JP2006516196A - Diagnosis method of susceptibility to osteoporosis or osteoporosis based on haplotype association - Google Patents

Abstract

Disclosed is a method for diagnosing osteoporosis or susceptibility to osteoporosis based on the detection of at-risk haplotypes associated with BMP2.

Description

Detailed Description of the Invention

Related Art This application claims the benefit of US Provisional Application No. 60 / 440,899 filed on January 16, 2003 and the benefit of US Provisional Application No. 60 / 450,652 filed on February 27, 2003. Insist. 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 the 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. In particular, postmenopausal women, estrogen deficiency and corresponding bone loss during menstruation increase both the likelihood of osteoporotic fractures and the number of potential sites So that the risk is highest. But 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 lead to osteoporosis. Nutrients (especially calcium, vitamin D, and vitamin K intake), hormone levels, age, gender, race, weight, activity level, and genetic factors all influence differences in individual bone density Effect. Currently, approved drugs for treating osteoporosis act as inhibitors of bone resorption. Treatment regimes include such methods as the use of hormone replacement therapy (HRT), selective estrogen receptor modulators, calcitonin, and biophosphonates. However, these treatments do not individually reduce risk and have consistent results. Furthermore, BMD improves when several therapeutic agents are administered simultaneously, others do not show improved efficacy or even reduced efficacy when combined.

  Clearly, as life expectancy is extended and health and economic concerns regarding osteoporosis are growing, a method to eliminate the risk of this late-onset disease is highly desirable. Therefore, early diagnosis of this disease or susceptibility to this disease is desired.

SUMMARY OF THE INVENTION As described herein, certain combinations of genetic markers (“haplotypes”) occur more frequently than expected in patients with osteoporosis and phenotypes associated with susceptibility to osteoporosis. It was discovered that it exists. Markers included in the haplotypes described herein relate to genomic regions that direct the expression of human bone morphogenetic protein 2 (BMP2).

  In certain embodiments, the invention detects the presence or absence of a risky haplotype comprising a haplotype selected from the group consisting of haplotype I, haplotype II, haplotype a, haplotype b, haplotype c, haplotype d and combinations thereof. Wherein the presence of a haplotype is an indicator of osteoporosis or susceptibility to osteoporosis, wherein the susceptibility to osteoporosis or osteoporosis in an individual is diagnosed. In certain embodiments, the present invention provides an analysis for the presence of a first nucleic acid molecule in a sample, wherein the sample is a second nucleic acid molecule comprising one or more of the haplotypes described herein. The present invention relates to an analysis including a contacting step. In certain embodiments, the step of determining the presence or absence of a haplotype comprises enzymatic amplification of nucleic acid from an individual. In certain embodiments, the step of determining the presence or absence of a haplotype further comprises electrophoretic analysis. For example, in certain embodiments, the step of determining the presence or absence of a haplotype includes restriction enzyme fragment length polymorphism analysis. In another embodiment, determining the presence or absence of a haplotype comprises sequence analysis.

  In another aspect, the present invention includes detecting the presence or absence of a haplotype at risk, including haplotype I, wherein the presence of the haplotype is indicative of osteoporosis or osteoporosis susceptibility in an individual. The present invention relates to a method for diagnosing sex. In certain embodiments, determining the presence or absence of a haplotype includes enzymatic amplification of nucleic acid from the individual. In certain embodiments, determining the presence or absence of a haplotype further comprises electrophoretic analysis. For example, in certain embodiments, the step of determining the presence or absence of a haplotype includes restriction enzyme fragment length polymorphism analysis. In another embodiment, determining the presence or absence of a haplotype comprises sequence analysis.

  In another embodiment, the invention includes detecting the presence or absence of a haplotype at risk, including haplotype II, wherein the presence of the haplotype is indicative of osteoporosis or osteoporosis susceptibility in an individual. The present invention relates to a method for diagnosing sex. In certain embodiments, determining the presence or absence of a haplotype includes enzymatic amplification of nucleic acid from the individual. In certain embodiments, determining the presence or absence of a haplotype further comprises electrophoretic analysis. For example, in certain embodiments, the step of determining the presence or absence of a haplotype includes restriction enzyme fragment length polymorphism analysis. In another embodiment, determining the presence or absence of a haplotype comprises sequence analysis.

  In another embodiment, the invention provides a kit for analyzing a sample for the presence of haplotypes associated with osteoporosis, wherein the haplotype comprises two or more specific alleles, and wherein the kit is 2 It relates to a kit comprising one or more nucleic acids capable of detecting the presence or absence of one or more specific alleles, thereby indicating the presence or absence of a haplotype in a sample. In certain embodiments, the nucleic acid comprises a contiguous nucleotide sequence that is completely complementary to the region containing the particular allele of the haplotype.

  In another embodiment, the present invention provides that the haplotype comprises two or more specific alleles and a) one or more labeled nucleic acids capable of detecting one or more specific alleles of the haplotype in separate containers; and b A reagent kit for analyzing a sample for the presence of haplotypes associated with osteoporosis. In certain embodiments, the labeled nucleic acid comprises a contiguous nucleotide sequence that is completely complementary to the region containing a particular allele of a haplotype.

