WO2008050855A1 - Gene sensitive to bone/joint disease and use thereof - Google Patents

Gene sensitive to bone/joint disease and use thereof Download PDF

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WO2008050855A1
WO2008050855A1 PCT/JP2007/070893 JP2007070893W WO2008050855A1 WO 2008050855 A1 WO2008050855 A1 WO 2008050855A1 JP 2007070893 W JP2007070893 W JP 2007070893W WO 2008050855 A1 WO2008050855 A1 WO 2008050855A1
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base
bone
represented
joint
sequence
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PCT/JP2007/070893
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French (fr)
Japanese (ja)
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Shiro Ikegawa
Hideyuki Mototani
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Riken
Takeda Pharmaceutical Company Limited
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to identification of genes related to bone and joint diseases such as osteoarthritis and polymorphisms in the genes correlated with the diseases, and bone-joint diseases based on the genes. Prevention, treatment, diagnosis of genetic susceptibility to the disease, and the like. Background art
  • Osteoarthritis (hereinafter also referred to as "OA"! /) Is a bone / joint disease with chronic arthritis. Degeneration of cartilage causes destruction of cartilage and proliferative changes in bone and cartilage. It is a sick disease. The number of patients is 5 to 7 million in Japan alone, and it is said that more than 80% of people over 60 years old have symptoms of osteoarthritis in the knee * elbow * hip joint and spine. It is a common disease. At present, symptomatic treatment that suppresses pain, such as administration of non-steroidal anti-inflammatory analgesics, hyaluronic acid, and steroids, is the main treatment method for OA.
  • CaM intracellular calcium binding protein
  • Non-patent Document 1 An intracellular calcium binding protein called calmodulin (hereinafter also referred to as "CaM”) is ubiquitously expressed in various tissues including chondrocytes. It is known that an increase in intracellular calcium concentration promotes chondrocyte differentiation (Non-patent Document 1). Major soft It has been reported that the expression of a gene encoding aggrecan, one of the bone substrates, is increased by mechanical stimulation. This expression is suppressed in the presence of a CaM inhibitor (Non-patent Document 2). Based on these findings, a model has been proposed that CaM activates transcription of the aggrecan gene via calcineurin and CaM-dependent kinase II.
  • Patent Document 1 International Publication No. 2006/014013 Pamphlet
  • Non-Patent Document 1 Tomita et al., “Journal 'of' Biological 'Chemistry (J • Biol. Chem.)” (USA), 2002, Vol. 277,. 42214-42218
  • Non-Patent Document 2 “Valhmu and Raia”, “Biochemical. Journal”, 2002, Vol. 361,. 689-696
  • Non-Patent Document 3 Mototani et al., "Human 'Molecular' Genetics (Hum. Mol. Genet.) J, (UK), 2005, Vol. 14, p. 1009-1017
  • the purpose of the present invention is to identify genes involved in the onset / progression of bone / joint diseases including osteoarthritis and elucidate their functions, thereby providing novel and fundamental prevention of the diseases. ⁇ To provide a means of treatment. Another object of the present invention is to provide a simple and highly accurate diagnostic method for genetic susceptibility to bone and joint diseases.
  • CALM2 and CALM3 other than CALM1 are the genes encoding CaM, and that they form the CaM gene family.
  • CALM2 was found to have the highest expression level.
  • CALM2 gene expression was elevated in osteoarthritis (Hip Osteoarthritis; also referred to as “HOA”) and knee osteoarthritis (hereinafter referred to as “KOA”) cartilage. Therefore, the present inventors conducted polymorphic discovery on the promoter region, intron 1 and all exons and their flanking regions of the CALM2 gene in order to investigate whether CALM2 is an OA susceptibility gene.
  • nucleic acid comprising a contiguous base sequence of about 15 bases or more;
  • the bases represented by base numbers 21, 3967, 5227, 6210, 712 1, 7791 and 16127 are cytosine, guanine, adenine, adenine, adenine, thymine and A nucleic acid comprising a base sequence of a haplotype which is adenine and lacks a base represented by base numbers 6913-6914;
  • Bone and joint diseases are osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis
  • a bone / joint disease prophylactic / therapeutic agent comprising a substance that activates the calcium / calmodulin pathway, characterized by administration;
  • the above-mentioned bone / joint disease is selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, arthropathy due to sports, and congenital bone disease [6] The agent described in;
  • the polymorphism in the CALM2 gene correlates with bone / joint diseases
  • the polymorphism can be used for simple determination of genetic susceptibility to bone / joint diseases.
  • bone / joint diseases particularly diseases related to degeneration / disappearance of cartilage matrix, reduced production ability, and reduced chondrocyte differentiation ability Prophylactic and therapeutic effects can be obtained.
  • FIG. 1 is a diagram showing a comparison of calmodulin gene expression levels in chondrocytes.
  • FIG. 2 is a diagram showing the expression of calmodulin gene in articular cartilage by microarray.
  • FIG. 3 shows the results of polymorphism discovery in the CALM2 gene region. Polymorphism discovery was performed using the genomic structure of the CALM 2 gene region and genomic DNA of 48 Japanese. The discovery region is all exons of the CALM2 gene and its flanking region, promoter region, and intron 1.
  • the method for diagnosing genetic susceptibility to bone / joint disease of the present invention is a polymorphism in the subject's CALM2 gene and / or linkage disequilibrium with it. It is characterized by detecting a polymorphism in a state.
  • “/ gene” polymorphism is a change of one or more bases (substitution, deletion, insertion, transposition, inversion, etc.) on genomic DNA, This refers to changes that occur at a frequency of 1% or more in the population. For example, one base is replaced with another base (SNP), 1 to several tens of bases are deleted or inserted. (DIP), where the sequence with 2 to several tens of bases as one unit is repeatedly present! /, With a different number of repetitions (repeating units with 2 to 4 bases are microsatellite polymorphisms, (A variable number of t andem repeat (VNTR)).
  • the polymorphism that can be used in the diagnostic method of the present invention may be any type as long as the above conditions are satisfied, but is preferably SNP.
  • the polymorphism in the base represented by base number 5227 and the base represented by base number 5606 is a novel SNP s found in the present invention, and is conventionally known
  • the base shown by base number 5227 is guanine
  • base number While the base represented by 5606 is adenine
  • the base represented by base number 5227 is adenine
  • the base represented by base number 5606 is guanine.
  • AC073283 (VERSION: AC073283.8, GI: 17432471, updated April 30, 2005)” (in this specification, simply abbreviated as “AC073283.8”).
  • base sequence complementary strand sequence
  • the base number 103843 to 122722 in the base sequence represented by AC073283.8
  • the distribution IJ is shown in SEQ ID NO: 1.
  • nucleotides (bases) such as polymorphic sites are all represented by the base number of AC 073283.8.
  • an SNP [“117496G> A” in the base represented by base number 117496 in the base sequence (complementary chain sequence) represented by GenBank accession No. AC07 3283.8
  • SNP ““117496G> A” in the base represented by base number 117496 in the base sequence (complementary chain sequence) represented by GenBank accession No. AC07 3283.8
  • it means "" base number in AC073283.8 "" base of major allele ">” base of minor allele ".
  • allele base notation it means deletion, and "N (N) N" (N is any base) means insertion.
  • 117496G> A has a major allele (G allele)
  • 117117A> G has a minor allele (G allele) allele frequency. It was found to be significantly higher in the HOA patient population without AD. That is, the 117496G and 117117G alleles are HOA sensitive (susceptible) alleles without AD.
  • Bone and joint diseases associated with reduced 'disappearance' production ability and chondrocyte differentiation ability such as osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders, congenital bone system Diseases [eg, bone system diseases with reduced cartilage formation ⁇ congenital bone system diseases complicated with osteoarthritis (e.g., achondroplasia, multiple epiphyseal dysplasia, vertebral epidysplasia, diaphysis) It is strongly suggested that it is sensitive to diseases such as acrodysplasia, Stickler syndrome, pseudochondral dysplasia, etc.).
  • cartilage matrix production ability 'Diseases associated with abnormally enhanced chondrocyte differentiation ability such as congenital bone system diseases [eg, bone system diseases with increased cartilage formation (eg, multiple exostoses, unilateral) 117496G allele and 117117G allele may be protective against diseases such as hypertrophy, Oriere disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor.
  • congenital bone system diseases eg, bone system diseases with increased cartilage formation (eg, multiple exostoses, unilateral) 117496G allele and 117117G allele may be protective against diseases such as hypertrophy, Oriere disease, Maftucci syndrome, etc.]
  • osteochondroma bone tumor
  • cartilage tumor cartilage tumor.
  • nucleotide sequence represented by GenBank accession No. AC073283.8
  • polymorphism at the base represented by base number 117496 or 117117
  • the polymorphisms in the state one whose allele frequency is significantly higher in any bone / joint disease patient population than in any bone / joint disease non-affected population should also be used in the diagnostic method of the present invention. Can do.
  • the “linkage disequilibrium coefficient D ′” means that for two SNPs, each allele of the first SNP is (A, a), each allele of the second SNP is (B, b), and four haplotypes ( If each frequency of AB, Ab, aB, ab) is P, P, P, P, then
  • the bone / joint disease patient population and the bone / joint disease non-affected population are each composed of sufficient numbers to give statistically reliable results, the size (number of samples), There are no particular restrictions on the background of each sample (eg, birthplace, age, gender, disease, etc.).
