JP2006515992A - Human osteoporosis gene - Google Patents

Abstract

  The role of human BMP2 nucleic acid in osteoporosis is disclosed. Also disclosed are methods of using polymorphisms of BMP2 nucleic acid, alone or in combination with other assays, for diagnosis of osteoporosis or susceptibility to osteoporosis, prediction of clinical course, and treatment.

Description

Detailed Description of the Invention

RELATED APPLICATION This application is a continuation of US patent application Ser. No. 10 / 346,723, filed Jan. 16, 2003, and claims priority to the above application. The above application is a continuation-in-part of U.S. Patent Application No. 09 / 952,360 filed on September 13, 2001, which is also a continuation-in-part of International Application No. PCT / IB01 / 01667, Claim priority to the above international application. The above international application designated the United States and was filed on September 12, 2001 and published in English. The international application is a continuation-in-part of US patent application Ser. No. 09 / 661,887 filed on Sep. 14, 2000. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION Osteoporosis is a debilitating disease characterized by low bone mass and bone tissue deterioration, as defined by a decrease in bone density (BMD). The direct consequences of microstructural degradation faced are that fractures are more likely and the skeleton is more likely to become brittle, which ultimately leads to high mortality, high morbidity on a global scale. , And incur high medical costs. Among menopausal women, estrogen deficiency and the corresponding decrease in bone mass during menstruation increases both the likelihood of osteoporotic fractures and the number of potential sites So that the risk is highest. In addition, older women are not the only groups with high demographic risks. Young women with malnutrition, amenorrhea, or lack of exercise are at risk of inhibiting bone mass growth at an early age. In addition, androgens play a role in increasing bone mass during puberty, and elderly or hypogonadic men who have not fully developed bone are at risk for osteoporosis.

  What is needed to find a cure for this disease is complicated by the fact that there are many factors that cause osteoporosis. Nutrients (especially calcium, vitamin D, and vitamin K intake), hormone levels, age, gender, race, weight, activity level, and genetic factors all contribute to differences in bone density between individuals It becomes. Currently approved drugs for treating osteoporosis act as inhibitors of bone resorption, including hormone replacement therapy (HRT), selective estrogen receptor modulators, calcitonin, and bisphosphonates (Biophosphonate). However, these treatments do not individually reduce risk and have consistent results; BMD improves when several therapeutics are given simultaneously, others improve when combined. Not shown or further reduces efficacy. Clearly, as life expectancy has increased and health and economic concerns regarding osteoporosis have grown, a method to eliminate the risk of this late onset disease is highly desirable. Early diagnosis of this disease or a predisposition to this disease is desired.

SUMMARY OF THE INVENTION As described herein, through human association analysis, polymorphisms in human bone morphogenetic protein 2 (BMP2) nucleic acid sequences are associated with multiple osteoporosis phenotypes. It has been revealed. Specifically, one or more single nucleotide polymorphisms within the nucleotide sequence encoding the BMP2 gene product has been shown to be associated with osteoporosis. Accordingly, the present invention provides an isolated nucleic acid molecule comprising a BMP2 nucleic acid of SEQ ID NO: 1 having at least one altered nucleotide as shown in Table 2, and the gene product encoded thereby (herein (Referred to as “mutant BMP2 nucleic acid or gene” or “mutant BMP2 gene product”).

  A number of polymorphisms have been observed in BMP2 nucleic acids as reported in Table 2. Thus, in a specific embodiment, an isolated nucleic acid molecule of the invention can have one of these nucleotide polymorphisms, or a combination thereof. These polymorphisms may be part of a group of other polymorphisms in BMP2 nucleic acids that contribute to the presence of osteoporosis, absence of osteoporosis, or the severity of osteoporosis. In some embodiments, the nucleic acid molecule comprises a polymorphism at least at nucleotide position 3747 and the gene product comprises a polymorphism at nucleotide position 3747 (amino acid change is serine to alanine).

  The present invention further provides a method for assaying a sample for the presence of nucleic acid molecules comprising all or part of BMP2 in the sample. In this method, the sample is a nucleotide sequence encoding a BMP2 polypeptide (eg, SEQ ID NO: 1 or a complement of SEQ ID NO: 1, which comprises at least one polymorphism shown in Table 2); Contacting a second nucleic acid molecule comprising a nucleotide sequence encoding SEQ ID NO: 2 comprising at least one polymorphism shown in Table 2 or a fragment or derivative thereof under conditions suitable for selective hybridization; included. In some embodiments, the nucleic acid molecule comprises a polymorphism at least at nucleotide position 3747 and the gene product comprises a polymorphism at nucleotide position 3747 (amino acid change is serine to alanine). The invention further provides a method for assaying a sample for the expression level of a BMP2 polypeptide, or a fragment or derivative thereof. This method involves detecting (directly or indirectly) the expression level of a BMP2 polypeptide, fragment or derivative thereof.

  The invention also relates to a vector comprising an isolated nucleic acid molecule of the invention operably linked to regulatory sequences, as well as a recombinant host cell comprising the vector. The invention also provides methods for preparing polypeptides (BMP2 polypeptides) encoded by the isolated nucleic acid molecules described herein. This method includes the step of culturing the recombinant host cell of the present invention under conditions suitable for the expression of the nucleic acid molecule.

  The present invention further provides isolated polypeptides (eg, BMP2 polypeptides) encoded by the isolated nucleic acid molecules of the present invention, as well as fragments or derivatives thereof. In a particular embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2 and comprises at least one polymorphism shown in Table 2. In another embodiment, the polypeptide comprises at least a polymorphism at nucleotide position 3747, which results in an amino acid change from serine to alanine. The present invention also relates to an isolated polypeptide comprising an amino acid sequence having greater than about 90% identity to the amino acid sequence of SEQ ID NO: 2, which comprises at least one polymorphism described herein . In another embodiment, the amino acid sequence is about 95% identical to the amino acid sequence of SEQ ID NO: 2.

  The invention also provides for assaying for the presence of an antibody or antigen-binding fragment thereof that selectively binds to a polypeptide of the invention, as well as a polypeptide encoded by an isolated nucleic acid molecule of the invention in a sample. Concerning the method. This method includes contacting the sample with an antibody that specifically binds to the encoded polypeptide.

  The present invention further relates to a method for diagnosing osteoporosis or a predisposition to osteoporosis. Methods for diagnosing an individual's predisposition to osteoporosis include detecting the presence of mutations in BMP2, and detecting changes in BMP2 polypeptide expression, such as the presence of various splicing variants of BMP2 polypeptide The process of carrying out is included. The change in expression can be quantitative, qualitative, and can also be quantitative and qualitative. The methods of the present invention can accurately diagnose osteoporosis at the time of or before the onset of the disease, either alone or in combination with other assays, eg, bone metabolic marker assays (eg, bone scans), and thus osteoporosis Can be reduced or minimized.

  The invention further relates to assays for identifying agents that alter (eg, increase or inhibit) the activity or expression of one or more BMP2 polypeptides. For example, a cell, cell fraction, or solution containing a BMP2 polypeptide or fragment or derivative thereof can be contacted with the agent being tested and the level of BMP2 polypeptide expression or activity can be assessed. The activity or expression of one or more BMP2 polypeptides can be assessed simultaneously (eg, a cell, cell fraction, or solution contains one or more types of BMP2 polypeptides such as various splicing variants) And the level of various polypeptides or splicing variants can be assessed).

  In another aspect, the invention relates to an assay for identifying a polypeptide that interacts with one or more BMP2 polypeptides. For example, in a yeast two-hybrid system, a first vector comprising a DNA binding domain and a nucleic acid encoding a BMP2 polypeptide, splicing variant, or fragment or derivative thereof is used, and a nucleic acid encoding a transcriptional activation domain. And a second vector comprising a nucleic acid that further encodes a polypeptide, splicing variant, or fragment or derivative thereof (eg, BMP2 polypeptide binding factor or receptor) that may be capable of interacting with a BMP2 polypeptide Is done. Factors that interact with a BMP2 polypeptide or a fragment or derivative thereof by incubation of the yeast containing both the first vector and the second vector under appropriate conditions and thus alter the activity or expression of the BMP2 polypeptide Polypeptides that can be identified are identified.

  Agents that increase or inhibit BMP2 polypeptide expression or activity are also included in the invention. BMP2 and / or polypeptide-containing cells, or BMP2 polypeptide is contacted with a factor that increases or inhibits BMP2 or polypeptide expression or activity, thereby altering BMP2 polypeptide expression or activity (increased) Methods) are also included in the present invention.

  Furthermore, the present invention relates to a pharmaceutical composition comprising an agent that alters the activity of the nucleic acid of the present invention, the polypeptide of the present invention, and / or the BMP2 polypeptide. The present invention further includes a factor that changes the activity of the nucleic acid of the present invention, the polypeptide of the present invention, the BMP2 polypeptide, or the factor that changes the activity of the nucleic acid, the polypeptide, and / or the BMP2 polypeptide. It relates to a method of treating osteoporosis by administering a BMP2 therapeutic agent such as a composition. In another aspect, the BMP2 therapeutic is a human BMP2 nucleic acid or gene product thereof from a healthy individual who does not have osteoporosis.

  In a further aspect, the present invention relates to a method for diagnosing an individual's susceptibility to osteoporosis. This method includes detecting a polymorphism in the human BMP2 gene of SEQ ID NO: 1. Here, the presence of a “G” at nucleotide position 122247 of AL035668; the presence of a “G” allele at position 118920 of AL035668; the presence of a “T” allele at position 167584 of AL035668; the “G” allele at position 138476 of AL035668 The presence of the gene; the presence of the “T” allele at position 167584 of AL035668; the presence of the “T” allele of TSC0428253; or the presence of the “G” allele of TSC0293456 is “T” at nucleotide 122247 of AL035668; AL035668 “A” allele at position 118920; AL035668 at position 167584 “C” allele; AL035668 at position 138476 “A” allele; AL035668 at position 167584 “C” allele; TSC0428253 “C” allele Gene; or an indicator of susceptibility to osteoporosis compared to an individual having the presence of an “A” allele in TSC0293456. In certain embodiments, the polymorphism is detected in a sample derived from a source selected from the group consisting of blood, serum, cells, and tissue.

  In another aspect, the invention relates to a method of diagnosing an individual's susceptibility to osteoporosis. This method includes detecting a polymorphism in the human BMP2 gene of SEQ ID NO: 1. Here, the polymorphism is from T to G at position 122247 of AL035668; A to G at position 118920 of AL035668; C from T to position 167584 of AL035668; A to G at position 138476 of AL035668; from C at position 167584 of AL035668 T: selected from the group consisting of C to T of TSC0428253; A to G of TSC0293456, and combinations thereof. In certain embodiments, the polymorphism is detected in a sample derived from a source selected from the group consisting of blood, serum, cells, and tissue.

  In another aspect, the invention relates to AL035668 at position 122247 T from AL; AL035668 at position 121366 C to T; AL035668 at position 118920 A to G; AL035668 at position 167584 from C to T; AL035668 at position 138476 An isolated nucleic acid molecule comprising a nucleic acid having SEQ ID NO: 1 comprising one or more nucleic acid changes selected from the group consisting of A to G; and combinations thereof.

  In another aspect, the invention relates to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of alanine at amino acid position 37, serine at amino acid position 94, serine at amino acid position 189, or a combination thereof.

  In yet another aspect, the present invention relates to a method for diagnosing susceptibility to osteoporosis. This method includes detecting a polypeptide described herein that is an indicator of susceptibility to osteoporosis. In certain embodiments, for determining an amino acid at a position selected from the group consisting of positions 37, 94, 1891, and combinations thereof, the sample is an antibody specific for either a control amino acid or a variant amino acid. And contacting with. In another aspect, the invention relates to a pharmaceutical composition comprising a polypeptide described herein. In another aspect, the invention relates to a method of treating osteoporosis in an individual, comprising administering to the individual a therapeutically effective amount of an isolated polypeptide described herein. Is included.

  In another aspect, the invention provides: a) serine at amino acid position 37, alanine at amino acid position 37, alanine at amino acid position 94, serine at amino acid position 94, arginine at amino acid position 189, serine at amino acid position 189, or a A kit comprising a combination of at least one antibody selected from the group consisting of antibodies specific for BMP2 protein; and b) a control BMP2 protein sample.

Detailed Description of the Invention As described herein, applicants have completed a genome-wide scan for patients with various forms of osteoporosis on chromosome 20 associated with osteoporosis. One region was identified. To date, there is no known association analysis of human osteoporosis that shows any relationship to this region of the chromosome. Based on the association analysis performed, applicants discovered a direct relationship between BMP2 and osteoporosis. Although BMP2 nucleic acids derived from normal individuals are known, there are no studies that directly examine the association between BMP2 and osteoporosis. Furthermore, there are no variants that have been reported to be associated with osteoporosis. Association analysis is relevant to patients with various osteoporosis phenotypes, ie hip, spine, complex, and complex severe phenotypes (eg, spinal compression fractures, hip fractures, other osteoporosis) Based on a wide-area genome scan that covers patients with low load fractures. The data obtained by the association analysis identified one region on chromosome 20, specifically the BMP2 gene. Mutant BMP2 nucleic acids have nucleotide changes that have not been previously reported and this has been observed in a population of patients (see Tables 2, 3, and 4 for polymorphic changes). See

  All nucleotide positions are shown for SEQ ID NO: 1 or for GenBank accession number AL035668. The polymorphism at nucleotide position 3747 appears statistically more frequently in the osteoporosis test population than in the control population.

Nucleic acid of the present invention
BMP2 nucleotides, portions thereof, and variants Accordingly, the present invention relates to an isolated nucleic acid molecule comprising a human BMP2 nucleic acid having at least one nucleotide change and associated with an indicator of osteoporosis. The term “variant BMP2” as used herein refers to an isolated nucleic acid molecule in chromosome 20 having at least one nucleotide that is altered. The variants of BMP2 of the present invention are associated with susceptibility to a number of osteoporosis phenotypes. The invention includes a portion or fragment of an isolated nucleic acid molecule comprising a change (eg, cDNA, including a fragment encoding a mutant BMP2 polypeptide (eg, a polypeptide having SEQ ID NO: 2)). Gene, or fragment thereof). In some embodiments, the isolated nucleic acid molecule comprises a group consisting of any one or combination of polymorphisms shown in Tables 2, 3, and 4 of the BMP2 gene comprising at least the nucleotide 3747 polymorphism. Contains the polymorphism that is selected. In a particular embodiment for therapeutic purposes, for example, the nucleic acid comprises the sequence of SEQ ID NO: 1, which represents the human BMP2 nucleic acid of a healthy individual.

