccccct acatgctaga cctgtatcgc aggcactcag 3900
gtcagccggg ctcacccgcc ccagaccacc ggttggagag ggcagccagc cgagccaaca 3960
ctgtgcgcag cttccaccat gaaggtgagg catggagcag ggcgtggggg cggggagtca 4020
ccctgcaaag ccctccaccg tgggcagact gcagccgtcc ctgtagaggc agcttggccg 4080
gggcaccagc ggacgtttcc actcttgctt ctgtactatc gtttctgaat ctgattttaa 4140
ctcactgctt gtgtggtggg ggagccaggg attccccttt agtaactccg caccctcttc 4200
ctggcttgca gccagaagag ctactcctcc tggaagaatt ggagagaaat caagtgatgg 4260
ggaagatgag ggcaaaaggc atgcctctag tcagctaaac gtgcaagaat tccacagagg 4320
gaaaaggaga aaaagggagg cagattgaga tttctttaag tctgtttgga agcttttgct 4380
ctataaatct gccgcttaag ccagggtttt agggtagaca gagccaaggg cagagttttc 4440
agagatagta ttgaaaaatc aaagcccagg gccccaaagt ctttctaatt tatagttgat 4500
ctgggcctgg tttggaagat tttgaatccc aatctaatcc ccgtgggaga tcaatactac 4560
aatcaatctt attgtttcca caatgacttt cttgtcctgt gcttaaatct gagataggct 420
ctgagtagag acaaggcaag ccttcagata aaagcgtttg tagcagctgc ctgttttttt 4080
ttcatgtgca ccgaaatgtg gatttttttt tcttttatga tactacatgt ggtttttcta 4740
aggtgggata tttctgcttg tttcatcaga agggcattta gtggactgga aatgtcttac 4800
agcagctatt gaggtctgct gtacctaagt tcttagagca attagtcaaa aatatgttcc 4860
acttcaattc tttttctaca cttttaaatg cttctttggc ttaatacatt taaaatagag 4920
catgggtttc ttcaattcct agaaaagagt acaaaagtgt atatcacaga gcaaccactt 4980
ggcagatatt t ggggagttg ggagtgaagt tctctttctt gcctttccct gcttaggtgg 5040
taaatttcaa gtgggaaatt tacactgata atagactaat gggaaatggc acttccagat 5100
gttttctccc agtgtgaagg gtgacttata cttgtgagag tatttgttgg taatgggaat 5160
aagtcccaaa ggcaagccac atagcagaag atacgttctc attgaggcag ctacacatta 5220
cgacggggac actgaattga tcatcagttc atttacaagc acatttctaa gtgaggtgct 5280
ctctgctagc agaaatcaga tttgaaaggc agtaagatct cactccactc tttcagaatt 5340
CA 02512239 2005-06-29
WO 2004/065938 PCT/US2004/000990
3/7
catccaatga aagcagaaat cacctgttgt catatgtaaa atttgtgtgt atgtgtacat 5400
tctgccatct taaccctgaa atgattatag atccagctaa tcattcccag gtaatgctga 5460
ttagaatact tttttttttg tataggaatg taataagaac aactgtttta gacacctctt 5520
ctggaaattt agcatggaag ctctcaactt tatttttaag gcctggaaga tgctgtgtct 5580
ctgttacaac ttaaaaggaa gatcatttaa gttagttaac acctaaaaca ttccattgtg 5640
tgaggatttt atcagtgatg tctgcatatt ctcatcattc atctagaagt ggtttgatca 5700
gaactaaaca ggctacacgt tattcaactg tgttatttta acttaaaaag catgcttgag 5760
tttataaaat cagaatttat atctttgtga gtgtaaatgt tacctgagaa acagtacaga 5820
agtgaccaac ttgattaaaa tcaacttgta ataacttcag gtcttaatgc agttagataa 5880
tggagaaaag ctatgtaatt ttgccccaaa tttcaactaa tccatttctt gtctcattat 5940
gactaatata tcatccttaa tctggatgga tatagcactt ttttcaagac taatcattgt 6000
tgtatacacc caggatttgc ttttgataaa catccttgtg ccatgcatgc cacgaaaaaa 6060
gtttttggta aaccatgtga tgaaggttgc tggctcaaga acagaattta gtttctacag 6120
