WO2004065939A2

WO2004065939A2 - Methods for diagnosing osteoporosis or a susceptibility to osteoporosis based on haplotype association

Info

Publication number: WO2004065939A2
Application number: PCT/US2004/000991
Authority: WO
Inventors: Unnur Styrkarsdottir; Jean-Baptiste Cazier; Jeffrey R. Gulcher
Original assignee: Decode Genetics Ehf.
Priority date: 2003-01-16
Filing date: 2004-01-15
Publication date: 2004-08-05
Also published as: WO2004065939A3; JP2006516196A; EP1585838A2; CA2512549A1

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 many 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 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 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, B711 l/rs235764,

B12845/rsl5705, P9313, B10631, D35548, rsl 116867, TSC0278787, D35548 and TSC0271643; wherein the presence of the haplotype is indicative of susceptibility to osteoporosis. In a particular embodiment, detennining 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 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/rsl5705, P9313, B10631, D35548, rslll6867, 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 B711 l/rs235764 and B12845/rsl5705. 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: rsl 116867,

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 BMD). The BMD values represent the lowest 10^th 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 markers 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 markers 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 make up the particular haplotypes can be performed by a variety of methods 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 an 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 particular chromosomal location, one member of a population has an adenine and another member of the population has a thymine at the same position, then this position is a polymorphic site, and, more specifically, the polymorphic site is a SNP. Polymorphic sites can allow for differences in sequences based on substitutions, insertions or deletions. Each version of the sequence with respect to the polymorphic site is referred to herein as an "allele" of the polymorphic site. Thus, in the previous example, the SNP allows for 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:l. 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 make 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 shift, the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a susceptibility to osteoporosis can be a synonymous 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, in 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 in situ hybridizations, can be used (see Current Protocols in Molecular Biology, Ausubel, F. et al, eds., John Wiley & Sons, including all supplements through 1999). For example, a biological sample from a test subject (a "test sample") of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed for, or carrying a defect for, osteoporosis (the "test individual"). The individual can be an adult, child, or fetus. The test sample can be from any source that contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs. A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling. The DNA, RNA, or cDNA sample is then examined to determine whether a polymorphism in 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 particular 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 NO: 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 markers 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 susceptibility to osteoporosis.

In another hybridization method, Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F. et al, eds., John Wiley & Sons, supra) 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. Bioconjug. Chem., 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 markers 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 flanking 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 polymorphic 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. Nature, 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 polymorphic 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 Current Protocols in Molecular Biology, supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified 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 complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3 '-most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).

With the addition of such analogs as locked nucleic acids (LNAs), the size of primers and probes can be reduced to as few as 8 bases. LNAs are a novel class of bicyclic DNA analogs in which the 2' and 4' positions in the furanose ring are joined via an O-methylene (oxy-LNA), S-methylene (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 in T_m 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 T_m could be increased considerably.

In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual, can be used to identify polymorphisms in a 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 "Genechips™," have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods (Fodor, S. et al, 1991. Science, 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 particular 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 Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are incorporated by reference herein. In brief, a target nucleic acid sequence, which includes one or more previously identified polymorphic markers, is amplified by well known amplification techniques, e.g., PCR. Typically this involves the use of primer sequences that are complementary to the two strands of the target sequence, both upstream and downstream, from the polymorphic 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 scamied to determine the position on the array to which the target sequence hybridizes. The hybridization data obtained from the scan is typically in the fonn 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 arrays so that varying, optimal conditions can be used during the hybridization of the target to the array. For example, it will often be desirable to provide for the detection of those polymorphisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows for the separate optimization of hybridization conditions for each situation.

Additional descriptions of use of oligonucleotide arrays for detection of polymoφhisms 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 polymoφhic site associated with BMP2. Representative methods include, for example, direct manual sequencing (Church and Gilbert, 1988. Proc. Natl. Acad. Sci. USA, 81 :1991-1995; Sanger, F. et al, 1977. Proc. Natl. Acad. Sci. USA, 74:5463-5467; Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymoφhism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V. et al, 1989. Proc. Natl. Acad. Sci. USA, 86:232-236), mobility shift analysis (Orita, M. et al, 1989. Proc. Natl Acad. Sci. USA, 86:2766- 2770), restriction enzyme analysis (Flavell, R. et al, 1978. Cell, 15:25-41; Geever, R. et al, 1981. Proc. Natl. Acad. Sci. USA, 78:5081-5085); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton, R. et al, 1985. Proc. 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. coli 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 change 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 in 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 etal, U.S. Pat. No. 4,376,110) such as immunoblotting (see also Current Protocols in Molecular Biology, particularly chapter 10).