  An isolated nucleic acid molecule of the present invention can be RNA (eg, mRNA), and can also be DNA, such as cDNA and genomic DNA. A DNA molecule can be double-stranded or single-stranded; single-stranded RNA or DNA can be either the coding strand, ie the “sense strand”, or the non-coding strand, ie the “antisense” strand obtain. A nucleic acid molecule can include the entire coding sequence of a gene or a portion thereof, and further includes non-coding sequences such as introns and non-coding 3 ′ and 5 ′ sequences (eg, including regulatory sequences and other flanking sequences). It can also be included. In addition, the nucleic acid molecule can be fused to a marker sequence, affinity tag, or other gene or sequence (eg, a sequence encoding a polypeptide that aids in isolation or purification of the polypeptide). Such sequences include, but are not limited to, a sequence encoding a glutathione-S-transferase (GST) fusion protein and a sequence encoding a hemagglutinin A (HA) polypeptide marker derived from influenza.

  An “isolated” nucleic acid molecule, as used herein, is separated from the nucleic acid that is normally adjacent (in the genomic sequence) to the gene or nucleotide sequence and / or other transcription. A nucleic acid molecule that has been completely or partially purified from a rendered sequence (eg, in an RNA library). For example, an isolated nucleic acid of the invention can be a complex cellular environment that exists in nature, or a culture medium if produced by recombinant techniques, or a chemical precursor if chemically synthesized. Alternatively, it can be substantially isolated with respect to other compounds. In some examples, the isolated material forms part of a composition (eg, a crude extract containing other substances), a buffer system, or a reagent mixture. In other situations, the material can be purified to be essentially homogeneous, as measured, for example, by column chromatography such as polyacrylamide gel electrophoresis (PAGE) or HPLC. An isolated nucleic acid molecule of the present invention can comprise at least about 50, 80, or 90% (on a molar basis) of all macromolecular species present. With respect to genomic DNA, the term “isolated” can also mean a nucleic acid molecule that is separated from the chromosome with which the genomic DNA is naturally associated. For example, an isolated nucleic acid molecule contains less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotides adjacent to the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived. obtain.

  A 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. Isolated nucleic acid molecules also include recombinant DNA molecules in heterologous host cells or organisms, as well as partially or substantially purified DNA molecules in solution. “Isolated” nucleic acid molecules also include 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. Thus, recombinant DNA contained in a vector is included in the definition of “isolated” as used herein. Such isolated nucleotide sequences can be used, for example, by in situ hybridization (eg, using chromosomes) to isolate homologous sequences (eg, from other mammalian species) in the production of the encoded polypeptide. ) Useful as a probe for gene mapping or for detecting expression of a gene in a tissue (eg, human tissue), eg, by Northern blot analysis or other hybridization techniques.

  The present invention is also not necessarily found in nature but nevertheless a BMP2 polypeptide (e.g. having the amino acid sequence of SEQ ID NO: 2 and at least one of the multiples shown in Tables 2, 3, and 4). Relates to a nucleic acid molecule encoding a polypeptide comprising a type). Thus, for example, a DNA molecule comprising a sequence that encodes a BMP2 polypeptide of the present invention due to the degeneracy of the genetic code, although differing from the naturally occurring nucleotide sequence, is also subject of the present invention. The invention also includes variants of the nucleotide sequences of the invention (eg, those that encode portions, analogs or derivatives of BMP2 polypeptides). Such variants may exist naturally, for example in the case of allelic variants, and for example in the case of those induced by various mutagens and mutagenic processes, It cannot exist in nature. Contemplated variations include, but are not limited to, one or more nucleotide additions, deletions, and substitutions. These can result in conservative or non-conservative amino acid changes, including additions and deletions. Nucleotide (and / or resulting amino acid) changes can be silent or conservative. That is, they need not necessarily change the properties or activity of the BMP2 polypeptide. In some embodiments, the nucleotide sequence is a fragment that includes one or more polymorphic microsatellite markers. In another embodiment, the nucleotide sequence is a fragment that includes one or more single nucleotide polymorphisms in the BMP2 gene.

  Other variations of the nucleic acid molecules of the invention include, for example, labeling, methylation, internucleotide modifications, such as uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoamidate, carbamate), charge Bonds (eg, phosphorothioate, phosphorodithioate), pendant moieties (eg, polypeptides), intercalators (eg, acridine, psoralen), chelators, alkylating agents, and modified bonds (eg, alpha anomers) Nucleic acid). Also included are synthetic molecules that mimic nucleic acid molecules with respect to their ability to bind to designed sequences through hydrogen bonding and other chemical interactions. Such molecules include, for example, molecules in which phosphate bonds in the molecular backbone are replaced with peptide bonds.

  The present invention also provides nucleic acid molecules (eg, herein) that hybridize to the nucleotide sequences described herein under high stringency hybridization conditions (eg, conditions for selective hybridization). Nucleic acid molecules that specifically hybridize to the nucleotide sequence encoding the described polypeptide and optionally have the activity of the polypeptide. In certain embodiments, the invention includes at least one polymorphism shown in Tables 2, 3, and 4 under high stringency hybridization and wash conditions (eg, conditions for selective hybridization). To a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1 or its complement, or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 comprising at least one polymorphism shown in Tables 2, 3, and 4 And variants described herein that hybridize to In some embodiments, the variant that hybridizes under high stringency hybridization has the activity of BMP2.

  Such nucleic acid molecules can be detected and / or isolated by allele specific hybridization or sequence specific hybridization (eg, under conditions of high stringency). “Specific hybridization”, as used herein, refers to a manner in which a first nucleic acid does not hybridize to any nucleic acid other than a second nucleic acid (eg, the first nucleic acid is The ability of a first nucleic acid to hybridize to a second nucleic acid when it has a higher complementarity to the second nucleic acid than to all other nucleic acids in the sample being hybridized) . “Stringency conditions” for hybridization is a terminology that refers to incubation and wash conditions that allow hybridization of a particular nucleic acid to a second nucleic acid, eg, temperature and buffer concentration conditions. The first nucleic acid is completely (ie, 100%) complementary to the second nucleic acid, and the first nucleic acid and the second nucleic acid are not completely complementary. (For example, 70%, 75%, 85%, 90%, 95%) may be shared. For example, certain high stringency conditions can be used to distinguish perfectly complementary nucleic acids from less complementary nucleic acids. “High stringency conditions”, “moderate stringency conditions”, and “low stringency conditions” for nucleic acid hybridization are described in Current Protocols in Molecular Biology (Ausubel, FM et al., “Current Protocols in Molecular Biology ", John Wiley & Sons, (1998), the entire teachings of which are incorporated herein by reference), as described on pages 2.10.1-2.10.16 and 6.3.1-6.3.6. Yes. The exact conditions that determine the stringency of hybridization are ionic strength (eg, 0.2 × SSC, 0.1 × SSC), temperature (eg, room temperature, 42 ° C., 68 ° C.), and destabilizing agents such as formamide or Not only the concentration of denaturing agents such as SDS, but also a subset of the sequence within the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences, and other non-identical sequences It also depends on factors such as frequency. Thus, equivalent conditions can be determined by changing one or more of these parameters while maintaining the same degree of identity or similarity between the two nucleic acid molecules. Typically, conditions are used in which sequences that are 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 each other. . By changing the hybridization conditions from the level of stringency at which no hybridization occurs to the level at which hybridization is first observed, a given sequence is selected from the most complementary sequence in the sample (eg, selective The conditions for hybridizing can be determined.

Exemplary conditions are described in Krause, MH and SAAaronson, Methods in Enzymology, 200: 546-556 (1991). Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons, (1998) also describes the determination of washing conditions for moderate or low stringency conditions. Washing is usually a process in which the conditions are set to determine the minimum level of hybrid complementarity. In general, starting from the lowest temperature at which only homologous hybridization occurs, and each final wash temperature decreases by 1 ° C. (while keeping the SSC concentration constant), the maximum between hybridizing sequences Increases mismatch percentage by 1%. Usually, doubling the concentration of SSC increases _Tm by about 17 ° C. Using these guidelines, the wash temperature can be empirically determined for high stringency, moderate stringency, or low stringency, depending on the level of mismatch expected.

  For example, a low stringency wash can include a 10 minute wash at room temperature in a solution containing 0.2 × SSC / 0.1% SDS. A moderate stringency wash may include a 15 minute wash at 42 ° C. in a pre-warmed (42 ° C.) solution containing 0.2 × SSC / 0.1% SDS. And high stringency washing can include washing for 15 minutes at 68 ° C. in a pre-warmed (68 ° C.) solution containing 0.1 × SSC / 0.1% SDS. Furthermore, washing can be performed repeatedly or continuously until the desired result is obtained, as is known in the art. Equivalent conditions are given as examples, as is known in the art, while maintaining the same degree of complementarity between the target nucleic acid molecule used and the primer or probe (eg, the sequence to be hybridized). It can be determined by changing one or more parameters to be changed.

  The percent identity between two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (eg, gaps can be introduced into the sequence of the 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 these sequences (ie, percent identity) ) = Number of identical positions / total number of positions × 100). In certain embodiments, the length of the 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 It is. The actual comparison of the two sequences can be done by well-known methods, for example using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al., Nucleic Acids Res., 25: 389-3402 (1997). If BLAST and Gapped BLAST programs are utilized, the default parameters for the respective program (eg, NBLAST) can be used. See the ncbi.nlm.nih.gov website on the World Wide Web. In certain embodiments, the parameters for sequence comparison can be set to score = 100, character length = 12, or can be varied (eg, W = 5, or W = 20).

  Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm 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 the ALIGN program is utilized to compare 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, including ADVANCE and ADAM described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10: 3-5, and Pearson and Lipman. (1988) PNAS, 85: 2444-8.

  In another embodiment, the percent identity between two amino acid sequences is either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4, and 2, This can be done using the GAP program of the GCG software package (Accelrys, Cambridge, UK) using either 3 or 4 length weights. In yet another embodiment, the percent identity between two nucleic acid sequences is performed using the GAP program in the GCG software package, using a gap weight of 50 and a length weight of 3. obtain.

  In accordance with the present invention, a high stringency of a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1 and comprising at least one polymorphism shown in Tables 2, 3, and 4 and its complement An isolated nucleic acid molecule comprising a fragment or portion that hybridizes under conditions is provided. In addition, an isolation comprising a fragment or portion that hybridizes under conditions of high stringency to a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 2, a polymorphic variant thereof, or a fragment or portion thereof Also provided is a nucleic acid molecule. The nucleic acid fragments of the present 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 encoding the antigenic polypeptides described herein, eg, 30 or more nucleotides in length, are particularly useful, for example, in the production of antibodies as described below.

Probes and Primers In a related aspect, the nucleic acid fragments of the present invention are used as probes or primers in assays such as those described herein. A “probe” or “primer” is an oligonucleotide that hybridizes in a base-specific manner to the complementary strand of a nucleic acid molecule. In addition to DNA and RNA, such probes and primers include polypeptide nucleic acids (PNA) as described in Nielsen et al., Science, 254, 1497-1500 (1991).

  The probe or primer includes SEQ ID NO: 1, which includes at least one polymorphism shown in Tables 2, 3, and 4, and its complement, or an amino acid sequence selected from SEQ ID NO: 2. At least about 15, typically about 20-25, and in some embodiments about 40, 50, or 75 nucleic acid molecules comprising a contiguous nucleotide sequence selected from A region of a nucleotide sequence that hybridizes to a contiguous nucleotide is included. In certain embodiments, a probe or primer can include 100 or fewer nucleotides. For example, certain embodiments include 6 to 50 nucleotides, or such as 12 to 30 nucleotides. In other embodiments, the probe or primer is at least 70% identical to a contiguous nucleotide sequence or to the complement of a contiguous nucleotide sequence, eg, in certain embodiments, at least 80% identical , In other embodiments, at least 85% identical, at least 90% identical, and in other embodiments, at least 95% identical, or against, or of contiguous nucleotide sequences It can even hybridize selectively to complements. In many cases, the probe or primer further includes a label, eg, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

  The nucleic acid molecules of the invention as described above can be identified and isolated using standard molecular biology techniques and the sequence information provided in SEQ ID NO: 1. For example, the nucleic acid molecule is based on one or more sequences provided in SEQ ID NO: 1 (and optionally including at least one polymorphism shown in Tables 2, 3, and 4) and / or complements thereof. Can be amplified and isolated by polymerase chain reaction using synthetic designed oligonucleotide primers. In general, PCR Technology: Principles and Applications for DNA Amplification (HAErlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Innis et al., Academic Press, San Diego, CA) , 1990); Mattila et al., Nucleic Acids Res., 19: 4967 (1991); Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (McPherson et al., IRL Press, Oxford); See 4,683,202. 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 ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4: 560 (1989), Landegren et al., Science, 241: 1077 (1988)), transcription amplification (Kwoh , Proc. Natl. Acad. Sci. USA, 86: 1173 (1989)), and replication of self-maintaining sequences (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874 (1990)), and nucleic acids Sequence amplification method based on (NASBA). The latter two amplification methods include isothermal reactions based on isothermal transcription, which allows both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) to be about 30 and 100 to 1, respectively. Produced as an amplification product at a rate.

  The amplified DNA can be labeled, eg, radiolabeled, and used as a probe to screen a cDNA library derived from human cells. The cDNA can be derived from mRNA, which can be included in zap express (Stratagene, La Jolla, Calif.), ZIPLOX (Gibco BRL, Gaithesburg, MD), or other suitable vector. Corresponding clones can be isolated, DNA can be obtained after in vivo excision, and the cloned insert can be used in the art to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. Depending on the recognized method, it can be sequenced in either direction or in both directions. For example, direct analysis of the nucleotide sequence of the nucleic acid molecules of the present invention can be performed using well-known methods that are commercially available. See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd edition, CSHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). In addition, fluorescence methods can also be used to analyze nucleic acids (Chen et al., Genome Res. 9: 492 (1999) and polypeptides. These methods or similar methods can be used to encode polypeptides and polypeptides. DNA can be isolated, sequenced and further characterized.