  In yet another aspect, the invention provides a reagent kit for analyzing a sample for the presence of a haplotype associated with osteoporosis, wherein the haplotype comprises two or more specific alleles, wherein the kit A primer extension that comprises one or more nucleic acids comprising a nucleotide sequence that is at least partially complementary to a portion of the nucleotide sequence of the BMP2 gene, wherein the nucleic acid is capable of detecting one or more specific alleles of a haplotype The present invention relates to a reagent kit that can act as a primer for the reaction.

  In another embodiment, the present invention relates to a BMP2-related, at-risk haplotype that is more frequently present in individuals who are not susceptible to osteoporosis compared to individuals who are not susceptible to osteoporosis. A method of diagnosing and identifying susceptibility to osteoporosis in an individual, comprising the step of a risky haplotype significantly increasing the risk. In certain embodiments, the significant increase is at least about 20%. In another embodiment, a significant increase is identified as an odds ratio of at least about 1.2.

  In another embodiment, the present invention relates to TSC0898956, B420, B8463, D20S846, TSC0191642, P4337, D20S892, B5048, B9082, D20S59, B7111 / rs235764, B12845 / rs15705, P9313, B10631, D35548, rs1116867, TSC048787D, 3635 Diagnosing susceptibility to osteoporosis in an individual, wherein the presence of the haplotype is indicative of susceptibility to osteoporosis, wherein the presence of the haplotype in the individual includes two or more alleles selected from the group consisting of On how to do. In certain embodiments, the step of determining the presence or absence of a haplotype further comprises electrophoretic analysis. For example, in certain embodiments, the step of determining the presence or absence of a haplotype includes restriction enzyme fragment length polymorphism analysis. In another embodiment, determining the presence or absence of a haplotype includes sequence analysis.

  In yet another embodiment, the present invention provides a step of obtaining a nucleic acid sample from an individual; and TSC0898956, B420, B8463, D20S846, TSC0191642, P4337, D20S892, B5048, B9082, D20S59, B7111 / rs235764, B12845 / rs15705, P9313, B10631 Analyzing a nucleic acid sample for the presence or absence of a haplotype comprising two or more alleles selected from the group consisting of D35548, rs1116867, TSC0278787, D35548 and TSC0271643, wherein the presence of the haplotype is indicative of susceptibility to osteoporosis And a method for diagnosing susceptibility to osteoporosis in an individual. In certain embodiments, the allele is selected from the group consisting of TSC0898956, B420, B8463, D20S846 and TSC0191642. In certain embodiments, the allele is selected from the group consisting of P4337, D20S892, B5048, B9082, and D20S59. In another embodiment, the haplotype comprises B7111 / rs235764 and B12845 / rs15705. In certain embodiments, the allele is selected from the group consisting of P9313, B10631 and D35548. In certain embodiments, the allele is selected from the group consisting of rs1116867, TSC0278787 and D35548. In another embodiment, the allele is selected from the group consisting of TSC0271643, P9313 and B7111.

Detailed Description of the Invention As described herein, Applicant has identified genes associated with an osteoporosis phenotype and a gene region associated with the expression of human bone morphogenetic protein 2 (BMP2) located on chromosome 20. Linkage analysis with a specific combination of markers (“haplotype”) was completed. The results presented herein represent the first proof of the haplotype used to indicate osteoporosis or susceptibility to osteoporosis. Based on related studies conducted, Applicants have found a direct relationship between BMP2-related haplotypes and osteoporosis. In particular, it has been discovered that certain haplotypes appear in patients with osteoporosis and phenotypes associated with osteoporosis susceptibility more frequently than expected. Based on this association, methods for diagnosing osteoporosis, for example, in combination with bone turnover marker analysis (eg, bone scan) are described herein. Furthermore, the method based on the detection of at least one haplotype described herein is a diagnosis of susceptibility to osteoporosis.

Diagnosis and Screening Assays of the Present Invention The present invention relates to diagnosis of susceptibility to osteoporosis or osteoporosis by detecting specific genetic markers that appear frequently in individuals with osteoporosis or individuals highly susceptible to osteoporosis, or The present invention relates to a method for assisting simple diagnosis. Diagnostic assays can be designed to assess BMP2. The combination of genetic markers is referred to herein as a “haplotype” and the present invention describes a method wherein the detection of a particular haplotype is an indication of osteoporosis or susceptibility to osteoporosis. Detection of specific genetic markers constituting a specific haplotype can be performed by various methods described herein and various methods known in the art. For example, genetic markers can be detected at the nucleic acid level, for example by direct sequencing, or at the amino acid level if the genetic marker affects the coding sequence of BMP2, such as BMP2 protein or a specific BMP2 variant protein. It can be detected by an immunoassay based on the antibody it recognizes.