  • bone-joint diseases include osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders, osteochondroma, bone tumor, chondroma Ulcers, congenital bone system diseases, etc., but preferably osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, arthropathy due to sports, cartilage substrate degeneration / disappearance Congenital bone system diseases related to decreased production ability and chondrocyte differentiation ability, etc., more preferably osteoarthritis (OA), more preferably osteoarthritis of the hip (hip joint OA, HOA) or deformability Knee arthropathy (knee joint OA, KOA), particularly preferably HOA, most preferably HOA without acetabular dysplasia (AD).
  • OA osteoarthritis
  • HOA hip joint OA
  • KOA deformability Knee arthropathy
  • the non-affected group of bone / joint diseases is usually a group of patients other than bone / joint diseases at a medical institution, Or, a group of subjects diagnosed as having a bone or joint disease in a mass screening in a certain region and being diagnosed as having a bone or joint disease are preferably used.
  • NCBL SNP database http: ⁇ www.ncbi.nlm
  • the NCBL SNP database contains all the CALM2 gene etason and their flanking region, promoter region, and intron 1.
  • Haplotype III includes the 117496A allele, which is the only insensitive allele of the above-mentioned bone 'joint disease, among the five haplotypes. Therefore, this haplotype is not only OA (particularly primary OA), but also bone and joint diseases related to degeneration / disappearance / decreased ability to produce cartilage and decreased ability to differentiate chondrocytes, such as osteoporosis, rheumatoid arthritis, arthritis.
  • Synovitis eg bone system diseases with reduced cartilage formation
  • congenital bone system diseases eg : Achondroplasia, multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.
  • Achondroplasia multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.
  • haplotype III the other seven polymorphisms in haplotype III are all in a completely linked state with the 117496A allele. Therefore, if an allele that is different from haplotype III is detected in these polymorphic sites, it is a haplotype other than haplotype III, and therefore has a very high probability of having 117496G, which is one of the susceptibility alleles of the present invention. .
  • 122702T allele exists only in haplotype II, 118756C allele only in haplotype V, 106596G allele only in haplotype IV, 116513G allele, 115810-115809TG allele, 115602G allele and 114932C allele haplotype II or Since these alleles are present in IV, genetic susceptibility to bone and joint diseases can be determined with high accuracy by detecting these alleles.
  • the polymorphism detected in the diagnostic method of the present invention may be any one of the polymorphisms described above, or may be two or more.
  • these may be sequentially performed until a haplotype other than haplotype III is identified.
  • it is necessary to detect the polymorphism of 117496G> A.
  • any known SNP detection method can be used to detect a polymorphism.
  • the power to do S As a classic detection method, for example, a genomic DNA extracted from a subject's cells or the like is used as a sample, and a partial base sequence of the base sequence represented by GenBank accession number AC073283.8, which About 1 including the base at the polymorphic site (preferably, the base represented by the base number 117496 or 117117, or the base represented by the base numbers 122702, 118756, 11 6513, 115810 to 115809, 115602, 1114932 or 106596)
  • a nucleic acid comprising a continuous base sequence of 5 to about 500 bases is used as a probe.
  • Hybridization is performed while accurately controlling the nuance, and only the sequence that is completely complementary to the probe is detected, or the base of the polymorphic site in the nucleic acid and the nucleic acid is changed to another base. Replaced nucleic acid ! / Using a mixed probe with one of the labels labeled and the other unlabeled, hybridization is performed while gradually lowering the reaction temperature from the denaturation temperature, and the sequence that is completely complementary to one probe is hybridized first. And a method for preventing cross reaction with a probe having a mismatch.
  • the labeling agent for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used.
  • the radioisotope for example, [ 125 I], [ m I], [3 ⁇ 4], [ 14 C] and the like are used.
  • the above enzyme those which are stable and have high specific activity are preferred.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • the luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • detection of polymorphism is performed by various methods described in WO 03/023063, for example, RFLP method, PCR-SSCP method, ASO hybridization, direct sequence method, ARMS method, denaturing agent Gradient gel electrophoresis, RNaseA cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic 'allele specific' neutral It can be carried out by a dialysis method, a node lock probe method, a UCAN method, a nucleic acid hybridization method using a DNA chip or a DNA microarray, an ECA method, etc. (WO 03/023063, page 17, page 5) Line to page 28, line 20).
  • TaqMan PCR method and A more detailed description will be given of the method of the vanvader.
  • the TaqMan PCR method uses a fluorescently labeled allele-specific oligonucleotide (TaqMan probe) and PCR using Taq DNA polymerase.
  • the TaqMan probe is a partial base sequence of the base sequence represented by GenBank accession number AC073283.8, which is a continuous base of about 15 to about 30 bases including the bases of any of the polymorphic sites described above.
  • An oligonucleotide consisting of a sequence is used.
  • the probe is labeled with a fluorescent dye such as FAM or VIC at the 5 'end and labeled with a quencher (quenching substance) such as 3' end force STAMRA. Fluorescence is not detected due to absorption.
  • probes for both alleles and label them with fluorescent dyes having different fluorescence wavelengths (for example, one allele is FAM and the other is VIC) for batch detection.
  • the 3 'end is phosphorylated to prevent PCR extension from the TaqMan probe.
  • the hybridized TaqMan probe is cleaved by the 5 'nuclease activity of Taq DNA polymerase, the fluorescent dye is released and is not affected by quenching, and fluorescence is detected.
  • the fluorescence intensity increases exponentially due to the vertical amplification.
  • an allele-specific oligonucleotide (about 15 to about 30 bases in length; If the A allele is FAM, the G allele is labeled 5 'with VIC, and the 3' end is! /, And both are labeled with TAMRA), the subject's dienotype is AA or GG. If present, the strong fluorescence intensity of FAM or VIC is recognized, and the other fluorescence is hardly recognized. On the other hand, if the subject's dienotypic force SAG, both FAM and VIC fluorescence are detected.
  • the invader method uses an allele-specific oligonucleotide (a (Rel probe) itself is not labeled, has a non-complementary sequence (flap) on the 5 'side of the base of the polymorphic site, and has a complementary sequence specific to the 3' side on the 3 'side .
  • a (Rel probe) itself is not labeled, has a non-complementary sequence (flap) on the 5 'side of the base of the polymorphic site, and has a complementary sequence specific to the 3' side on the 3 'side .
  • an oligonucleotide having a specific complementary sequence on the 3 'side of the polymorphic site of the saddle type Invader probe; any base corresponding to the polymorphic site at the 5' end of the probe is optional.
  • the 5 'side has a sequence that can take a hairpin structure, and when the hairpin structure is formed, the sequence that is 3' side continuous from the base paired with the 5 'end base is the flap of the allele probe.
  • a FRET (fluorescence resonance energy transfer) probe characterized in that it is a complementary sequence.
  • the 5 'end of the FRET probe is fluorescently labeled (for example, FAM, VIC, etc.), and a quencher (for example, TAMRA, etc.) is bound in the vicinity thereof, and fluorescence is not detected as it is (hairpin structure). Les.
  • the 3 'end of the invader probe enters the polymorphic site when the three partners bind complementarily.
  • the enzyme that recognizes the structure of this polymorphic site cleavase
  • the single-stranded part of the allele probe ie, the 5 'side flap part from the base of the polymorphic site
  • the flap is complementary to the FRET probe. Binds and the flap polymorphic site enters the hairpin structure of the FRET probe.
  • the polymorphism As a result of examining the polymorphism as described above, it was significantly higher in any bone / joint disease patient population than in any bone / joint disease non-affected population, and possessed alleles! / , Especially if the allele is determined to be a homozygote.
  • the person can be diagnosed with a high genetic susceptibility to the bone and joint disease.
  • GenBank accession number AC073283.8 For example, in the base sequence represented by GenBank accession number AC073283.8,
  • Subjects are osteoarthritis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy related to bone / joint diseases associated with degeneration / disappearance of cartilage matrix, decreased production ability, and reduced chondrocyte differentiation ability , Sports joint disorders, congenital bone system diseases [eg, reduced cartilage formation! /, Bone system diseases ⁇ congenital bone system diseases complicated by osteoarthritis (eg, soft bone aplasia, Multiple epiphyseal dysplasia, vertebral epidysodysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)] can be diagnosed .
  • the subject may have a disease associated with abnormal production of chondrocytes, such as a congenital bone disease [for example, a bone disease with increased cartilage formation (eg, Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor, and other diseases.
  • a congenital bone disease for example, a bone disease with increased cartilage formation (eg, Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]]
  • osteochondroma bone tumor, cartilage tumor, and other diseases.
  • Subjects are osteoarthritis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy related to bone / joint diseases associated with degeneration / disappearance of cartilage matrix, decreased production ability, and reduced chondrocyte differentiation ability , Sports joint disorders, congenital bone system diseases [eg, reduced cartilage formation! /, Bone system diseases ⁇ congenital bone system diseases complicated by osteoarthritis (eg, soft bone aplasia, Multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)].
  • congenital bone system diseases eg, reduced cartilage formation! /
  • Bone system diseases ⁇ congenital bone system diseases complicated by osteoarthritis eg, soft bone aplasia, Multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dys
  • the subject may have a disease associated with abnormal enhancement of cartilage matrix production ability / chondrocyte differentiation ability, such as congenital bone disease [eg, bone disease with increased cartilage formation (eg, : Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor, and other diseases.
  • congenital bone disease eg, bone disease with increased cartilage formation (eg, : Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]
  • osteochondroma bone tumor, cartilage tumor, and other diseases.
  • Subjects are osteoarthritis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy related to bone / joint diseases associated with degeneration / disappearance of cartilage matrix, decreased production ability, and reduced chondrocyte differentiation ability , Sports joint disorders, congenital bone system diseases [eg, reduced cartilage formation! /, Bone system diseases ⁇ congenital bone system diseases complicated by osteoarthritis (eg, soft bone aplasia, Multiple epiphyseal dysplasia, vertebral epidysodysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)] can be diagnosed .