  An antisense nucleic acid molecule of the invention is a nucleotide sequence of SEQ ID NO: 1, comprising at least one polymorphism shown in Tables 2, 3, and 4, and / or its complement, and / or Part of SEQ ID NO: 1 that includes at least one polymorphism shown in Tables 2, 3, and 4 or can be designed using its complement and use procedures known in the art Can be constructed using chemical synthesis and enzymatic ligation reactions. For example, an antisense nucleic acid molecule (eg, an antisense oligonucleotide) is a double-stranded physical stability formed to increase the biological stability of the molecule or formed between an antisense nucleic acid and a sense nucleic acid. It can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase sex. For example, phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, antisense nucleic acid molecules can be biologically produced using expression vectors in which the nucleic acid molecule is subcloned in the antisense orientation (ie, RNA transcribed from the inserted nucleic acid molecule is of interest In the antisense direction relative to the target nucleic acid).

  In general, the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels and as chromosomal markers that are labeled to map related gene locations. The nucleic acid sequence can also be hybridized to a patient's endogenous DNA sequence to identify a genetic disorder (e.g., predisposition to osteoporosis or susceptibility to osteoporosis) and to, for example, a relevant DNA sequence, It can also be used as a probe to find relevant DNA sequences or to subtract known sequences from a sample. The nucleic acid sequence is further used to derive primers for gene fingerprinting, to elicit anti-polypeptide antibodies using immunization techniques, and to elicit anti-DNA antibodies or elicit an immune response. It can be used as an antigen. A portion or fragment of a nucleotide sequence identified herein (and the corresponding complete gene sequence) can be used in a number of ways as a polynucleotide reagent. For example, these sequences (i) map their respective genes onto chromosomes; thereby to determine gene regions that are associated with genetic disease; (ii) individuals from trace biological samples Can be used to identify (tissue classification); and (iii) to aid forensic identification of biological samples. Furthermore, the nucleotide sequences of the present invention can be used to identify and express a recombinant polypeptide for analytical, characterization, or therapeutic use, or for the corresponding polypeptide to be constitutive during tissue differentiation. Or as a marker for tissues expressed in either disease states. The nucleic acid sequences can further be used as reagents for screening and / or diagnostic assays described herein, and of kits for use in screening and / or diagnostic assays described herein. It can also be included as a component (eg, a reagent kit).

Vector Another aspect of the invention is SEQ ID NO: 1 selected from the group consisting of at least one polymorphism shown in Tables 2, 3, and 4 and their complements or parts It relates to a nucleic acid construct comprising a nucleic acid molecule. Yet another aspect of the present invention relates to a nucleic acid construct comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2 or a polymorphic variant thereof. Constructs include vectors (eg, expression vectors) into which the sequences of the invention are inserted in sense or antisense orientation. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication, and mammalian episomal vectors). Other vectors (eg, mammalian non-episomal vectors), when introduced into a host cell, integrate into the host cell's genome and are thereby replicated along with the host genome. In addition, certain vectors, called expression vectors, can direct the expression of genes to which they are operably linked. In general, expression vectors that are useful in recombinant DNA technology are often in the form of plasmids. However, the present invention is intended to include other forms of expression vectors such as viral vectors that perform equivalent functions (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses).

  The recombinant expression vector of the present invention can include a nucleic acid molecule of the present invention in a form suitable for expression of the nucleic acid molecule in a host cell. This means that the recombinant expression vector contains one or more regulatory sequences selected based on the host cell used for expression, and is operably linked to the nucleic acid sequence to be expressed. . In a recombinant expression vector, it is operably or operably linked to express the nucleotide sequence in a manner (eg, in an in vitro transcription / translation system or when the vector is introduced into a host cell). In host cells) is intended to mean that the nucleotide sequence of interest is linked to regulatory sequence (s). The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include regulatory sequences that direct constitutive expression of nucleotide sequences in many types of host cells, and regulatory sequences that direct expression of nucleotide sequences only in specific host cells (eg, tissue-specific regulatory sequences). Is mentioned. It will be appreciated by those skilled in the art that the design of the expression vector can vary depending on factors such as the choice of the host cell to be transformed and the level of expression of the desired polypeptide. The expression vectors of the invention can be introduced into a host cell, thereby producing a polypeptide, including a fusion polypeptide, encoded by the nucleic acid molecules described herein.

  The recombinant expression vectors of the invention can be used in prokaryotic or eukaryotic cells, for example bacterial cells such as E. coli, insect cells (eg, using baculovirus expression vectors), yeast cells, or mammalian cells. Can be designed for expression of the polypeptides of the present invention. Appropriate host cells are discussed further in Goeddel (supra). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

  Another aspect of the invention relates to a host cell into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms mean not only a particular subject cell, but also the progeny or future progeny of such a cell. Such progeny may not actually be identical to the parental cell, as certain modifications may occur in future generations, either due to mutations or environmental effects, but these are still Included within the scope of this term as used in the specification.

  The host cell can be any prokaryotic or eukaryotic cell. For example, the nucleic acid molecules of the invention can be expressed in bacterial cells (eg, E. coli), insect cells, yeast or mammalian cells (eg, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

  Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to various art-recognized techniques for introducing foreign nucleic acid molecules (eg, DNA) into host cells. This includes calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Supra) and other laboratory manuals.

  For stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small percentage of cells are known to be able to integrate foreign DNA into their genome. . In order to identify and select these integrants, a gene that encodes a selectable marker (eg, for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Selectable markers can include selectable markers that confer resistance to drugs such as G418, hygromycin, and methotrexate. A nucleic acid molecule encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (eg, cells that have incorporated the selectable marker gene survive, but other cells die).

  A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (ie, express) a polypeptide of the invention. Thus, the present invention further provides a method for producing a polypeptide using the host cell of the present invention. In one embodiment, the method comprises culturing a host cell of the invention, into which a recombinant expression vector encoding a polypeptide of the invention has been introduced, in a suitable culture medium in which the polypeptide is produced. The process of carrying out is included. In another aspect, the method further comprises isolating the polypeptide from the medium or the host cell.

  The host cells of the invention can also be used to obtain non-human transgenic animals. For example, in certain embodiments, a host cell of the invention is a fertilized oocyte into which a nucleic acid molecule of the invention (eg, an exogenous BMP2 gene or an exogenous nucleic acid encoding a BMP2 polypeptide) has been introduced. Or embryonic stem cells. Such a host cell then creates a non-human transgenic animal into which the exogenous nucleotide sequence has been introduced into the genome, or a homologous recombinant animal in which the endogenous nucleotide sequence has been altered. Can be used. Such animals are useful for studying the nucleotide sequence, and the function and / or activity of the polypeptides encoded by the sequence, and for identifying and / or evaluating modulators for their activity. As used herein, a “transgenic animal” is a non-human animal, eg, a mammal, eg, a rodent such as a rat or mouse, one or more of these animals The cells contain a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, pigs, goats, chickens, and amphibians. A transgene is a gene product that is integrated into the genome of a cell that maintains the genome of an adult animal that generates the transgenic animal, and thereby is encoded in one or more cell types or tissues of the transgenic animal Exogenous DNA that directs the expression of. As used herein, a “homologous recombinant animal” is a non-human animal, eg, a mammal, eg, a mouse, in which the endogenous gene is an endogenous gene and an animal cell (eg, The embryonic cells of the animal prior to the development of the animal) have been altered by homologous recombination with exogenous DNA molecules introduced into the animal.

  Methods for producing transgenic animals, particularly animals such as mice, by embryo manipulation and microinjection are common in the art, e.g., U.S. Patent Nos. 4,736,866 and 4,870,009, No. 4,873,191 and Hogan, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). Methods for constructing homologous recombination vectors and homologous recombination animals are further described in Bradley (1991) Current Opinion in Bio / Technology, 2: 823-829, and PCT International Publication Nos. WO90 / 11354, WO91 / 01140, It is described in WO92 / 0968 and WO93 / 04169. Non-human transgenic animal clones described herein are also described in Wilmut et al., (1997) Nature, 385: 810-813, and PCT International Publication Nos. WO97 / 07668 and WO97 / 07669. It can be produced according to the methods that are being used.

Polypeptides of the invention The invention also includes isolated BMP2 polypeptides, such as proteins and variants thereof, and polypeptides encoded by the nucleotide sequences described herein (eg, other splicing). Mutant). The term “polypeptide” refers to a polymer of amino acids and is not of a particular length. Thus, peptides, oligopeptides, and proteins are included in the definition of polypeptide. As used herein, a polypeptide is, if it is isolated from recombinant and non-recombinant cells, if it is substantially free of cellular material, or if it is chemically synthesized. If so, it is said to be “isolated” or “purified” if it is substantially free of chemical precursors or other compounds. However, the polypeptide can be connected to another polypeptide not normally associated with it in the cell, which is still “isolated” or “purified”.

  The polypeptides of the invention can be purified to homogeneity. However, it is understood that preparations that are not purified until the polypeptide is homogeneous are useful. An important feature is that the preparation can still perform the desired function of the polypeptide, even in the presence of significant amounts of other components. Accordingly, the present invention includes various degrees of purity. In some embodiments, the phrase “substantially free of cellular material” includes less than about 30% (dry weight) of other proteins (ie, contaminating proteins) and less than about 20% of other proteins. Polypeptide preparations having less than about 10% other proteins or less than about 5% other proteins are included.

  If the polypeptide is produced recombinantly, it can be substantially free of culture medium. That is, the culture medium occupies less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation. The phrase “substantially free of chemical precursors or other compounds” includes preparations of polypeptides that are separated from chemical precursors or other compounds involved in their synthesis. . In some embodiments, the phrase “substantially free of chemical precursors or other compounds” includes less than about 30% (dry weight) of chemical precursors or other compounds, Polypeptide preparations having less than about 20% other compounds, less than about 10% chemical precursors or other compounds, and less than about 5% chemical precursors or other compounds are included .

  In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO: 1, comprising at least one polymorphism shown in Tables 2, 3, and 4, and their complements and portions An amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence is included. However, the present invention also includes sequence variants. Variants include substantially homologous polypeptides encoded at the same locus in an organism, ie allelic variants, as well as other splicing variants. Variants are also derived from other loci of the organism but are SEQ ID NO: 1 and include at least one polymorphism shown in Tables 2, 3, and 4, and their complements and A nucleotide sequence encoding SEQ ID NO: 2 or a polymorphic variant thereof having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of a portion Also included are polypeptides having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide selected from the group consisting of a body. Variants also include polypeptides that are substantially homologous to or identical to these polypeptides, but derived from another organism, ie, orthologs. Variants also include polypeptides that are substantially homologous to or identical to these polypeptides that are produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by recombinant methods.

  As used herein, two polypeptides (or regions of polypeptides) have an amino acid sequence of 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 about 90% or more homologous or identical when substantially homologous or identical. A substantially homologous amino acid sequence, according to the present invention, is SEQ ID NO: 1, comprising at least one polymorphism shown in Tables 2, 3, and 4, or a part thereof, Encoded by a nucleic acid molecule that hybridizes under stringent conditions more specifically described above, or against a nucleic acid sequence encoding SEQ ID NO: 2 or a portion thereof, or a polymorphic variant thereof. , Encoded by a nucleic acid molecule that hybridizes under stringent conditions more specifically described therefor.

  The present invention includes a poly having a low degree of identity but sufficient similarity to perform one or more of the same functions performed by the polypeptides encoded by the nucleic acid molecules of the present invention. Peptides are also included. Similarity is determined by conservative amino acid substitutions or by structural similarity. Such conservative substitutions are those that substitute a given amino acid in a polypeptide with another amino acid of similar properties. Conservative substitutions are probably phenotypically silent. Usually, replacement of one of the aliphatic amino acids Ala, Val, Leu, and Ile with another; exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu Exchange, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg, and substitution between the aromatic residues Phe and Tyr are considered conservative substitutions. Guidance on which amino acid changes are probably phenotypically silent can be found in Bowie et al., Science 247: 1306-1310 (1990).

  A variant polypeptide may differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations, or any combination thereof. Furthermore, variant polypeptides may be fully functional or may lack function in one or more activities. Mutants with full function usually include only conservative changes or changes in non-critical residues or non-critical regions. Functional variants may also include substitutions of similar amino acids that result in no or minor changes in function. Alternatively, such substitutions can have some positive or negative impact on function. Nonfunctional variants usually include one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions within critical residues or heavy regions, Or a deletion is included.

  Amino acids essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scan mutagenesis (Cunningham et al., Science, 244: 1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro. Sites important for polypeptide activity can be determined by structural analysis, such as crystallography, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol., 244: 899-904 (1992); de Vos et al. Science, 255: 306-312 (1992).

  The present invention also includes polypeptide fragments of the polypeptides of the present invention. The fragment may be derived from a polypeptide encoded by a nucleic acid molecule comprising SEQ ID NO: 1 and comprising at least one polymorphism shown in Tables 2, 3, and 4, or a portion thereof and complements thereof. However, the present invention also includes fragments of variants of the polypeptides described herein. As used herein, a fragment includes at least 6 consecutive amino acids. Useful fragments include those that retain one or more of the biological activities of the polypeptide, as well as fragments that can be used as immunogens to generate polypeptide-specific antibodies.

  Biologically active fragments (eg peptides with a length of 6, 9, 12, 15, 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids ) Include domains, segments or motifs identified by analysis of polypeptide sequences using well-known methods, eg, signal peptides, extracellular domains, one or more transmembrane segments or loops, ligand binding regions, A zinc finger domain, DNA binding domain, acylation site, glycosylation site, or phosphorylation site may be included.

  Fragments can be discontinuous (not fused to other amino acids or polypeptides) or can be present in larger polypeptides. Furthermore, several fragments can be contained within a single larger polypeptide. In certain embodiments, a fragment designed for expression in a host is fused to a heterologous pre-polypeptide and pro-polypeptide region fused to the amino terminus of the polypeptide fragment, and to the carboxyl terminus of the fragment. There can be additional regions.

  Accordingly, the present invention provides a chimeric or fusion polypeptide. These include a polypeptide of the invention operably linked to a heterologous protein or polypeptide having an amino acid sequence that is not substantially homologous to the polypeptide of the invention. “Operably linked” indicates that the polypeptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide. In certain embodiments, the fusion polypeptide does not affect the function of the polypeptide itself. For example, the fusion polypeptide can be a GST-fusion polypeptide, in which the polypeptide sequence is fused to the C-terminus of the GST sequence. Other types of fusion polypeptides include, but are not limited to, enzyme fusion polypeptides, such as β-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, and Ig fusions. Not. Such fusion polypeptides, particularly poly-His fusions, can facilitate the purification of recombinant polypeptides. In certain host cells (eg, mammalian host cells), expression and / or secretion of the polypeptide can be increased by using a heterologous signal sequence. Thus, in another aspect, the fusion polypeptide includes a heterologous signal sequence at its N-terminus.