  In certain embodiments, this assay is used in the context of biological samples (eg, blood, serum, cells, tissues), thereby determining whether the individual is suffering from or at risk of developing osteoporosis ( Whether or not 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, nucleic acid sequence changes can be assayed in a biological sample. Such an assay can be used for prognostic or predictive purposes, thereby allowing prophylactic treatment of the individual prior to the onset of symptoms associated with osteoporosis.

Diagnostic Assays In certain embodiments of the invention, diagnosis of susceptibility to osteoporosis is performed by detecting a haplotype associated with BMP2, as described herein. BMP2-related haplotypes describe a series of genetic markers related to BMP2. In certain embodiments, a haplotype can include one or more markers, two or more markers, three or more markers, four or more markers, or five or more markers. Genetic markers are specific “alleles” at the “polymorphic site” associated with BMP2. Nucleotide positions that may have more than one sequence within a population (either a natural population or a synthetic population, such as a library of synthetic molecules) are referred to herein as “polymorphic sites”. When a polymorphic site is one nucleotide long, the site is referred to as a single nucleotide polymorphism (“SNP”). For example, if one member of the population has adenine at a particular chromosomal location and another member of the population has thymine at the same location, this location is a polymorphic site, and more specifically, a polymorphic site. The type site is SNP. Polymorphic sites can take into account differences in sequences based on substitutions, insertions or deletions. Each version of a sequence with respect to a polymorphic site is referred to herein as an “allele” of the polymorphic site. Thus, in the previous example, the SNP takes into account both the adenine allele and the thymine allele.

  Typically, a reference sequence is referred to for a specific sequence. Alleles that differ from the reference are referred to as “variant” alleles. For example, the reference BMP2 sequence is denoted herein by SEQ ID NO: 1. The term “variant BMP2” as used herein refers to a BMP2 sequence that differs from SEQ ID NO: 1 but otherwise substantially the same. The genetic markers that make up the haplotypes described herein are BMP2 variants. Variants of BMP2 used for the determination of haplotypes disclosed in the present specification are associated with susceptibility to a number of osteoporosis phenotypes.

  Additional variants can include changes that affect a polypeptide, eg, a BMP2 polypeptide. These sequence differences result in a single nucleotide or more than one nucleotide insertion or deletion that results in a frameshift when compared to the reference nucleotide sequence; at least one nucleotide that results in a change in the encoded amino acid. A change; at least one nucleotide change resulting in the generation of an immature stop codon; a deletion of several nucleotides resulting in a deletion of one or more amino acids encoded by the nucleotide; a coding sequence interruption in the reading frame Insertion of one or several nucleotides, such as by unequal recombination or gene conversion; replication of all or part of the sequence; transposition; or rearrangement of the nucleotide sequence. Such sequence changes alter the polypeptide encoded by the BMP2 nucleic acid. For example, if a change in the nucleic acid sequence causes a frameshift, the frameshift can result in a change in the encoded amino acid and / or can result in the generation of an immature stop codon that results in the production of a truncated polypeptide. Alternatively, a polymorphism associated with susceptibility to osteoporosis can be a synonymous change in one or more nucleotides (ie, a change that does not result in a change in the BMP2 amino acid sequence). Such polymorphisms can, for example, alter splice sites, affect mRNA stability or migration, or otherwise affect polypeptide transcription or translation. A polypeptide encoded by a reference nucleotide sequence is a “reference” polypeptide having a specific reference amino acid sequence, and a polypeptide encoded by a variant allele is a “variant” polypeptide having a variant amino acid sequence. Called.

  A haplotype is a specific allele at a combination of genetic markers, eg, polymorphic sites. The haplotypes described herein are associated with susceptibility to osteoporosis and / or osteoporosis. Therefore, the detection of the presence or absence of a haplotype in this specification is an indicator of osteoporosis, susceptibility to osteoporosis or lack thereof. Therefore, detection of the presence or absence of these haplotypes is necessary for the purposes of the present invention to detect osteoporosis or susceptibility to osteoporosis. The haplotypes described herein are a combination of various genetic markers, such as SNP and microsatellite. Thus, haplotype detection can be accomplished by known methods for detecting sequences at polymorphic sites.

  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 include genomic DNA, such as blood samples, amniotic fluid samples, cerebrospinal fluid samples, or tissue samples derived from skin, muscle, buccal or conjunctival mucosa, placenta, digestive tract, or other organs. It can be derived from any source including. 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 haplotype alleles can be indicated by sequence-specific hybridization of nucleic acid probes specific for a particular allele. Sequence specific probes can be directed to hybridizing to genomic DNA, RNA or cDNA. A “nucleic acid probe” as used herein can be a DNA probe or an RNA probe that hybridizes to a complementary sequence. Methods of designing probes that cause sequence-specific hybridization only when a particular allele is present in the genomic sequence from the test sample are known to those skilled in the art.

  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 allele included in the haplotypes described herein, or the probe , Which may be a complementary sequence to such a sequence. 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 (see below). 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, the sample contains the allele, which is present in the nucleic acid probe. This process can be repeated for other markers that make up the haplotype, or multiple probes can be used simultaneously to detect more than one marker simultaneously. Detection of a specific marker of a haplotype in a sample is an indication that the source of the sample has a specific haplotype and thus is susceptible to osteoporosis or osteoporosis.