cattaatgag catttatttg aaaaaagacc ataaagaccc aatcataaga attacctgtt 6180
gggttttctt tgtaggtgtg atcgaatggt ttggtggaat tactcgacga gatatcatga 6240
tagcattctt tcaaccaata tgagtataat gcgaccatat cataggggat ctgagacaga 6300
attatcagtt gtatttttcc tattgaattt tgtctagtcc tttctccagt ggcttttatt 6360
tgggagaata tcagctttgc taaaatgtta ttgttttcaa gatcattaaa aagtgcttca 6420
gctacataga cctttggaaa ctgccattga acatagaaaa gtcagttctg caagtggaaa 6480
gagtgttttg tgtattgctg tagttggaaa cacattgaaa ctggttgact tcactggccc 6540
tccaaaaagt ctttatgctt ttttgtcaga tgggagagag aaagaccagg tgcttcttgt 6600
tctcctcact ctgaaggaca cagtcttctt tctacatgaa ataactggat tatttgcctc 6660
tgtgactgaa gctttcaaat agagattaac cctctttcca caaatataat tattatgaaa 6720
atatccatat aatagaaaag ttcaagaaat aactattgcc ctgcattaga gactttgtgg 6780
cacaaattcc cccgtgcaaa caacagattt ggacacatag atccaccaaa accaatactt 6840
acctggtatg gttccctagt ggccccaggt atttcattgt cattacagag gccacattaa 6900
gtaggaaaat tactctattt ggaaatggtt gttgagattg aggctttggt gtccagtgat 6960
acttccttgg cactgacatt ttccgttcca cctgtttttt agtggttccc ctaaatttct 7020
cttaatccct ttgcagtgaa ctattttgcg ttcttagact tgctctttgt gtattttcac 7080
tgagacaata agagaatatt tcatcattcc gaaggtgttg gtgttaaggg tgggcagagg 7140
ccaaatcagg gttgttgatg acaaccatgc tctctattcc tttatttgcc attcccttgt 7200
tgtatttttt ttaaaatgga atgtttttaa ccttttgtat ttgatatttt ttttctcctt 7260
gatcagttgt ctgttatttt attatctgga aaatcttata ttatactcag cctctttcat 7320
tttgtgttag ggcagtgact tccagcctta ctgattgcca gcatatcccc aggttttgtt 7380
gttgttgttg ttgttttact ggagattttt tagcccaaag tgtgttttaa aatcctcgaa 744D
gcataacggt aacttacttt tttgataaaa cttaccatac tttatttaga acaaaagggc 7500
agccacaaaa tagcagtggc tccttataaa atagacacat tccagtgggc cccgtcactt 7560
ttctgctcat ttctgtctgt tctgtccatc atacctaagt catatatttc tgttcattta 7620
gttgggacag aactcaccca atgttatcat tgtactaaat ataaatgtgc ccctaatggt 7680
tttgactttt gcttaagttt ttgagtcctc atgtatgtta ggtagtgcca tctagtagcc 7740
agaaatttgg gaactggctg ggcatgatgg ctaatacctg taatcccagc actttgggag 7800
gcttaggtgg gtggatcact tgaggtcagg agttccagac cagcctggcc aacatggtga 7860
aaccacatct ctactaaaat ataaaaaaat agccaggtat gatggcccat gcctgtaatc 7920
cgagctaatt gggaggctga gatgggagga ctgcttgaac ctgggaggtg gaggctgctg 7980
tgagccaaga ttgtgccact gcactccagc ctgggcaaca gagtgagacc ctgtgtcaca 8040
aaaacaagaa acaaaacaaa acaaaagaca agaaacctga gaagcgcagt agattcaatt 8100
atatatatct acttttaatt tgctagctct gtgaccttag gaaagttaca taacctctct 8160
gaact gcaac tgtttcattt acaaaatgga gataatgata gtttttctct aattggtttg 8220
ttgtgagata attcatataa agctgatggt gccagattac actcaaaaaa agcattcagc 8280
tgtcattatc attatgactt cttttgttaa tgttatagcc tttecttctc tagggaaaag 8340
gaggccagag tggacctagg ctgactgaga gaattcagct cagtcttttg aattattttg 8400
aggtagagga atgattgata tagtatagat tattaaatta ggacttcact tttggagaaa 8460
