Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR DIAGNOSING OSTEOPOROSIS OR A SUSCEPTIBILITY TO
OSTEOPOROSIS BASED ON HAPLOTYPE ASSOCIATION
RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No.
60/440,899, filed on January 16, 2003, and claims the benefit of U.S.
Provisional
Application No. 60/450, 652, filed on February 27, 2003. .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 corresponding decrease in bone mass
experienced during menopause increase both the probability of osteoporotic
fracture
and the number of potential fracture sites. However, aging women are not the
only
demographic group at risk. Young women who are malnourished, amenorrheic, 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 puberty, so
elderly or hypogonadal men face the risk of osteoporosis if their bones were
insufficiently developed.
The need to find a cure for this disease is complicated by the fact that there
are maazy contributing factors that lead to osteoporosis. Nutrition
(particularly
calcium, vitamin D and vitamin K intake), hormone levels, age, sex, race, body
weight, activity level, and genetic factors all influence the variance seen in
bone
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mineral density among individuals. Currently, the drugs approved to treat
osteoporosis act as inhibitors of bone reabsorption. Treatment regimens
include
methods such as hormone replacement therapy (HRT), the use of selective
estrogen
receptor modulators, calcitonin, and biophosphonates. However, these
treatments
may not individually reduce risk with consistent results. Moreover, while some
therapies improve BMD when co-administered, others show no improvement or
even loss of 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 detection of a
susceptibility to the
disease is therefore desirable.
SUMMARY OF THE INVENTION
As described herein, it has been discovered that particular combinations of
genetic markers ("haplotypes"), are present at a higher than expected
frequency in
patients with phenotypes associated with osteoporosis and a susceptibility to
osteoporosis. The markers that are included in the haplotypes described herein
are
associated with the genomic region that directs expression of the human bone
morphogenetic protein 2 (BMP2).
In one embodiment, the invention is directed to a method of diagnosing
osteoporosis or a susceptibility to osteoporosis in an individual, comprising
detecting the presence or absence of an at-risk haplotype, comprising a
haplotype
selected from the group consisting of: haplotype I, haplotype II, haplotype a,
haplotype b, haplotype c, haplotype d and combinations thereof; wherein the
presence of the haplotype is indicative of osteoporosis or a susceptibility to
osteoporosis. In a particular embodiment, the invention is directed to
assaying for
the presence of a first nucleic acid molecule in a sample, comprising
contacting said
sample with a second nucleic acid molecule comprising the one or more
haplotypes
described herein. In one embodiment, determining the presence or absence of
the
haplotype comprises enzymatic amplification of nucleic acid from the
individual. In
a particular embodiment, determining the presence or absence of the haplotype
further comprises electrophoretic analysis. For example, in one embodiment,
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determining the presence or absence of the haplotype comprises restriction
fragment
length polymorphism analysis. In another embodiment, determining the presence
or
absence of the haplotype comprises sequence analysis. .
In another embodiment, the invention is directed to a method of diagnosing
osteoporosis or a susceptibility to osteoporosis in an individual, comprising
detecting the presence or absence of an at-risk haplotype comprising haplotype
I,
wherein the presence of the haplotype is indicative of osteoporosis or a
susceptibility
to osteoporosis. In a particular embodiment, determining the presence or
absence of
the haplotype comprises enzymatic amplification of nucleic acid from the
individual.
In a particular embodiment, determining the presence or absence of the
haplotype
fiu-ther comprises electrophoretic analysis. For example, in one embodiment,
determining the presence or absence of the haplotype comprises restriction
fragment
length polymorphism analysis. In another embodiment, determining the presence
or
absence of the haplotype comprises sequence analysis.
In another embodiment, the invention is directed to a method of diagnosing
osteoporosis or a susceptibility to osteoporosis in an individual, comprising
detecting the presence or absence of an at-risk haplotype comprising haplotype
II,
wherein the presence of the haplotype is indicative of osteoporosis or a
susceptibility
to osteoporosis. In a particular embodiment, determining the presence or
absence of
the haplotype comprises enzymatic amplification of nucleic acid from the
individual.
In a particular embodiment, determining the presence or absence of the
haplotype
further comprises electrophoretic analysis. For example, in one embodiment,
determining the presence or absence of the haplotype comprises restriction
fragment
length polymorphism analysis. In another embodiment, determining the presence
or
absence of the haplotype comprises sequence analysis.
In another embodiment, the invention is directed to a kit for assaying a
sample for the presence of a haplotype associated with osteoporosis, wherein
the
haplotype comprises two or more specific alleles, and wherein the kit
comprises one
or more nucleic acids capable of detecting the presence or absence of two or
more of
the specific alleles, thereby indicating the presence or absence of the
haplotype in the
sample. In a particular embodiment, the nucleic acid comprises a contiguous
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nucleotide sequence that is completely complementary to a region comprising
specific allele of the haplotype.
In another embodiment, the invention is directed to a reagent kit for assaying
a sample for the presence of a haplotype associated with osteoporosis, wherein
the
haplotype comprises two or more specific alleles, comprising in separate
containers: .
a) one or more labeled nucleic acids capable of detecting one or more specific
alleles
of the haplotype; and b) reagents for detection of said label. In a particular
embodiment, the labeled nucleic acid comprises a contiguous nucleotide
sequence
that is completely complementary to a region comprising specific allele of the
haplotype.
In yet another embodiment, the invention is directed to a reagent kit for
assaying a sample for the presence of a haplotype associated with
osteoporosis,
wherein the haplotype comprises two or more specific alleles, wherein the kit
comprises one or more nucleic acids comprising a nucleotide sequence that is
at
least partially complementary to a part of the nucleotide sequence of the BMP2
gene,
and wherein the nucleic acid is capable of acting as a primer for a primer
extension
reaction capable of detecting one or more of the specific alleles of the
haplotype.
In another embodiment, the invention is directed to a method for the
diagnosis and identification of susceptibility to osteoporosis in an
individual,
comprising: screening for an at-risk haplotype associated with BMP2 that is
more
frequently present in an individual susceptible to osteoporosis compared to an
individual who is not susceptible to osteoporosis wherein the at-risk
haplotype
increases the risk significantly. In a particular embodiment, the significant
increase
is at least about 20%. In another embodiment, the significant increase is
identified
as an odds ratio of at least about 1.2.
In another embodiment, the invention is directed to a method.for diagnosing
a susceptibility to osteoporosis in an individual, comprising determining the
presence or absence in the individual of a haplotype, comprising two or more
alleles
selected from the group consisting of: TSC0898956, B420, B8463, D20S846,
TSC0191642, P4337, D20S892, B5048, B9082, D20S59, B7111/rs235764,
B12845/rs15705, P9313, B10631, D35548, rs1116867, TSC0278787, D35548 and
TSC0271643; wherein the presence of the haplotype is indicative of
susceptibility to
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osteoporosis. In a particular embodiment, determining the presence or absence
of
the haplotype fiu-ther comprises electrophoretic analysis. For example, in one
embodiment, determining the presence or absence of the haplotype comprises
restriction fragment length polymorphism analysis. In another embodiment,
determining the presence or absence of the haplotype comprises sequence
analysis.
In yet another embodiment, the invention is directed to a method for
diagnosing a susceptibility to osteoporosis in an individual, comprising
obtaining a
nucleic acid sample from the individual; and analyzing the nucleic acid sample
for
the presence or absence of a haplotype comprising two or more alleles selected
from
the group consisting of: TSC0898956, B420, B8463, D20S846, TSC0191642,
P4337, D20S892, B5048, B9082, D20S59, B7111/rs235764, B12845/rs15705,
P9313, B10631, D35548, rs1116867, TSC0278787, D35548 and TSC0271643,
wherein the presence of the haplotype is indicative of susceptibility to
osteoporosis.
In a particular embodiment, the alleles are selected from the group consisting
of:
TSC0898956, B420, B8463, D20S846 and TSC0191642. In a particular
embodiment, the alleles are selected from the group consisting of: P4337,
D20S892,
B5048, B9082 and D20S59. In a different embodiment, the haplotype comprises
B7111/rs235764 and B12845/rs15705. In a particular embodiment, the alleles are
selected from the group consisting of: P9313, B10631 and D35548. In a
particular
embodiment, the alleles are selected from the group consisting of: rs1116867,
TSC0278787 and D35548. In another embodiment, the alleles are selected from
the
group consisting of: TSC0271643, P9313 and B7111.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a tabular presentation of haplotype association data for haplotypes
a, b and c for various phenotypes (as indicated, including BMP from spine and
hip,
osteoporotic fracture, weight corrected BMD). Data are also presented for pre-
and
post-menopausal patients.
FIG. 2 is a tabular presentation of haplotype association data for haplotype I
and haplotype II. Data are presented for fracture and weight corrected BMD for
hip
and spine.
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FIG. 3 is a tabular presentation of haplotype d for various phenotypes (as
indicated, including BMD from spine and hip, osteoporotic fracture, weight
corrected BIVff~). The BMD values represent the lowest 10"' percentile in all
cases.
Data are also presented for pre- and post-menopausal patients.
DETAILED DESCRIPTION OF THE INVENTION
As described herein, Applicant has completed linkage analysis between
osteoporosis phenotypes and particular combinations of genetic markers
("haplotypes") associated with the genomic region, located on chromosome 20,
that
directs expression of the human bone morphogenetic protein 2 (BMP2). The
results
shown here represent the first demonstration of haplotypes used to indicate
osteoporosis or a susceptibility to osteoporosis. Based on the linkage studies
conducted, Applicant has discovered a direct relationship between the BMP2-
associated haplotypes and osteoporosis. In particular, it has been discovered
that
particular haplotypes appear at higher than expected frequencies in patients
with
phenotypes associated with osteoporosis and a susceptibility to osteoporosis.