  In another hybridization method, Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F. et al., John Wiley & Sons, supra) is a polymorphism associated with susceptibility to osteoporosis. Used to identify the presence of For Northern analysis, RNA test samples are obtained from the individual by appropriate means. Specific hybridization of a nucleic acid probe to RNA from an individual, as described above, is indicative of a particular allele that is complementary to the probe.

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

  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, P. et al., 1994. Bioconjug. Chem., 5: 3-7). PNA probes can be designed to specifically hybridize to molecules in a sample suspected of containing one of the haplotype genetic markers implicated in susceptibility to osteoporosis. Hybridization of a PNA probe is a diagnosis of susceptibility to osteoporosis or osteoporosis.

  In certain embodiments of the invention, diagnosis of susceptibility to osteoporosis or osteoporosis associated with BMP2 or a haplotype associated with 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. This technique can utilize commercially available techniques such as, for example, TaqMan® (Applied Biosystems, Foster City, Calif.), Which can identify polymorphisms and haplotypes. 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 invention, analysis by restriction digestion can be used to detect a particular allele if the allele results in the creation or elimination of a restriction site related to the reference sequence. A test sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify a genomic BMP2 region (including flanking sequences, if necessary) in a test sample from a 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 a particular allele is present or absent in the sample.

  Sequence analysis can also be used to detect specific alleles at polymorphic sites related to BMP2. A test sample of DNA or RNA is obtained from the test individual. PCR or other suitable methods can be used to amplify BMP2, and / or its flanking sequences, if desired. In this way, the presence of a particular allele is directly detected by sequencing polymorphic sites of genomic DNA in the sample.

  Allele-specific oligonucleotides are also dot-blot with amplified allele-specific oligonucleotide (ASO) probes to detect the presence of specific alleles at polymorphic sites related to BMP2. It can be used through the use of hybridization (see, eg, Saiki, R. et al., 1986, Nature, 324: 163-166). An “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, or approximately 15-30 base pairs, Specifically hybridizes to BMP2, which includes specific alleles at polymorphic sites as shown by the haplotypes described herein. Allele-specific oligonucleotide probes specific for a particular polymorphism in BMP2 can be prepared using standard methods (see Current Protocols in Molecular Biology (supra)). PCR can be used to amplify entire BMP2 or fragments thereof, and flanking sequences of the genome. 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 indicative of a particular allele at the polymorphic site associated with BMP2.

  An allele-specific primer hybridizes to a site on the target DNA that overlaps with the polymorphic site 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 a distant site on the opposite strand. 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, eg, 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 are incorporated herein by reference in their 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 can hybridize to the array. Hybridization detection is the detection of a specific allele in the nucleic acid of interest. 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 herein incorporated by reference). Incorporated in the above). 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 polymorphic site) that are complementary to the duplex of the target sequence. Asymmetric PCR techniques can also be used. The amplified target typically incorporates a label that is then hybridized to the array under appropriate conditions that allow sequence-specific hybridization. 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 polymorphic site, the array can include multiple detection blocks, thus analyzing multiple specific polymorphisms. be able to. In another arrangement, the 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 the 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 that are 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 specific alleles at polymorphic sites related to BMP2. Representative methods include, for example, direct manual sequencing (Church and Gilbert, 1988. Proc. Natl. Acad. Sci. USA, 81: 1991-1995; Sanger, F. et al., 1977. Proc. Natl. Acad. Sci., USA, 74: 5463-5467; Beavis et al., 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, V. et al., 1989. Proc. Natl. Acad. Sci. USA, 86: 232-236); Mobility shift analysis (Orita, M. et al., 1989. Proc. Natl. Acad. Sci. USA, 86: 2766-2770), restriction enzyme analysis (Flavell, R. et al., 1978. Cell, 15: 25-41; Geever, R. et al., 1981. Proc. Natl. Acad Sci. USA, 78: 5081-5085); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton, R. et al., 1985. Proc. Natl. Acad. Sci. USA, 85: 4397-4401 ); RNase protection assay (Myers, R. et al., 1985. Science, 230: 1242-1246); like E. coli mutS protein Use of recognizing a polypeptide mismatched nucleotides; as well as allele-specific PCR.

  In another aspect of the invention, diagnosing susceptibility to osteoporosis is when the genetic markers included in the haplotypes described herein result in altered expression of the polypeptide (eg, altered amino acid sequence or level of expression). Can also be performed by testing the expression and / or composition of the BMP2 polypeptide. Such detection can be performed using a variety of methods including enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. 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, 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 that corresponds to the test sample (eg, derived from the same type of cells) and is derived from an individual who is not suffering from osteoporosis or susceptibility to osteoporosis. Similarly, the presence of one or more different splicing variants in a test sample or the presence of significantly different amounts of multiple different splicing variants in a test sample compared to a control sample is indicative of susceptibility to osteoporosis. It is. A change in the expression or composition of a polypeptide in a test sample relative to a control sample can be indicative of a particular allele if the allele associated with the reference changes the splice site. 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 the polypeptide encoded by the mutant BMP2 or an antibody that specifically binds to the polypeptide encoded by the reference allele is encoded by the mutant BMP2 allele. To be identified in the test sample, or that the polypeptide encoded by the reference allele is not present in the test sample.

