CA2512549A1 - Methods for diagnosing osteoporosis or a susceptibility to osteoporosis based on haplotype association - Google Patents

Abstract

Methods for diagnosis of osteoporosis or a susceptibility to osteoporosis based on detection of at risk haplotypes associated with BMP2 are disclosed.

Description

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 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, 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 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 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.

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 _'7_ 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 _g_ 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 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 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 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 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 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 (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 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 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 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), 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).

WO 2004/065939 ' PCT/US2004/000991 =19-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 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 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 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"

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.

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).

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 =26-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.

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 _28_ 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 WO 2004/065939 ' PCT/US2004/000991 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 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.

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.

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.

"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 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.

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.

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 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 tgtcttcta gcgttg ctgcttccc caggtcctc ctgggc ggcgcgget 3704 CysLeuLeu AlaLeu LeuLeuPro GlnValLeu LeuGly GlyAlaAla ggcctcgtt ccggag ctgggccgc aggaagttc gcggcg gcgtcgtcg 3752 GlyLeuVal ProGlu LeuGlyArg ArgLysPhe AlaAla AlaSerSer ggccgcccc tcatcc cagccctct gacgaggtc ctgagc gagttcgag 3800 GlyArgPro SerSer GlnProSer AspGluVal LeuSer GluPheGlu ttgcggctg ctcagc atgttcggc ctgaaacag agaccc acccccagc 3848 LeuArgLeu LeuSer MetPheGly LeuLysGln ArgPro ThrProSer agggacgcc gtggtg cccccctac atgctagac ctgtat cgcaggcac 3896 ArgAspAla ValVal ProProTyr MetLeuAsp LeuTyr ArgArgHis tcaggtcag ccgggc tcacccgcc ccagaccac cggttg gagagggca 3944 SerGlyGln ProGly SerProAla ProAspHis ArgLeu GluArgAla 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 ccggggtat cacgccttt tactgc cacggagaa tgccctttt cctctg 12409 ProGlyTyr HisAlaPhe TyrCys HisGlyGlu CysProPhe ProLeu getgatcat ctgaactcc actaat catgccatt gttcagacg ttggtc 12457 AlaAspHis LeuAsnSer ThrAsn HisAlaIle ValGlnThr LeuVal aactctgtt aactctaag attcct aaggcatgc tgtgtcccg acagaa 12505 AsnSerVal AsnSerLys IlePro LysAlaCys CysValPro ThrGlu ctcagtget atctcgatg ctgtac cttgacgag aatgaaaag gttgta 12553 LeuSerAla Ile5erMet LeuTyr LeuAspGlu AsnGluLys ValVal ttaaagaac tatcaggac atggtt gtggagggt tgtgggtgt cgctag 12601 LeuLysAsn TyrGlnAsp MetVal ValGluGly CysGlyCys Arg 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 <400> 2 Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys G1n Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp 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 Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Tle Pro Thr Glu Glu Phe Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala 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 Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys 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 Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg <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 Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe Tle Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Tle Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser Lys Arg His 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 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 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 Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg

Claims (38)

1. A method of diagnosing osteoporosis or a susceptibility to osteoporosis in an individual, comprising detecting the presence or absence of at least one 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.

2. A method for assaying the presence of a first nucleic acid molecule in a sample, comprising contacting said sample with a second nucleic acid molecule comprising the haplotype of Claim 1.

3. The method of Claim 1, wherein determining the presence or absence of the haplotype comprises enzymatic amplification of nucleic acid from the individual.

4. The method of Claim 3, wherein determining the presence or absence of the haplotype further comprises electrophoretic analysis.

5. The method of Claim 1, wherein determining the presence or absence of the haplotype comprises restriction fragment length polymorphism analysis.

6. The method of Claim 1, wherein determining the presence or absence of the haplotype comprises sequence analysis.

7. A method of diagnosing osteoporosis or a susceptibility to osteoporosis in an individual, comprising detecting the presence or absence of at least one at-risk haplotype comprising haplotype I, wherein the presence of the haplotype is indicative of osteoporosis or a susceptibility to osteoporosis.