Methods for the diagnosis of osteoporosis based on this association, in
combination
with, for example, bone turnover marker assays (e.g., bone scans), are
described
herein. Additionally, methods based on the detection of at least one haplotype
described herein is diagnostic of a susceptibility to osteoporosis.
DIAGNOSTIC AND SCREENING ASSAYS OF THE INVENTION
The present invention pertains to methods of diagnosing or aiding in the
diagnosis of osteoporosis or a susceptibility to osteoporosis by detecting
particular
genetic marlcers that appear more frequently in individuals with osteoporosis
or who
are susceptible to osteoporosis. Diagnostic assays can be designed for
assessing
BMP2. Such assays can be used alone or in combination with other assays, e.g.,
bone turnover marker assays (e.g., bone scans). Combinations of genetic
marlcers
are referred to herein as "haplotypes," and the present invention describes
methods
whereby detection of particular haplotypes is indicative of osteoporosis or a
susceptibility to osteoporosis. The detection of the particular genetic
markers that
malce up the particular haplotypes can be performed by a variety of methods
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described herein and known in the art. For example, genetic markers can be
detected at the nucleic acid level, e.g., by direct sequencing or at the amino
acid
level if the genetic marker affects the coding sequence of BMP2, e.g., by
immunoassays based on antibodies that recognize the BMP2 protein or a
particular
BMP2 variant protein.
In one embodiment, the assays are used in the context of a biological sample
(e.g., blood, serum, cells, tissue) 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
prognostic (or predictive) assays for determining whether an individual is
susceptible to developing osteoporosis. For example, variations in a nucleic
acid
sequence can be assayed in a biological sample. Such assays can be used for
prognostic or predictive purposes to thereby allow for the prophylactic
treatment of
axi individual prior to the onset of symptoms associated with osteoporosis.
DIAGNOSTIC ASSAYS
In one embodiment of the invention, diagnosis of a susceptibility to
osteoporosis is made by detecting a haplotype associated with BMP2 as
described
herein. The BMP2-associated haplotypes describe a set of genetic markers
associated with BMP2. In a certain embodiment, the haplotype can comprise one
or
more markers, two or more markers, three or more markers, four or more
markers,
or five or more markers. The genetic markers are particular "alleles" at
"polymorphic sites" associated with BMP2. A nucleotide position at which more
than one sequence is possible in a population (either a natural population or
a
synthetic population, e.g., a library of synthetic molecules), is referred to
herein as a
"polymorphic site". Where a polymorphic site is a single nucleotide in length,
the
site is referred to as a single nucleotide polymorphism ("SNP"). For example,
if at a
pa1-ticular chromosomal location, one member of a population has an adenine
and
another member of the population has a thymine at the same position, then this
position is a polymorphic site, and, more specifically, the polymorphic site
is a SNP.
Polymorphic sites can allow for differences in sequences based on
substitutions,
insertions or deletions. Each version of the sequence with respect to the
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polymorphic site is referred to herein as an "allele" of the polymorphic site.
Thus, in
the previous example, the SNP allows for both an adenine allele and a thymine
allele.
Typically, a reference sequence is referred to for a particular sequence.
Alleles that differ from the reference are referred to as "variant" alleles.
For
example, the reference BMP2 sequence is described herein by SEQ ID NO:1. The
term, "variant BMP2", as used herein, refers to a BMP2 sequence that differs
from
SEQ ID NO:l, but is otherwise substantially similar. The genetic markers that
malce
up the haplotypes described herein are BMP2 variants. The variants of BMP2
that
are used to determine the haplotypes disclosed herein~of the present invention
are
associated with a susceptibility to a number of osteoporosis phenotypes.
Additional variants can include changes that affect a polypeptide, e.g., the
BMP2 polypeptide. These sequence differences, when compared to a reference
nucleotide sequence, can include the insertion or deletion of a single
nucleotide, or
of more than one nucleotide, resulting in a frame shift; the change of at
least one
nucleotide, resulting in a change in the encoded amino acid; the change of at
least
one nucleotide, resulting in the generation of a premature stop codon; the
deletion of
several nucleotides, resulting in a deletion of one or more amino acids
encoded by
the nucleotides; the insertion of one or several nucleotides, such as by
unequal
recombination or gene conversion, resulting in an interruption of the coding
sequence of a reading frame; duplication of all or a part of a sequence;
transposition;
or a rearrangement of a nucleotide sequence. 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 sluft, the frame shift can result in a
change in
the encoded amino acids, andlor can result in the generation of a premature
stop
codon, causing generation of a truncated polypeptide. Alternatively, a
polymorphism associated with a susceptibilitST to osteoporosis can be a
synonymous
change in one or more nucleotides (i.e., a change that does not result in a
change in
the BMP2 amino acid sequence). Such a polymorphism can, for example, alter
splice sites, affect the stability or transport of mRNA, or otherwise affect
the
transcription or translation of the polypeptide. The polypeptide encoded by
the
reference nucleotide sequence is the "reference" polypeptide with a particular
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reference amino acid sequence, and polypeptides encoded by variant alleles are
referred to as "variant" polypeptides with variant amino acid sequences.
Haplotypes are a combination of genetic markers, e.g., particular alleles at
polymorphic sites. The haplotypes described herein are associated with
osteoporosis
and/or a susceptibility to osteoporosis. Therefore, detection of the presence
or
absence of the haplotypes herein is indicative of osteoporosis, a
susceptibility to
osteoporosis or a lack thereof. Detection of the presence or absence of these
haplotypes, therefore, is necessary for the purposes of the invention, iri
order to
detect osteoporosis or a susceptibility to osteoporosis. The haplotypes
described
herein are a combination of various genetic markers, e.g., SNPs and
microsatellites.
Therefore, detecting haplotypes can be accomplished by methods known in the
art
for detecting sequences at polymorphic sites.
In a first method of diagnosing a susceptibility to osteoporosis,
hybridization
methods, such as Southern analysis, Northern analysis, or iTZ 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, br 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, or tissue sample from skin, muscle, buccal or
conjunctiva)
n lucosa, 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 an allele of the haplotype can be indicated by sequence-specific.
hybridization of a nucleic acid probe specific for the pauicular allele. A
sequence-
specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA. A
"nucleic acid probe", as used herein, can be a DNA probe or an RNA probe that
hybridizes to a complementary sequence. One of skill in the art would know how
to
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design such a probe such that sequence specific hybridization will occur only
if a
particular allele is present in a genomic sequence from a test sample.
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
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
N0:1,
optionally comprising at least one allele contained in the haplotypes
described
herein, or the probe can be the complementary sequence of such a sequence.
Other
suitable probes for use in the diagnostic assays of the invention are
described herein.
The hybridization sample is maintained under conditions that are sufficient to
allow specific hybridization of the nucleic acid probe to BMP2. "Specific
hybridization", as used herein, indicates exact hybridization (e.g., with no
mismatches). Specific hybridization can be performed under high stringency
conditions or moderate stringency conditions (see below). 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 the sample contains the allele that is present in the nucleic
acid probe.
The process can be repeated for the other marlcers that make up the haplotype,
or
multiple probes can be used concurrently to detect more than one marker at a
time.
Detection of the particular markers of the haplotype in the sample is
indicative that
the source of the sample has the particular haplotype and therefore has
osteoporosis
or a susceptibiliy to osteoporosis.
In another hybridization method, Northern analysis (see Current Protocols in
Molecular Biology, Ausubel, F. et al., eds., Jolm Wiley & Sons, sups°a)
is used to
identify the presence of a polymorphism associated with a susceptibility to
osteoporosis. For Northern analysis, a test sample of RNA is obtained from the
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individual by appropriate means. Specific hybridization of a nucleic acid
probe, as
described above, to RNA from the individual is indicative of a particular
allele
complementary to the probe.
For representative examples of use of nucleic acid probes, see, for example,
U.S. Patents No. 5,288,611 and 4,851,330.
Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a
nucleic acid probe in the hybridization methods described above. PNA is a DNA
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. et al., 1994.
Baocohjug.
C7Zern., 5:3-7). The PNA probe can be designed to specifically hybridize to a
molecule in a sample suspected of containing one of the genetic marlcers of
the
haplotypes associated with a susceptibility to osteoporosis. Hybridization of
the
PNA probe is diagnostic for osteoporosis or a susceptibility to osteoporosis.
In one embodiment of the invention, diagnosis of osteoporosis or a
susceptibility to osteoporosis associated with BMP2 or a haplotype associated
with
osteoporosis, can be made by expression analysis using quantitative PCR
(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 polymorphisms and haplotypes. 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, analysis by restriction digestion can be
used to detect a particular allele if the allele results in the creation or
elimination of a
restriction site relative to a reference sequence. A test sample containing
genomic
DNA is obtained from the individual. Polymerase chain reaction (PCR) can be
used
to amplify the genomic BMP2 region (including flankitlg sequences if
necessary) in
the test sample from the test individual. RFLP analysis is conducted as
described
(see Current Protocols in Molecular Biology, supra). The digestion pattern of
the
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relevant DNA fragment indicates the presence or absence of the particular
allele in
the sample.
Sequence analysis can also be used to detect specific alleles at polymorphic
sites associated with BMP2. A test sample of DNA or RNA is obtained from the
test individual. PCR or other appropriate methods can be used to amplify BMP2
and/or its flanking sequences, if desired. The presence of a specific allele
is thus
detected directly by sequencing the polymorphic site of the genomic DNA in the
sample.