  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. The diagnosis of a specific allele that is an indicator and causes the differential expression. 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. In another aspect, the level or amount and composition of the polypeptide can be assessed in a test sample and in a control sample.

  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, at least one of SEQ ID NO: 2 included in the haplotype described herein) Antibodies that bind to one genetic marker), 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, etc. . 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 a BMP2 nucleic acid comprising a haplotype that is at risk of being more frequently present in an individual who has or is susceptible to osteoporosis. Primers for nucleic acid amplification of these regions may be included. Primers can be designed using a portion of the nucleic acid adjacent to an SNP that is indicative of osteoporosis. In certain embodiments, the primer is a region of BMP2 nucleic acid associated with the haplotypes at risk for osteoporosis shown in Table 1, ie more specifically haplotype I, haplotype II, haplotype a, haplotype b, haplotype c or haplotype d. It is designed to amplify. 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).

Haplotype Screening The present invention further relates to methods for diagnosing and identifying susceptibility to osteoporosis in an individual by identifying at-risk haplotypes in BMP2. In certain embodiments, the risky haplotype is one that confers a significant risk of osteoporosis. In some embodiments, significance associated with a haplotype 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 with a variety of factors, including the particular disease, haplotype, and often environmental factors.

  The present invention also relates to a method of diagnosing osteoporosis or susceptibility to osteoporosis in an individual. This method involves a haplotype at risk associated with a BMP2 nucleic acid that is more frequently present in individuals susceptible to osteoporosis (affected) compared to the frequency of its presence in healthy individuals (control) Screening. Here, the presence of a haplotype is an indication 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, X. et al., 1999. Genome Res., 9: 492- 498), PCR, LCR, nested PCR, and other techniques for nucleic acid amplification can be used. In some 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 haplotype or a higher frequency of haplotype compared to a healthy control individual is an indicator that the individual has osteoporosis or is susceptible to osteoporosis.

  Haplotype analysis involves defining candidate susceptibility loci using rod values. The defined regions are then hyperfinely mapped using microsatellite markers with an average spacing between markers less than 100 kb. All available microsatellite markers found in public databases and mapped within the area can be used. In addition, microsatellite markers identified within the deCODE Genetics sequence set of the human genome can be used.

  The frequency of haplotypes in patients and controls using the expectation maximization algorithm can be assessed (Dempster, A. et al., 1977. J.R. Stat. Soc. B, 39: 1-389). Implementations of this algorithm that can manipulate lost genotype and phase uncertainty can be used. Under the null hypothesis, patients and controls are assumed to have the same frequency. Using a likelihood approach, alternatives where a candidate risky haplotype can have a higher frequency in the patient than the control while the proportion of other haplotype frequencies is assumed to be the same in both groups The hypothesis is tested. The likelihood is maximized separately under both hypotheses and the statistical significance is assessed using the corresponding 1-df likelihood ratio statistics.

  To look for haplotypes at risk in 1-lod drop, for example, the association of all possible combinations of genotyped markers, where these markers are substantive Can be studied. The combined patient and control groups can be randomly divided into two sets that are the same in size as the original group of patients and controls. The haplotype analysis is then repeated and the most significant p-value recorded is measured. This randomization scheme can be repeated over 100 times to construct an empirical p-value distribution.

Nucleic acids and polypeptides of the invention All nucleotide positions are relative to SEQ ID NO: 1 or GenBank No. AL035668 as indicated. The nucleic acids, polypeptides and antibodies described herein can be used in methods of diagnosing susceptibility to osteoporosis and in kits useful for diagnosing susceptibility to osteoporosis. The reference amino acid sequence of BMP2 is represented by SEQ ID NO: 2.

  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 present invention may have its naturally occurring complex cellular environment, or 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 determined, 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 comprises 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 to isolate homologous sequences (eg, from other mammalian species), eg, in the production of encoded polypeptides (eg, by in situ hybridization using chromosomes). ) 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 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 nucleotide sequences containing polymorphic sites related to the haplotypes described. In certain embodiments, the present invention includes polymorphisms included in at least one of the haplotypes described herein under high stringency hybridization and wash conditions (eg, conditions for selective hybridization). Composition or expression provided by a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1 comprising at least one allele at the type site or its complement, or an allele included in a haplotype described herein Included are variants described herein that hybridize to a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2, which contains a change in level.