  EP-A-0,464,533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. Fc is useful for therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0,232,262). In drug discovery, for example, human proteins are fused with Fc moieties for the purpose of high-throughput screening assays to identify antagonists. Bennett et al., J. Mol. Rec., 8: 52-58 (1995) and Johanson et al., J. Biol. Chem., 270, 16: 9459-9471 (1995). Accordingly, the present invention also includes a soluble fusion comprising a polypeptide of the present invention and various portions of the constant region of an immunoglobulin heavy or light chain of various subclasses (IgG, IgM, IgA, IgE). Polypeptides are also included.

  Chimeric or fusion polypeptides can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding various polypeptide sequences are ligated together in frame according to conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be performed using anchor primers that produce complementary overhangs between two consecutive nucleic acid fragments, which are then annealed and amplified again to yield a chimeric nucleic acid sequence. (See Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (eg, a GST protein). A nucleic acid molecule encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide.

  An isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or any known protein synthesis method. Can be synthesized using. In certain embodiments, the polypeptide is produced by recombinant DNA technology. For example, a nucleic acid molecule encoding a polypeptide is cloned into an expression vector, the expression vector is introduced into a host cell, and the polypeptide is 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, the polypeptides of the invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using art-recognized methods. The polypeptides of the present invention may be used to raise antibodies or elicit an immune response. The polypeptide is also used as a reagent, eg, a labeling reagent, in an assay to quantitatively determine the level of polypeptide or molecule (eg, receptor or ligand) that binds to it in a biological fluid. obtain. Polypeptides can also be used as markers for cells or tissues, where the corresponding polypeptides are preferentially expressed either constitutively, during tissue differentiation, or in disease states. Polypeptides are used to isolate corresponding binding partners, e.g. receptors or ligands, e.g. in interaction capture assays, and to screen for peptides or small molecule antagonists or agonists of binding interactions. obtain.

Antibodies of the Invention Polyclonal and / or monoclonal antibodies that specifically bind to one form of the gene product but not to the other form of the gene product are also provided. Antibodies that bind to a portion of either the variant or control gene product that includes the polymorphic site (s) are also provided. According to the present invention, the polypeptide and polypeptide fragment of the present invention, for example, having the amino acid sequence encoded by SEQ ID NO: 2 and comprising at least one polymorphism shown in Tables 2, 3, and 4, or a part thereof Or having an amino acid sequence encoded by a nucleic acid molecule comprising all or part of SEQ ID NO: 1 and comprising at least one polymorphism shown in Tables 2, 3, and 4. An antibody is provided. The term “antibody” as used herein means an immunoglobulin molecule and is an immunologically active molecule of an immunoglobulin molecule, ie, a molecule comprising an antigen binding site that specifically binds an antigen. Say part. Molecules that specifically bind to a polypeptide of the invention bind to the polypeptide or a fragment thereof, but are not substantial to other molecules in a sample, eg, a biological sample that naturally contains the polypeptide. Molecules that do not bind together. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab ′) ₂ fragments that can be generated by treating an antibody with an enzyme such as pepsin. The present invention provides polyclonal and monoclonal antibodies that bind to the polypeptides of the present invention. The term “monoclonal antibody” or “monoclonal antibody composition” as used herein includes only one of the antigen binding sites capable of immunoreacting with a particular epitope of a polypeptide of the invention. Refers to a population of antibody molecules. Thus, a monoclonal antibody composition usually displays a single binding affinity for a particular polypeptide of the invention that immunoreacts therewith.

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

  Any of the many well-known protocols used to fuse lymphocytes and immortalized cells can be utilized for the purpose of generating monoclonal antibodies to the polypeptides of the present invention (eg, Current Protocols in Immunology, previous See Galfre et al. (1977) Nature, 266: 55052; RHKenneth, Monoclonal Antibodies: A New Dimension In Biological Analysis, Plenum Publishing Corp., New York, New York (1980); and Lerner (1981) Yale J. Biol. Med., 54: 387-402). Furthermore, it will be appreciated by those skilled in the art that there are many variations of such methods that are also useful.

  Instead of preparing a hybridoma that secretes a monoclonal antibody, a monoclonal antibody against a polypeptide of the invention can be obtained by screening a recombinant combinatorial immunoglobulin library (eg, an antibody phage display library) with the polypeptide, and By isolating a member of an immunoglobulin library that binds to the polypeptide, it can be identified and isolated. Kits for creating and screening phage display libraries are commercially available (eg, Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP® Phage Display Kit, catalog number 240,612) . In addition, examples of methods and reagents that are particularly suitable for use in generating and screening antibody display libraries include, for example, US Pat. No. 5,223,409; PCT International Publication No. WO92 / 18619; PCT International Publication No. WO91 / 17271; PCT International Publication Number WO92 / 20791; PCT International Publication Number WO92 / 15679; PCT International Publication Number WO93 / 01288; PCT International Publication Number WO92 / 01047; PCT International Publication Number WO92 / 09690; PCT International Publication Number WO90 / 02809; Fuchs et al. (1991) Bio / Technology, 9: 1370-1372; Hay et al. (1992) Hum. Antibod. Hibridomas, 3: 81-85; Huse et al. (1989) Science, 246: 1275-1281; Griffiths et al. ( 1993) EMBO J., 12: 725-734.

  In addition, recombinant antibodies, such as chimeric antibodies and humanized monoclonal antibodies, which contain both human and non-human portions, which can be made using standard recombinant DNA techniques, are of the present invention. Included in the range. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

In general, the antibodies of the invention (eg, monoclonal antibodies) can be used to isolate the polypeptides of the invention by standard techniques such as affinity chromatography or immunoprecipitation. Polypeptide-specific antibodies can facilitate the purification of naturally occurring polypeptides from cells and the recombinantly produced polypeptides expressed in host cells. Furthermore, an antibody specific for a polypeptide of the invention detects the polypeptide (eg, in a cell lysate, cell supernatant, or tissue sample) to assess the amount and pattern of expression of the polypeptide. Can be used for. Antibodies can be used diagnostically, for example, to monitor protein concentration in tissues as part of a clinical trial procedure, to determine the effectiveness of a given treatment regimen. Detection can be facilitated by binding of 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. Examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S, ³² P, ³³ P, ¹⁴ C, or ³ H.

Diagnosis and Screening Assays of the Invention The present invention also relates to a diagnosis of osteoporosis or susceptibility to osteoporosis associated with the presence of a BMP2 nucleic acid or gene product in an individual, or a method to aid such a diagnosis. Diagnostic assays can be designed to assess BMP2 gene expression or to assess the activity of the BMP2 polypeptides of the invention. Such assays can be used alone or in combination with other assays, eg, bone metabolic marker assays (eg, bone scans). In certain embodiments, this assay is used in the context of biological samples (eg, blood, serum, cells, tissues, synovial fluid), thereby determining whether the individual is suffering from or is at risk of developing osteoporosis. Is determined (has predisposition to osteoporosis or susceptibility to osteoporosis). The present invention also provides a prognostic (ie, predictive) assay for determining whether an individual is susceptible to developing osteoporosis. For example, genetic alterations can be assayed in a biological sample. Such an assay can thereby be used for prognostic or predictive purposes to prophylactically treat an individual prior to the onset of symptoms associated with osteoporosis. Another aspect of the present invention is an assay for monitoring the effect of a factor (eg, a drug, compound, or other factor) on the expression or activity of a polypeptide of the present invention, and further to a BMP2 polypeptide. It relates to assays for identifying agents that bind. These and other assays and factors are described in further detail in the following sections.

Diagnostic Assays The nucleic acids, polypeptides, and antibodies described herein can be used in methods of diagnosing susceptibility to osteoporosis or osteoporosis, and in kits useful for diagnosing osteoporosis or susceptibility to osteoporosis.

  In certain embodiments of the invention, diagnosis of susceptibility to osteoporosis is made by detecting a polymorphism of BMP2 as described herein. A polymorphism is an insertion or deletion of one nucleotide or more than one nucleotide that results in a frameshift; at least one nucleotide change that results in a change in the encoded amino acid; at least one nucleotide that results in the creation of an early termination codon Several nucleotide deletions that result in deletion of one or more amino acids encoded by the nucleotide; disruption of the coding sequence of the gene, such as by unequal recombination or inversion of the gene BMP2 changes, such as insertion of one or several nucleotides; duplication of an entire gene or part of a gene; transposition of an entire gene or part of a gene; or rearrangement of an entire gene or part of a gene It can be. One or more such changes can be present in a gene. Such sequence changes alter the polypeptide encoded by the BMP2 nucleic acid. For example, if a nucleic acid sequence change causes a frameshift, the creation of a termination codon at an earlier position can result in a change in the encoded amino acid and / or production of a truncated polypeptide. Can occur. Alternatively, the polymorphism associated with susceptibility to osteoporosis can be a synonymous change of one or more nucleotides (ie, a change that does not change the polypeptide encoded by the BMP2 gene). Such polymorphisms can, for example, alter splicing sites, affect mRNA stability or transport, or otherwise affect gene transcription or translation. A BMP2 nucleic acid having any of the above changes is also referred to herein as a “variant nucleic acid”, or sometimes a “mutant gene”.

  In the first method for diagnosing susceptibility to osteoporosis, hybridization methods such as Southern analysis, Northern analysis, or in situ hybridization can be used (Current Protocols in Molecular Biology, Ausubel, F. et al., Ed., John Wiley). & Sons (see all supplements since 1999)). For example, a biological sample from a genomic DNA, RNA, or cDNA test subject (“test sample”) is suspected of having or is susceptible to osteoporosis. Or an individual predisposed to osteoporosis or having an osteoporotic disorder ("test individual"). An individual can be an adult, a child, or a fetus. Test samples such as blood samples, amniotic fluid samples, cerebrospinal fluid samples, synovial fluid samples, or tissue samples derived from skin, muscle, buccal or conjunctival mucosa, placenta, digestive tract, or other organs Such as from any source, including genomic DNA. Test samples of DNA derived from fetal cells or tissues can be obtained by any suitable method, such as by amniocentesis or chorion sampling. DNA, RNA, or cDNA samples are then tested to determine if a polymorphism is present in BMP2. The presence of a polymorphism can be indicated by hybridization of a gene in genomic DNA, RNA, or cDNA to a nucleic acid probe. A “nucleic acid probe” as used herein can be a DNA probe or an RNA probe. The nucleic acid probe includes at least one polymorphism in BMP2. The probe can be any of the nucleic acid molecules described above (eg, genes or nucleic acids, fragments, vectors containing genes, etc.).

  In order to diagnose susceptibility to osteoporosis, a hybridization sample is formed by contacting a test sample containing BMP2 with at least one nucleic acid probe. Non-limiting examples of probes for detecting mRNA or genomic DNA are labeled nucleic acid probes that can hybridize to the mRNA or genomic DNA sequences described herein. A nucleic acid probe can be, for example, a full-length nucleic acid molecule or a portion thereof, eg, stringent to appropriate mRNA or genomic DNA, eg, at least 15, 30, 50, 100, 250, or 500 nucleotides in length. Sufficient oligonucleotide to specifically hybridize under mild conditions. For example, the nucleic acid probe can be the entire SEQ ID NO: 1 or a portion thereof, optionally including at least one polymorphism shown in Tables 2, 3, and 4, or a complement thereof. Or may be part of them. Other probes suitable for use in the diagnostic assays of the invention are described herein.

  The hybridization sample is maintained under conditions sufficient to effect specific hybridization of the nucleic acid probe to BMP2. “Specific hybridization” as used herein indicates exact hybridization (eg, does not include mismatches). Specific hybridization can be performed under conditions of high stringency or moderate stringency, eg, as described above. In certain embodiments, 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, BMP2 has a polymorphism, which is present in the nucleic acid probe. One or more nucleic acid probes can also be used simultaneously in this method. Specific hybridization of any one of the nucleic acid probes is an indicator of the polymorphism in BMP2, and thus is a diagnosis of susceptibility to osteoporosis.

  In another hybridization method, Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F. et al., Ed., John Wiley & Sons, supra) has been linked to susceptibility to osteoporosis. Used to identify the presence of a type. For Northern analysis, RNA test samples are obtained from the individual by appropriate means. Specific hybridization of a nucleic acid probe to RNA derived from an individual as described above is an indicator of polymorphism in BMP2, and thus a diagnosis of susceptibility to osteoporosis.

  See, eg, US Pat. Nos. 5,288,611 and 4,851,330 for representative examples of the use of nucleic acid probes.

  Alternatively, peptide nucleic acid (PNA) probes can be used in place of nucleic acid probes in the hybridization methods described above. PNA is a DNA mimic with a peptide-like inorganic backbone, for example, having an organic base (A, G, C, T, or U) attached to the glycine nitrogen via a methylene carbonyl linker. N- (2-aminoethyl) glycine units (see, for example, Nielsen, PE et al., Bioconjugate Chemistry, 1994, 5, American Chemical Society, p. 1 (1994)). PNA probes can be designed to specifically hybridize to genes that have polymorphisms associated with susceptibility to osteoporosis. Hybridization of the PNA probe to BMP2 is a diagnosis of susceptibility to osteoporosis.

  In certain aspects of the invention, diagnosis of susceptibility to BMP2-related osteoporosis or osteoporosis can be made by expression analysis using quantitative PCR (dynamic temperature cycling). In certain embodiments, the diagnosis of osteoporosis is performed in combination with a bone metabolic marker assay (eg, a bone scan) by detecting an allele associated with at least one BMP2. Commercially available techniques such as, for example, TaqMan® (Applied Biosystems, Foster City, Calif.) Can be utilized for this technique, for example, to identify polymorphisms. This technique can assess whether there is a change in the expression or composition of the polypeptide encoded by BMP2, or whether there are splicing variants. Furthermore, the expression of the variant can be quantified as a physical or functional difference.

  In another method of the present invention, if a restriction site is created or disappeared due to a mutation or polymorphism in the gene, the analysis of the mutation by restriction digestion is performed by using a mutant gene containing the polymorphism (s). Can be used to detect the singular (s). A test sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify BMP2 (and flanking sequences, if necessary) in a test sample of genomic DNA from the test individual. RFLP analysis is performed as described (see Current Protocols in Molecular Biology (supra)). The digestion pattern of the relevant DNA fragment indicates whether the mutation or polymorphism is present or absent in BMP2, and thus indicates whether this susceptibility to osteoporosis is present or absent.