agttcagata tcattgttgt cttatttttc ttcactttcc cacatttttg cagccatagc 8520
tccatccatt tggttaagaa cttagaagct cacaaactcg ggtcaaagac aggtcgaaat 8580
cctcaaatcc cttaagaact tcagcttatt caggaaggga tatttacaga aaactagcaa 8640
ttgtataagt ctccaaaaaa gcatacatta cttgaggatc catatatttt tggcatcctc 8700
agggttgctg tgatgattta tagaaggttt gtttatttaa tttactttat ttcaaatagg 8760
ttttaatttt tgtaccctta agaaaagatt cgtactcttc cctggcagat taaagaaaat 8820
gagcgtatat tccctaacct tggccagtta ctttcctggg tttgagggtt tctgtgaacg 8880
tctaacttac ctctgtgacc tgtttctgca accaggggtg ttgcaatgga tgcttttgtc 8940
ttgaggatgg gacctttcaa gaaacagatt cactgaggtg cagtgggaag gtcagagaaa 9000
gatcttcgta tcgcctatta ttatttgctc gtctattttt tctcctttct taaggccact 9060
CA 02512239 2005-06-29
WO 2004/065938 PCT/US2004/000990
4/7
aactgattct cctttgctaa ggctgcctac ttccactgag accttgaacc acatgaaatt 9120
gttgttgtct gtgtttctgg tcaaatagtg gcaattttgt atgattcaat cttgtcattt 9180
aattttttgg gaggttatta ttctatttca tacctttttt atacccatct tctttacttc 9240
atttacctgt ccctcatact tgacttgtag cttgtccctt cactgtcatc gtctggccat 9300
gtgggtgtgt acgtgtgtgc gagagagaga atgtgtgaga atgtatgttt ctttatgcat 9360
tgggatttag ggtttttctt gcaattgtga tttctctggg cacttttgtt aatatagcta 9420
gtcagcgagt gctctagata attttccttg cctccccctc tttgaaagaa aagagggtgt 9480
tcttagatgt attcttatca gataagccag tagctcaggt gctggtctgg ctttggtgtc 9540
attggggtct gaggttgctg acttttacct tctctgctga aaaattacct tcagcagaaa 9600
cgtctgaatt gcaaggagaa ggagaaaaaa acaggccaaa cacagtcctt ggtactcctt 9660
gggagccact gagaagagtc caggttcaaa tggtcagaag gttattttaa tgattgtgtc 9720
tggcctaaag taccattagc ttccagtgga gtttagaatg tggatggatc ctgaaaggta 9780
ttccccagag gtttggatta ataggcacaa gggaacccta aaggactcta ttggcctgat 9840
actccccata tccacgtaga agagctttag aagaaccttc tgttctgaga ccctggctgg 9900
gcccacccag agctggccca ttcaactctt actcctttgc caccactaat ggttcttcta 9960
ctagttttta tattatttaa caaaaaggca ctttaaaaat gcactcctgg caatctatac 10020
tggaatatga aaaacatgct gcaaaacctt gacactccaa gtgtggtctt acagttccca 10080
gaatcccctc cttgaggagc tgctagaaat gctgaatctc aagcatctcc ccagacctac 10140
tgaatcagag cctgcatctg aagctttacg gtgtacaagc tgttttatgt gaaggctgaa 10200
gtttgaaaag cactgcatta aagcgttagt ttggtataaa ctgccctgac tgaacttggt 10260
gtgtccactt agcttgcatg atgactgttg ctttgatgat gaaggcttac acgggtagat 10320
cctttgagtg agtgatctga catgattctc ctttgctaag gcatctagat tcagtgcaca 10380
acttacagct gtttgtcttt aggggaaata caactgtaaa attaataaaa acatagtctc 10440
ttcttatgat aacatggaac gatggcaaaa tagattttgt tagcacttgg gtaggaattc 10500
tgaatgaagc aggcaaattc tgttggcagt gaaatgatag gatgtggtaa agttagaata 10560
aaataaactt aaatgtctca aactctcatg gtatatacta ccagtttaat aataatgttg 10620
tacctttgat gatttgcaga ctacaagcat tcaaggtgct gtgttatata ttacttgctt 10680
ggagaataat acttcttaaa aattgaaatt cagaaatttt aaatcagaca aagcttttgt 10740