Allele-specific oligonucleotides can also be used to detect the presence of a
particular allele at a polymorpluc site associated with BMP2, through the use
of dot-
blot hybridization of amplified oligonucleotides with allele-specific
oligonucleotide
(ASO) probes (see, for example, Saiki, R. et al., 1986. Natm°e, 324:163-
166). An
"allele-specific oligonucleotide" (also referred to herein as an "allele-
specific
oligonucleotide probe") is an oligonucleotide of approximately 10-50 base
pairs or
approximately 15-30 base pairs, that specifically hybridizes to BMP2, and that
contains a specific allele at a pol5nnorphic site as indicated by the
haplotypes
described herein. 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). PCR can be used to amplify all
or a
fragment of BMP2, as well as genomic flanking sequences. The DNA containing
the amplified BMP2 (or fragment of the gene) is dot-blotted, using standard
methods
(see CLU-rent Protocols in Molecular Biology, supra), and the blot is
contacted with
the oligonucleotide probe. The presence of specific hybridization of the probe
to the
amplified BMP2 is then detected. Specific hybridization of an allele-specific
oligonucleotide probe to DNA from the individual is indicative of a specific
allele at
a polymorphic site associated with BMP2.
An allele-specific primer hybridizes to a site on target DNA overlapping a
polymorphic site and only primes amplification of an allelic form to which the
primer exhibits perfect complementarity (Gibbs, R. et al., 1989. Nucleic Acids
Res.,
17:2437-2448). This primer is used in conjunction with a second primer, which
hybridizes at a distal site on the opposite strand. Amplification proceeds
from the
two primers, resulting in a detectable product, which indicates the particular
allelic
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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 oomplementarity to a distal site. The single-base mismatch
prevents
amplification and no detectable product is formed. The method works best when
the
mismatch is included in the 3'-most position of the oligonucleotide aligned
with the
polymorphism because this position is most destabilizing to elongation from
the
primer (see, e.g., WO 93/22456).
With the addition of such analogs as locked nucleic acids (LNAs), the size of
primers and probes can be reduced to as few as 8 bases. LNAs are a novel class
of
bicyclic DNA analogs in which the 2' and 4' positions in the furanose ring are
joined
via an O-methylene (oxy-LNA), S-methylene (thio-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 iri Tm are also obtained when LNA monomers
are used in combination with standard DNA or RNA monomers. For primers and
probes, depending on where the LNA monomers are included (e.g., the 3' end,
the
5'end, or in the middle), the Tm could be increased considerably.
In another embodiment, arrays of oligonucleotide probes that are
complementary to target nucleic acid sequence segments from an individual, can
be
used to identify polynorphisms in a BMP2 nucleic acid. For example, in one
embodiment, an oligonucleotide array can be used. Oligonucleotide arrays
typically
comprise a plurality of different oligonucleotide probes that are coupled to a
surface
of a substrate in different known locations. These oligonucleotide arrays,
also
described as "GenechipsTM," have been generally described in the art, for
example,
U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and
92/10092. These arrays can generally be produced using mecha~ucal 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. Scievrce, 251:767-773; Pirrung et al., U.S. Pat. No.
5,143,854
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(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
allowed to hybridize with the array. Detection of hybridization is a detection
of a
pal-ticular allele in the nucleic acid of interest. Hybridization and scanning
are
generally carried out by methods described herein and also in, e.g., published
PCT
ApplicationNos. 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
polylnorphic markers, is amplified by well known amplification techniques,
e.g.,
PCR. Typically this involves the use of primer sequences that are
complementary to
the two strands of the target sequence, both upstream and downstream, from the
polylnorphic site. Asymmetric PCR techniques can also be used. Amplified
target,
generally incorporating a label, is then allowed to hybridize with the array
under
appropriate conditions that allow for sequence-specific hybridization. Upon
completion of hybridization and washing of the array, the array is scamled 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 polymorphic site, arrays can include multiple detection
blocks,
and thus be capable of analyzing multiple, specific polymorphisms. In
alternate
arrangements, it will generally be understood that detection blocks can be
grouped
within a single array or in multiple, separate allays 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
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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 U.S. Patents 5,858,659 and
5,837,832,
_ the entire teachings of which are incorporated by reference herein.
Other methods of nucleic acid analysis can be used to detect a particular
allele at a polynorphic site associated with BMP2. Representative methods
include,
for example, direct manual sequencing (Church and Gilbert, 1988. P~°oe.
Natl. Acad.
Sci. USA, 81:1991-1995; Sanger, F. et al., 1977. Pnoc. Natl. Acad. Sci. USA,
74:5463-5467; Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescent
sequencing; single-stranded conformation polymorphism assays (SSCP); clamped
denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis
(DGGE) (Sheffield, V. et al., 1989. P~oc. Natl. Acad. Sci. USA, 86:232-236),
mobility shift analysis (Orita, M. et al., 1989. P~oc. Natl. Acad. Sci. USA,
86:2766-
2770), restriction enzyme analysis (Flavell, R. et al., 1978. Cell, 15:25-41;
Geever,
R. et al., 1981. Ps°oc. Natl. Acad. Sci. USA, 78:5081-5085);
heteroduplex analysis;
chemical mismatch cleavage (CMC) (Cotton, R. et al., 1985. P~°oc. Natl.
Acad. Sci.
USA, 85:4397-4401); RNase protection assays (Myers, R. et al., 1985. Science,
230:1242-1246); use of polypeptides that recognize nucleotide mismatches, such
as
E. colt mutS protein; and allele-specific PCR.
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 in those instances where the genetic marker contained in a
haplotype described herein results in a change in the expression of the
polypeptide
(e.g., an altered amino acid sequence or a chaazge in expression levels). 'A
variety of
methods can be used to make such a detection, including enzyme linked
immunosorbent assays (ELISA), 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
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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 to
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 who is not affected by osteoporosis or a
susceptibility to 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, can be indicative of a specific allele ilz the instance where the
allele alters a
splice site relative to the reference. Various means of examining expression
or
composition of the polypeptide encoded by BMP2 can be used, including
spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and
immunoassays
(e.g., David et al., LT.S. Pat. No. 4,376,110) such as immunoblotting (see
also
Current Protocols in Molecular Biology, particularly chapter 10).
For example, in one embodiment, alz 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
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end-labelhzg 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 variant BMP2, or an antibody that
specifically
~ binds to a polypeptide encoded by a reference allele, can be used to
identify the
presence in a test sample of a polypeptide encoded by a variant BMP2 allele,
or the
absence in a test sample of a polypeptide encoded by the reference allele.
In one embodiment of this method, the level or amount of polypeptide
encoded by BMP2 in a test sample is compared 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 sample, 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 particular allele responsible for causing the
difference in
expression. Alternatively, the composition of the polypeptide encoded by BMP2
in
a test sample is compared with the composition of the polypeptide encoded by
BMP2 in a control sample. 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.
Kits 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 RFLP analysis), allele-specific
oligonucleotides,
antibodies which bind to altered or to non-altered (native) BMP2 polypeptide
(e.g.,
to SEQ ID N0:2 and comprising at least one genetic marker included in the
haplotypes described herein), means for amplification of nucleic acids
comprising
BMP2, 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).
Kits (e.g., reagent kits) useful in the methods of diagnosis comprise
components useful in any of the methods described herein, including for
example,
hybridization probes or primers as described herein (e.g., labeled probes or
primers),
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reagents for detection of labeled molecules, restriction enzymes (e.g., for
RFLP
analysis), allele-specific oligonucleotides, antibodies that bind to altered
or to
non-altered (native) BMP2 polypeptide, means for amplification of nucleic
acids
comprising a BMP2, or means for analyzing the nucleic acid sequence of a BMP2
nucleic acid or for analyzing the amino acid sequence of a BMP2 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 an at-risk haplotype that is more
frequently present in an individual having osteoporosis or is susceptible to
osteoporosis. The primers can be designed using portions of the nucleic acids
flanking SNPs that are indicative of osteoporosis. In a certain embodiment,
the
primers are designed to amplify regions of the BMP2 nucleic acid associated
with an
at-risk haplotype for osteoporosis, shoran in Table 1, or more particularly
haplotype
I, haplotype II, haplotype a, haplotype b, haplotype c or haplotype d.
Additionally,
lcits 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).
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HAPLOTYPE SCREENING
,The invention further pertains to a method for the diagnosis and
identification of susceptibility to osteoporosis in an individual, by
identifying an
at-risk haplotype in BMP2. In one embodiment, the at-risk haphotype is one
that
confers a significant risk of osteoporosis. In one embodiment, significance
associated with a haplotype is measured by an odds ratio. In a further
embodiment,
the significance is measured by a percentage. In one embodiment, a significant
risk
is measured as an odds ratio of at least about 2.2, including by not limited
to: 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. In a further embodiment, an odds ratio
of at least
1.2 is significant. In a further embodiment, an odds ratio of at least about
1.5 is
significant. In a further embodiment, a significant increase in risk is at
least about
1.7 is significant. In a further embodiment, a significant increase in risk is
at least
about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. In a further
embodiment, a significant increase in risk is at least about 50%. It is
understood
however, that identifying whether a risk is medically significant may also
depend on
a variety of factors, including the specific disease, the haplotype, and
often,
environmental factors.
The invention also pertains to methods of diagnosing osteoporosis or a
susceptibility to osteoporosis in an individual, comprising screening for an
at-risk
haphotype associated with the BMP2 nucleic acid that is more frequently
present in
an individual susceptible to osteoporosis (affected), compared to the
frequency of its
presence in a healthy individual (control), wherein the presence of the
haplotype is
indicative of osteoporosis or susceptibility to osteoporosis. Standard
techniques for
genotyping for the presence of SNPs and/or microsatellite markers that are
associated with osteoporosis can be used, such as fluorescent based techniques
(Chen, X. et al., 1999. Ge~onze Res., 9:492-498), PCR, LCR, Nested PCR and
other
techniques for nucleic acid amplification. In one embodiment, the method
comprises assessing in an individual the presence or frequency of a specific
SNP
allele or microsatehlite allele associated with the BMP2 nucleic acid that are
associated with osteoporosis, wherein an excess or higher frequency of the
haplotype
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compared to a healthy control individual is indicative that the individual has
osteoporosis or is susceptible to osteoporosis.