  Sequence analysis can also be used to detect specific polymorphisms in BMP2. A test sample of DNA or RNA is obtained from the test individual. PCR or other suitable methods can be used to amplify the gene, and / or its flanking sequences, if desired. The sequence of BMP2, or gene fragment, or cDNA or cDNA fragment, or mRNA or mRNA fragment is determined using standard methods. The sequence of a gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment, where appropriate, is a gene, cDNA (eg, SEQ ID NO: 1, at least as shown in Tables 2, 3, and 4 Compared to the known nucleic acid sequence of the mRNA). The presence of a specific polymorphism in BMP2 indicates that the individual is susceptible to osteoporosis.

  Allele-specific oligonucleotides are also used through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes to detect the presence of polymorphisms in BMP2. (See, for example, Saiki, R. et al. (1986) Nature (London) 324: 163-166). An “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, or approximately 15-30 base pairs, Specifically hybridizes to BMP2, including polymorphisms associated with susceptibility to osteoporosis. Allele-specific oligonucleotide probes specific for a particular polymorphism in BMP2 can be prepared using standard methods (see Current Protocols in Molecular Biology (supra)). In order to identify polymorphisms in genes associated with susceptibility to osteoporosis, a test sample of DNA is obtained from the individual. PCR can be used to amplify whole BMP2 or a fragment thereof, and its flanking sequences. DNA containing amplified BMP2 (or a fragment of that gene) is dot-blotted using standard methods (see Current Protocols in Molecular Biology (supra)) and the blot is the oligonucleotide probe. Contacted with. The presence of specific hybridization of the probe to the amplified BMP2 is then detected. Specific hybridization to DNA from an individual of an allele-specific oligonucleotide probe is an indicator of specific polymorphisms in BMP2, and thus an indicator of susceptibility to osteoporosis.

  An allele-specific primer hybridizes to a site on the target DNA that overlaps with the polymorphism and primes only the amplification of the allelic form of the primer that exhibits complete complementarity thereto (Gibbs, R. et al., 1989. Nucleic Acids Res., 17: 2437-2448). This primer is used in combination with a second primer that hybridizes to the remote site. Amplification proceeds from the two primers and yields a detectable product, indicating that a particular allelic form is present. Controls are usually performed with a second pair of primers. One of the pair shows a single base mismatch at the polymorphic site and the other shows complete complementarity to the remote site. A single base mismatch prevents amplification and no detectable product is formed. This method works best when the mismatch is contained in the most 3 'position of the oligonucleotide that is aligned with the polymorphism. This is because this position most destabilizes extension from the primer (see, eg, International Publication No. WO93 / 22456).

By adding analogs such as locked nucleic acids (LNA), the size of primers and probes can be reduced to as little as 8 bases. LNA is a bicyclic ring in which the 2 'and 4' positions of the furanose ring are connected via O-methylene (oxy-LNA), S-methylene (thio-LNA), or aminomethylene (amino-LNA) moieties A new class of DNA analogues. All these LNA variants share a common affinity for complementary nucleic acids, far exceeding the maximum reported for DNA analogs. For example, all specific oxy-LNA nonamers are in contrast to 28 ° C for both DNA and RNA with respect to the corresponding DNA nonamer when complexed with complementary DNA or RNA. Are shown to have melting points of 64 ° C. and 74 ° C., respectively. A substantial increase in T _m is also obtained when LNA monomers are used in combination with standard DNA or RNA monomers. Depending on where the LNA monomer is included (eg, at the 3 ′ end, 5 ′ end, or in the middle) for the primer and probe, the T _m can be significantly increased.

  In another embodiment, an array of oligonucleotide probes that are complementary to a target nucleic acid sequence segment from an individual can be used to identify polymorphisms in a BMP2 nucleic acid. For example, in some embodiments, oligonucleotide arrays can be used. Oligonucleotide arrays typically include a plurality of different oligonucleotide probes bound to the surface of the substrate at various known locations. These oligonucleotide arrays are also described as “Genechip®” and are commonly used in the art, for example, US Pat. No. 5,143,854 and PCT International Patent Publication Nos. WO 90/15070 and 92/10092. It is described in. These arrays are usually mechanical synthesis methods or photospecific synthesis methods incorporating a combination of photolithography and solid phase oligonucleotide synthesis methods (Fodor, S. et al., 1991. Science, 251: 767- 773; Pirrung et al., US Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070); and Fodor, S. et al., PCT International Publication No. WO 92/10092 and US Pat. No. 5,424,186, their respective teachings. Which is incorporated herein by reference in its entirety). Techniques for the synthesis of these arrays using mechanical synthesis methods are described, for example, in US Pat. No. 5,384,261, the entire teachings of which are incorporated herein by reference. In another example, a linear array can be utilized.

  Once the oligonucleotide array is prepared, the nucleic acid of interest is hybridized with the array and scanned for polymorphisms. Hybridization and scanning are generally described herein and, for example, published PCT application numbers WO92 / 10092 and WO95 / 11995, and US Pat. No. 5,424,186, the entire teachings of which are hereby incorporated by reference. The method described in (1). Briefly, a target nucleic acid sequence containing one or more previously identified polymorphic markers is amplified by well-known amplification techniques, such as PCR. Typically, this technique involves the use of primer sequences (both upstream and downstream of the polymorphism) that are complementary to the duplex of the target sequence. Asymmetric PCR techniques can also be used. The amplified target usually incorporates a label that is then hybridized to the array under appropriate conditions. When array hybridization and washing are complete, the array is scanned to determine the location on the array where the target sequence has hybridized. Hybridization data obtained by scanning is usually in the form of fluorescence intensity as a function of array position.

  With respect to a single detection block, for example, although primarily described for the detection of a single polymorphism, the array may contain multiple detection blocks, thus analyzing multiple specific polymorphisms Can do. In another arrangement, detection blocks can be grouped in a single array or in multiple different arrays so that various optimal conditions can be used during hybridization of a target to the array. For example, it is often desirable to provide detection of these polymorphisms contained in a G-C rich stretch of genomic sequences separate from polymorphisms contained in A-T rich segments. Thereby, the hybridization conditions for each situation can be optimized separately.

  Further descriptions of the use of oligonucleotide arrays for polymorphism detection are found, for example, in US Pat. Nos. 5,858,659 and 5,837,832, the entire teachings of which are incorporated herein by reference. Can be issued.

  Other nucleic acid analysis methods can be used to detect BMP2 polymorphisms. 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., 74: 5463-5467; Beavis et al., US Pat. No. 5,288,644); automated fluorescent sequencing; single strand conformation polymorphism assay (SSCP); clamp denaturing gel electrophoresis (CDGE) Denaturant gradient gel electrophoresis (DGGE) (Sheffield, VC et al. (19891) 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 et al. (1978) Cell 15:25; Geever et al. (1981) Proc. Natl. Acad. Sci. USA, 78: 5081); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al. (1985) Proc. Natl. Acad. Sci. USA, 85: 4397-4401); RNase protection assay (Myers, RM et al., (1985) Science 230: 1242); E. coli mutS tongue Use of recognizing the polypeptide of mismatch of nucleotides, such as click quality; as well as allele-specific PCR.

  In certain embodiments of the invention, diagnosis of susceptibility to a disease or condition associated with BMP2 nucleic acid (eg, osteoporosis) or a disease or condition associated with BMP2 nucleic acid (eg, osteoporosis) is also quantitative PCR. It can be done by expression analysis by (dynamic temperature cycling). In certain embodiments, the diagnosis of the disease itself, eg, osteoporosis, is performed in combination with an assay for bone metabolic markers (eg, a bone scan) by detecting at least one allele associated with BMP2. This technique utilizing TaqMan® can be used to identify polymorphisms and to identify whether a patient is homozygous or heterozygous. This technique can assess whether there is a change in the expression or composition of the polypeptide encoded by the BMP2 nucleic acid, or whether there are splicing variants encoded by the BMP2 nucleic acid. Furthermore, the expression of the variant can be quantified as a physical or functional difference.

  In another aspect of the invention, diagnosis of susceptibility to osteoporosis can also be performed by various methods including enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence, and expression of BMP2 polypeptide and / or Or it can be done by testing the composition. A test sample from an individual is evaluated whether there is a change in the expression of the polypeptide encoded by BMP2 and / or a change in the composition of the polypeptide encoded by BMP2. A change in the expression of a polypeptide encoded by BMP2 can be, for example, a quantitative change in the expression of the polypeptide (ie, the amount of polypeptide produced), and a change in the composition of the polypeptide encoded by BMP2. Is a qualitative change in the expression of a polypeptide (eg, expression of a mutant BMP2 polypeptide, or expression of another splicing variant). In certain embodiments, the diagnosis of susceptibility to osteoporosis is made by detecting a particular splicing variant encoded by BMP2, or a particular pattern of splicing variants.

  Both such changes (quantitative and qualitative) can exist. A “change” in the expression or composition of a polypeptide, as used herein, is an expression or composition in a test sample as compared to the expression or composition of a polypeptide encoded by BMP2 in a control sample. It means change. A control sample is a sample corresponding to a test sample (eg, derived from the same type of cells) and a sample derived from an individual not suffering from osteoporosis. Similarly, the presence of one or more different splicing variants in a test sample or the presence of a significantly different amount of multiple different splicing variants in a test sample compared to a control sample is an indicator of susceptibility to osteoporosis It is. A change in the expression or composition of the polypeptide in the test sample compared to the control sample is an indication of susceptibility to osteoporosis. Various means for testing the expression or composition of the polypeptide encoded by BMP2 can be used, including spectroscopic, colorimetric, electrophoresis, isoelectric focusing, and immunoblotting (Current Protocols in Molecular Biology, in particular, see also Chapter 10) immunoassays (eg David et al., US Pat. No. 4,376,110).

For example, in certain embodiments, an antibody (eg, an antibody having a detectable label) that can bind to a polypeptide (eg, as described above) may be used. The antibody may be polyclonal or monoclonal. An intact antibody, or a fragment thereof (eg, Fab or F (ab ′) ₂ ) can be used. The term “labeled” when used with respect to a probe or antibody, directly labels the probe or antibody by binding (ie, 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 primary antibodies using fluorescently labeled secondary antibodies, and end labeling of DNA probes with biotin to allow detection with fluorescently labeled streptavidin Is included.

  Western blot analysis using the above antibody that specifically binds to a polypeptide encoded by a mutant BMP2 or an antibody that specifically binds to a polypeptide encoded by a gene that is not a mutant is a polymorphic BMP2 or To identify that the polypeptide encoded by mutant BMP2 is present in the test sample, or that the polypeptide encoded by a non-polymorphic or non-mutant gene is not present in the test sample Can be used. The presence of a polypeptide encoded by a polymorphic gene or a mutant gene, or the absence of a polypeptide encoded by a gene that is not a polymorphic gene or a mutant, is a diagnosis of susceptibility to osteoporosis. is there.

  In some embodiments of this method, the level or amount of polypeptide encoded by BMP2 in the test sample is compared to the level or amount of polypeptide encoded by BMP2 in the control sample. The level or amount of polypeptide in the test sample that is greater or less than the level or amount of polypeptide in the control sample, as statistically significant, is indicative of a change in the expression of the polypeptide encoded by BMP2. It is an index and a diagnosis of susceptibility to osteoporosis. Alternatively, the composition of the polypeptide encoded by BMP2 in the test sample is compared to the composition of the polypeptide encoded by BMP2 in the control sample. The difference in the composition of the polypeptide in the test sample compared to the composition of the polypeptide in the control sample is a diagnosis of susceptibility to osteoporosis. In another aspect, the level or amount and composition of the polypeptide can be assessed in a test sample and in a control sample. Difference in the amount or level of polypeptide in the test sample compared to the control sample; difference in composition in the test sample compared to the control sample; or both difference in amount or level and difference in composition may result in osteoporosis Is an indicator of susceptibility to.

  Kits useful in diagnostic methods include components useful in any of the methods described herein, including, for example, hybridization probes, restriction enzymes (eg, for RFLP analysis), Allele-specific oligonucleotide, altered BMP2 polypeptide or unchanged (natural) BMP2 polypeptide (eg, SEQ ID NO: 2, at least one of the multiples shown in Tables 2, 3, and 4) Antibodies that bind to a type), means for amplification of nucleic acids containing BMP2, or means for analyzing the nucleic acid sequence of BMP2 or for analyzing the amino acid sequence of a BMP2 polypeptide. In addition, the kit can provide reagents for assays used in combination with the methods of the invention, eg, bone metabolic marker assays (eg, bone scans).

  Kits that are useful in diagnostic methods (eg, reagent kits) include components that are useful in any of the methods described herein, such as those described herein. Hybridization probes or primers (eg, labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (eg, for RFLP analysis), allele-specific oligonucleotides, altered BMP2 poly An antibody that binds to a peptide or an unmodified (natural) BMP2 polypeptide, a means for amplification of a nucleic acid containing BMP2, or a nucleic acid sequence of a BMP2 nucleic acid as described herein Or means for analyzing the amino acid sequence of the BMP2 polypeptide. In certain embodiments, a kit for diagnosing osteoporosis or susceptibility to osteoporosis comprises one or more polymorphic sites that are more frequently present in individuals who have or are susceptible to osteoporosis, Primers for nucleic acid amplification of regions in the BMP2 nucleic acid can be included. Primers can be designed using a portion of the nucleic acid adjacent to an SNP that is indicative of osteoporosis. In addition, the kit can provide reagents for assays used in combination with the methods of the invention, eg, bone metabolic marker assays (eg, bone scans).

  The invention further relates to methods for diagnosing and identifying susceptibility to osteoporosis in an individual by identifying risk markers in BMP2, such as SNPs. In certain embodiments, the risk marker is one that confers a significant risk of osteoporosis. In certain embodiments, significance associated with a marker is measured by an odds ratio. In a further aspect, significance is measured by percentage. In some embodiments, significant risk is measured as an odds ratio that is at least about 2.2, including but not limited to 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. In a further aspect, an odds ratio of at least 1.2 is significant. In a further aspect, an odds ratio of at least about 1.5 is significant. In a further aspect, the significant increase in risk is significant at least about 1.7. In a further aspect, the significant increase in risk is at least about 20% and about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, and 98%. In a further aspect, the significant increase in risk is at least about 50%. However, it is understood that the identification of whether a risk is medically significant can also vary depending on a variety of factors, including the particular disease, markers, and often environmental factors.