  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%, 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, F. et al., “Current Protocols in Molecular Biology ", John Wiley & Sons, (1998), the entire teachings of which are incorporated herein by reference), and are described on pages 2.10.1-2.10.16 and 6.3.1-6.3.6. ing. 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 describing the determination of wash conditions for moderate or low stringency conditions are Kraus, M. and Aaronson, S., Methods Enzymol., 200: 546-556 (1991); and Ausubel F. et al., "Current Protocols in Molecular Biology", John Wiley & Sons, (1998). 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. Non-limiting examples of such mathematical algorithms are described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993). Such algorithms are incorporated in the NBLAST and XBLAST programs (version 2.0) as described in Altschul, S. et al., Nucleic Acids Res., 25: 3389-3402 (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 Tolllis, A. and Robotti, C., 1994. Comput. Appl. Biosci., 10: 3-5. ADAM and FASTA described in Pearson, W. and Lipman, D., 1988. Proc. Natl. Acad. Sci. USA, 85: 2444-8.

  In another embodiment, the percent identity between two amino acid sequences 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.

  According to the present invention, also to a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1 and comprising at least one allele included in one or more haplotypes described herein, and its complement Thus, an isolated nucleic acid molecule comprising a fragment or portion that hybridizes under conditions of high stringency is provided. In addition, according to the present invention, a fragment or portion that hybridizes under high stringency conditions 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 an isolated nucleic acid molecule comprising 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.

  The nucleic acid fragments of the 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, P. et al., 1991. Science, 254: 1497-1500.

  A probe or primer includes SEQ ID NO: 1, including at least one allele included in one or more haplotypes described herein, and a contiguous nucleotide sequence derived from its complement. Included are regions of a nucleotide sequence that hybridize to at least about 15, typically about 20-25, and in some embodiments, about 40, 50, or 75 contiguous nucleotides of a nucleic acid molecule. The present invention also includes an isolated fragment or portion that hybridizes under high stringency conditions 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. Nucleic acid molecules are provided. 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 to or against 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 includes one or more sequences provided in SEQ ID NO: 1 (and optionally includes at least one allele included in one or more haplotypes described herein) and / or Alternatively, it can be amplified and isolated by polymerase chain reaction using synthetic oligonucleotide primers designed based on their complements. 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, P. et al., 1991. Nucleic Acids Res., 19: 4967-4973; Eckert, K. and Kunkel, T., 1991. PCR Methods and Applications, 1: 17-24; PCR (McPherson et al. Ed., IRL Press, Oxford); and US Pat. No. 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; Wu, D. and Wallace, R., 1989. Genomics, 4: 560-469, Landegren, U. et al., 1988. Science, 241: 1077-1080 ), Transcription amplification (Kwoh, D. et al., 1989. Proc. Natl. Acad. Sci. USA, 86: 1173-1177), and replication of self-maintaining sequences (Guatelli, J. et al., 1990. Proc Nat. Acad. Sci. USA, 87: 1874-1878), and nucleic acid based sequence amplification methods (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. 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 invention can be performed using well-known methods that are commercially available. For example, see 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, X. et al., 1999. Genome Res. 9: 492-498) and polypeptides. Using these or similar methods, polypeptides and DNA encoding the polypeptides can be isolated, sequenced, and further characterized.

  In general, the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels and as chromosomal markers that are labeled to map related gene locations. The nucleic acid sequence may 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.

  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, optionally comprising at least one allele included in the haplotype described herein, 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 as described more specifically therefor.

  A variant polypeptide can 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 are identified by methods known in the art, such as site-directed mutagenesis or alanine scan mutagenesis (Cunningham, B. and Wells, J., 1989. Science, 244: 1081-1085). obtain. 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 are determined by structural analysis, for example, crystallography, nuclear magnetic resonance, or photoaffinity labeling (Smith, L. et al., 1992. J. Mol. Biol., 244: 899-904; de It can also be determined by Vos, A. et al., 1992. Science, 255: 306-312.

  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 present 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. The present invention provides an antibody against a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a mutant BMP2 polypeptide. 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 hybridoma technology (Kohler, G. And Milstein, C., 1975. Nature, 256: 495-497), human B cell hybridoma technology (Kozbor, D. et al., 1983. Immunol. Today, 4:72), EBV-hybridoma technology (Cole et al., 1985). Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp77-96), or can be used to prepare monoclonal antibodies by trioma technology.

  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 to generate monoclonal antibodies to the polypeptides of the invention (see, eg, Current Protocols in Immunology, supra; Galfre, G. et al., 1977. Nature, 266: 550-552; Kenneth, R., Monoclonal Antibodies A New Dimension In Biological Analysis, Plenum Publishing Corp., New York, New York (1980); and Lerner, E. 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, P. et al., 1991. Biotechnology (NY), 9: 1369-1372; Hay, B. et al., 1992. Hum. Antibodies Hibridomas, 3: 81-85; Huse, W. et al., 1989. Science, 246: 1275- 1281; Griffiths, A. et al., 1993. EMBO J., 12: 725-734.

  In addition, recombinant antibodies, such as chimeric antibodies and humanized monoclonal antibodies, that 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, an antibody of the invention (eg, a monoclonal antibody) detects a polypeptide (eg, 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.

  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 haplotypes A haplotype spanning the BMP2 nucleic acid sequence associated with osteoporosis was identified.