For example, in one embodiment, an antibody capable of binding to the polypeptide (e.g., as described above), e.g., an antibody with a detectable label, can be used. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

Western blot analysis, using an antibody as described above that specifically binds to a polypeptide encoded by a 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 NO: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, shown in Table 1, or more particularly haplotype I, haplotype II, haplotype a, haplotype b, haplotype c or haplotype d. 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).

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 haplotype 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 haplotype 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. Genome 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 microsatellite 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 lOOkb. 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. Stat. Soc. B, 39:1-389). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis where a candidate at-risk-haplotype is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups is tested. Likelihoods are maximized separately under both hypotheses and a corresponding 1-df likelihood ratio statistics is used to evaluate the statistic significance. To look for at-risk-haplotypes in the 1-lod drop, 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 kits useful for diagnosis of a susceptibility to osteoporosis. The reference amino acid sequence for BMP2 is described by SEQ ID NO: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 medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography 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 kb, 1 kb, 0.5 kb 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 in vivo and in vitro RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means.

Therefore, recombinant DNA contained in a vector are included in the definition of "isolated" as used herein. Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in 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 under 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 polymoφhic 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:l comprising at least one allele at a polymorphic site contained in at least one of the haplotypes described herein polymoφhism, 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 of temperature and buffer concentration, that permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid can be perfectly (i.e., 100%) complementary to the second, or the first and second can share some degree of complementarity that is less than perfect (e.g., 70%, 75%, 85%, 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 Current Protocols in Molecular Biology (Ausubel, F. et al., "Current Protocols in Molecular Biology", John Wiley & Sons, (1998), the entire teachings of which are incoφorated by reference herein). The exact conditions that determine the stringency of hybridization depend not only on ionic strength (e.g., 0.2XSSC, 0.1XSSC), 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 detennined by varying one i or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules. Typically, conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions that will allow a given sequence to hybridize (e.g., selectively) with the most complementary sequences in the sample can be detennined. Exemplary conditions that describe the determination of wash conditions for moderate or low stringency conditions are described in Kraus, M. and Aaronson, S., Methods Enzymol, 200:546-556 (1991); and in, Ausubel, F. et al, "Current Protocols in Molecular Biology", John 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 T_m 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

0.1XSSC/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 puφoses (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 pmposes 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 algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incoφorated 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 algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incoiporated 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, 85:2444-8.

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.T 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 NO:2, a polymoφhic 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 can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic polypeptides described herein, are particularly useful, such as for the generation of antibodies as described below.

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 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 NO:2, a polymoφhic 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 contiguous 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:l (and optionally comprising at least one allele contained in one or more haplotypes described herein) and/or the complement thereof. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Eriich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al, Academic Press, San Diego, CA, 1990); Mattila, P. et al, 1991. Nucleic Acids Res., 19:4967-4973; Eckert, K. and Kunkel, T., 1991. PCR Methods and 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. Genomics, 4:560-469; Landegren, U. et al, 1988. Science, 241:1077-1080), transcription amplification (Kwoh, D. et al, 1989. Proc. Natl Acad. Sci. USA, 86:1173-1177), and self-sustained sequence replication (Guatelli, J. et al, 1990. Proc. Nat. Acad. Sci. USA, 57:1874-1878) and nucleic acid based sequence amplification (NASBA)_t 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, Gaithesburg, MD) or other suitable vector. Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. For example, the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well known methods that are commercially available. See, for example, Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al , Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

Additionally, fluorescence methods are also available for analyzing nucleic acids (Chen, X. et al, 1999. Genome 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 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 fingeφrinting, 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:l and optionally comprising at least one allele contained in the haplotypes 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 NO:2 portion thereof or polymoφhic 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 truncations or a combination of any of these. Further, variant polypeptides can be fully functional or can lack function in one or more activities. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function.