  The present invention also relates to a method of diagnosing osteoporosis or susceptibility to osteoporosis in an individual. This method screens for risk markers associated with BMP2 nucleic acids that are more frequently present in individuals susceptible to osteoporosis (affected) compared to their frequency of presence in healthy individuals (control). The process of carrying out is included. Here, the presence of the marker is an indicator of susceptibility to osteoporosis or osteoporosis. Standard techniques for genotyping for the presence of SNPs and / or microsatellite markers associated with osteoporosis, such as fluorescence-based techniques (Chen et al., 1999. Genome Res., 9: 492), PCR, LCR, Nested PCR, and other techniques for nucleic acid amplification can be used. In certain embodiments, the method includes assessing in an individual the presence or frequency of a particular SNP allele or microsatellite allele associated with a BMP2 nucleic acid associated with osteoporosis. Here, an excess of markers or a high frequency of markers compared to a healthy control individual is an indicator that the individual has osteoporosis or is susceptible to osteoporosis. See Tables 2, 3, and 4 for SNPs and markers that can be used as screening tools.

Screening Assays and Factors Identified Thereby According to the present invention, a method for identifying the presence of a nucleotide that hybridizes to a nucleic acid of the present invention and further for identifying the presence of a polypeptide encoded by the nucleic acid of the present invention (Also referred to herein as a “screening assay”). In certain embodiments, the presence (or absence) of a nucleic acid molecule of interest (eg, a nucleic acid having significant homology to a nucleic acid of the invention) in a sample causes the sample to contain a nucleic acid (eg, A nucleic acid having the sequence of SEQ ID NO: 1 and comprising at least one polymorphism shown in Tables 2, 3, and 4, or a complement thereof, or an amino acid having the sequence of SEQ ID NO: 2. By contacting the encoding nucleic acid, or a fragment or variant of such nucleic acid) under stringent conditions as described above, and then evaluating the sample for the presence (or absence) of hybridization Can be evaluated. In certain embodiments, high stringency conditions are conditions suitable for selective hybridization. In another embodiment, the sample containing the nucleic acid molecule of interest is a contiguous nucleotide sequence (eg, as described above) that is at least partially complementary to a portion of the nucleic acid molecule of interest (eg, a mutant BMP2 nucleic acid). The contacted sample is evaluated for the presence or absence of hybridization. In certain embodiments, a nucleic acid comprising a contiguous nucleotide sequence is completely complementary to a portion of the nucleic acid molecule of interest.

  In any of these embodiments, the entire nucleic acid of interest or a portion thereof can be amplified prior to hybridization.

  In another aspect, the presence (or absence) of a polypeptide of interest, such as a polypeptide of the invention or a fragment or variant thereof, in a sample specifically hybridizes the sample to the polypeptide of interest. It can be assessed by contacting with an antibody (eg, an antibody such as those described above) and then evaluating the sample for the presence (or absence) of binding of the antibody to the polypeptide of interest.

  In another aspect, the invention alters (eg, increases or decreases) the activity of the polypeptides described herein, or otherwise interacts with the polypeptides herein. Methods are provided for identifying agents that act (eg, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules, or other drugs, or ribozymes). For example, such an agent can be a factor that binds to a polypeptide described herein (eg, a BMP2 binding factor); for example, a factor that has a stimulatory or inhibitory effect on the activity of a polypeptide of the invention. A factor that alters (eg, increases or inhibits) the ability of a polypeptide of the invention to interact with a BMP2 binding agent (eg, a receptor or other binding agent); or post-translational processing of a BMP2 polypeptide; (Eg, factors that alter proteolytic processing to direct the polypeptide to another location in the cell from where it is normally synthesized, eg, to the cell surface; more polypeptides A factor that alters proteolytic processing to be released from the cell).

  In certain embodiments, the invention binds to a polypeptide (or biologically active portion (s) thereof) described herein or is polypeptide (or biologically active thereof). Assays for screening candidate or test agents that modulate the activity of the active moiety (s), as well as factors that can be identified by this assay, are provided. Test factors can be obtained using any of a number of techniques in combinatorial library methods known in the art, including combinatorial library methods such as biological libraries; Possible parallel solid phase or liquid phase libraries; synthetic library methods requiring deconvolution; “one bead one compound” library method; and synthetic library methods using affinity chromatography selection. Biological library approaches are limited to polypeptide libraries, but the other four approaches can be applied to small molecule libraries of polypeptides, non-peptide oligomers, or compounds (Lam, KS (1997) Anticancer Drug Des., 12: 145).

  In some embodiments, comprises a BMP2 polypeptide (eg, SEQ ID NO: 2, comprising at least one polymorphism shown in Tables 2, 3, and 4), or an active fragment or derivative thereof (described above) A cell, cell lysate, or solution that is or expresses can be contacted with the agent being tested to identify an agent that alters the activity of the BMP2 polypeptide. Alternatively, the polypeptide can be contacted directly with the agent being tested. The level (amount) of BMP2 activity is assessed (eg, the level (amount) of BMP2 activity is measured directly or indirectly) and the level of activity in the control (ie, under conditions in which there is no agent being tested) Level of activity of the BMP2 polypeptide or active fragment or derivative thereof). A factor that alters the activity of a BMP2 polypeptide if the level of activity in the presence of the factor differs from the level of activity in the absence of the factor by a statistically significant amount. . An increase in the level of BMP2 activity compared to the control indicates that this factor is a factor that increases BMP2 activity (it is an agonist of BMP2 activity). Similarly, a decrease in the level of BMP2 activity compared to the control indicates that this factor is a factor that inhibits BMP2 activity (an antagonist of BMP2 activity). In another embodiment, the level of activity of the BMP2 polypeptide or derivative or fragment thereof in the presence of the agent being tested is compared to a previously established control level. A level of activity in the presence of a factor that differs by a statistically significant amount from the control level indicates that this factor alters BMP2 activity.

  The invention also includes factors that alter the expression of BMP2 nucleic acid (eg, antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules, or other drugs, or Ribozymes), factors that alter (eg, increase or decrease) gene expression (eg, transcription or translation), or that otherwise interact with nucleic acids described herein It also relates to the assay to be performed, as well as the factors that can be identified by this assay. For example, a solution containing a nucleic acid encoding a BMP2 polypeptide (eg, a BMP2 gene) can be contacted with the agent being tested. The solution can include, for example, cells containing nucleic acids or cell lysates containing nucleic acids. Alternatively, the solution can be another solution containing elements essential for transcription / translation of nucleic acids. Cells not suspended in solution can also be used if desired. The level and / or pattern of BMP2 expression (eg, the level and / or pattern of mRNA or expressed protein, eg, the level and / or pattern of various splicing variants) and the level of expression in the control And / or pattern (ie, the level and / or pattern of expression of BMP2 under the absence of the agent being tested). If the level and / or pattern in the presence of the factor differs from the level and / or pattern in the absence of the factor in a statistically significant amount or manner, then the factor is expressed in BMP2 It is a factor that changes Increased expression of BMP2 indicates that this factor is an agonist of BMP2 activity. Similarly, inhibition of BMP2 expression indicates that this factor is an antagonist of BMP2 activity. In another embodiment, the level and / or pattern of the BMP2 polypeptide (s) (eg, various splicing variants) in the presence of the agent being tested is a previously established control level and / or pattern. Compared with If the level and / or pattern in the presence of the factor differs from the control level and / or pattern in a statistically significant amount or manner, this indicates that the factor alters the expression of BMP2.

  In another aspect of the invention, an agent that alters the expression of a BMP2 nucleic acid or that otherwise interacts with a nucleic acid described herein is a BMP2 nucleic acid operably linked to a reporter gene. It can be identified using cells, cell lysates, or solutions that contain nucleic acid encoding the promoter region. After contact with the agent being tested, the level of expression of the reporter gene (eg, the level of mRNA or expressed protein) is assessed and the level of expression in the control (ie, conditions where the agent being tested is not present) The level of expression of the reporter gene below). If the level in the presence of the factor differs in a statistically significant amount or manner from the level in the absence of the factor, then the factor is a gene operably linked to the BMP2 gene promoter It is a factor that alters the expression of BMP2, as shown by its ability to alter the expression of. Increased reporter expression indicates that this factor is an agonist of BMP2 activity. Similarly, inhibition of reporter expression indicates that this factor is an antagonist of BMP2 activity. In another embodiment, the level of reporter expression in the presence of the agent being tested is compared to a pre-established control level. If the level in the presence of the factor differs from the control level in a statistically significant amount or manner, it indicates that the factor alters BMP2 expression.

  Factors that alter the amount of various splicing variants encoded by BMP2 (eg, factors that increase the activity of the first splicing variant and inhibit the activity of the second splicing variant), and Factors that are agonists of the activity of one splicing variant and antagonists of the activity of the second splicing variant can be identified using these methods described above.

In other embodiments of the invention, the assay can be used to assess the effect of a test agent on the activity of a polypeptide associated with a BMP2 binding agent. For example, cells expressing a compound that interacts with BMP2 (referred to herein as a “BMP2 binding agent”, which can be a polypeptide or other molecule that interacts with BMP2, such as a receptor) The ability of the test factor to be contacted with BMP2 in the presence of the test factor to alter the interaction between BMP2 and the BMP2 binding factor is determined. Alternatively, cell lysates or solutions containing BMP2 binding agents can be used. A factor that binds to BMP2 or a BMP2 binding factor interferes with or enhances the ability of BMP2 to bind to, associate with, or otherwise interact with a BMP2 binding factor, Interaction can be changed. Determination of the ability of a test agent to bind to BMP2 or a BMP2 binding agent can be performed, for example, by coupling the test agent with a radioisotope or enzyme label, resulting in ¹²⁵ I, ³⁵ S, ³² P, ³³ P The binding of the test agent to the polypeptide can be determined by detecting either directly or indirectly labeled at ¹⁴ C, or ³ H, and the radioisotope directly emits radiation. It can be counted or detected by scintillation counting. Alternatively, the test agent can be enzyme labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzyme label can be detected by measuring the conversion of the appropriate substrate to product. It is also within the scope of the present invention to determine the ability of a test agent to interact with a polypeptide without any label of the interactant. For example, a microphysiometer can be used to detect the interaction of a test agent with BMP2 or BMP2 binding agent without labeling any of the test agent, BMP2, or BMP2 binding agent (McConnell, HM et al. (1992) Science, 257: 1906-1912). As used herein, a “microphysiometer” (eg, Cytosensor®) is a rate at which a cell acidifies its environment using a photoaddressable potentiometric sensor (LAPS). Is an analytical instrument for measuring This change in acidification rate can be used as an indicator of the interaction between the ligand and the polypeptide. See the “Examples” section for a discussion of known BMP2 binding partners. Thus, these receptors can be used to screen for compounds that are BMP2 receptor agonists for use in the treatment of osteoporosis or compounds that are BMP2 receptor antagonists for the study of osteoporosis. The linkage data provided herein provides for the first time such correction for osteoporosis. Drugs can be designed to modulate BMP2 receptor activation and then used to modulate signal transduction pathways and downstream gene transcription events.

  In another aspect of the invention, the assay can be used to identify polypeptides that interact with one or more BMP2 polypeptides described herein. For example, the yeast two-hybrid system (Fields, S. and Song, O., Nature 340: 245-246 (1989)) is used to identify polypeptides that interact with one or more BMP2 polypeptides. obtain. In such a yeast two-hybrid system, the vector is constructed based on the flexibility of a transcription factor having two functional domains: a DNA binding domain and a transcription activation domain. If the two domains are separated but fused to two different proteins that interact with each other, transcriptional activation can occur and specific markers (eg, nutritional markers such as His and Ade) , Or a colored marker such as lacZ) can be used to identify the presence of interaction and transcriptional activation. For example, the method of the present invention uses a first vector comprising a DNA binding domain and a nucleic acid further encoding a BMP2 polypeptide, splicing variant, or fragment or derivative thereof, and a nucleic acid encoding a transcriptional activation domain. And a second nucleic acid encoding a polypeptide that may be capable of interacting with a BMP2 polypeptide, splicing variant, or a fragment or derivative thereof (eg, a BMP2 polypeptide binding factor or receptor). A vector is used. Incubation under the appropriate conditions of the yeast containing the first and second vectors (eg, conjugation conditions as used in Clontech's Matchmaker® system) yields colonies that express the marker of interest. Identified. These colonies can be tested to identify the polypeptide (s) that interact with the BMP2 polypeptide or a fragment or derivative thereof. Such polypeptides can be useful as factors that alter the activity of expression of a BMP2 polypeptide, as described above.

  In one or more embodiments of the above assay methods of the present invention, the assay may be further automated to facilitate separation of the complex form from the uncomplexed form of one or both polypeptides. To accommodate, it may be desirable to immobilize either BMP2, a BMP2 binding agent, or other components of the assay on a solid support. Binding of the test agent to the polypeptide or interaction of the polypeptide with the binding agent in the presence and absence of the test agent should be performed in any container suitable for containing the reactants. Can be broken. Examples of such containers include microtiter plates, test tubes, or microcentrifuge tubes. In certain embodiments, a fusion protein (eg, a GST fusion protein) can be provided, to which a domain capable of binding BMP2 or a BMP2 binding agent to a matrix or other solid support has been added.

  In another embodiment, a cell, cell lysate, or solution containing a nucleic acid encoding BMP2 is contacted with a test agent and an appropriate mRNA or polypeptide (eg, splicing mutation) in the cell, cell lysate, or solution. In the method in which the expression of the body (s) is determined, the modulator of expression of the nucleic acid molecule of the invention is determined. The level of expression of the appropriate mRNA or polypeptide (s) in the presence of the test factor is compared to the level of expression of the mRNA or polypeptide (s) in the absence of the test factor . The test factor can then be identified as a regulator of expression based on this comparison. For example, if mRNA or polypeptide expression is greater (statistically significantly greater) in the presence of the test agent than in the absence of it, the test agent is a stimulator of mRNA or polypeptide expression. Or identified as a facilitator. Alternatively, if mRNA or polypeptide expression is less (statistically significantly less) in the presence of the test agent than in the absence of it, the test agent is an inhibitor of mRNA or polypeptide expression. Identified as The level of mRNA or polypeptide expression in a cell can be determined by the methods described herein for detecting mRNA or polypeptide.