gcatggccca cttaaatggc tattttgaaa taatgatagt ggatatagaa ggattattct 10800
~taataggat gagactgttc cttttgtcat ggagatcata atcatatttt tgtaaatttt 10860
tattattttt ttggttttgt gtccatcctg cacactatta ctgggtaggt acatggtttt 10920
ttaacatggt ttatctttca aaactataaa ggcattgcaa acagaagaca ggtcatttat 10980
~tttcttcca aaagcatcta aaatgagatt ttgatatttg aggtcataaa gaggtgagag 11040
~acagacaac agttgggaaa gctatttctc ttgaaattgt ttggccttaa ttactacagt 11100
~tcctagtac cacccatacg tttccaaaga agtagatccc tgtaaatgcc tt tgtctctg 11160
~acttttgag taaaatagta gggtgtgctt tgcaaaatgt catcgttgat gttgagtttc 11220
~gagtcttta attaggaagc tgaaatctgt atat,cgagat ttgtaaatca tctaaattgc 11280
igagtaatgt tttagaatac tgcttaaggg attggcatta aagccttttt taaaaaagaa 11340
~tgcaataat ttcctcaaat cctcactcat tagacctcta ctaactatag tgctgacttt 11400
ttttttttt taccctaaag tctggaattc caaagaaatg cttcaccatt tcccccatta 11460
tatagccac ctggaagcag tattcatgta ttagatcaaa aacacaacaa agaattatga 11520
aggttgttt cctggtatgc aatgcatgat gacatgaact tacagaacag agagaaggga 11580
gctccatgt ttatttaaag aggaaatttt tattttctgg ttacctactt ttacatgggt 11640
acatcaaat cccacgatga ggtttaaaaa ttctcataga taatcaaacg tcattacttg 11700
cttactgaa attcagactt ttcttttttc ttccctgttt ttctctatca aattagaatc 11760
ttggaagaa ctaccagaaa cgagtgggaa aacaacccgg agattcttct ttaatttaag 11820
tctatcccc acggaggagt ttatcacctc agcagagctt caggttttcc gagaacagat 11880
caagatgct ttaggaaaca atagcagttt ccatcaccga attaatattt atgaaatcat 11940
aaacctgca acagccaact cgaaattccc cgtgaccaga cttttggaca ccaggttggt 12000
aatcagaat gcaagcaggt gggaaagttt tgatgtcacc cccgctgtga tgcggtggac 12060
gcacaggga cacgccaacc atggattcgt ggtggaagtg gcccacttgg aggagaaaca 12120
ggtgtctcc aagagacatg ttaggataag caggtctttg caccaagatg aacacagctg 12180
tcacagata aggccattgc tagtaacttt tggccatgat ggaaaagggc atcctctcca 12240
3aaagagaa aaacgtcaag ccaaacacaa acagcggaaa cgccttaagt ccagctgtaa 12300
~gacaccct ttgtacgtgg acttcagtga cgtggggtgg aatgactgga ttgtggctcc 12360
~cggggtat cacgcctttt actgccacgg agaatgccct tttcctctgg ctgatcatct 12420
~actccact aatcatgcca ttgttcagac gttggtcaac tctgttaact ctaagattcc 12480
~aggcatgc tgtgtcccga cagaactcag tgctatctcg atgctgtacc ttgacgagaa 12540
~aaaaggtt gtattaaaga actatcagga catggttgtg gagggttgtg ggtgtcgcta 12600
:acagcaaa attaaataca taaatatata tatatatata tattttagaa aaaagaaaaa 12660
~caaacaaa caaaaaaacc ccaccccagt tgacacttta atatttccca atgaagactt 12720
~tttatgga atggaatgga aaaaaaaaca gctattttga aaatatattt atatctacga 12780
CA 02512239 2005-06-29
WO 2004/065938 PCT/US2004/000990
5/7
aaagaagttg ggaaaacaaa tattttaatc agagaattat tccttaaaga tttaaaatgt 12840
atttagttgt acattttata tgggttcaac cccagcacat gaagtataat ggtcagattt 12900
attttgtatt tatttactat tataaccact ttttaggaaa aaaatagcta atttgtattt 12960