Haplotype analysis involves defining a candidate susceptibility locus using
LOD scores. The defined regions are then ultra-fine mapped with microsatellite
markers with an average spacing between markers of less than 1001cb. All
usable
microsatellite markers that found in public databases and mapped within that
region
can be used. In addition, microsatellite markers identified within the deCODE
genetics sequence assembly of the human genome can be used.
The frequencies of haplotypes in the patient and the control groups using an
expectation-maximization algorithm can be estimated (Dempster A. et al., 1977.
J.
R. Stcrt. Soc. B, 39:1-389). An implementation of this algoritlnn that can
handle
missing genotypes a.nd uncertainty with the phase can be used. Under the null
hypothesis, the patients and the controls are assumed to have identical
frequencies.
Using a likelihood approach, an alternative hypothesis where a candidate
at-risk-haplotype is allowed to have a higher frequency in patients than
controls,
while the ratios of the frequencies of other haplotypes are assumed to be the
same in
both groups is tested. Likelihoods are maximized separately under both
hypotheses
and a coiTesponding 1-df likelihood ratio statistics is used to evaluate the
statistic
significance.
To look for at-risk-haplotypes in the 1-lod drop, for example, association of
all possible combinations of genotyped markers is studied, provided those
markers
span a practical region. The combined patient and control groups can be
randomly
divided into two sets, equal in size to the original group of patients and
controls.
The haplotype analysis is then repeated and the most significant p-
value.registered is
determined. This randomization scheme can be repeated, for example, over 100
times to construct an empirical distribution of p-values.
NUCLEIC ACIDS AND POLYPEPTIDES OF THE INVENTION
All nucleotide positions are relative to SEQ ID NO:1 or GenBank number
AL035668, as indicated. The nucleic acids, polypeptides and antibodies
described
herein can be used in methods of diagnosis of a susceptibility to
osteoporosis, as
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well as in lcits useful for diagnosis of a susceptibility to osteoporosis. The
reference
amino acid sequence for BMP2 is described by SEQ ID N0:2.
An "isolated" nucleic acid molecule, as used herein, is one that is separated
from nucleic acids that normally flank the gene or nucleotide sequence (as in
genomic sequences) and/or has been completely or partially purified from other
transcribed sequences (e.g., as in an RNA library). For example, an isolated
nucleic
acid of the invention can be substantially isolated with respect to the
complex
cellular milieu in which it naturally occurs, or culture medimn when produced
by
recombinant techniques, or chemical precursors or other chemicals when
chemically
synthesized. In some instances, the isolated material will form part of a
composition
(for example, a crude extract containing other substances), buffer system or
reagent
mix. In other circumstances, the material can be purified to essential
homogeneity,
for example as determined by polyacrylamide gel electrophoresis (PAGE) or
column
cluomatography such as HPLC. An isolated nucleic acid molecule of the
invention
can comprise at least about 50, 80 or 90% (on a molar basis) of all
macromolecular
species present. With regard to genomic DNA, the term "isolated" also can
refer to
nucleic acid molecules that are separated 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 lcb, 1 kb, 0.5 lcb or
0.1 kb of
the nucleotides that flank the nucleic acid molecule in the genomic DNA of the
cell
from which the nucleic acid molecule is derived.
The nucleic acid molecule can be fused to other coding or regulatory
sequences and still be considered isolated. Thus, recombinant DNA contained in
a
vector is included in the definition of "isolated" as used herein. Also,
isolated
nucleic acid molecules include recombinant DNA molecules in heterologous host
cells or heterologous organisms, as well as partially or substantially
purified DNA
molecules in solution. "Isolated" nucleic acid molecules also encompass ifz
vivo and
i~z vita°o 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
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example, in the manufacture of the encoded polypeptide, as probes for
isolating
homologous sequences (e.g., from other mammalian species), for gene mapping
(e.g., by ifZ situ hybridization with chromosomes), or for detecting
expression of the
gene in tissue (e.g., human tissue), such as by Northern blot analysis or
other
hybridization techniques.
The invention also pertains to nucleic acid molecules that hybridize Lender
high stringency hybridization conditions, such as for selective hybridization,
to a
nucleotide sequence described herein (e.g., nucleic acid molecules that
specifically
hybridize to a nucleotide sequence containing a polymorphic site associated
with a
haplotype described herein). 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 ID NO:1 comprising at least one allele
at a
polymorphic site contained in at least one of the haplotypes described herein
polymorphism, or the complement thereof, or a nucleotide sequence encoding an
amino acid sequence of SEQ ID NO:2 comprising an altered composition or
expression level as the result of an allele contained in a haplotype described
herein.
Such nucleic acid molecules can be detected and/or isolated by allele- or
sequence-specific hybridization (e.g., under high stringency conditions).
"Specific
hybridization," as used herein, refers to the ability of a first nucleic acid
to hybridize
to a second nucleic acid in a manner such that the first nucleic acid does not
hybridize to any nucleic acid other than to the second nucleic acid (e.g.,
when the
first nucleic acid has a higher complementarity to the second nucleic acid
than to any
other nucleic acid in a sample wherein the hybridization is to be performed).
"Stringency conditions" for hybridization is a term of art that refers to the
incubation
and wash conditions, e.g., conditions o~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%, 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"
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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 Curs°ent
Ps°otocols in Molecula~°
Biology (Ausubel, F. et al., "Curref7t P~°otocols ifz Molecular
Biology", John Wiley
& Sons, (1998), the entire teachings of which are incorporated by reference
herein).
The exact conditions that determine the stringency of hybridization depend not
only
on ionic strength (e.g., 0.2XSSC, O.1XSSC), temperature (e.g., room
temperature,
42°C, 68°C) and the concentration of destabilizing agents such
as formamide or
denaturing agents such as SDS, but also on factors such as the length of the
nucleic
acid sequence, base composition, percent mismatch between hybridizing
sequences
and the frequency of occurrence of subsets of that sequence within other non-
identical sequences. Thus, equivalent conditions can be determined by varying
one
i
or more of these parameters while maintaining a similar degree of identity br
similarity between the two nucleic acid molecules. Typically, conditions are
used
such that sequences at least about 60%, at least about 70%, 'at least about
80%, at
least about 90% or at least about 95% or more identical to each other remain
hybridized to one another. Ey varying hybridization conditions from a level of
stringency at which no hybridization occurs to a level at which hybridization
is first
observed, 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 that describe the determination of wash conditions for
moderate or low stringency conditions are described in Kraus, M. and Aaronson,
S.,
Methods Enzyf~2ol., 200:546-556 (1991); and in, Ausubel, F. et al.,
"Cm°f°ent
P~°otocols in Molecula~° Biology", Jolm Wiley & Sons, (1998).
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.
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For example, a low stringency wash can comprise washing in a solution
containing 0.2XSSC/0.1% SDS for 10 minutes at room temperature; a moderate
stringency wash can comprise washing in a pre-warmed solution (42°C)
solution
containing 0.2XSSC/0.1% SDS for 15 minutes at 42°C; and a high
stringency wash
can comprise washing in pre-warmed (68°C) solution containing
O.1XSSC/0.1%SDS for 15 minutes at 68°C. Furthermore, washes can be
performed
repeatedly or sequentially to obtain a desired result as known in the art.
Equivalent
conditions can be determined by varying one or more of the parameters given as
an
example, as known in the art, while maintaining a similar degree of
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.g.,
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
the two sequences is a function of the number of identical positions shared by
the
sequences (i. e., % identity = # of identical positions/total # of positions x
100). In
certain embodiments, the length of a sequence aligned for comparison purposes
is at
least 30%, at least 40%, at least 60%, at least 70%, at least 80% 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
algoritlun. A non-limiting example of such a mathematical algoritlun is
described in
Marlin, S. and Altschul, S., P~°oc. Natl. Acad. Sci. USA, 90:5873-5877
(1993). Such
an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0)
as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (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 algoritlun of Myers and Miller, CABIOS (1989).
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Such an algorithm is incorporated into the ALIGN program (version 2.0), which
is
part of the GCG sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue table, a
gap
length penalty of 12, and a gap penalty of 4 can be used. Additional
algorithms for
sequence analysis are known in the art and include ADVANCE and ADAM as
described in Torellis, A. and Robotti, C., 1994. Comput. Appl. Biosci., 10:3-
5; and
FASTA described in Pearson, W. and Lipman, D., 1988. Proc. Natl. Acad. Sci.
USA,
8 5 :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 portion that hybridizes under highly stringent
conditions to a
nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO:
l
and comprising at least one allele contained in one or more haplotypes
described
herein, and the complement thereof. The invention 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 ID N0:2, a pol5nnorphic variant thereof, or a fragment or
portion
thereof. The nucleic acid fragments of the invention are at least about 15, at
least
about 18, 20, 23 or 25 nucleotides, and ca.n be 30, 40, 50, 100, 200 or more
nucleotides in length. Longer fragments, for example, 30 or more nucleotides
in
length, which encode antigeuc polypeptides described herein, are particularly
useful, such as for the generation of antibodies as described below.
The nucleic acid fragments of the invention are used as probes or primers in
assays such as those described herein. "Probes" or "primers" are
oligonucleotides
that hybridize in a base-specific mamier to a complementary strand of nucleic
acid
molecules. In addition to DNA and RNA, such probes and primers include
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polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., 1991.
Science,
254:1497-1500.
A probe or primer comprises a region of nucleotide sequence that hybridizes
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 from SEQ, ID NO:l and comprising at least one allele
contained
in one or more haplotypes described herein, and the complement thereof. The
invention 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 ID N0:2, a polymorphic
variant thereof, or a fragment or portion 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 80% identical in certain embodiments, at least 85% identical
in
other embodiments, at least 90% identical, and in other embodiments at least
95%
identical, or even capable of selectively hybridizing to the coiltiguous
nucleotide
sequence or to the complement of the contiguous nucleotide sequence. Often,
the
probe or primer further comprises a label, e.g., radioisotope, fluorescent
compound,
enzyme, or enzyme co-factor.