  The invention further relates to a novel factor identified by the screening assay described above. Accordingly, it is within the scope of the present invention to further use the factors identified as described herein in appropriate animal models. For example, factors identified as described herein (eg, test factors that are modulators, antisense nucleic acid molecules, specific antibodies, or polypeptide binding agents) used such factors. It can be used in animal models to determine the efficacy, toxicity, or side effects of treatment. Alternatively, factors identified as described herein can be used in animal models to determine the mechanism of action of such factors. The present invention further relates to the use of the novel factors identified by the above screening assays for the treatments described herein. In addition, an agent identified as described herein may include a polypeptide or gene herein to alter the activity of a polypeptide encoded by BMP2, or to alter the expression of BMP2. It can be used by contacting the identified agent as described in the document (or contacting the cell containing the polypeptide or gene with the identified agent).

Pharmaceutical Compositions The invention also includes a nucleic acid described herein, in particular a nucleotide encoding a polypeptide described herein; a polypeptide described herein ( For example, comprising a factor that alters (eg, increases or inhibits) the activity of a BMP2 polypeptide described herein. For example, a polypeptide, protein, fragment, fusion protein or prodrug thereof of the present invention, or a nucleotide or nucleic acid construct (vector) containing the nucleotide of the present invention, or a factor that alters the activity of a BMP2 polypeptide is a pharmaceutical composition. Can be formulated with a physiologically acceptable carrier or excipient. The carrier and composition can be sterile. The formulation should be tailored to the mode of administration.

  Suitable pharmaceutically acceptable carriers include water, salt solution (eg, NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin, These include carbohydrates (eg, lactose, amylose or starch, dextrose), magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid ester, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, and combinations thereof. It is not limited. Pharmaceutical preparations, if desired, to act on lubricants, preservatives, stabilizers, wetting agents, emulsifiers, osmotic pressure, without reacting adversely with active factors It can be mixed with auxiliaries such as salts, buffers, colorants, flavors, and / or fragrances.

  If desired, the composition can also include minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate and the like.

  Methods for introducing these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral, and intranasal. Other suitable introduction methods may include gene therapy (below), rechargeable or biodegradable devices, particle accelerators (“gene guns”), and sustained release polymer devices. The pharmaceutical compositions of the invention can also be administered as part of a combination therapy with other reagents.

  The composition can be formulated according to routine procedures as a pharmaceutical composition adapted for human administration. For example, a composition for intravenous administration is usually a solution in a sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to reduce pain at the site of the injection. In general, the components are either frozen separately or mixed together in a unit dosage form, for example, frozen in a sealed container such as an ampoule or bag that presents the amount of active agent. Supplied as a dry powder or water free concentrate. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  For topical application, non-sprayable forms, viscous to semi-solid or solid forms containing carriers compatible with topical application and having a kinematic viscosity greater than water can be used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, emulsions, ointments, powders, enemas, lotions, sols, coatings, ointments, aerosols and the like. These can be sterilized or mixed with adjuvants such as preservatives, stabilizers, wetting agents, buffers, or salts to affect osmotic pressure, if desired. The factor can be mixed into the cosmetic formulation. For topical application, the active ingredients are packaged in squeeze bottles, optionally combined with a solid or liquid inert carrier material, or compressed volatile, usually gaseous, high pressure Also suitable are sprayable aerosol preparations which are mixed with a gas, for example compressed air.

  The reagents described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, as well as sodium, potassium, ammonium, calcium, Examples thereof include salts formed using free carboxyl groups such as those derived from ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, and procaine.

  The factor is administered in a therapeutically effective amount. The amount of a factor that is therapeutically effective in the treatment of a particular disorder or symptom varies depending on the nature of the disorder or symptom and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The exact dose used in the formulation will also vary depending on the route of administration and the severity of the symptoms of osteoporosis and should be determined according to the judgment of the physician and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Optionally, such container (s) may be accompanied by a notice in a format prescribed by a government agency that controls the manufacture, use, or sale of a pharmaceutical or biological product. This notice reflects government approval of manufacture, use, or sale for human administration. The pack or kit may display information regarding the mode of administration, the order of administration of the drugs (eg, separately, sequentially or simultaneously), and the like. The pack or kit may also include means for keeping the patient informed of the treatment. The pack or kit can be a single unit dose of the combination therapy, or it can be a plurality of unit doses. Specifically, the factors can be individualized, mixed together in any combination, and can be present in one vial or in a tablet. Factors gathered in blister packs or other dispersing means are envisioned by the present invention. For the purposes of the present invention, the unit dose varies with the individual pharmacokinetics of each reagent and is intended to mean a dose administered at an FDA approved dose over a standard time course. The

Therapeutic Methods The present invention also relates to methods of treatment (prophylactic and / or therapeutic) for osteoporosis or susceptibility to osteoporosis using a BMP2 therapeutic agent. A “BMP2 therapeutic” is a factor (eg, a BMP2 agonist or antagonist) that alters (eg, increases or inhibits) the activity of a BMP2 polypeptide and / or the expression of BMP2, as described herein. ).

  A BMP2 therapeutic agent may be an activity or gene expression of a BMP2 polypeptide, eg, by providing additional BMP2 polypeptide or by up-regulating transcription or translation of the BMP2 gene; after translation of the BMP2 polypeptide By altering the processing; by altering the transcription of the BMP2 splicing variant; or by interfering with the activity of the BMP2 polypeptide (eg, by binding to the BMP2 polypeptide), or of the BMP2 gene It can be altered by various means such as by down-regulating transcription or translation. Exemplary BMP2 therapeutic agents include the following: nucleic acids described herein or fragments or derivatives thereof, specifically nucleotides encoding the polypeptides described herein, and Such a nucleic acid (eg, a nucleic acid encoding a BMP2 polypeptide or an active fragment or derivative thereof, such as a gene, cDNA, and / or mRNA, or oligonucleotide; eg, SEQ ID NO: 1, optionally Comprising at least one polymorphism shown in Tables 2, 3, and 4, or a nucleic acid encoding SEQ ID NO: 2, optionally at least one polymorphism shown in Tables 2, 3, and 4 Or a fragment or derivative thereof); a polypeptide described herein (eg, at least one polymorphism shown in Tables 2, 3, and 4) Including, but not limited to, other splicing variants encoded by SEQ ID NO: 2 and / or BMP2, or fragments or derivatives thereof; other polypeptides (eg, BMP2 receptor); BMP2R2, BMPR1A, and BMPR1B Non-mutated BMP2 binding agents; peptidomimetics; fusion proteins or prodrugs thereof; antibodies (eg, antibodies to mutant BMP2 polypeptides, or antibodies to non-mutated BMP2 polypeptides, as described above, or specific encoded by BMP2 Antibodies to splicing variants); ribozymes; other small molecules; and others that alter (eg, increase or inhibit) BMP2 gene expression or polypeptide activity, or others that regulate transcription of BMP2 splicing variants Factors (eg which splicing variants are expressed Factors influencing the amount of each splice variants factors or express, influence whether).

  BMP2R2, BMPR1A, and BMPR1B are cell surface receptors that bind to BMP2 protein. BMP2 protein binds to these receptors and then stimulates specific intracellular responses (Fujii, M. et al., Mol. Biol. Cell, 10 (11) 3801-13 (1999); Massague, J., Annu. Rev. Biochem., 67: 753-91 (1998); Chen, D. et al., J. Cell Biol., 142 (1): 295-305 (1998); and Kirsch, T. et al., Nature Structural Biology. , 7 (6): 492-496 (2000)). There are also known direct inhibitors of BMP2, such as noggin and chordin (at least noggin; chordin is a known inhibitor of other BMPs) (Zimmerman, LB et al., Cell, 86: 599-606 (1996); Aspenberg, P. et al., J. Bone Miner. Res., 16 (3): 497-500 (2001); and Dale., L. et al., Bioessays, 21 (9): 751-60 (1999). )).

  More than one MP2 therapeutic can be used concurrently with another therapeutic if desired.

  BMP2 therapeutic agents that are nucleic acids are used in the treatment of osteoporosis or the susceptibility to osteoporosis. The term “treatment”, as used herein, not only alleviates the symptoms associated with the disease, but also prevents or delays the onset of the disease, as well as the severity of the symptoms of the disease. Or to reduce the frequency. A treatment is designed to alter (eg, inhibit or increase), replace or supplement the activity of a BMP2 polypeptide in an individual. For example, a BMP2 therapeutic agent may be used to up-regulate or increase the expression or utility of a BMP2 gene or a specific splicing variant of BMP2, or vice versa. It can be administered to down-regulate or reduce sex. Upregulation or increase in expression or utility of a natural BMP2 nucleic acid or a particular splicing variant can interfere with or compensate for the expression or activity of the defective gene or another splicing variant. Down-regulation or reduction of expression or utility of a natural BMP2 nucleic acid or a particular splicing variant can minimize the expression or activity of the defective gene or a particular splicing variant, thereby reducing the defective gene or the particular splicing variant. The effect of splicing variants can be minimized. In certain embodiments, the BMP2 therapeutic is a healthy gene or gene product (SEQ ID NO: 2), for example, BMP2 nucleic acids and gene products that are not associated with osteoporosis.

  The BMP2 therapeutic agent (s) can be administered in a therapeutically effective amount (eg, by alleviating the symptoms associated with the disease, preventing or delaying the onset of the disease, and / or the severity or frequency of the symptoms of the disease. In an amount sufficient to treat the disease, such as by lowering. The amount that is therapeutically effective in the treatment of a particular individual's disorder or condition will vary depending on the symptoms and severity of the disease and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The exact dose used in the formulation will also vary according to the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the physician and the circumstances of each patient . Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  In some embodiments, a nucleic acid encoding a BMP2 polypeptide, such as a nucleic acid of the invention (e.g., SEQ ID NO: 1, optionally comprising at least one polymorphism shown in Tables 2, 3, and 4), or Another nucleic acid encoding a BMP2 polypeptide, or a splicing variant, derivative or fragment thereof, such as a nucleic acid encoding SEQ ID NO: 2 and comprising at least one polymorphism shown in Tables 2, 3, and 4. Can be used either alone or in a pharmaceutical composition as described above, for example, a cDNA encoding BMP2 or a BMP2 polypeptide can be used either on its own or in a vector Can be introduced into cells (either in vitro or in vivo) to produce the native BMP2 polypeptide, if necessary, gene or cDNA, or gene or cD Cells that have been transformed with a vector containing NA can be introduced (or reintroduced) into an individual suffering from the disease, and thus cells or mutants that are deficient in expression and activity of native BMP2 in nature Cells that have BMP2 expression and activity, or cells that have disease-related BMP2 splicing variant expression, can have BMP2 polypeptide, or an active fragment of BMP2 polypeptide (or a different mutation in BMP2 polypeptide). In certain embodiments, a nucleic acid encoding a BMP2 polypeptide, or an active fragment or derivative thereof, can be introduced into an expression vector, such as a viral vector, wherein the vector is an animal Other gene transfer systems may be used, including viral and non-viral transfer systems. Alternatively, gene transfer methods other than viruses, such as calcium phosphate coprecipitation, mechanical techniques (eg, microinjection); membrane fusion mediated introduction via liposomes, or direct DNA incorporation can also be used. .

  Alternatively, in another aspect of the invention, a nucleic acid of the invention; a nucleic acid complementary to a nucleic acid of the invention; or a portion of such a nucleic acid (eg, an oligonucleotide as described below) It can be used in “sense” therapy. Here, nucleic acids (eg, oligonucleotides) that specifically hybridize to BMP2 mRNA and / or genomic DNA are administered or made in situ. Antisense nucleic acids that specifically hybridize to mRNA and / or DNA inhibit BMP2 polypeptide expression, for example, by inhibiting translation and / or transcription. Antisense nucleic acid binding is obtained by conventional base-pair complementarity or, for example, in the case of binding to a DNA duplex, is through a specific interaction in the deep groove of the double helix. obtain.

  The antisense construct of the present invention can be delivered, for example, as an expression plasmid as described above. When the plasmid is transcribed in the cell, this results in an RNA that is complementary to a portion of the mRNA and / or DNA encoding the BMP2 polypeptide. Alternatively, the antisense construct can be an oligonucleotide probe that is made ex vivo and introduced into a cell. This in turn inhibits expression by hybridizing to BMP2 mRNA and / or genomic DNA. In certain embodiments, the oligonucleotide probe is a modified oligonucleotide that is resistant to endogenous nucleases, eg, exonucleases and / or endonucleases, thereby stabilizing the oligonucleotide probe in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate, and methylphosphonate analogs of DNA (US Pat. Nos. 5,176,996, 5,264,564, and 5,256,775). See also). In addition, general techniques for constructing oligomers useful for antisense therapy are also described, for example, by Van der Krol et al. ((1988) Biotechniques 6: 958-976); and Stein et al. ((1988) Cancer Res. 48: 2659-2668). For antisense DNA, oligodeoxyribonucleotides from the translation initiation site, eg, the region between −10 and +10 of the BMP2 sequence, can be utilized.

  To perform antisense therapy, an oligonucleotide (mRNA, cDNA, or DNA) that is complementary to the mRNA encoding BMP2 is designed. Antisense oligonucleotides bind to the BMP2 mRNA transcript and prevent translation. Absolute complementarity is not necessary. Sequence “complementarity” to a portion of RNA, as used herein, is sufficient complementarity so that the sequence can hybridize to RNA to form a stable duplex. It shows that it has. In the case of double stranded antisense nucleic acids, one strand of double stranded DNA can be tested in this way, or triplex formation can be assayed. The ability to hybridize depends on both the degree of complementarity of the antisense nucleic acid and the length of the antisense nucleic acid, as described in detail above. In general, the longer the nucleic acid that hybridizes, the more base mismatches it may contain with RNA, and still a stable duplex (or in some cases triple) ) Can be formed. One skilled in the art can ascertain an acceptable degree of mismatch by using standard procedures.

  Oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives, or modified versions thereof, single stranded or double stranded. Oligonucleotides can be modified with a base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. Oligonucleotides include other additional groups such as peptides (eg, to target host cell receptors in vivo) or cross the cell membrane (eg, Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; see PCT International Publication No. WO88 / 09810)) or cross the blood brain barrier (See, eg, PCT International Publication No. WO89 / 10134) Factors that facilitate transport, or cleavage factors induced by hybridization (see, eg, Krol et al., (1988) BioTechniques 6: 958-976. Or an intercalating agent (see, for example, Zon, (1988) Pharm. Res. 5: 539-549). For this purpose, the oligonucleotide can also be linked to another molecule (eg a peptide, a hybridization-induced cross-linking agent, a transport factor, a hybridization-induced cleavage agent).