atatgtaatc aaaagaagta tcgggtttgt acataatttt ccaaaaattg tagttgtttt 13020
cagttgtgtg tatttaagat gaaaagtcta catggaaggt tactctggca aagtgcttag 13080
cacgtttgct tttttgcagt gctactgttg agttcacaag ttcaagtcca gaaaaaaaaa 13140
gtggataatc cactctgctg actttcaaga ttattatatt attcaattct caggaatgtt 13200
gcagagtgat tgtccaatcc atgagaattt acatccttat taggtggaat atttggataa 13260
gaaccagaca ttgctgatct attatagaaa ctctcctcct gccccttaat ttacagaaag 13320'
aataaagcag gatccataga aataattagg aaaacgatga acctgcagga aagtgaatga 13380
tggtttgttg ttcttctttc ctaaattagt gatcccttca aaggggctga tctggccaaa 13440
gtattcaata aaacgtaaga tttcttcatt attgatattg tggtcatata tatttaaaat 13500
tgatatctcg tggccctcat caagggttgg aaatttattt gtgttttacc tttacctcat 13560
ctgagagctc tttattctcc aaagaaccca gttttctaac tttttgccca acacgcagca 13620
aaattatgca catcgtgttt tctgcccacc ctctgttctc tgacctatca gcttgctttt 13680
ctttccaagg ttgtgtgttt gaacacattt ctccaaatgt taaacctatt tcagataata 13740
aatatcaaat ctctggcatt tcattctata aagtccaacc tgtaagagaa aatggtgcat 13800
ttgtatagcg cttacaatga tgaccttgtg tttgcatttt tgtttctgaa gttatatatt 13860
ttagaggggg tgggggaaag gtaatgaatg gctggaaaat tgcaggcaag ttatttgata 13920
agtcatattt gcactaaagg tgttaccagt gatttagtat ttttcaaatg aacttctttg 13980
gggcagaaag atttaaggga aaactaaagc ctacaaaaca agcaaaacct ggataacccg 14040
agataaagtt tcagagataa tagcccatgc aacagaggca acggtgccag aaaattagaa 14100
agggaaagtg tcggagatca gcttctataa gaacatctgc cagttggact gacgcccaaa 14160
cagaatgaag tcaaattagg ctgctcagat tgaacactta ccagagtgtc agggcttctg 14220
taccctgggt tagaatcaga ccaaggaagg gttcagcaga tgttcataag agcagggcac 14280
ccacaactac ccactatttt actggcagta ttttaggtca gtttccagga ctttgcatcc 14340
cctctgatcc tgccatgcat gattggtgaa acctacctct aatctccttg gaattggcta 14400
aaaaacagtg tgtttataat ggaacagact gttataatca aattcttcct aggaattaac 14460
ttttgatgac tatgagctta gttacagttc ggaggttatg aggttatgta aaccttatct 14520
ttaaatgtgc atgacagtta tcttttacta atgctggtta acttttaaaa tcttgcagct 14580
cctttttatc tctagttcta ttgttcttga ttaggtgaga accattagat catacccaac 14640
tgaggggatt ggggtcttgt ttgttctcca gctgttcttc accctctatt gccatggaca 14700
tgaaggacag actgcacggt cttaacatgt taaaacgaat gacccatgtt ttctcatat 14759
<210> 2
<211> 396
:212> PRT
:213> Homo Sapiens
:400> 2
Zet Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val
1 5 10 15
~eu Leu Gly Gly Ala A1a Gly Leu Val Pro Glu Leu Gly Arg Arg Lys
20 25 30
she Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu
35 40 45
'al Leu Ser G1u Phe G1u Leu Arg Leu Leu Ser Met Phe Gly Leu Lys
50 55 60
a1n Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu
70 75 g0
sp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp
85 90 95
is Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe
100 105 110
is His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr
115 120 125
hr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe
130 135 140
1e Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala
45 150 155 160
CA 02512239 2005-06-29
WO 2004/065938 PCT/US2004/000990
6/7
Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn I1e Tyr Glu Ile
165 170 175 .
Tle Lys Pro Ala Thr A1a Asn Ser Lys Phe Pro Val Thr Arg Leu Leu
180 185 190
Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp
195 200 205
Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His
210 215 220
Gly Phe Val Val Glu Va1 Ala His Leu Glu Glu Lys G1n Gly Val Ser
225 230 235 240
Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Sex
245 250 255
Trp Ser Gln T1e Arg Pro Leu Leu Val Thr Phe G1y His Asp Gly Lys
260 265 270
Gly His Pro Leu His Lys Arg Glu Lys Arg Gln Ala Lys His Lys Gln
275 280 285
Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val Asp
290 295 300
Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro G1y Tyr
305 310 315 320
His Ala Phe Tyr Cys His Gly G1u Cys Pro Phe Pro Leu Ala Asp His
325 330 335
Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val
340 345 350
Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala
355 360 365
Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn
370 375 380
Tyr Gln Asp Met Val Val Glu G1y Cys Gly Cys Arg
385 390 395
<210> 3
<211> 281
<212> PRT
<213> Homo Sapiens
<400> 3
G1u Ser Leu G1u Glu Leu Pro Glu Thr Ser Gly Lys Thr Thr Arg Arg
1 5 10 15
Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu G1u Phe Tle Thr Ser
20 25 30
A1a G1u Leu Gln Val Phe Arg Glu G1n Met Gln Asp Ala Leu Gly Asn
35 40 45
Asn Ser Ser Phe His His Arg Ile Asn Tle Tyr Glu Ile Ile Lys Pro
50 55 60
Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu Asp Thr Arg
65 70 75 80
Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp Val Thr Pro
85 90 95
A1a Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His Gly Phe Val
100 105 110
Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser Lys Arg His
115 120 125
Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser Trp Ser Gln
130 135 140
Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys Gly His Pro
145 150 155 160
Leu His Lys Arg G1u Lys Arg Gln Ala Lys His Lys Gln Arg Lys Arg
165 170 175
Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp
180 185 190
CA 02512239 2005-06-29
WO 2004/065938 PCT/US2004/000990
7/7
Val Gly Trp Asn Asp Trp Ile Va1 Ala Pro Pro Gly Tyr His Ala Phe
195 200 205
Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser
210 215 220
Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys
225 230 235 240
Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met
245 250 255
Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp
260 265 270
Met Val Val G1u G1y Cys Gly Cys Arg
275 280