  “Haplotype I”, “Haplotype II”, “Haplotype a”, “Haplotype b”, “Haplotype c” and “Haplotype d” are shown in Table 1 below. Each haplotype contains alleles at one or more polymorphic sites (haplotype I contains 4 SNPs and 1 microsatellite; haplotype II contains 3 SNPs and 2 microsatellite; haplotype a has 2 Haplotype b contains 3 SNPs; haplotype c contains 3 SNPs; and haplotype d contains 3 SNPs).

  Actual haplotypes include the markers listed in Table 1.

Allele number: A = 0, C = 1, G = 2, T = 3 for SNP allele; for microsatellite allele: CEPH sample 1347-02 (CEPH genomics repository) is used as a reference The lower allele of each microsatellite in this sample is set to 0, and all other alleles in other samples are numbered with respect to this reference. Thus, in CEPH sample 1347-02, allele 1 is 1 bp longer than the lower allele, in CEPH sample 1347-02, allele 2 is 2 bp longer than the lower allele, and allele in CEPH sample 1347-02. 3 is 3 bp longer than the lower allele, allele 4 is 4 bp longer than the lower allele in CEPH sample 1347-02, and allele-1 is lower than the lower allele in CEPH sample 1347-02 1 bp shorter, allele-2 in CEPH sample 1347-02 is 2 bp shorter than the lower allele, etc.

Haplotype analysis Haplotypes were identified as described above, and haplotype analysis was performed as described elsewhere (Stefansson, H. et al., 2002, Am. J. Hum. Genet., 71: 877-92).

Osteoporosis phenotype and control samples In the haplotype analysis, several different osteoporosis phenotypes were used, including those used for linkage analysis and other osteoporosis related phenotypes. The relationship between various phenotypes and haplotypes a, b and c is shown in FIGS. Haplotypes I and II are shown in FIG.

  For association analysis, the material collected for linkage analysis and all sporadic individuals with Z values lower than -1 SD were used. The control group included two randomly collected groups from the general population, one group used BMD measurements and questionnaire information, and the other group did not use medical information. These groups served as an unrelated population within 5 meiotic events, collected randomly based on controls; the total number of members in both groups was 1272.

  BMD of all participants, patients and relatives was measured using dual energy X-ray absorptiometry in the lumbar spine (L2-L4) and total hip joint (proximal thigh) and whole body (QDR) in anteroposterior view 4500A, Hologic, Waltham, MA). Body weight and height were measured at the time of BMD measurement. All participants completed a detailed questionnaire regarding their medical history, menstrual period, current and past medications (including hormone replacement therapy (HRT)) and all previous fractures and traumas.

  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.

FIG. 1 tabulates haplotype-related data for haplotypes a, b, and c (including BMP from spinal column and hip joint, osteoporotic fracture, weight-corrected BMD, as shown) for various phenotypes. is there. Data are also shown for pre-menopausal and post-menopausal patients. FIG. 2 is a table showing haplotype-related data of haplotype I and haplotype II. Data are shown for hip and spinal fractures and weight corrected BMD. FIG. 3 is a tabular representation of haplotype d for various phenotypes (including BMD from spinal column and hip joint, osteoporotic fracture, weight corrected BMD, as shown). BMD values show the 10th lowest percentile in all cases. Data are also shown for pre-menopausal and post-menopausal patients.

[Sequence Listing]

Claims (38)