The nucleic acid molecules of the invention such as those described above
can be identified and isolated using standard molecular biology techniques and
the
sequence information provided in SEQ ID NO:1. For example, nucleic acid
molecules can be amplified and isolated by the polymerase chain reaction using
synthetic oligonucleotide primers designed based on one or more of the
sequences
provided in SEQ ID NO:1 (and optionally comprising at least one allele
contained in
one or more haplotypes described herein) and/or the complement thereof. See
generally PCR Techf~ology: Prifzciples and Applicatiof~s for DNA
Amplifrcatiofa (ed.
H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods
and ApplacatioaZS (Eds. Innis, et al., Academic Press, San Diego, CA, 1990);
Mattila,
P. et al., 1991. Nucleic Acids Res., 19:4967-4973; Eclcert, K. and Kunkel, T.,
1991.
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PCR Methods a>zd Applications, 1:17-24; PCR (eds. McPherson et al., IRL Press,
Oxford); and U.S. Patent 4,683,202. The nucleic acid molecules can be
amplified
using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate
vector and characterized by DNA sequence analysis.
Other suitable amplification methods include the ligase chain reaction (LCR;
see Wu, D. and Wallace, R., 1989. Ger~ornics, 4:560-469; Landegren, U. et al.,
1988.
Scie>7ce, 241:1077-1080), transcription amplification (Kwon, D. et al., 1989.
Pr~oc.
Natl. Acad. Sci. USA, 86:1173-1177), and self sustained sequence replication
(Guatelli, J. et al., 1990. Pr°oc. Nat. Acad. Sci. USA, X7:1874-1878)
and nucleic acid
based sequence amplification (NASBA)~ The latter two amplification methods
involve isothermal reactions based on isothermal transcription, which produce
both
single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) as the
amplification products in a ratio of about 30 and 100 to 1, respectively.
The amplified DNA can be labeled, for example radiolabeled, and used as a
probe for screening a cDNA library derived from human cells. The cDNA can be
derived from mRNA and contained in zap express (Stratagene, La Jolla, CA),
ZIPLOX (Gibco BRL, Gaithesbuxg, MD) or other suitable vector. CoiTesponding
clones can be isolated, DNA can obtained following i>? vivo excision, and the
cloned
insert 'can be sequenced in either or both orientations by art recognized
methods to
identify the correct reading frame encoding a polypeptide of the appropriate
molecular weight. For example, the direct analysis of the nucleotide sequence
of
nucleic acid molecules of the present invention can be accomplished using well
lcnown methods that are commercially available. See, for example, Sambrook et
al..,
Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New Yorlc 1989);
Zyslcind et al., Reconabinant DNA Labor°atory Manual, (Acad. Press,
1988)).
Additionally, fluorescence methods are also available for analyzing nucleic
acids
(Chen, X. et al., 1999. Gehome Res., 9:492-498) and polypeptides. Using these
or
similar methods, the polypeptide and the DNA encoding the polypeptide can be
isolated, sequenced and further characterized.
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
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to compare with endogenous DNA 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 DNA sequences or to subtract out known
sequences
from a sample. The nucleic acid sequences can further be used to derive
primers for
genetic fmgelprinting, to raise anti-polypeptide antibodies using immunization
techniques, and as an antigen to raise anti-DNA antibodies or elicit immune
responses.
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 80-85%, 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 ID NO:1 and optionally comprising at least one allele
contained
in the haphotypes described herein, under stringent conditions as more
particularly
described above or will be encoded by a nucleic acid molecule hybridizing to a
nucleic acid sequence encoding SEQ ID N0:2 portion thereof or polymorphic
variant thereof, under stringent conditions as more particularly described
thereof.
A variant polypeptide can differ in amino acid sequence by one or more
substitutions, deletions, insertions, inversions, fusions, and tTUncations or
a
combination of any of these. Further, variant polypeptides can be fully
functional or
can lack function in one or more activities. Fully functional variants
typically
contain only conservative variation or variation in non-critical residues or
in
non-critical regions. Functional variants can also contain substitution of
similar
amino acids that result in no change or an insignificant change in function.
Ahtematively, such substitutions can positively or negatively affect function
to. some
degree. Non-functional variants typically contain one or more non-conservative
amino acid substitutions, deletions, insertions, inversions, or truncation or
a
substitution, insertion, inversion, or deletion in a critical residue or
critical region.
Amino acids that are essential for function can be identified by methods
k110Wn 111 the art, such as site-directed mutagenesis or alanine-scanning
lnutagenesis
(Cunningham, B and Wells, J., 1989. Scievrce, 244:1081-1085). The latter
procedure
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introduces single alanine mutations at every residue in the molecule. The
resulting
mutant molecules axe then tested for biological activity if2 vit~~o. Sites
that are
critical for polypeptide activity can also be determined by structural
analysis, for
example, by crystallization, nuclear magnetic resonance or photoaffmity
labeling
(Smith, L. et al., 1992. J. Mol. Biol., 224:899-904; de Vos, A. et al., 1992.
Science,
255:306-312).
The isolated polypeptide can be purified from cells that naturally express it,
purified from cells that have been altered to express it (recombinant), or
synthesized
using known protein synthesis methods. In one embodiment, the polypeptide is
produced by recombinant DNA techniques. For example, a nucleic acid molecule
encoding the polypeptide is cloned into an expression vector, the expression
vector
introduced into a host cell and the polypeptide expressed in the host cell.
The
polypeptide can then be isolated from the cells by an appropriate purification
scheme
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-recogiuzed 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 cam also be used as markers for cells
or tissues
in which the corresponding polypeptide is preferentially expressed, either
constitutively, during tissue differentiation, or in a diseased state. The
polypeptides
can be used to isolate a corresponding binding 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.
ANTIBODIES OF THE INVENTION
Polyclonal and/or monoclonal antibodies that specifically bind one form of
the gene product but not to the other form of the gene product are also
provided.
Antibodies are also provided that bind a portion of either the variant or the
reference
gene product that contains the polymorphic site or sites. The invention
provides
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antibodies to polypeptides having an amino acid sequence of SEQ ID N0:2 or a
variant BMP2 polypeptide. The term "antibody" as used herein refers to
immunoghobuhin molecules and immunologicalhy active portions of immunoghobulin
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 pohypeptide 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 of immtmologically 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 pohypeptide 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.
Polyclonal antibodies can be prepared as described above by immunizing a
suitable subject with a desired immunogen, e.g., pohypeptide 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 pohypeptide can be isolated from the mammal (e.g., from
the
blood) and further purified by well-known techniques, such as protein A
chromatography, to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the antibody titers are highest, antibody-producing
cells
can be obtained from the subject and used to prepare monoclonal antibodies by
standard techniques, such as the hybridoma technique (Kohher, G. and Milstein,
C.,
1975. Nature, 256:495-497),~the human B cell hybridoma technique (Kozbor, D.
et
al., 1983. If~af~auf7ol. Today, 4:72), the EBV-hybridoma technique (Cole et
al., 1985.
Mo~ocloy~al Af~rtibodies and Cance~~ Thej°apy, Alan R. Liss, Inc., pp.
77-96) or trioma
techniques.
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The technology for producing hybridomas is well known (see generally
Cuf°refzt Protocols ifs Immunology (1994) Coligan et al. (eds.) Jolm
Wiley & Sons,
Inc., New York, NY). Briefly, an immortal cell (typically a myehoma) 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.
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 pohypeptide of the invention (see, e.g.,
Cut°f°efzt Ps°otocols ih
Inzmu~ology, sups°a; Gahfre, G. et al., 1977. Nature, 266:550-552;
Kenneth, R., in
Mofzoclohal Antibodies A New Dif~ae~zsioyz IT7 Biological Analyses, Plenum
Publishing Corp., New York, New York (1980); and Lerner, E., 1981. Yale J.
Biol.
Med., 54:387-402). Moreover, the ordinarily skilled worker will appreciate
that
there are marry variations of such methods that also would be useful.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal. antibody to a pohypeptide of the invention can be identified and
isolated
by screening a recombinant combinatorial immunoglobuhin 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 Phannacia
Recombif~a~zt Phage
Afztibody System, Catalog No. 27-9400-O1; and the Stratagene SurfZAPTM Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods and
reagents
particularly amenable for use in generating and screening antibody display
library
can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No.
WO
92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791;
PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT
Publication No. WO 90/02809; Fuchs, P. et al., 1991. Biotechnology (NY),
9:1369-1372; Hay, B. et al., 1992. Hum. Antibodies Hyb~°idor~aas, 3:81-
85; Huse, W.
et al., 1989. Science, 246:1275-1281; Griffiths, A. et al., 1993. EMBO J.,
12:725-734.
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Additionally, recombinant antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made using standard recombinant DNA techniques, axe within the scope of the
invention. Such chimeric and humanized monoclonal antibodies can be produced
by
recombinant DNA techniques known in the art.
In general, antibodies of the invention (e.g., a monoclonal antibody) can be
used to detect a 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, allcaline 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 1251, isih 3sS, 32P, 33P,'4C or 3H.
The invention will be further described by the following non-limiting
example. The teachings of all publications cited herein are incorporated
herein by
reference in their entirety.
EXEMPLIFICATION
Ide~ztificatiori of BMP2 Haplotypes.
Haplotypes spanning the BMP2 nucleic acid sequence that are associated to
osteoporosis have been identified.
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"Haplotype I", "Haplotype II", "Haplotype a", "Haplotype b", "Haplotype c"
and "Haplotype d" are described below in Table 1. Each haplotype comprises
alleles
at more than one polymorphic site (haplotype I comprises 4 SNPs and a
microsatellite; haplotype II comprises 3 SNPs and 2 microsatellites; haplotype
a
comprises 2 SNPs; haplotype b comprises 3 SNPs; haplotype c comprises 3 SNPs;
and haplotype d comprises 3 SNPs).