  • the subject may have a disease associated with abnormal production of chondrocytes, such as a congenital bone disease [for example, a bone disease with increased cartilage formation (eg, Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor, and other diseases.
  • a congenital bone disease for example, a bone disease with increased cartilage formation (eg, Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]]
  • osteochondroma bone tumor, cartilage tumor, and other diseases.
  • polymorphisms at the bases indicated by base numbers 117496 and 117117 in the base arrangement IJ (complementary strand sequence) represented by GenBank accession number AC073283.8 are first seen in the present invention.
  • New polymorphisms released (117496A and 117117G are new alleles) and marker polymorphisms that can be used to diagnose genetic susceptibility to bone and joint diseases
  • the present invention also provides a partial base sequence of the base sequence (complementary strand sequence) represented by GenBank accession number AC073283.8, (a) a base represented by base number 117496 (provided that the base is A), or
  • a nucleic acid comprising a continuous base sequence of about 15 to about 500 bases (preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases).
  • a novel nucleic acid containing a strong SNP site can be preferably used to detect a polymorphism in the base in the above-described diagnostic method of the present invention.
  • Such a nucleic acid can be synthesized using a DNA / RNA automatic synthesizer based on the nucleotide sequence information represented by SEQ ID NO: 1.
  • haptic type III has an insensitive allele (117496A) for various bone and joint diseases, including OA (particularly HOA without AD).
  • the present invention also provides a base or base represented by base numbers 122702, 118756, 117496, 116513, 115602, 114932 and 106596 in the base sequence (complementary strand sequence) represented by GenBank accession No. AC073283.8.
  • a nucleic acid comprising a haplotype base sequence in which the sequences are G, A, A, A, T, and A, respectively, and the bases represented by base numbers 115810 to 115809 are deleted.
  • Such a nucleic acid can also be used as a probe for detecting an insensitive haplotype for bone / joint diseases such as OA (particularly HOA without AD) in the diagnostic method of the present invention.
  • a nucleic acid is probed with a nucleic acid containing all or part of the base sequence represented by SEQ ID NO: 1 from genomic DNA isolated from a human cell or tissue carrying the CALM2 gene having the haplotype structure. It can be isolated by using as Alternatively, the nucleic acid can be isolated from a genomic DNA isolated from a human cell or tissue having a CALM2 gene having an arbitrary haplotype structure by isolating the corresponding part of the nucleic acid according to the same method.
  • a polymorphism present in the CALM2 gene and / or its surrounding region, and / or 117117A> G or a linkage disequilibrium coefficient with the polymorphism D ′ of 0.9 or more, preferably a complete linkage Polymorphism in the CALM2 gene and / or its surrounding region that is in a state (ie, D ′ l), and one allele frequency power is more arbitrary than in the population without any bone 'joint disease It comprises one or more nucleic acid probes and / or primers capable of detecting each of one or more polymorphisms selected from the group consisting of high polymorphisms in the bone / joint disease patient population.
  • the nucleic acid probe used in the diagnostic kit of the present invention is a nucleic acid that hybridizes with genomic DNA in a region containing the base of the polymorphic site to be detected in the diagnostic method of the present invention.
  • its length is not particularly limited. For example, about 15 bases or more, preferably about 15 to about 500 bases, more preferably about 15 to about 200 bases, and even more preferably about 15 to about 50 bases.
  • the probe may contain additional sequences suitable for detection of polymorphisms (not complementary to genomic DNA! /, IJ).
  • the allele probe used in the invader method has an additional sequence called a flap at the 5 ′ end of the base at the polymorphic site.
  • the probe may be a suitable labeling agent such as a radioisotope (eg, 125 I, m I, 3 H, 14 C, etc.), an enzyme (eg, 0 galactosidase, 0 dalcosidase, alkaline phosphatase, Oxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluoreoretsen isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) Also good.
  • a radioisotope eg, 125 I, m I, 3 H, 14 C, etc.
  • an enzyme eg, 0 galactosidase, 0 dalcosidase, alkaline phosphatase, Oxidase, malate dehydrogenase, etc.
  • fluorescent substances eg, fluorescamine,
  • a quencher quenching substance that absorbs fluorescence energy emitted by the fluorescent substance may be further bound in the vicinity of the fluorescent substance (eg, FAM VIC).
  • the fluorescent substance eg, FAM VIC
  • the fluorescent substance and the quencher are separated and the fluorescence is detected.
  • the nucleic acid probe used in the diagnostic kit of the present invention is GenBank accessio. n No.
  • the base sequence (complementary strand sequence) represented by AC073283.8
  • the polymorphic site represented by base numbers 117496 and / or 117117, or base numbers 122702, 118756, 116513, 115 810 to 115809, 115602, 114932
  • the base represented by base number 117496 is G or A,
  • base represented by base number 117117 is A or G
  • base represented by base number 122702 is C or T
  • base represented by base number 118756 is G or C
  • base represented by base number 116513 is A or G
  • the base represented by base numbers 115810 to 115809 is a deleted force, or TG,
  • base represented by base number 115602 is A or G
  • the base represented by the base number 114932 is T or C
  • the base represented by base number 106596 is A or G,
  • a nucleic acid having one of the bases for each polymorphic site can be used, or two types of nucleic acids having a base corresponding to each allele can be used.
  • the base at the polymorphic site ie, the 3 ′ terminal base
  • the base at the polymorphic site may be any base.
  • the nucleic acid primer used in the diagnostic kit of the present invention is designed so as to specifically amplify a genomic DNA region containing the base of the polymorphic site to be detected in the diagnostic method of the present invention. Anything is acceptable.
  • a partial base sequence of the base sequence represented by GenBank access ion No. AC073283.8, which hybridizes to a part of the complementary strand sequence 5 'to the base of the polymorphic site to be detected.
  • About 15 to about 50 bases preferably about 15 to about 30 bases, and hybridizes to a part of the sequence 3 ′ from the base of the polymorphic site with about 15 to about 50 bases.
  • a base preferably a combination with a nucleic acid containing a base sequence of about 15 to about 30 bases, and the fragment length of the nucleic acid amplified by them is about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably Is A pair of nucleic acids having about 50 to about 200 bases is included.
  • the primer may contain additional sequences suitable for polymorphism detection (non-complementary to genomic DNA! /, Sequences), such as a linker sequence.
  • the primer may be a suitable labeling agent such as a radioisotope (eg, 125 ⁇ , 1 31 I, 3 H, 14 C, etc.), an enzyme (eg, ⁇ -galatatosidase, ⁇ -darcosidase, alkaline phosphatase). Phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescene isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) It may be labeled.
  • a radioisotope eg, 125 ⁇ , 1 31 I, 3 H, 14 C, etc.
  • an enzyme eg, ⁇ -galatatosidase, ⁇ -darcosidase, alkaline phosphatase.
  • the nucleic acid primer used in the diagnostic kit of the present invention is a partial base sequence of the base sequence represented by GenBank Accession No. AC073283.8 and represented by Base Nos. 11 7496 and / or 117117. Selected from the group consisting of base numbers 122702, 118756, 116513, 115810 to 115809, 115602, 114932 and 106596, more preferably 117496 and / or 117117.
  • a pair of nucleic acids that can amplify a continuous base sequence of about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably about 50 to about 200 bases, including the bases of the polymorphic site.
  • the nucleic acid probe or primer used in the diagnostic kit of the present invention may be DNA or RNA, and may be single-stranded or double-stranded. In the case of a double strand, any of a double-stranded DNA, a double-stranded RNA, and a DNA / RNA nobled may be used. Therefore, when describing a nucleic acid having a certain base sequence in this specification, unless otherwise specified, a single-stranded nucleic acid having the base sequence, a single-stranded nucleic acid having a sequence complementary to the base sequence, and those If it is used to include all double-stranded nucleic acids that are hybrids, it should be rationalized.
  • the nucleic acid probe or primer can be synthesized according to a conventional method using a DNA / RNA automatic synthesizer based on the information of the base sequence represented by AC073283.8, for example.
  • the nucleic acid probe and / or primer are each separately (or mixed if possible) in water or in an appropriate buffer (eg, TE buffer) at an appropriate concentration (eg, 2 X to Can be dissolved at a concentration of 20 X at 1-50 M) and stored at about -20 ° C. it can.
  • an appropriate buffer eg, TE buffer
  • concentration eg, 2 X to Can be dissolved at a concentration of 20 X at 1-50 M
  • the diagnostic kit of the present invention may further include other components necessary for carrying out the method as a component depending on the polymorphism detection method.
  • the kit when the kit is for detecting a polymorphism by TaqMan PCR, the kit contains 10 X PCR reaction buffer, lO X MgCl aqueous solution, 10 X dNTPs aqueous solution, Taq DNA polymerase (5 U / L) and the like. Further can be included.
  • the diagnostic kit of the present invention is a bone / joint disease associated with degeneration / disappearance / decreased production ability of cartilage matrix, or reduced ability to differentiate chondrocytes, such as osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis.
  • Synovitis eg, bone disease with reduced cartilage formation
  • congenital bone disease with osteoarthritis eg, cartilage
  • Aplasia multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.
  • the diagnostic kit of the present invention can be used for diseases associated with abnormal enhancement of cartilage matrix production ability 'chondrocyte differentiation ability, such as congenital bone disease (eg, bone disease with enhanced soft bone formation ( For example, multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.)], osteochondroma, bone tumor, cartilage tumor, and other genetic susceptibility diagnosis.