8. A method for assaying the presence of a first nucleic acid molecule in a sample, comprising contacting said sample with a second nucleic acid molecule comprising the haplotype of Claim 7.

9. The method of Claim 7, wherein determining the presence or absence of the haplotype comprises enzymatic amplification of nucleic acid from the individual.

10. The method of Claim 9, wherein determining the presence or absence of the haplotype further comprises electrophoretic analysis.

11. The method of Claim 7, wherein determining the presence or absence of the haplotype comprises restriction fragment length polymorphism analysis.

12. The method of Claim 7, wherein determining the presence or absence of the haplotype comprises sequence analysis.

13. A method of diagnosing osteoporosis or a susceptibility to osteoporosis in an individual, comprising detecting the presence or absence of at least one at-risk haplotype comprising haplotype II, wherein the presence of the haplotype is indicative of osteoporosis or a susceptibility to osteoporosis.

14. A method for assaying the presence of a first nucleic acid molecule in a sample, comprising contacting said sample with a second nucleic acid molecule comprising the haplotype of Claim 13.

15. The method of Claim 13, wherein determining the presence or absence of the haplotype comprises enzymatic amplification of nucleic acid from the individual.

16. The method of Claim 15, wherein determining the presence or absence of the haplotype further comprises electrophoretic analysis.

17. The method of Claim 13, wherein determining the presence or absence of the haplotype comprises restriction fragment length polymorphism analysis.

18. The method of Claim 13, wherein determining the presence or absence of the haplotype comprises sequence analysis.

19. A kit for assaying a sample for the presence of at least one 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 one or more of the specific alleles, thereby indicating the presence or absence of the haplotype in the sample.

20. The kit of Claim 19, wherein the nucleic acid comprises at least one contiguous nucleotide sequence that is completely complementary to a region comprising at least one specific allele of the haplotype.

21. A reagent kit for assaying a sample for the presence of at least one 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.

22. The reagent kit of Claim 21, wherein the labeled nucleic acid comprises at least one contiguous nucleotide sequence that is completely complementary to a region comprising at least one specific allele of the haplotype.

23. A reagent kit for assaying a sample for the presence of at least one haplotype associated with osteoporosis, wherein the haplotype comprises two or more specific alleles, wherein the kit comprises one or more nucleic acids comprising at least one nucleotide sequence that is at least partially complementary to a part of the nucleotide sequence of BMP2, and wherein the nucleic acid is capable of acting as a primer for a primer extension reaction capable of detecting two or more of the specific alleles of the haplotype.

24. A method for the diagnosis and identification of susceptibility to osteoporosis in an individual, comprising: screening for at least one 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.

25. The method of Claim 24, wherein the significant increase is at least about 20%.

26. The method of Claim 25, wherein the significant increase is identified as an odds ratio of at least about 1.2.

27. A method for diagnosing a susceptibility to osteoporosis in an individual, comprising determining the presence or absence in the individual of at least one haplotype comprising two or more alleles selected from the group consisting of: TSC0898956, B420, B8463, D20S846, TSC0191642, P4337, D20S892, 85048, 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.

28. The method of Claim 27, wherein determining the presence or absence of the haplotype comprises enzymatic amplification of nucleic acid from the individual.

29. The method of Claim 28, wherein determining the presence or absence of the haplotype further comprises electrophoretic analysis.

30. The method of Claim 27, wherein determining the presence or absence of the haplotype further comprises restriction fragment length polymorphism analysis.

31. The method of Claim 27, wherein determining the presence or absence of the haplotype further comprises sequence analysis.

32. 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 at least one 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.

33. The method of Claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of: TSC0898956, B420, B8463, D20S846 and TSC4191642.

34. The method of Claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of: P4337, D20S892, B5048, B9082 and D20S59.

35. The method of Claim 32, wherein the haplotype comprises B7111/rs235764 or B12845/rs15705.

36. The method of Claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of: P9313, B10631 and D35548.

37. The method of Claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of: rs1116867, TSC0278787 and

38. The method of Claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of: TSC0271643, P9313 and B7111.