The actual haplotypes involve the markers listed in Table 1.
Table 1.
Haplotypes
linked
to osteoporosis.
haplotype marker type allelepos. AL035668haplotype
allele
hapl TSC0898956 SNP 1 114671 C
hapl 8420 SNP 0 118920 A
hapl B8463 SNP 3 126963 T
hapl D20S846 microsatellite6 135601-136526
hapl TSC0191642 SNP 3 139007 T
hapll P4337 SNP 3 112887 T
hapll D20S892 microsatellite10 121625-121661
hapll B5048 SNP 1 123548 C
hapll B9082 SNP 2 127582 G
hapll D20S59 microsatellite6 162787-162827
hap-a B7111/rs235764SNP 2 125611 G
hap-a B128451rs15705SNP 1 131345 C
hap-b P9313 SNP 3 117863 T
hap-b B10631 SNP 2 129131 G
hap-b D35548 SNP 3 167584 T
hap-c rs1116867 SNP 0 1.49529 A
hap-c TSC0278787 SNP 0 154077 A
hap-c D35548 SNP 3 167584 T
hap-d TSC0271643/rs965291 SNP 3 upstream T
hap-d P9313 SNP 3 117863 T
hap-d 87111 SNP 2 125611 G
Alleles #'s: For SNP alleles A = 0, C = 1, G = 2, T = 3; for microsatellite
alleles: the
CEPH sample 1347-02 (CEPH genomics repository) is used as a reference, the
lower
allele of each microsatellite in this sample is set at 0 and all other alleles
in other
samples are numbered according in relation to this reference. Thus allelel is
1 by
longer than the lower allele in the CEPH sample 1347-02, allele 2 is 2 by
longer than
the lower allele in the CEPH sample 1347-02, allele 3 is 3 by longer than the
lower
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allele in the CEPH sample 1347-02, allele 4 is 4 by longer than the lower
allele in
the CEPH sample 1347-02, allele -1 is 1 by shorter than the lower allele in
the
CEPH sample 1347-02, allele -2 is 2 by shorter than the lower allele in the
CEPH
sample 1347-02, and so on.
Haplotype analysis
Haplotypes were identified as described above and haplotype analysis was
performed as described elsewhere (Stefansson, H. et al., 2002. Af~a. J. Hztna.
Genet.,
71:877-92).
Plzet~otypes and cor~t~ol samples for Osteoporosis
Several different osteoporotic phenotypes were used in the haplotype
analysis; including phenotypes used in linkage analysis as well as other
osteoporosis-
related phenotypes. The relationship between various phenotypes and haplotypes
a,
b and c are shown in FIG. 1 and FIG. 3. Haplotypes I and II are shown in FIG.
2.
For association analysis, the material collected for the linkage analysis was
used, as well as all sporadic individuals with a Z-score less than -1 SD. The
control
group comprised two randomly collected groups from the general population; one
with BMD measurements and questionnaire infounation, the other with no medical
information. These groups served as randomly collected population based
controls,
L
unrelated within 5 meiotic events; the total number of members in both groups
was
1272.
The BMD of all participants, patients as well as relatives, was determined
using dual energy X-ray absorptiometry at the lumbar spine (L2-L4) in
posterior-anterior projection, and total hip (proximal end of femur) and whole
body
(QDR 4500A, Hologic, Waltham, MA). Weight and height were measured at the
time of BMD measurement. All participants completed a detailed questionnaire
regarding their medical history, menstrual periods, current and past
medications
(including hormone replacement therapy (HRT)), and history of all fractures
and
tramna.
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While this invention has been particularly shown and described with
reference to preferred embodiments thereof, it will be understood by those
skilled in
the art that various changes in form and details 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
Cazier, Jean-Baptiste
Gulcher, Jeffrey
<120> Methods for Diagnosing Osteoporosis or a
Susceptibility to Osteoporosis Based on Haplotype
Association
<130> 2345.2052-003
<150> 60/440,899
<151> 2003-O1-16
<150> 60/450,652
<151> 2003-02-27
<160> 3
<170> FastSEQ for Windows Version 4.0
<210> 1
<2l1> 14759
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (3639)...(3984)
<221> CDS
<222> (11757)...(12601)
<400> 1
ccttggtttt ggggatcatt tgggcaagcc cgaggtgctg tgcatggggg ctcctggaat 60
cctgggaagg gcagaaagcc ttggccccag actcatcgtg cagcagctct gagcagtatt 120
tcggctgagg agtgacttca gtgaatattc agctgaggag tgacttggcc acgtgtcaca 180
gocctacttc 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 tgccaaaata 780
cgctgtaatt gaggtgtttt gttttgtttt gttttttgaa atcgtatatt accgaaaaac 840
ttcaaactga aagttgaata acgggcccag cggggaaata agaggccaga ccctgaccct 900
goatttgtcc tggatttcgc ctccagagtc cccgcgaggg tccggcgcgc cagctgatct 960
ctcctttgag 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
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2/8
gcgccgggga ctccggagcc gatccctagc gccgcgatgc ggagcaccta ctgcaggaga 1560
tcgggggcct gggacgcgct ggccgaggtg tgatcggacc ccaggctagc cacaaagggc 1620
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 ggagacttgg 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 ggtcgacc atg gtg gcc ggg acc cgc 3656
Met Val Ala Gly Thr Arg
1 5
tgtcttcta gcgttg ctgcttccc caggtcctc ctgggc ggcgcgget 3704
CysLeuLeu AlaLeu LeuLeuPro GlnValLeu LeuGly GlyAlaAla
10 15 20
ggcctcgtt ccggag ctgggccgc aggaagttc gcggcg gcgtcgtcg 3752
GlyLeuVal ProGlu LeuGlyArg ArgLysPhe AlaAla AlaSerSer
25 30 35
ggccgcccc tcatcc cagccctct gacgaggtc ctgagc gagttcgag 3800
GlyArgPro SerSer GlnProSer AspGluVal LeuSer GluPheGlu
40 45 50
ttgcggctg ctcagc atgttcggc ctgaaacag agaccc acccccagc 3848
LeuArgLeu LeuSer MetPheGly LeuLysGln ArgPro ThrProSer
55 60 65 70
agggacgcc gtggtg cccccctac atgctagac ctgtat cgcaggcac 3896
ArgAspAla ValVal ProProTyr MetLeuAsp LeuTyr ArgArgHis
75 80 85
tcaggtcag ccgggc tcacccgcc ccagaccac cggttg gagagggca 3944
SerGlyGln ProGly SerProAla ProAspHis ArgLeu GluArgAla
90 95 100
CA 02512549 2005-07-05
WO 2004/065939 PCT/US2004/000991
3/8
gcc agc cga gcc aac act gtg cgc agc ttc cac cat gaa g gtgaggcatg 3994
Ala Ser Arg Ala Asn Thr Val Arg Ser Phe His His Glu
105 110 115
gagcagggcg tgggggcggg gagtcaccct gcaaagccct ccaccgtggg cagactgcag 4054
ccgtccctgt agaggcagct tggccggggc accagcggac gtttccactc ttgcttctgt 4114
actatcgttt ctgaatctga ttttaactca ctgcttgtgt ggtgggggag ccagggattc 4174
ccctttagta actccgcacc ctcttcctgg cttgcagcca gaagagctac tcctcctgga 4234
agaattggag agaaatcaag tgatggggaa gatgagggca aaaggcatgc ctctagtcag 4294
ctaaacgtgc aagaattcca cagagggaaa aggagaaaaa gggaggcaga ttgagatttc 4354
tttaagtctg tttggaagct tttgctctat aaatctgccg cttaagccag ggttttaggg 4414
tagacagagc caagggcaga gttttcagag atagtattga aaaatcaaag cccagggccc 4474
caaagtcttt ctaatttata gttgatctgg gcctggtttg gaagattttg aatcccaatc 4534
taatccccgt gggagatcaa tactacaatc aatcttattg tttccacaat gactttcttg 4594
tcctgtgctt aaatctgaga taggctctga gtagagacaa ggcaagcctt cagataaaag 4654
cgtttgtagc agctgcctgt ttttttttca tgtgcaccga aatgtggatt tttttttctt 4714
ttatgatact acatgtggtt tttctaaggt gggatatttc tgcttgtttc atcagaaggg 4774
catttagtgg actggaaatg tcttacagca gctattgagg tctgctgtac ctaagttctt 4834
agagcaatta gtcaaaaata tgttccactt caattctttt tctacacttt taaatgcttc 4894
tttggcttaa tacatttaaa atagagcatg ggtttcttca attcctagaa aagagtacaa 4954
aagtgtatat cacagagcaa ccacttggca gatatttggg gagttgggag tgaagttctc 5014
tttcttgcct ttccctgctt aggtggtaaa tttcaagtgg gaaatttaca ctgataatag 5074
actaatggga aatggcactt ccagatgttt tctcccagtg tgaagggtga cttatacttg 5134
tgagagtatt tgttggtaat gggaataagt cccaaaggca agccacatag cagaagatac 5194
gttctcattg aggcagctac acattacgac ggggacactg aattgatcat cagttcattt 5254
acaagcacat ttctaagtga ggtgctctct gctagcagaa atcagatttg aaaggcagta 5314
agatctcact ccactctttc agaattcatc caatgaaagc agaaatcacc tgttgtcata 5374
tgtaaaattt gtgtgtatgt gtacattctg ccatcttaac cctgaaatga ttatagatcc 5434