  • congenital bone disease eg, bone disease with enhanced soft bone formation ( For example, multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.)]
  • osteochondroma e.g, osteochondroma, bone tumor, cartilage tumor, and other genetic susceptibility diagnosis.
  • bone / joint diseases in particular, bone / joint diseases related to degeneration / disappearance / decreased production ability of cartilage matrix and reduced ability to differentiate chondrocytes [for example, osteoporosis, osteoarthritis, Rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders, congenital bone system diseases [eg, bone system diseases with reduced soft bone formation ⁇ congenital bones with osteoarthritis Susceptibility to systematic diseases (eg, achondroplasia, multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia)]]]
  • the subject who is determined to be susceptible has a Ca / CaM pathway in the process of onset and / or progression of bone / joint diseases associated with degeneration / disappearance of the cartilage matrix, decreased production capacity, and decreased chondrocyte
  • a bone 'joint disease prevention' therapeutic agent comprising a substance that activates the Ca / CaM pathway, characterized by being administered to a human having the allele of
  • bone / joint diseases include bone / joint diseases related to degeneration / disappearance / decreased production ability of cartilage matrix and decreased ability to differentiate chondrocytes, such as osteoporosis, osteoarthritis, rheumatoid arthritis, Arthritis, synovitis, metabolic arthropathy, sports joint disorders, congenital bone system diseases [eg bone system diseases with reduced cartilage formation ⁇ congenital bone system diseases complicated with osteoarthritis (eg: Achondroplasia, multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphy
  • the ⁇ rCa / CaM pathway '' means a series of signal transduction pathways shown in, for example, Non-Patent Document 2 (page 694, Scheme 1), and in addition to CaM, calcineurin, CaM-dependent kinase II ( CaMK II) etc.
  • Examples of the substance that activates the Ca / CaM pathway include a substance that promotes the expression and / or activity of one or more of CaM, calcineurin, and CaMKII.
  • Specific examples of such substances include CaM or a partial peptide thereof, calcineurin or a partial peptide thereof, CaMKII or a partial peptide thereof, and a nucleic acid encoding any one of the above proteins or peptides.
  • C aM and partial peptides thereof, and nucleic acids encoding them are described in Patent Document 1 above.
  • Calcineurin, CaMKII and their partial peptides, and nucleic acids encoding them can be similarly prepared based on the known amino acid sequence and base sequence of each subunit.
  • a screening method for a substance that promotes CaM expression and / or activity is described in Patent Document 1 above. Screening for substances that promote the expression of calcineurin and CaMKII can be performed in the same manner as CaM.
  • Calcineurin is a dephosphorylation enzyme of a transcription factor (eg, NFATcl) that is indicated to be involved in chondrocyte differentiation, and its activity is, for example, a cis element (eg, NFAT response element) whose expression is controlled by the transcription factor. It can be measured using the expression of a reporter gene under the control of a promoter containing Alternatively, a substrate analog of the enzyme can be synthesized and labeled with 32 P to directly measure dephosphorylation. If dephosphorylation is promoted in the presence of the test substance, the force S can be selected to select the substance as a calcineurin activity promoter.
  • a transcription factor eg, NFATcl
  • a cis element eg, NFAT response element
  • CaMKII is thought to activate (phosphorylate) cAMP response element-binding transcription factors to promote the production of cartilage substrate genes, and the activity is controlled, for example, by the promoter of the cartilage matrix gene.
  • a substrate analog of the enzyme can be synthesized and the phosphorylation of the substrate can be directly measured using 32 P-labeled ATP. If phosphorylation is promoted in the presence of the test substance, the substance can be selected as a CaMKII activity promoter.
  • test substances include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel. It may be! /, And it may be a publicly known one! /.
  • a substance that activates the Ca / CaM pathway when used as the prophylactic / therapeutic agent, it can be formulated according to conventional means.
  • the substance when the substance is a nucleic acid encoding CaM, calcineurin or CaM or a partial peptide thereof, the nucleic acid can be used alone or in a suitable vector such as a retroinoleless vector, an adenowinores vector, or an adenowinore associated betater. Then, it can be formulated according to conventional means.
  • the nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hard mouth gel catheter.
  • a substance that activates the Ca / CaM pathway may be administered orally as a tablet, capsule, elixir, microcapsule, or the like with sugar coating as needed. It can be used parenterally in the form of injections such as sterile solutions with other pharmaceutically acceptable liquids or suspensions.
  • a substance that activates the Ca / CaM pathway together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be produced by mixing in the required unit dosage form. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
  • binders such as gelatin, corn starch, tragacanth and gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • lubricants such as magnesium stearate
  • sweeteners such as sucrose, lactose or saccharin
  • flavoring agents such as peppermint, coconut oil or cherry.
  • a liquid carrier such as fats and oils in the above type of material.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice, such as dissolving or suspending active substances, naturally
  • aqueous solution for injection for example, isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used.
  • adjuvants such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80 TM, HCO-50)
  • oily liquid for example, sesame oil, soybean oil and the like are used, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • the prophylactic / therapeutic agent includes, for example, a buffer (for example, phosphate buffer solution, sodium acetate buffer solution), a soothing agent (for example, benzalkonium chloride, pro-in hydrochloride, etc.), a stable agent. You may mix
  • the prepared injection liquid is usually filled into a suitable ampoule.
  • the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rat, mouse, nomster, usagi, hidge, goat, pig, ushiki) ⁇ Uma ⁇ cat ⁇ i , Monkeys, chimpanzees, birds, etc.).
  • warm-blooded animals for example, rat, mouse, nomster, usagi, hidge, goat, pig, ushiki
  • the dose of the substance that activates the Ca / CaM pathway varies depending on the administration subject, target organ, symptom, administration method, etc.
  • patients with osteoarthritis As 60 kg
  • the single dose varies depending on the administration subject, target organ, symptom, administration method, etc.
  • it is usually treated with osteoarthritis patients (60 In terms of kg)
  • it is convenient to apply about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day.
  • the amount converted per 60 kg is measured with the force S.
  • bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below.
  • optical isomers for amino acids L form is shown unless otherwise specified.
  • DNA Deoxyribonucleic acid
  • RNA Ribonucleic acid
  • mRNA Messenger ribonucleic acid
  • SEQ ID NO: 1 the SNP site is an alternative shown in Appendix 2, Table 1 of “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (July 2002). This is indicated by the symbol.
  • nHAC Calmodulin Gene Expression in Chondrocytes by Quantitative PCR Normal human chondrocytes (nHAC) were purchased from Cambrex. After culturing nHAC with chondrocyte growth medium (Cambrex) to a density of about 90%, the cells were embedded in alginate beads and cultured with chondrocyte differentiation medium (Cambrex). The medium was replaced with fresh chondrocyte differentiation medium every 3 or 4 days. Three weeks after embedding in alginate beads, chondrocytes were isolated according to the method of Bloomberglmann et al. (J. Cell Sci. 107 (Ptl): 17-27 (1994)).
  • Calmodulin genes (CALM 1, CALM2 and CALM3) were quantified using the QuantiTect STBR Green PCR (Qiagen) on the ABI PRISM 7700 sequence detection system (Applied biosystems) according to the method in the package insert. It was. Table 1 shows the primers used for quantification of remission. The copy number of each gene was calculated using a calibration curve. The amount of each gene was standardized by interrupting the total RNA amount quantified using the GAPDH gene as an index.
  • CALM2 gene expression level was the highest in chondrocytes (Fig. 1). The amount was about 3 times that of the CALM1 gene. On the other hand, the expression level of the CALM3 gene was the lowest, about 1/3 that of the CALM1 gene. This result suggests that the CALM2 gene may contribute most to the total amount of calmodulin at the protein level.
  • Example 2 Comparison of expression levels of calmodulin gene in normal and OA articular cartilage by oligonucleotide microarray analysis
  • CALM1, CALM2, and CALM3 expression analyzes were performed using a microarray set (GeneChip U95 and U133, Affymetrix) consisting of oligonucleotide probes for approximately 60,000 target sequences.
  • a series of operations such as biotinylated cRNA preparation and array hybridization (also performed according to the Affymetrix GeneChip expression analysis manual.
  • RNA for osteoarthritic cartilage and normal articular cartilage was approved by the in-house ethics committee. Purchased from Direct Clinical Access Co., Ltd.
  • RNA 5-lOmg in ⁇ shape synthesize ⁇ rst strand cDNA using T7-poly ⁇ primer and: superscript II (Invitrogen), then b.coli Secona strand cDNA was synthesized using DNA polymerase i (Invitrogen and ligase (Invitrogen). Using the resulting double-stranded cDNA, biotinylated UTP and CTP (Enzo Diagn ostics) were born.
  • Cin vitro transcription (Ambioru Biotinylated cRNA was fragmented by incubating at 94 ° C for 30 minutes in a buffer containing Tris (pH 8.1), lOOmM potassium acetate, 30 mM magnesium acetate, and array hybridization was performed. Cleaning and cleaning Staining was performed using a dedicated Fluidics Station (Affymetrix), signals were detected using a dedicated Confocal Scanner (Molecular Dynamics), and array hybridization assay was performed using GeneChip analysis software. The median signal value of all genes was normalized as 1.
  • the polymorphisms in the CALM2 gene were genotyped for the hip osteoarthritis patient group and the control group, and the correlation analysis was performed using the results.
  • a TaqMan probe for dienotyping was prepared by Assays-by-Design service (Applied biosystems). Using this TaqMan probe, a genomic DNA collected from each individual was used as a sample to perform a dienotyping reaction on a 384 multiwell plate.