  Detecting the presence or absence of at least one risky haplotype comprising a haplotype selected from the group consisting of haplotype I, haplotype II, haplotype a, haplotype b, haplotype c, haplotype d and combinations thereof, wherein haplotype A method of diagnosing susceptibility to osteoporosis or osteoporosis in an individual, wherein the presence of is an indicator of susceptibility to osteoporosis or osteoporosis.   A method for analyzing the presence of a first nucleic acid molecule in a sample comprising the step of contacting said sample with a second nucleic acid molecule comprising a haplotype according to claim 1.   2. The method of claim 1, wherein determining the presence or absence of a haplotype comprises enzymatic amplification of nucleic acid from the individual.   4. The method of claim 3, wherein determining the presence or absence of a haplotype further comprises electrophoretic analysis.   The method of claim 1, wherein the step of determining the presence or absence of a haplotype further comprises restriction enzyme fragment length polymorphism analysis.   The method of claim 1, wherein determining the presence or absence of a haplotype comprises sequence analysis.   A method of diagnosing susceptibility to osteoporosis or osteoporosis in an individual, comprising detecting the presence or absence of at least one at-risk haplotype comprising haplotype I, wherein the presence of the haplotype is an indicator of osteoporosis or susceptibility to osteoporosis.   8. A method for analyzing the presence of a first nucleic acid molecule in a sample, the method comprising contacting the sample with a second nucleic acid molecule comprising a haplotype according to claim 7.   8. The method of claim 7, wherein determining the presence or absence of a haplotype comprises enzymatic amplification of nucleic acid from the individual.   The method of claim 9, wherein determining the presence or absence of a haplotype further comprises electrophoretic analysis.   8. The method of claim 7, wherein the step of determining the presence or absence of a haplotype comprises restriction enzyme fragment length polymorphism analysis.   8. The method of claim 7, wherein the step of determining the presence or absence of a haplotype comprises sequence analysis.   A method of diagnosing susceptibility to osteoporosis or osteoporosis in an individual, comprising detecting the presence or absence of at least one risky haplotype comprising haplotype II, wherein the presence of the haplotype is indicative of osteoporosis or susceptibility to osteoporosis.   14. A method for analyzing the presence of a first nucleic acid molecule in a sample, the method comprising contacting the sample with a second nucleic acid molecule comprising a haplotype according to claim 13.   14. The method of claim 13, wherein determining the presence or absence of a haplotype comprises enzymatic amplification of nucleic acid from the individual.   16. The method of claim 15, wherein determining the presence or absence of a haplotype further comprises electrophoretic analysis.   14. The method of claim 13, wherein the step of determining the presence or absence of a haplotype comprises restriction enzyme fragment length polymorphism analysis.   14. The method of claim 13, wherein determining the presence or absence of a haplotype includes sequence analysis.   A kit for analyzing a sample for the presence of at least one haplotype associated with osteoporosis, wherein the haplotype comprises two or more specific alleles, wherein the kit comprises one or more specific haplotypes A kit comprising one or more nucleic acids capable of detecting the presence or absence of an allele, thereby indicating the presence or absence of a haplotype in a sample.   20. The kit of claim 19, wherein the nucleic acid comprises at least one contiguous nucleotide sequence that is completely complementary to a region comprising at least one specific allele of a haplotype. Haplotypes contain two or more specific alleles in separate containers
a) one or more labeled nucleic acids capable of detecting one or more specific alleles of the haplotype; and
b) A reagent kit for analyzing a sample for the presence of at least one haplotype associated with osteoporosis, comprising a reagent for the detection of said label.   The reagent kit according to claim 21, wherein the labeled nucleic acid comprises at least one continuous nucleotide sequence that is completely complementary to a region containing at least one specific allele of the haplotype.   A reagent kit for analyzing a sample for the presence of at least one haplotype associated with osteoporosis, wherein the haplotype comprises two or more specific alleles, said kit comprising part of the nucleotide sequence of BMP2 For a primer extension reaction comprising one or more nucleic acids comprising at least one nucleotide sequence that is at least partially complementary to and wherein the nucleic acids are capable of detecting two or more specific alleles of a haplotype Reagent kit that can act as a primer.   Screening for at least one at-risk haplotype associated with BMP2, which is more frequently present in individuals who are more susceptible to osteoporosis compared to individuals who are not as susceptible to osteoporosis, wherein the at-risk haplotype is A method for diagnosing and identifying susceptibility to osteoporosis in an individual that significantly increases risk.   25. The method of claim 24, wherein the significant increase is at least about 20%.   26. The method of claim 25, wherein the significant increase is identified as an odds ratio of at least about 1.2.   TSC0898956, B420, B8463, D20S846, TSC0191642, P4337, D20S892, B5048, B9082, D20S59, B7111 / rs235764, B12845 / rs15705, P9313, B10631, D35548, rs1116867, TSC0278787, D35548 and TSC0271643 A method for diagnosing susceptibility to osteoporosis in an individual, comprising the step of determining the presence or absence in at least one haplotype individual comprising the above allele, wherein the presence of the haplotype is an indicator of susceptibility to osteoporosis.   28. The method of claim 27, wherein determining the presence or absence of a haplotype comprises enzymatic amplification of nucleic acid from the individual.   30. The method of claim 28, wherein determining the presence or absence of a haplotype further comprises electrophoretic analysis.   28. The method of claim 27, wherein the step of determining the presence or absence of a haplotype further comprises restriction enzyme fragment length polymorphism analysis.   28. The method of claim 27, wherein determining the presence or absence of a haplotype further comprises sequence analysis.   Obtaining nucleic acid samples from an individual; and TSC0898956, B420, B8463, D20S846, TSC0191642, P4337, D20S892, B5048, B9082, D20S59, B7111 / rs235764, B12845 / rs15705, P9313, B10631, D35548, rs1116867, TSC0278787, 716 Analyzing the nucleic acid sample for the presence or absence of at least one haplotype comprising two or more alleles selected from the group consisting of wherein the presence of the haplotype is indicative of susceptibility to osteoporosis A method for diagnosing susceptibility.   33. The method of claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of TSC0898956, B420, B8463, D20S846 and TSC0191642.   35. The method of claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of P4337, D20S892, B5048, B9082 and D20S59.   33. The method of claim 32, wherein the haplotype comprises B7111 / rs235764 or B12845 / rs15705.   33. The method of claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of P9313, B10631 and D35548.   33. The method of claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of rs1116867, TSC0278787 and D35548.   33. The method of claim 32, wherein the haplotype comprises two or more alleles selected from the group consisting of TSC0271643, P9313 and B7111.