agctaatcat tcccaggtaa tgctgattag aatacttttt tttttgtata ggaatgtaat 5494
aagaacaact gttttagaca cctcttctgg aaatttagca tggaagctct caactttatt 5554
tttaaggcct ggaagatgct gtgtctctgt tacaacttaa aaggaagatc atttaagtta 5614
gttaacacct aaaacattcc attgtgtgag gattttatca gtgatgtctg catattctca 5674
tcattcatct agaagtggtt tgatcagaac taaacaggct acacgttatt caactgtgtt 5734
attttaactt aaaaagcatg cttgagttta taaaatcaga atttatatct ttgtgagtgt 5794
aaatgttacc tgagaaacag tacagaagtg accaacttga ttaaaatcaa cttgtaataa 5854
cttcaggtct taatgcagtt agataatgga gaaaagctat gtaattttgc cccaaatttc 5914
aactaatcca tttcttgtct cattatgact aatatatcat ccttaatctg gatggatata 5974
gcactttttt caagactaat cattgttgta tacacccagg atttgctttt gataaacatc 6034
cttgtgccat gcatgccacg aaaaaagttt ttggtaaacc atgtgatgaa ggttgctggc 6094
tcaagaacag aatttagttt ctacagcatt aatgagcatt tatttgaaaa aagaccataa 6154
agacccaatc ataagaatta cctgttgggt tttctttgta ggtgtgatcg aatggtttgg 6214
tggaattact cgacgagata tcatgatagc attctttcaa ccaatatgag tataatgcga 6274
ccatatcata ggggatctga gacagaatta tcagttgtat ttttcctatt gaattttgtc 6334
tagtcctttc tccagtggct tttatttggg agaatatcag ctttgctaaa atgttattgt 6394
tttcaagatc attaaaaagt gcttcagcta catagacctt tggaaactgc cattgaacat 6454
agaaaagtca gttctgcaag tggaaagagt gttttgtgta ttgctgtagt tggaaacaca 6514
ttgaaactgg ttgacttcac tggccctcca aaaagtcttt atgctttttt gtcagatggg 6574
agagagaaag accaggtgct tcttgttctc ctcactctga aggacacagt cttctttcta 6634
catgaaataa ctggattatt tgcctctgtg actgaagctt tcaaatagag attaaccctc 6694
tttccacaaa tataattatt atgaaaatat ccatataata gaaaagttca agaaataact 6754
attgccctgc attagagact ttgtggcaca aattcccccg tgcaaacaac agatttggac 6814
acatagatcc accaaaacca atacttacct ggtatggttc cctagtggcc ccaggtattt 6874
cattgtcatt acagaggcca cattaagtag gaaaattact ctatttggaa atggttgttg 6934
agattgaggc tttggtgtcc agtgatactt ccttggcact gacattttcc gttccacctg 6994
ttttttagtg gttcccctaa atttctctta atccctttgc agtgaactat tttgcgttct 7054
tagacttgct ctttgtgtat tttcactgag acaataagag aatatttcat cattccgaag 7114
gtgttggtgt taagggtggg cagaggccaa atcagggttg ttgatgacaa ccatgctctc 7174
tattccttta tttgccattc ccttgttgta ttttttttaa aatggaatgt ttttaacctt 7234
ttgtatttga tatttttttt ctccttgatc agttgtctgt tattttatta tctggaaaat 7294
cttatattat actcagcctc tttcattttg tgttagggca gtgacttcca gccttactga 7354
ttgccagcat atccccaggt tttgttgttg ttgttgttgt tttactggag attttttagc 7414
CA 02512549 2005-07-05
WO 2004/065939 PCT/US2004/000991
4/8
ccaaagtgtg ttttaaaatc ctcgaagcat aacggtaact tacttttttg ataaaactta 7474
ccatacttta tttagaacaa aagggcagcc acaaaatagc agtggctcct tataaaatag 7534
acacattcca gtgggccccg tcacttttct gctcatttct gtctgttctg tccatcatac 7594
ctaagtcata tatttctgtt catttagttg ggacagaact cacccaatgt tatcattgta 7654
ctaaatataa atgtgcccct aatggttttg acttttgctt aagtttttga gtcctcatgt 7714
atgttaggta gtgccatcta gtagccagaa atttgggaac tggctgggca tgatggctaa 7774
tacctgtaat cccagcactt tgggaggctt aggtgggtgg atcacttgag gtcaggagtt 7834
ccagaccagc ctggccaaca tggtgaaacc acatctctac taaaatataa aaaaatagcc 7894
aggtatgatg gcccatgcct gtaatccgag ctaattggga ggctgagatg ggaggactgc 7954
ttgaacctgg gaggtggagg ctgctgtgag ccaagattgt gccactgcac tccagcctgg 8014
gcaacagagt gagaccctgt gtcacaaaaa caagaaacaa aacaaaacaa aagacaagaa 8074
acctgagaag cgcagtagat tcaattatat atatctactt ttaatttgct agctctgtga 8134
ccttaggaaa gttacataac ctctctgaac tgcaactgtt tcatttacaa aatggagata 8194
atgatagttt ttctctaatt ggtttgttgt gagataattc atataaagct gatggtgcca 8254
gattacactc aaaaaaagca ttcagctgtc attatcatta tgacttcttt tgttaatgtt 8314
atagcctttc cttctctagg gaaaaggagg ccagagtgga cctaggctga ctgagagaat 8374
tcagctcagt cttttgaatt attttgaggt agaggaatga ttgatatagt atagattatt 8434
aaattaggac ttcacttttg gagaaaagtt cagatatcat tgttgtctta tttttcttca 8494
ctttcccaca tttttgcagc catagctcca tccatttggt taagaactta gaagctcaca 8554
aactcgggtc aaagacaggt cgaaatcctc aaatccctta agaacttcag cttattcagg 8614
aagggatatt tacagaaaac tagcaattgt ataagtctcc aaaaaagcat acattacttg 8674
aggatccata tatttttggc atcctcaggg ttgctgtgat gatttataga aggtttgttt 8734
atttaattta ctttatttca aataggtttt aatttttgta cccttaagaa aagattcgta 8794
ctcttccctg gcagattaaa gaaaatgagc gtatattccc taaccttggc cagttacttt 8854
cctgggtttg agggtttctg tgaacgtcta acttacctct gtgacctgtt tctgcaacca 8914
ggggtgttgc aatggatgct tttgtcttga ggatgggacc tttcaagaaa cagattcact 8974
gaggtgcagt gggaaggtca gagaaagatc ttcgtatcgc ctattattat ttgctcgtct 9034
attttttctc ctttcttaag gccactaact gattctcctt tgctaaggct gcctacttcc 9094
actgagacct tgaaccacat gaaattgttg ttgtctgtgt ttctggtcaa atagtggcaa 9154
ttttgtatga ttcaatcttg tcatttaatt ttttgggagg ttattattct atttcatacc 9214
ttttttatac ccatcttctt tacttcattt acctgtccct catacttgac ttgtagcttg 9274
tcccttcact gtcatcgtct ggccatgtgg gtgtgtacgt gtgtgcgaga gagagaatgt 9334
gtgagaatgt atgtttcttt atgcattggg atttagggtt tttcttgcaa ttgtgatttc 9394
tctgggcact tttgttaata tagctagtca gcgagtgctc tagataattt tccttgcctc 9454
cccctctttg aaagaaaaga gggtgttctt agatgtattc ttatcagata agccagtagc 9514
tcaggtgctg gtctggcttt ggtgtcattg gggtctgagg ttgctgactt ttaccttctc 9574
tgctgaaaaa ttaccttcag cagaaacgtc tgaattgcaa ggagaaggag aaaaaaacag 9634
gccaaacaca gtccttggta ctccttggga gccactgaga agagtccagg ttcaaatggt 9694
cagaaggtta ttttaatgat tgtgtctggc ctaaagtacc attagcttcc agtggagttt 9754
agaatgtgga tggatcctga aaggtattcc ccagaggttt ggattaatag gcacaaggga 9814
accctaaagg actctattgg cctgatactc cccatatcca cgtagaagag ctttagaaga 9874
accttctgtt ctgagaccct ggctgggccc acccagagct ggcccattca actcttactc 9934
ctttgccacc actaatggtt cttctactag tttttatatt atttaacaaa aaggcacttt 9994
aaaaatgcac tcctggcaat ctatactgga atatgaaaaa catgctgcaa aaccttgaca 10054
ctccaagtgt ggtcttacag ttcccagaat cccctccttg aggagctgct agaaatgctg 10114
aatctcaagc atctccccag acctactgaa tcagagcctg catctgaagc tttacggtgt 10174
acaagctgtt ttatgtgaag gctgaagttt gaaaagcact gcattaaagc gttagtttgg 10234
tataaactgc cctgactgaa cttggtgtgt ccacttagct tgcatgatga ctgttgcttt 10294
gatgatgaag gcttacacgg gtagatcctt tgagtgagtg atctgacatg attctccttt 10354
gctaaggcat ctagattcag tgcacaactt acagctgttt gtctttaggg gaaatacaac 10414
tgtaaaatta ataaaaacat agtctcttct tatgataaca tggaacgatg gcaaaataga 10474
ttttgttagc acttgggtag gaattctgaa tgaagcaggc aaattctgtt ggcagtgaaa 10534
tgataggatg tggtaaagtt agaataaaat aaacttaaat gtctcaaact ctcatggtat 10594
atactaccag tttaataata atgttgtacc tttgatgatt tgcagactac aagcattcaa 10654
ggtgctgtgt tatatattac ttgcttggag aataatactt cttaaaaatt gaaattcaga 10714
aattttaaat cagacaaagc ttttgtgcat ggcccactta aatggctatt ttgaaataat 10774
gatagtggat atagaaggat tattctgtaa taggatgaga ctgttccttt tgtcatggag 10834
atcataatca tatttttgta aatttttatt atttttttgg ttttgtgtcc atcctgcaca 10894
ctattactgg gtaggtacat ggttttttaa catggtttat ctttcaaaac tataaaggca 10954
ttgcaaacag aagacaggtc atttattttt cttccaaaag catctaaaat gagattttga 11014
tatttgaggt cataaagagg tgagagaaca gacaacagtt gggaaagcta tttctcttga 11074
aattgtttgg ccttaattac tacagtgtcc tagtaccacc catacgtttc caaagaagta 11134
CA 02512549 2005-07-05
WO 2004/065939 PCT/US2004/000991
518
gatccctgta aatgcctttg tctctggact tttgagtaaa atagtagggt gtgctttgca 11194
aaatgtcatc gttgatgttg agtttcagag tctttaatta ggaagctgaa atctgtatat 11254
cgagatttgt aaatcatcta aattgcagag taatgtttta gaatactgct taagggattg 11314
gcattaaagc cttttttaaa aaagaaatgc aataatttcc tcaaatcctc actcattaga 11374
cctctactaa ctatagtgct gacttttttt tttttttacc ctaaagtctg gaattccaaa 11434
gaaatgcttc accatttccc ccattattat agccacctgg aagcagtatt catgtattag 11494
atcaaaaaca caacaaagaa ttatgaaagg ttgtttcctg gtatgcaatg catgatgaca 11554
tgaacttaca gaacagagag aagggaggct ccatgtttat ttaaagagga aatttttatt 11614
ttctggttac ctacttttac atgggttaca tcaaatccca cgatgaggtt taaaaattct 11674