  • i-CALM2-10 0 3 354 357 0.004 1 .00 0 5 364 369 0 .01 0 .99 0.51 0.51 i 1 CALM2-11 264 87 5 356 0 • 7F 2 ⁇ 2 94 9 375 0 .15 0 • 9F 0.62 ⁇ - CALM2-12 353 3 0 356 0.004 1, .00 371 1 0 372 0. 001 1 • 00 0.30
  • the hip osteoarthritis group was divided into a patient group with and without acetabular dysplasia, and a correlation analysis was performed according to the method of Example 4.
  • i-CALM2-5 and i_CALM2-6 showed a significant correlation in patients without acetabular dysplasia (when P was 0.05).
  • i-CALM2-6, frequency power of minor alleles was high in the case group (Table 4). This result suggests that the CALM2 gene is a susceptibility gene for osteoarthritis of the hip without acetabular dysplasia.
  • Example 7 Haplotype angular segregation using osteoarthritis patient group without acetabular dysplasia
  • the haplotype structure in each population was estimated.
  • the method of Excoffier et al. [Excoffier and ⁇ latkin M (1995) Maximum-likelihood estimation of molecular haplotype frequency in a diploid population. Mol Biol Evol 12: 921-927] was used as a reference. As a result, it has become clear that there are mainly five types of haplotypes in this area. The total frequency of those haplotypes accounted for 95% of the total.
  • haplotype III showed a significant correlation with hip osteoarthritis without acetabular dysplasia (when the significance level was set to P 0.05).
  • the frequency of haplotype III was higher in the control group (Table 6). This result, together with Example 5, suggests that the CALM2 gene is a susceptibility gene for osteoarthritis of the hip without acetabular dysplasia.

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Abstract

Disclosed is a method for the diagnosis on the genetic sensitivity to a bone/joint disease, particularly a disease selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, a joint disorder induced by a sporting activity, osteochondroma, bone tumor, cartilage tumor and a congenital constitutional disorder of bone, which comprises detecting the polymorphism occurring in a nucleotide at nucleotide No. 5227 and/or a nucleotide at nucleotide No. 5606 in the nucleotide sequence depicted in SEQ ID NO:1. Also disclosed is a prophylactic/therapeutic agent for a bone/joint disease comprising a substance capable of activating the calcium-calmodulin pathway, particularly a substance capable of expressing and/or enhancing the activity of at least one protein selected from the group consisting of calmodulin, calcineurin and calmodulin-dependent kinase II, which can be administered to a human patient who is determined to have an allele sensitive to the disease by the above-mentioned diagnosis method.

Description

明 細 書  Specification
骨 ·関節疾患感受性遺伝子およびその用途  Bone and joint disease susceptibility genes and their uses
技術分野  Technical field
[0001] 本発明は、変形性関節症などの骨 ·関節疾患 (bone and joint diseases)に関連する 遺伝子および該疾患と相関する該遺伝子内の多型の同定、並びにそれらに基づく 骨'関節疾患の予防'治療、該疾患に対する遺伝的感受性の診断などに関する。 背景技術  [0001] The present invention relates to identification of genes related to bone and joint diseases such as osteoarthritis and polymorphisms in the genes correlated with the diseases, and bone-joint diseases based on the genes. Prevention, treatment, diagnosis of genetic susceptibility to the disease, and the like. Background art
[0002] 骨 ·関節の生活習慣病は、直接命に影響を及ぼすことは少ないが、痛みや歩行障 害などのために日常生活動作（ADL)に支障をきたすことから、高齢者の QOLを損な う最大の原因となっている。また、これらの疾患は加齢とともに発症頻度が急増し且 つ慢性に経過することから、国民医療経済にも重大な負担を課すこととなり、高齢化 社会においては社会全体で克服すべき重要な課題である。世界保健機関（WHO) も 21世紀の最初の 10年を "骨と関節の 10年（the Bone and Joint Decade; BJD)"と位 置付けて骨 ·関節疾患の制圧に乗り出してレ、る。  [0002] Lifestyle-related diseases of bones and joints have little direct effect on life, but daily life movement (ADL) is hindered due to pain and gait disturbance. It is the biggest cause of damage. In addition, since the incidence of these diseases increases rapidly with age and continues to be chronic, it imposes a significant burden on the national health care economy. In an aging society, it is an important issue to be overcome by society as a whole. It is. The World Health Organization (WHO) has also taken the first decade of the 21st century as the “Bone and Joint Decade (BJD),” and has embarked on the suppression of bone and joint diseases.
[0003] 変形性関節症 (osteoarthritis；以下、「OA」とも!/、う）は慢性の関節炎を伴う骨 ·関節 疾患で、軟骨の退行変性により軟骨の破壊と骨や軟骨の増殖性変化をきたす病気 である。その患者数は日本だけでも 500万〜 700万人にのぼり、 60歳以上では、 80%以 上が膝 *肘*股関節や脊椎に変形性関節症の症状を呈すると言われている、代表的 な common diseaseである。現在のところ、 OAの根本的な治療手段はなぐ非ステロイ ド性消炎鎮痛剤やヒアルロン酸、ステロイド剤の投与など、痛みを抑制する対症療法 が中心となっている。進行した場合、関節鏡視下手術 ·骨切り術 ·人工関節置換など の手術適応となるが、人工関節には寿命があるため 55歳位まではこの手術を避ける ことが望ましいと考えられている。従って、関節の退行性変化を抑制する治療方法の 開発が望まれている。  [0003] Osteoarthritis (hereinafter also referred to as "OA"! /) Is a bone / joint disease with chronic arthritis. Degeneration of cartilage causes destruction of cartilage and proliferative changes in bone and cartilage. It is a sick disease. The number of patients is 5 to 7 million in Japan alone, and it is said that more than 80% of people over 60 years old have symptoms of osteoarthritis in the knee * elbow * hip joint and spine. It is a common disease. At present, symptomatic treatment that suppresses pain, such as administration of non-steroidal anti-inflammatory analgesics, hyaluronic acid, and steroids, is the main treatment method for OA. When advanced, surgical indications such as arthroscopic surgery, osteotomy, and artificial joint replacement are applicable. However, it is considered desirable to avoid this surgery until about 55 years old because the artificial joint has a life span. . Therefore, development of treatment methods that suppress degenerative changes in joints is desired.
[0004] カルモジュリン (以下、「CaM」ともいう）と呼ばれる細胞内カルシウム結合タンパク質 は、軟骨細胞を含む種々の組織でュビキタスに発現している。細胞内カルシウム濃 度の上昇が軟骨細胞分化を促進することが知られている（非特許文献 1)。主要な軟 骨基質の 1つであるァグリカンをコードする遺伝子の発現は力学的刺激によって上昇 する力 この発現は CaM阻害剤の存在下で抑制されることが報告されている（非特許 文献 2)。これらの知見に基づいて、 CaMがカルシニューリンや CaM依存性キナーゼ II を介してァグリカン遺伝子の転写を活性化するとのモデルが提唱されている。 [0004] An intracellular calcium binding protein called calmodulin (hereinafter also referred to as "CaM") is ubiquitously expressed in various tissues including chondrocytes. It is known that an increase in intracellular calcium concentration promotes chondrocyte differentiation (Non-patent Document 1). Major soft It has been reported that the expression of a gene encoding aggrecan, one of the bone substrates, is increased by mechanical stimulation. This expression is suppressed in the presence of a CaM inhibitor (Non-patent Document 2). Based on these findings, a model has been proposed that CaM activates transcription of the aggrecan gene via calcineurin and CaM-dependent kinase II.
[0005] 本発明者らは、ゲノムワイド連鎖解析の結果から、 CaMをコードする遺伝子の 1つで ある CALM1遺伝子における多型が OAと相関することを見出し、 OA感受性の SNPお よびノ、プロタイプを同定するとともに、 OA軟骨における該遺伝子の発現が正常軟骨 のそれに比して高いこと、 CALM1遺伝子のプロモーター領域における OA感受性ァ レルが該遺伝子の転写活性を低下させること等を見出して、 CaM経路の異常が OA の発症や進展に深く関与している可能性を提唱している（特許文献 1、非特許文献 3 )。 [0005] From the results of genome-wide linkage analysis, the present inventors have found that a polymorphism in the CALM1 gene, which is one of the genes encoding CaM, is correlated with OA. As well as identifying the type, we found that the expression of the gene in OA cartilage is higher than that in normal cartilage, and that the OA-sensitive allele in the promoter region of the CALM1 gene decreases the transcription activity of the gene. Proposed the possibility that abnormalities in the pathway are deeply involved in the onset and progression of OA (Patent Document 1, Non-Patent Document 3).
特許文献 1：国際公開第 2006/014013号パンフレット  Patent Document 1: International Publication No. 2006/014013 Pamphlet
非特許文献 1 :富田（Tomita)ら著， 「ジャーナル'ォヴ 'バイオロジカル 'ケミストリー (J • Biol. Chem.)」，（米国)， 2002年，第 277巻， . 42214-42218  Non-Patent Document 1: Tomita et al., “Journal 'of' Biological 'Chemistry (J • Biol. Chem.)” (USA), 2002, Vol. 277,. 42214-42218
非特許文献 2 :ヴァルームおよびレイァ (Valhmu and Raia)著，「バイオケミカル 'ジャー ナル (Biochem. J.)j , 2002年，第 361巻， . 689-696  Non-Patent Document 2: “Valhmu and Raia”, “Biochemical. Journal”, 2002, Vol. 361,. 689-696
非特許文献 3 :本谷 (Mototani)ら著， 「ヒューマン 'モレキュラー'ジエネテイクス（Hum. Mol. Genet.)j , (英国)， 2005年，第 14巻， p. 1009-1017  Non-Patent Document 3: Mototani et al., "Human 'Molecular' Genetics (Hum. Mol. Genet.) J, (UK), 2005, Vol. 14, p. 1009-1017
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0006] 本発明の目的は、変形性関節症をはじめとする骨 ·関節疾患の発症 ·進行に関与 する遺伝子を同定し、その機能を解明することにより、当該疾患の新規且つ根本的な 予防 ·治療手段を提供することである。また、本発明の別の目的は、骨'関節疾患に 対する遺伝的感受性の簡便且つ高確度な診断方法を提供することである。  [0006] The purpose of the present invention is to identify genes involved in the onset / progression of bone / joint diseases including osteoarthritis and elucidate their functions, thereby providing novel and fundamental prevention of the diseases. · To provide a means of treatment. Another object of the present invention is to provide a simple and highly accurate diagnostic method for genetic susceptibility to bone and joint diseases.