Alternatively, such substitutions 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 known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham, B and Wells, J., 1989. Science, 244:1081-1085). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro. Sites that are critical for polypeptide activity can also be determined by structural analysis, for example, by crystallization, nuclear magnetic resonance or photoaffinity 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-recognized methods. The polypeptides of the present invention can be used to raise antibodies or to elicit an immune response. The polypeptides can also be used as a reagent, e.g., a labeled reagent, in assays to quantitatively determine levels of the polypeptide or a molecule to which it binds (e.g. , a receptor or a ligand) in biological fluids. The polypeptides can also be used as markers for cells or tissues in which the corresponding polypeptide is preferentially expressed, either constitutively, during tissue differentiation, or in a diseased state. The polypeptides can be used to isolate a corresponding binding 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 polymoφhic site or sites. The invention provides antibodies to polypeptides having an amino acid sequence of SEQ ID NO:2 or a variant BMP2 polypeptide. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. A molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g. , a biological sample that naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments that can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using an immobilized polypeptide. If desired, the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography, to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (Kohler, G. and Milstein, C, 1975. Nature, 256:495-497), the human B cell hybridoma technique (Kozbor, D. et al, 1983. Immunol. Today, 4:72), the EBV-hybridoma technique (Cole et al, 1985. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell (typically a myeloma) is fused to a lymphocyte (typically a splenocyte) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.

Any of the many well known protocols used for fusing lymphocytes and immortalized cells can be applied for the puφose of generating a monoclonal antibody to a polypeptide of the invention (see, e.g., Current Protocols in

Immunology, supra; Galfre, G. et al, 1977. Nature, 266:550-552; Kenneth, R., in Monoclonal Antibodies A New Dimension In 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 many variations of such methods that also would be useful.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfLA?™ 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 Hybridomas, 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, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

In general, antibodies of the invention (e.g., a monoclonal antibody) can be used to 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, alkaline phosphatase, β-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, ¹³¹1, ³⁵S, ³²P, ³³P, ¹⁴C or ³H.

The invention will be further described by the following non-limiting example. The teachings of all publications cited herein are incoφorated herein by reference in their entirety.

EXEMPLIFICATION

Identification ofBMP2 Haplotypes.

Haplotypes spanning the BMP2 nucleic acid sequence that are associated to osteoporosis have been identified. "Haplotype I", "Haplotype IF, "Haplotype a", "Haplotype b", "Haplotype c" and "Haplotype d" are described below in Table 1. Each haplotype comprises alleles at more than one polymoφhic 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 allele pos. AL035668 haplotype

# allele apl TSC0898956 SNP 1 114671 C hapl B420 SNP 0 118920 A hapl B8463 SNP 3 126963 T hapl D20S846 microsatellite 6 135601-136526 hapl TSC0191642 SNP 3 139007 T hapll P4337 SNP 3 112887 T hapll D20S892 microsatellite 10 121625-121661 hapll B5048 SNP 1 123548 C hapll B9082 SNP 2 127582 G hapll D20S59 microsatellite 6 162787-162827 hap-a B7111/rs235764 SNP 2 125611 G hap-a B12845/rs15705 SNP 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 B7111 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 allele 1 is 1 bp longer than the lower allele in the CEPH sample 1347-02, allele 2 is 2 bp longer than the lower allele in the CEPH sample 1347-02, allele 3 is 3 bp longer than the lower allele in the CEPH sample 1347-02, allele 4 is 4 bp longer than the lower allele in the CEPH sample 1347-02, allele -1 is 1 bp shorter than the lower allele in the CEPH sample 1347-02, allele -2 is 2 bp 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. Am. J. Hum. Genet., 71 :877-92).

Phenotypes and control 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 information, the other with no medical information. These groups served as randomly collected population based controls, 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 absoφtiometry 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 trauma. 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.

Claims

CLAΓMSWhat is claimed is:

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 polymoφhism 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 detennining 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 polymoφhism 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 polymoφhism 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, B5048, B9082, D20S59, B711 l/rs235764, B12845/rsl5705, ^■ P9313, B10631, D35548, rsl 116867, 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, B711 l/rs235764, B12845/rsl5705, P9313, B10631, D35548, rsl 116867, 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 TSC0191642.

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 B711 l/rs235764 or B12845/rsl5705.

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: rsl 116867, TSC0278787 and D35548

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