catagataat caaacgtcat tacttggctt actgaaattc agacttttct tttttcttcc 11734
ctgtttttct ctatcaaatt ag as tct ttg gaa gaa cta cca gaa acg agt 11785
Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser
120 125
ggg aaa aca acc cgg aga ttc ttc ttt aat tta agt tct atc ccc acg 11833
Gly Lys Thr Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr
130 135 140
gag gag ttt atc acc tca gca gag ctt cag gtt ttc cga gaa cag atg 11881
Glu Glu Phe Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met
145 150 155
caa gat get tta gga aac aat agc agt ttc cat cac cga att aat att 11929
Gln Asp Ala Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile
160 165 170
tat gaa atc ata aaa cct gca aca gcc aac tcg aaa ttc ccc gtg acc 11977
Tyr Glu Ile Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr
175 180 185
aga ctt ttg gac acc agg ttg gtg aat cag aat gca agc agg tgg gaa 12025
Arg Leu Leu Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu
190 195 200 205
agt ttt gat gtc acc ccc get gtg atg cgg tgg act gca cag gga cac 12073
Ser Phe Asp Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His
210 215 220
gcc aac cat gga ttc gtg gtg gaa gtg gcc cac ttg gag gag aaa caa 12121
Ala Asn His Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln
225 230 235
ggt gtc tcc aag aga cat gtt agg ata agc agg tct ttg cac caa gat 12169
Gly Val Ser Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp
240 245 250
gaa cac agc tgg tca cag ata agg cca ttg cta gta act ttt ggc cat 12217
Glu His Ser Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His
255 260 265
gat gga aaa ggg cat cct ctc cac aaa aga gaa aaa cgt caa gcc aaa 12265
Asp Gly Lys Gly His Pro Leu His Lys Arg Glu Lys Arg Gln Ala Lys
270 275 280 285
cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag aga cac cct ttg 12313
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu
290 295 300
tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg att gtg get ccc 12361
Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro
305 310 315
CA 02512549 2005-07-05
WO 2004/065939 PCT/US2004/000991
6/8
ccggggtat cacgccttt tactgc cacggagaa tgccctttt cctctg 12409
ProGlyTyr HisAlaPhe TyrCys HisGlyGlu CysProPhe ProLeu
320 325 330
getgatcat ctgaactcc actaat catgccatt gttcagacg ttggtc 12457
AlaAspHis LeuAsnSer ThrAsn HisAlaIle ValGlnThr LeuVal
335 340 345
aactctgtt aactctaag attcct aaggcatgc tgtgtcccg acagaa 12505
AsnSerVal AsnSerLys IlePro LysAlaCys CysValPro ThrGlu
350 355 360 365
ctcagtget atctcgatg ctgtac cttgacgag aatgaaaag gttgta 12553
LeuSerAla Ile5erMet LeuTyr LeuAspGlu AsnGluLys ValVal
370 375 380
ttaaagaac tatcaggac atggtt gtggagggt tgtgggtgt cgctag 12601
LeuLysAsn TyrGlnAsp MetVal ValGluGly CysGlyCys Arg
385 390 395
tacagcaaaa ttaaatacat aaatatatat atatatatat attttagaaa aaagaaaaaa 12661
acaaacaaac aaaaaaaccc caccccagtt gacactttaa tatttcccaa tgaagacttt 12721
atttatggaa tggaatggaa aaaaaaacag ctattttgaa aatatattta tatctacgaa 12781
aagaagttgg gaaaacaaat attttaatca gagaattatt ccttaaagat ttaaaatgta 12841
tttagttgta cattttatat gggttcaacc ccagcacatg aagtataatg gtcagattta 12901
ttttgtattt atttactatt ataaccactt tttaggaaaa aaatagctaa tttgtattta 12961
tatgtaatca aaagaagtat cgggtttgta cataattttc caaaaattgt agttgttttc 13021
agttgtgtgt atttaagatg aaaagtctac atggaaggtt actctggcaa agtgcttagc 13081
acgtttgctt ttttgcagtg ctactgttga gttcacaagt tcaagtccag aaaaaaaaag 13141
tggataatcc actctgctga ctttcaagat tattatatta ttcaattctc aggaatgttg 13201
cagagtgatt gtccaatcca tgagaattta catccttatt aggtggaata tttggataag 13261
aaccagacat tgctgatcta ttatagaaac tctcctcctg ccccttaatt tacagaaaga 13321
ataaagcagg atccatagaa ataattagga aaacgatgaa cctgcaggaa agtgaatgat 13381
ggtttgttgt tcttctttcc taaattagtg atcccttcaa aggggctgat ctggccaaag 13441
tattcaataa aacgtaagat ttcttcatta ttgatattgt ggtcatatat atttaaaatt 13501
gatatctcgt ggccctcatc aagggttgga aatttatttg tgttttacct ttacctcatc 13561
tgagagctct ttattctcca aagaacccag ttttctaact ttttgcccaa cacgcagcaa 13621
aattatgcac atcgtgtttt ctgcccaccc tctgttctct gacctatcag cttgcttttc 13681
tttccaaggt tgtgtgtttg aacacatttc tccaaatgtt aaacctattt cagataataa 13741
atatcaaatc tctggcattt cattctataa agtccaacct gtaagagaaa atggtgcatt 13801
tgtatagcgc ttacaatgat gaccttgtgt ttgcattttt gtttctgaag ttatatattt 13861
tagagggggt gggggaaagg taatgaatgg ctggaaaatt gcaggcaagt tatttgataa 13921
gtcatatttg cactaaaggt gttaccagtg atttagtatt tttcaaatga acttctttgg 13981
ggcagaaaga tttaagggaa aactaaagcc tacaaaacaa gcaaaacctg gataacccga 14041
gataaagttt cagagataat agcccatgca acagaggcaa cggtgccaga aaattagaaa 14101
gggaaagtgt cggagatcag cttctataag aacatctgcc agttggactg acgcccaaac 14161
agaatgaagt caaattaggc tgctcagatt gaacacttac cagagtgtca gggcttctgt 14221
accctgggtt agaatcagac caaggaaggg ttcagcagat gttcataaga gcagggcacc 14281
cacaactacc cactatttta ctggcagtat tttaggtcag tttccaggac tttgcatccc 14341
ctctgatcct gccatgcatg attggtgaaa cctacctcta atctccttgg aattggctaa 14401
aaaacagtgt gtttataatg gaacagactg ttataatcaa attcttccta ggaattaact 14461
tttgatgact atgagcttag ttacagttcg gaggttatga ggttatgtaa accttatctt x.4521
taaatgtgca tgacagttat cttttactaa tgctggttaa cttttaaaat cttgcagctc 14581
ctttttatct ctagttctat tgttcttgat taggtgagaa ccattagatc atacccaact 14641
gaggggattg gggtcttgtt tgttctccag ctgttcttca ccctctattg ccatggacat 14701
gaaggacaga ctgcacggtc ttaacatgtt aaaacgaatg acccatgttt tctcatat 14759
<210> 2
<211> 396
<212> PRT
<2l3> Homo Sapiens
CA 02512549 2005-07-05
WO 2004/065939 PCT/US2004/000991
7/8
<400> 2
Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val
1 5 10 15
Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys
20 25 30
Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu
35 40 45
Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys
50 55 60
G1n Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu
65 70 75 80
Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp
85 90 95
His Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe
l00 105 110
His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr
115 120 125
Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Tle Pro Thr Glu Glu Phe
130 135 140
Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala
145 150 155 160
Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile
165 l70 175
Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu
l80 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 Val Ala His Leu Glu Glu Lys Gln Gly Val Ser
225 230 235 240
Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser
245 250 255
Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly 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 2g5
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 Gly Tyr
305 310 315 320
His Ala Phe Tyr Cys His Gly Glu 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 Gly Cys Gly Cys Arg
385 390 395
<210> 3
<211> 281
<212> PRT
<213> Homo Sapiens
<400> 3
Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr Thr Arg Arg
1 5 10 l5
CA 02512549 2005-07-05
WO 2004/065939 PCT/US2004/000991
8/8
Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe Tle Thr Ser
20 25 30
Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala Leu Gly Asn
35 40 45
Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Tle 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
Ala 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 l40
Tle Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys Gly His Pro
145 150 155 160
Leu His Lys Arg Glu Lys Arg Gln Ala Lys His Lys Gln Arg Lys Arg
165 l70 175
Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp
180 185 190
Val Gly Trp Asn Asp Trp Ile Val 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 Glu Gly Cys Gly Cys Arg
275 280