  Antisense molecules are delivered in vivo to cells that express BMP2. A number of methods can be used to deliver antisense DNA or RNA to cells. For example, antisense molecules can be injected directly into a tissue site, or modified antisense molecules designed to target the desired cells (eg, receptors expressed on the surface of target cells). Antisense linked to peptides or antibodies that specifically bind to the body or antigen) can be administered systemically. Alternatively, another embodiment utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter (eg, pol III or pol II). Using such a construct to transfect target cells in a patient results in the transcription of a sufficient amount of single stranded RNA to form a complementary base pair with the endogenous BMP2 transcript. Prevents the translation of BMP2 mRNA. For example, the vector can be introduced in vivo such that it is taken up by the cell and directs transcription of the antisense RNA. Such a vector can remain episomal or can be integrated into the chromosome so long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by the recombinant DNA technology methods described above that are standard in the art. For example, a plasmid, cosmid, YAC, or viral vector can be used to prepare a recombinant DNA construct that can be introduced directly into a tissue site. Alternatively, viral vectors can be used to selectively infect the desired tissue if administration can be by another route (eg, systemically).

  Expression of endogenous BMP2 can also be reduced by inactivating or “knocking out” BMP2 or its promoter using targeted homologous recombination (eg, Smithies et al., (1985) Nature 317: 230-234; Thomas & Capecchi (1987) Cell 51: 503-512; see Thompson et al. (1989) Cell 5: 313-321). For example, mutated non-functional BMP2 (or a completely unrelated DNA sequence) flanked by DNA homologous to endogenous BMP2 (either the coding or regulatory region of BMP2) expresses BMP2 in vivo Can be used with or without selectable markers and / or negative selectable markers to transfect cells. Insertion of the DNA construct by targeted homologous recombination results in inactivation of BMP2. The recombinant DNA construct can be administered directly or can be targeted in vivo to the required site using an appropriate vector as described above. Alternatively, the expression of unmutated BMP2 can be increased using similar methods. The targeted homologous recombination replaces the mutant BMP2 in the cell, as described above, with a DNA construct (eg, a nucleic acid having SEQ ID NO: 1) comprising a non-mutant functional BMP2 or part thereof. Can be used to insert into In another embodiment, targeted homologous recombination can be used to insert a DNA construct comprising a nucleic acid encoding a BMP2 polypeptide variant different from that present in the cell.

  Alternatively, endogenous BMP2 expression is reduced by targeting a deoxyribonucleotide sequence that is complementary to the regulatory region of BMP2 (ie, the BMP2 promoter and / or enhancer), so that BMP2 in target cells in the body Triple helix structures can be formed that interfere with transcription of proteins (generally Helene, C. (1991) Anticancer Drug Des., 6 (6): 569-84; Helene, C. et al., (1992) Ann, NY Acad. Sci., 660: 27-36; and Maher, LJ (1992) Bioassays 14 (12): 807-15). Similarly, antisense constructs described herein can be used to manipulate tissue in both in vivo and ex vivo tissue culture, for example by antagonizing one normal biological activity of the BMP2 protein. Can be used for tissue differentiation. In addition, antisense technology (eg, microinjection of antisense molecules, or transfection with plasmids whose transcripts are antisense to BMP2 mRNA or gene sequences) is responsible for the role of BMP2 in developmental events, It can be used to study the normal cellular function of BMP2 in adult tissue. Such techniques can be utilized in cell culture, but can also be used in the production of transgenic animals.

  In yet another aspect of the invention, other BMP2 therapeutics described herein can also be used in the treatment or prevention of osteoporosis. The therapeutic agent can be delivered in a composition as described above or by itself. They can be administered systemically or targeted to specific tissues. Therapeutic agents can be produced by various means including, for example, chemical synthesis, recombinant production, in vivo production (eg, transgenic animals such as Meade et al. US Pat. No. 4,873,316) and are described herein. Can be isolated using standard means, such as

  Any combination of the above treatment methods (eg, administration of a non-mutated BMP2 polypeptide, antisense therapy targeting a second variant encoded by BMP2, associated with antisense therapy targeting mutant BMP2 mRNA) Administration of a variant encoded by BMP2) can also be used.

  The invention is further described by the following non-limiting examples. The teachings of all publications cited herein are hereby incorporated by reference in their entirety.

Identification of BMP2 nucleic acids associated with osteoporosis and family composition Patients who have low impact fractures and / or who are taking bisphosphonates to treat osteoporosis Automatically treat as you are. Humans with low bone density (BMD) measurements are considered osteoporosis and they have been shown to have a substantially increased risk of fracture. BMD measurements are taken for both the hip and spine. For each BMD-measured person, a standardized BMD score is calculated (mean 0, standard deviation 1 for the population) and adjusted for gender, age, weight, and hormone replacement therapy (HRT). For the combined analysis, add the two measurements. Population BMD data from Iceland and the US are used for standardization and adjustment. For example, a human having a positive BMD score is above the average of his / her age, weight, and possibly HRT, and a human having a negative score is below average. If almost normal, a score of −1 corresponds roughly to the 16th percentile from the lowest.

  For analysis, we are humans with BMD measurements and / or severely affected humans and humans with whom we have genotypes. Start with a list of primary human status. We then use a genealogy database to create a family group that relates these primary humans using the limit distance of five meiotic events. This procedure yielded a group of 190 possible information for a total of 1215 primary humans.

Chain data
Four whole genome scans (GWS) were performed using osteoporosis phenotypes at various skeletal sites; hip, spine, and complex phenotypes. All GWS analyzes were placed in a 20 cM region on Chr20 between 10 cM and 30 cM based on the Marshfield map.

All analyzes were performed using the Allegro linkage program (Gudbjartsson et al., Nature Genetics, 25: 12-13, May 2000) developed at deCODE. For the allele sharing analysis, the GENEHUNTER S _pairs scoring function (Kruglyak et al., Am. J. Hum. Genet., 46: 1347-1363, 1996) is used. However, the family was weighted using a methodology that is a compromise between equal weighting between families and equal weighting between affected pairs. Allele sharing LOD scores were calculated using the “exponential model” described in Kong and Cox, Am. J. Hum. Genet., 61: 1179-1188 (1997).

Hip joint The phenotype used was HMD corrected BMD <-1 SD for age, gender, weight, and hip joint (whole hip joint). Hip fracture cases and bisphosphonate users were also considered affected even if the value was above −1 SD. A total of 346 morbidities were used for this analysis. GWS gave an LOD score of 3.1 using our standard set of markers. By adding 10 additional markers to the region of interest (between 11 cM and 39 cM), a LOD score of 3.3 was obtained.

The spinal phenotype was HRT corrected BMD <−1 SD for age, gender, weight, and lumbar spine (L2-L4). Cases of vertebral compression fractures and users of bisphosphonates were also considered affected even if the value was above −1 SD. A total of 402 affected humans were used for this analysis. The GWS gave a LOD score of 2.4 at the same position as the hip analysis using a standard set of markers, but a LOD score of 2.9 when using an additional set of markers.

Composite type The phenotype used was the sum of the corrected BMD <-1.5 SD. Vertebral compression fractures, hip fractures, other low-impact fractures associated with osteoporosis (at least 2 fractures), and bisphosphonate users (if available, BMD measurements before starting treatment) Use) is considered to be all affected. A total of 522 morbidities were used in this analysis. GWS gave an LOD score of 2.5 when using a standard set of markers, but an LOD score of 3.9 when using an additional set of markers within this region.

Complex Type Severe The phenotype used was the sum of age, gender, weight, and HMD corrected BMD <-2.3 SD. Vertebral compression fractures, hip fractures, other low-impact fractures associated with osteoporosis (at least two fractures), and bisphosphonate users are affected. The number of morbidities in this analysis was 290. GWS gave an LOD score of 3.8 when using the standard set, but an additional LOD score of 4.7 when using an additional 10 markers.

  Corticosteroid users and premenopausal women were excluded as affected in all analyses.

BMP2 gene
BMP2 nucleic acid is present in this region. There is only 5 kb between the marker D20S846 (which gives the highest LOD score) and the 3 ′ end of the gene. This gene has been sequenced and characterized in terms of exon / intron structure, promoter region, and transcription start site. This information is publicly available.

  A number of nucleotide changes are observed in the Icelandic population. These changes have not been previously described to the best of our knowledge (see Table 2).

  BMP2 binds to the receptors BMPR-IA or BMPR-IB, and BMPR-II, leading to the formation of receptor complex heterodimers and phosphorylation of BMPR-IA or BMPR-IB receptors. Once activated, these receptors subsequently phosphorylate SMAD1, SMAD5, or SMAD8, which then complex with SMAD4 and translocate to the nucleus where transcription of specific genes is affected. (Massague, J., Annu. Rev. Biochem., 67: 753-791 (1998); Chen, D. et al., J. Cell Biol., 142 (1): 295-305 (1998). SMAD6 and SMAD7 Has been shown to block signals by blocking activation of SMAD1, SMAD5, or SMAD8 by the BMP2 receptor and inhibit osteoblast differentiation (Miyazono, K., Bone, 25 (1 ): 91-93 (1999); Fujii, M. et al., Mol. Biol. Cell, 10 (11): 3801-3813 (1999)) BMP2 is Cbfal, alkaline phosphatase, and type I collagen (osteoblast specific) (Chen, D., et al., J. Cell Biol., 142 (1): 295-305 (1998)). Cbfal is a protein of osteoprotegerin (OPG). Regulate expression. Theoprotegerin is a glycoprotein secreted by osteoblasts that acts as a potent inhibitor of osteoblast differentiation and thus as a potent inhibitor of bone resorption (Thirunavukkarasu, K. et al., J. Biol. Chem., (2000)) Cbfal regulates osteoblast differentiation and bone formation Cbfal mRNA is markedly reduced (up to 50%) during human blast cell senescence ( Christiansen, M. et al., J. Gerontol. A Biol. Sci. Med. Sci., 55 (4): B194-200 (2000)).

Results and Discussion As a result of linkage analysis, this analysis indicates that this locus is associated with multiple osteoporosis phenotypes. Furthermore, mutations within the human BMP2 nucleic acid probably explain the phenotype in these families. Sporadic occurrences of osteoporosis, i.e., non-family relationships, can also be determined using the information contained herein.

  Osteoporosis can be caused by defects in the BMP2 nucleic acid as follows. Changes in BMP2 nucleic acids (transcriptional variants, splicing variants, protein variants, etc.) could lead to its effect on Cbfal via BMPR-IB and subsequent reduction of signaling pathways. This leads to less bone formation due to fewer, less active osteoblasts, and more bone resorption due to fewer OPGs and more osteoclasts . This leads to a reduction in bone tissue. This effect can become more important with increasing age as a significant decrease in Cbfal levels is associated with aging osteoblasts.

Direct sequencing of the BMP2 nucleic acid sequence reveals other polymorphisms Additional genetic markers were identified in the BMP2 nucleic acid by directly sequencing this region in various populations. These are listed in Table 3, along with the nucleotide positions compared to the sequence AL035668 in the previous SNP update file.

  Additional linkage data for various SNPs against the osteoporosis phenotype are listed in Table 4. Table 4 shows the p-value (p-value), relative risk (r), proportion of affected individuals (Aff.freq.carrier), number of affected individuals (#aff) for the relationship with the SNP phenotype List the percentage of controls (Ctrl.freq.carrier), number of controls (#ctrl), allele identification, and screened phenotype.

Although the invention has been particularly shown and described with respect to preferred embodiments thereof, various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will understand that this can be done therein.

[Sequence Listing]

Claims (11)

  Detecting at least one polymorphism in the human BMP2 gene of SEQ ID NO: 1, wherein the presence of “G” at nucleotide position 122247 of AL035668; presence of the “G” allele at position 118920 of AL035668; position of AL035668 Presence of “T” allele at 167584; Presence of “G” allele at position 138476 of AL035668; Presence of “T” allele at position 167584 of AL035668; Presence of “T” allele at TSC0428253; The presence of the "G" allele is "T" at nucleotide position 122247 of AL035668; "A" allele at position 118920 of AL035668; "C" allele at position 167584 of AL035668; "A" allele at position 138476 of AL035668 An individual having the presence of the "C" allele at position 167584 of AL035668; the "C" allele at TSC0428253; or the "A" allele of TSC0293456; A method for diagnosing susceptibility to osteoporosis in an individual, which is an index of susceptibility to osteoporosis compared to the body.   The method of claim 1, wherein a polymorphism is detected in a sample from a source selected from the group consisting of blood, serum, cells and tissues.   Detecting at least one polymorphism in the human BMP2 gene of SEQ ID NO: 1, wherein the polymorphism is T to G at position 122247 of AL035668; A to G at position 118920 of AL035668; at position 167584 of AL035668 Affection for osteoporosis in an individual selected from the group consisting of C to T; A to G at position 138476 in AL035668; C to T at position 167584 in AL035668; C to T in TSC0428253; A to G in TSC0293456 and combinations thereof How to diagnose sex.   4. The method of claim 3, wherein a polymorphism is detected in a sample from a source selected from the group consisting of blood, serum, cells and tissue.   AL 035668 at position 122247 from T to G; AL035668 at position 121366 from C to T; AL035668 at position 118920 from A to G; AL035668 at position 167584 from C to T; AL035668 from position A at position 138476 An isolated nucleic acid molecule comprising a nucleic acid having one or more nucleic acid changes selected from the group consisting of G and combinations thereof.   An isolated polypeptide comprising an amino acid sequence selected from the group consisting of alanine at amino acid position 37, serine at amino acid position 94, serine at amino acid position 189, or a combination thereof.   A method for diagnosing susceptibility to osteoporosis, comprising the step of detecting the polypeptide according to claim 6 which is an index of susceptibility to osteoporosis.   Determining an amino acid at a position selected from the group consisting of position 37, position 94, position 1891 and combinations thereof comprises contacting the sample with an antibody specific for either a reference amino acid or a variant amino acid; The method of claim 7.   A pharmaceutical composition comprising the polypeptide according to claim 6.   A method of treating osteoporosis in an individual, comprising administering to the individual a therapeutically effective amount of the isolated polypeptide of claim 6. a) specific for BMP2 protein comprising serine at amino acid position 37, alanine at amino acid position 37, alanine at amino acid position 94, serine at amino acid position 94, arginine at amino acid position 189, serine at amino acid position 189, or combinations thereof At least one antibody selected from the group consisting of antibodies; and
b) A kit containing a reference BMP2 protein sample.