課題を解決するための手段  Means for solving the problem
[0007] 本発明者らは、 CaMをコードする遺伝子には CALM1以外に CALM2および CALM3 があり、 CaM遺伝子ファミリーを形成していることに着目し、軟骨組織での各遺伝子の 発現量を調べたところ、 CALM2が最も発現量が大きいことが明らかとなった。さらに、 CALM2遺伝子の発現が変形性股関節症（Hip Osteoarthritis ;以下、「HOA」ともいう) および変形性膝関節症（Knee Osteoarthritis；以下、「KOA」ともいう）軟骨において 上昇していることを見出した。そこで、本発明者らは、 CALM2が OA感受性遺伝子で あるか否かを検討するため、 CALM2遺伝子のプロモータ領域、イントロン 1並びに全 ェクソンおよびそれらのフランキング領域に対して多型ディスカバリーを行い、得られ た多型について相関解析を行った。その結果、 HOA群と対照群との相関解析では有 意な相関は認められなかった力 HOA群を臼蓋形成不全症（Acetabular dysplasia; 以下、「AD」ともいう）の有無で分割した場合、 ADを伴わない HOA群で、有意な相関 を示す多型およびノヽプロタイプを同定することに成功した。 [0007] The present inventors examined the expression level of each gene in cartilage tissue, focusing on the fact that CALM2 and CALM3 other than CALM1 are the genes encoding CaM, and that they form the CaM gene family. However, CALM2 was found to have the highest expression level. further, We found that CALM2 gene expression was elevated in osteoarthritis (Hip Osteoarthritis; also referred to as “HOA”) and knee osteoarthritis (hereinafter referred to as “KOA”) cartilage. Therefore, the present inventors conducted polymorphic discovery on the promoter region, intron 1 and all exons and their flanking regions of the CALM2 gene in order to investigate whether CALM2 is an OA susceptibility gene. Correlation analysis was performed on the polymorphisms. As a result, when the HOA group was divided by the presence or absence of acetabular dysplasia (hereinafter referred to as “AD”), there was no significant correlation in the correlation analysis between the HOA group and the control group. In the HOA group without AD, we successfully identified polymorphisms and nodal protypes that showed significant correlation.
本発明者らは、これらの知見に基づいてさらに検討を重ねた結果、本発明を完成 するに至った。  As a result of further studies based on these findings, the present inventors have completed the present invention.
すなわち、本発明は、  That is, the present invention
[1]配列番号： 1で表される塩基配列中、塩基番号 5227で示される塩基（但し、該塩 基はアデニンである）もしくは塩基番号 5606で示される塩基（但し、該塩基はグァニ ンである）を含む該塩基配列の部分配列であって、約 15塩基以上の連続した塩基配 列を含む核酸；  [1] In the base sequence represented by SEQ ID NO: 1, the base represented by base number 5227 (provided that the base is adenine) or the base represented by base number 5606 (provided that the base is guanine) A nucleic acid comprising a contiguous base sequence of about 15 bases or more;
[2]配列番号： 1で表される塩基配列において、塩基番号 21、 3967、 5227、 6210、 712 1、 7791および 16127で示される塩基がそれぞれシトシン、グァニン、アデニン、アデ ニン、アデニン、チミンおよびアデニンであり、塩基番号 6913〜6914で示される塩基 が欠失して!/、るハプロタイプの塩基配列を含む核酸；  [2] In the base sequence represented by SEQ ID NO: 1, the bases represented by base numbers 21, 3967, 5227, 6210, 712 1, 7791 and 16127 are cytosine, guanine, adenine, adenine, adenine, thymine and A nucleic acid comprising a base sequence of a haplotype which is adenine and lacks a base represented by base numbers 6913-6914;
[3]配列番号： 1で表される塩基配列において、塩基番号 5227で示される塩基および /または塩基番号 5606で示される塩基における多型を検出することを含む、骨 ·関節 疾患に対する遺伝的感受性の診断方法；  [3] Genetic susceptibility to bone / joint diseases including detection of a polymorphism in the base represented by base number 5227 and / or the base represented by base number 5606 in the base sequence represented by SEQ ID NO: 1. Method of diagnosis;
[4]配列番号： 1で表される塩基配列において、塩基番号 21、 3967、 6210、 6913〜69 14、 7121、 7791および 16127で示される塩基もしくは塩基配列からなる群より選択され る 1以上の塩基もしくは塩基配列における多型を検出することを含む、骨 ·関節疾患 に対する遺伝的感受性の診断方法；  [4] In the base sequence represented by SEQ ID NO: 1, one or more selected from the group consisting of bases or base sequences represented by base numbers 21, 3967, 6210, 6913-6914, 7121, 7791 and 16127 A method of diagnosing genetic susceptibility to bone / joint disease, comprising detecting a polymorphism in a base or base sequence;
[5]骨 ·関節疾患が、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎、滑膜炎 、代謝性関節症、スポーツによる関節障害、骨軟骨腫、骨腫瘍、軟骨腫瘍および先 天性骨系統疾患からなる群より選択される上記 [3]または [4]記載の方法； [5] Bone and joint diseases are osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis The method according to the above [3] or [4], selected from the group consisting of metabolic arthropathy, sports joint damage, osteochondroma, bone tumor, cartilage tumor, and congenital bone disease;
[6]配列番号： 1で表される CALM2遺伝子の塩基配列中、塩基番号 5227で示される 塩基がグァニンである、および/または塩基番号 5606で示される塩基がグァニンで あるアレルを保有するヒトに投与することを特徴とする、カルシウム/カルモジュリン経 路を活性化する物質を含有してなる骨 ·関節疾患予防 ·治療剤；  [6] In a human having an allele in which the base represented by base number 5227 is guanine and / or the base represented by base number 5606 is guanine in the base sequence of CALM2 gene represented by SEQ ID NO: 1. A bone / joint disease prophylactic / therapeutic agent comprising a substance that activates the calcium / calmodulin pathway, characterized by administration;
[7]物質がカルモジュリン、カルシニューリンおよびカルモジュリン依存性キナーゼ II 力、らなる群より選択される 1以上のタンパク質の発現および/または活性を促進する ものである、上記 [6]記載の剤；並びに  [7] The agent described in [6] above, wherein the substance promotes the expression and / or activity of one or more proteins selected from the group consisting of calmodulin, calcineurin and calmodulin-dependent kinase II force; and
[8]骨 ·関節疾患が、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎、滑膜炎 、代謝性関節症、スポーツによる関節障害および先天性骨系統疾患からなる群より 選択される、上記 [6]記載の剤；  [8] The above-mentioned bone / joint disease is selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, arthropathy due to sports, and congenital bone disease [6] The agent described in;
などを提供する。  Etc.
発明の効果  The invention's effect
[0009] CALM2遺伝子における多型が骨 ·関節疾患と相関することから、該多型は骨 ·関節 疾患に対する遺伝的感受性の簡便な判定に利用することができる。また、該遺伝子 における感受性多型を有するヒトにおいて、 Ca/CaM経路を活性化することにより、骨 •関節疾患、特に軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能低下が関連 する疾患に対して予防 ·治療効果が得られうる。  [0009] Since the polymorphism in the CALM2 gene correlates with bone / joint diseases, the polymorphism can be used for simple determination of genetic susceptibility to bone / joint diseases. In addition, by activating the Ca / CaM pathway in humans with a susceptibility polymorphism in the gene, bone / joint diseases, particularly diseases related to degeneration / disappearance of cartilage matrix, reduced production ability, and reduced chondrocyte differentiation ability Prophylactic and therapeutic effects can be obtained.
図面の簡単な説明  Brief Description of Drawings
[0010] [図 1]軟骨細胞におけるカルモジュリン遺伝子の発現量の比較を示す図である。ヒト 軟骨細胞におけるカルモジュリン遺伝子（CALMl, CALM2, CALM3)の発現量を定 量的リアルタイム PCR法にて解析した (n=3)。  [0010] FIG. 1 is a diagram showing a comparison of calmodulin gene expression levels in chondrocytes. The expression level of calmodulin gene (CALMl, CALM2, CALM3) in human chondrocytes was analyzed by quantitative real-time PCR (n = 3).
[図 2]マイクロアレイによる関節軟骨におけるカルモジュリン遺伝子の発現を示す図で ある。 A)ヒト股関節正常軟骨 (n=l)および股関節 OA軟骨 (n=5)、 B)膝関節正常軟骨（ n=2)および膝関節 OA軟骨（n=4)におけるカルモジュリン遺伝子の発現量をマイクロ アレイ（GeneChip)を用いて解析した。両関節軟骨において、 CALM2量が最も高ぐ かつ OA軟骨で発現上昇して!/、る。 [図 3]CALM2遺伝子領域における多型ディスカバリーの結果を示す図である。 CALM 2遺伝子領域のゲノム構造と、 日本人 48人のゲノム DNAを用いて多型ディスカバリー を行った。ディスカバリー領域は CALM2遺伝子の全てのェクソンおよびそのフランキ ング領域、プロモータ領域、イントロン 1である。 FIG. 2 is a diagram showing the expression of calmodulin gene in articular cartilage by microarray. A) Micro-expression of calmodulin gene in human hip normal cartilage (n = l) and hip joint OA cartilage (n = 5), B) knee joint normal cartilage (n = 2) and knee joint OA cartilage (n = 4) Analysis was performed using an array (GeneChip). In both articular cartilages, the CALM2 level is the highest and the expression is increased in OA cartilage! FIG. 3 shows the results of polymorphism discovery in the CALM2 gene region. Polymorphism discovery was performed using the genomic structure of the CALM 2 gene region and genomic DNA of 48 Japanese. The discovery region is all exons of the CALM2 gene and its flanking region, promoter region, and intron 1.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0011] 本発明の骨 ·関節疾患に対する遺伝的感受性の診断方法（以下、単に「本発明の 診断方法」という場合がある）は、被験者の CALM2遺伝子における多型および/また はそれと連鎖不平衡状態にある多型を検出することを特徴とする。 [0011] The method for diagnosing genetic susceptibility to bone / joint disease of the present invention (hereinafter sometimes simply referred to as "diagnostic method of the present invention") is a polymorphism in the subject's CALM2 gene and / or linkage disequilibrium with it. It is characterized by detecting a polymorphism in a state.
本明細書にお!/、て「（遺伝子）多型」とは、ゲノム DNA上の 1または複数の塩基の変 化（置換、欠失、揷入、転位、逆位等）であって、その変化が集団内に 1 %以上の頻 度で存在するものをいい、例えば、 1個の塩基が他の塩基に置換されたもの（SNP)、 1〜数十塩基が欠失もしくは揷入されたもの（DIP)、 2〜数十塩基を 1単位とする配列 が繰り返し存在する部位にお!/、てその繰り返し回数が異なるもの（繰り返し単位が 2 〜4塩基のものをマイクロサテライト多型、数〜数十塩基のものを variable number of t andem repeat (VNTR)という）等が挙げられる。本発明の診断方法に利用することがで きる多型は、上記の条件を満たす限りいずれのタイプのものであってもよいが、好まし くは SNPである。  In this specification, “/ gene” polymorphism is a change of one or more bases (substitution, deletion, insertion, transposition, inversion, etc.) on genomic DNA, This refers to changes that occur at a frequency of 1% or more in the population. For example, one base is replaced with another base (SNP), 1 to several tens of bases are deleted or inserted. (DIP), where the sequence with 2 to several tens of bases as one unit is repeatedly present! /, With a different number of repetitions (repeating units with 2 to 4 bases are microsatellite polymorphisms, (A variable number of t andem repeat (VNTR)). The polymorphism that can be used in the diagnostic method of the present invention may be any type as long as the above conditions are satisfied, but is preferably SNP.
[0012] 本発明の診断方法において利用することができる多型（以下、「骨'関節疾患マー カー多型」という場合もある）は、  [0012] Polymorphisms that can be used in the diagnostic method of the present invention (hereinafter sometimes referred to as "bone 'joint disease marker polymorphism") are:
(1)配列番号： 1で表される塩基配列において、塩基番号 5227で示される塩基および /または塩基番号 5606で示される塩基における多型、あるいは該多型と連鎖不平衡 係数 D 'が 0.9以上の連鎖不平衡状態にある、好ましくは完全連鎖状態にある（即ち、 D ' =l)、該遺伝子内および/またはその周辺領域内に存在する多型であって、 (1) In the base sequence represented by SEQ ID NO: 1, the polymorphism in the base represented by base number 5227 and / or the base represented by base number 5606, or the polymorphism and linkage disequilibrium coefficient D ′ is 0.9 or more A polymorphism present in the gene and / or its surrounding region, preferably in a linkage disequilibrium state, preferably in a complete linkage state (ie D ′ = l),
(2)その一方のアレル頻度が、任意の骨 ·関節疾患非罹患者集団におけるよりも任意 の骨 ·関節疾患患者集団にぉレ、て有意に高!、ものであれば特に制限されなレ、。 (2) If the frequency of one allele is significantly higher in any bone / joint disease patient population than in any bone / joint disease non-affected population, it is not particularly limited. ,.
[0013] 配列番号： 1で表される塩基配列において、塩基番号 5227で示される塩基および 塩基番号 5606で示される塩基における多型は、本発明において見出された新規 SNP sであり、従来公知のアレルでは塩基番号 5227で示される塩基はグァニン、塩基番号 5606で示される塩基はアデニンであるのに対し、新たに見出されたアレルでは、塩基 番号 5227で示される塩基はアデニン、塩基番号 5606で示される塩基はグァニンであ る。これらの新規アレルはいずれもマイナーアレルである。尚、これらの塩基は、 Gen Bank accession No. AC073283 (VERSION: AC073283.8, GI: 17432471， 2005年 4月 30日更新)」（本明細書においては、単に「AC073283.8」と略記する場合もある）で表 される塩基配列（相補鎖配列）中、それぞれ塩基番号 117496および 117117で示され る塩基に相当する。本明細書では、 NCBI Nucleotide (GenBank)データベース上の 情報更新による配列不一致の混乱を避けるために、 AC073283.8で表される塩基配 列中、塩基番号 103843〜122722に相当する部分配列（相補鎖配歹 IJ)を配列番号： 1 に示した。これにより、将来、万一 AC073283.8の配列情報が参照不能となった場合 でも、 AC073283.8における塩基番号（N )を配列番号： 1で表される塩基番号（N = 12 [0013] In the base sequence represented by SEQ ID NO: 1, the polymorphism in the base represented by base number 5227 and the base represented by base number 5606 is a novel SNP s found in the present invention, and is conventionally known In the allele, the base shown by base number 5227 is guanine, base number While the base represented by 5606 is adenine, in the newly discovered allele, the base represented by base number 5227 is adenine and the base represented by base number 5606 is guanine. These new alleles are all minor alleles. These bases are referred to as “Gen Bank accession No. AC073283 (VERSION: AC073283.8, GI: 17432471, updated April 30, 2005)” (in this specification, simply abbreviated as “AC073283.8”). In the base sequence (complementary strand sequence) represented by the base number 117496 and 117117, respectively. In this specification, in order to avoid the confusion of sequence mismatch due to information update on the NCBI Nucleotide (GenBank) database, the partial sequence (complementary strand) corresponding to base numbers 103843 to 122722 in the base sequence represented by AC073283.8 The distribution IJ) is shown in SEQ ID NO: 1. As a result, even if the sequence information of AC073283.8 cannot be referred to in the future, the base number (N) in AC073283.8 is changed to the base number represented by SEQ ID NO: 1 (N = 12
0  0
2723-N )に換算することにより、本発明の実施に必要な配列情報の記載は担保され 2723-N), the description of the sequence information necessary for carrying out the present invention is guaranteed.
0 0
る。以下、本明細書において、多型部位等のヌクレオチド (塩基）の位置はすべて AC 073283.8の塩基番号により表記する。 The Hereinafter, in this specification, the positions of nucleotides (bases) such as polymorphic sites are all represented by the base number of AC 073283.8.
好ましい本発明の骨.関節疾患マーカー多型として、 GenBank accession No. AC07 3283.8で表される塩基配列（相補鎖配列）中、塩基番号 117496で示される塩基にお ける SNP[「117496G〉A」と略記する場合がある；以下、本明細書中で同様の表記があ る場合、「"AC073283.8における塩基番号""メジャーアレルの塩基"〉"マイナーァレ ルの塩基"」を意味する。アレルの塩基の表記力 の場合は欠失を意味し、 "N(N)N "(Nは任意の塩基)は揷入を意味する。 ]および塩基番号 117117で示される塩基にお ける SNP (117117A〉G)が挙げられる。後記実施例 5に示される通り、ケース—コント口 ール解析の結果、 117496G〉Aではメジャーアレル（Gアレル）、 117117A〉Gではマイ ナーアレル (Gアレル）のアレル頻度力 OA非罹患者集団におけるそれよりも ADを伴 わない HOA患者集団において有意に高いことが明らかとなった。即ち、 117496Gァレ ルおよび 117117Gアレルは、 ADを伴わない HOAの感受性（易罹患性）アレルである。  As a preferable bone and joint disease marker polymorphism of the present invention, an SNP [“117496G> A” in the base represented by base number 117496 in the base sequence (complementary chain sequence) represented by GenBank accession No. AC07 3283.8 Hereinafter, if there is a similar notation in this specification, it means "" base number in AC073283.8 "" base of major allele ">" base of minor allele ". In the case of allele base notation, it means deletion, and "N (N) N" (N is any base) means insertion. And SNP (117117A> G) in the base represented by base number 117117. As shown in Example 5 below, as a result of case-control analysis, 117496G> A has a major allele (G allele), and 117117A> G has a minor allele (G allele) allele frequency. It was found to be significantly higher in the HOA patient population without AD. That is, the 117496G and 117117G alleles are HOA sensitive (susceptible) alleles without AD.
ADの患者では、軟骨に過剰な負荷力 Sかかるために二次的に HOAを発症する可能性 が示唆されているので、上記多型は特別な原因が特定できない一次性の HOAの遺 伝的感受性を診断するのに有効である。 [0015] また、カルモジュリン（CaM)が軟骨基質合成 ·軟骨細胞分化を促進する機能を有す ることを考慮すれば、 117496Gアレルおよび 117117Gアレルは、 OA (特に一次性 OA) だけでなぐ軟骨基質の変性 '消失'産生能低下、軟骨細胞分化能低下が関連する 骨 ·関節疾患、例えば、骨粗鬆症、慢性関節リウマチ、関節炎、滑膜炎、代謝性関節 症、スポーツによる関節障害、先天性骨系統疾患 [例えば、軟骨形成の低下している 骨系統疾患 ·変形性関節症を合併する先天性骨系統疾患 (例:軟骨無形成症、多発 性骨端異形成症、脊椎骨端異形成症、骨幹端異形成症、 Stickler症候群、偽性軟骨 無形成症等）]などの疾患に対しても感受性を示すことが強く示唆される。一方、軟骨 基質の産生能 '軟骨細胞分化能の異常亢進が関連する疾患、例えば、先天性骨系 統疾患 [例えば、軟骨形成の亢進している骨系統疾患 (例：多発性外骨腫、片側肥 大、オリエール病、マフツチ症候群等）]、骨軟骨腫、骨腫瘍、軟骨腫瘍などの疾患に 対しては、 117496Gアレルおよび 117117Gアレルは保護的であり得る。 In AD patients, it has been suggested that HOA may develop secondarily due to the excessive loading force on cartilage S, so the above polymorphisms are inherited from primary HOA where no specific cause can be identified. It is effective for diagnosing sensitivity. [0015] Considering that calmodulin (CaM) has a function of promoting cartilage matrix synthesis and chondrocyte differentiation, the 117496G allele and 117117G allele are not only OA (particularly primary OA) but cartilage matrix. Bone and joint diseases associated with reduced 'disappearance' production ability and chondrocyte differentiation ability, such as osteoporosis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders, congenital bone system Diseases [eg, bone system diseases with reduced cartilage formation · congenital bone system diseases complicated with osteoarthritis (e.g., achondroplasia, multiple epiphyseal dysplasia, vertebral epidysplasia, diaphysis) It is strongly suggested that it is sensitive to diseases such as acrodysplasia, Stickler syndrome, pseudochondral dysplasia, etc.). On the other hand, cartilage matrix production ability 'Diseases associated with abnormally enhanced chondrocyte differentiation ability, such as congenital bone system diseases [eg, bone system diseases with increased cartilage formation (eg, multiple exostoses, unilateral) 117496G allele and 117117G allele may be protective against diseases such as hypertrophy, Oriere disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor.
[0016] GenBank accession No. AC073283.8で表される塩基配列（相補鎖配列）中、塩基 番号 117496または 117117で示される塩基における多型と、連鎖不平衡係数 D 'が 0.9 以上の連鎖不平衡状態にある多型のうち、一方のアレル頻度が任意の骨 ·関節疾患 非罹患者集団におけるよりも任意の骨 ·関節疾患患者集団において有意に高いもの も、本発明の診断方法に利用することができる。ここで「連鎖不平衡係数 D '」は、 2つ の SNPについて第一の SNPの各アレルを（A, a)、第二の SNPの各アレルを（B， b)とし 、 4つのハプロタイプ（AB， Ab， aB， ab)の各頻度を P , P , P , P とすると、下記式  [0016] In the nucleotide sequence (complementary strand sequence) represented by GenBank accession No. AC073283.8, the polymorphism at the base represented by base number 117496 or 117117, and the linkage disequilibrium with linkage disequilibrium coefficient D 'of 0.9 or more Among the polymorphisms in the state, one whose allele frequency is significantly higher in any bone / joint disease patient population than in any bone / joint disease non-affected population should also be used in the diagnostic method of the present invention. Can do. Here, the “linkage disequilibrium coefficient D ′” means that for two SNPs, each allele of the first SNP is (A, a), each allele of the second SNP is (B, b), and four haplotypes ( If each frequency of AB, Ab, aB, ab) is P, P, P, P, then
AB Ab aB ab  AB Ab aB ab
により得られる。  Is obtained.
D ' =(P P — P P )/Min [(P + P )(P + P )，（P + P )(P + P )]  D '= (P P — P P) / Min [(P + P) (P + P), (P + P) (P + P)]
AB ab Ab aB AB aB aB ab AB Ab Ab ab  AB ab Ab aB AB aB aB ab AB Ab Ab ab
[式中、 Min [(P + P )(P + P )，（P + P )(P + P )]は、（P + P )(P + P )と（  [Where, [Min [(P + P) (P + P), (P + P) (P + P)]] is (P + P) (P + P) and (
AB aB aB ab AB Ab Ab ab AB aB aB ab AB aB aB ab AB Ab Ab ab AB aB aB ab
P + P )(P + P )とのうち、値の小さい方をとることを意味する。 ] It means taking the smaller one of P + P) and (P + P). ]
AB Ab Ab ab  AB Ab Ab ab
[0017] 骨 ·関節疾患患者集団および骨 ·関節疾患非罹患者集団は、それぞれ統計学上信 頼し得る結果を与えるに十分な数からなる集団であれば、その大きさ（サンプル数）、 各サンプルの背景 (例えば、出身地、年齢、性別、疾患等）などに特に制限はない。 骨-関節疾患としては、例えば、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節 炎、滑膜炎、代謝性関節症、スポーツによる関節障害、骨軟骨腫、骨腫瘍、軟骨腫 瘍、先天性骨系統疾患等が挙げられるが、好ましくは骨粗鬆症、変形性関節症、慢 性関節リウマチ、関節炎、滑膜炎、代謝性関節症、スポーツによる関節障害、軟骨基 質の変性 ·消失 ·産生能低下、軟骨細胞分化能低下が関連する先天性骨系統疾患 等であり、より好ましくは変形性関節症 (OA)、いっそう好ましくは変形性股関節症 (股 関節 OA、 HOA)もしくは変形性膝関節症 (膝関節 OA、 KOA)、特に好ましくは HOA、 最も好ましくは臼蓋形成不全症（AD)を伴わない HOAである。また、倫理上、試料提 供者のインフォームド 'コンセントを必要とすることから、骨 ·関節疾患非罹患者集団と しては、通常、ある医療機関における骨 ·関節疾患以外の患者群、あるいはある地域 における集団検診におレ、て骨 ·関節疾患に罹患してレ、なレ、と診断された被験者群な どが好ましく用いられる。 [0017] If the bone / joint disease patient population and the bone / joint disease non-affected population are each composed of sufficient numbers to give statistically reliable results, the size (number of samples), There are no particular restrictions on the background of each sample (eg, birthplace, age, gender, disease, etc.). Examples of bone-joint diseases include osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders, osteochondroma, bone tumor, chondroma Ulcers, congenital bone system diseases, etc., but preferably osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, arthropathy due to sports, cartilage substrate degeneration / disappearance Congenital bone system diseases related to decreased production ability and chondrocyte differentiation ability, etc., more preferably osteoarthritis (OA), more preferably osteoarthritis of the hip (hip joint OA, HOA) or deformability Knee arthropathy (knee joint OA, KOA), particularly preferably HOA, most preferably HOA without acetabular dysplasia (AD). In addition, because ethics require the informed consent of the sample provider, the non-affected group of bone / joint diseases is usually a group of patients other than bone / joint diseases at a medical institution, Or, a group of subjects diagnosed as having a bone or joint disease in a mass screening in a certain region and being diagnosed as having a bone or joint disease are preferably used.
後記実施例 3に示される通り、多型ディスカバリーの結果、 CALM2遺伝子の全エタ ソンおよびそれらのフランキング領域、プロモータ領域並びにイントロン 1内には、 NC Bl SNPデータベース（http：〃 www.ncbi.nlm.nih.gov/SNP/)に登録された 8つの公知 多型（rsl353644、 rs878665、 rsl723484、 rs815815、 rs815816、 rsl7036325、 rsl02747 8および rs2454084)の他、上記 2つの骨.関節疾患マーカー多型（11496G〉Aおよび 1 17117A〉G)とともに、下記 8つの新規多型 [GenBank accession No. AC073283.8で表 される塩基配列（相補鎖配列）中、塩基番号 120456で示される塩基における SNP (T/ C)、塩基番号 118756で示される塩基における SNP (C/G)、塩基番号 115958で示さ れる塩基における SNP (C/T)、塩基番号 115084で示される塩基における SNP (G/A) 、塩基番号 114307で示される塩基における SNP (G/A)、塩基番号 106372で示される 塩基における SNP (G/A)、塩基番号 115810〜115809で示される塩基配列における 欠失（TG/—)および塩基番号 115303〜115302で示される塩基配列における揷入（G A/G[C]A) ]が見出された (表 2を参照）。これらの公知および新規多型のうち、マイナ 一アレル頻度（MAF)の比較的高い 8個の多型〔5個の公知多型 [ReiSNP ID: rsl3536 44 (122702C〉T)、 rsl723484 (116513A〉G)、 rs815815 (115602A〉G)、 rs815816 (1149 32T〉C)および rsl7036325 (106596A〉G) ]並びに 3個の新規多型（118756G〉C、 1174 960八ぉょび115810〜115809—〉下0〕を用ぃてハプロタィプ構造を推定した結果、 実施例 7に示される通り、 5つのコモンハプロタイプ (ノヽプロタイプ頻度の合計で全体 の約 95%を占める）が同定され（表 6参照）、そのうちハプロタイプ III (122702C, 11875 6G， 117496A, 116513A, 115810〜115809—， 115602A, 114932T, 106596A)が ADを 伴わない HOAと有意な相関を示し、コントロール（非 HOA群）に比べてその頻度は低 かった。ハプロタイプ IIIは、上記 5つのハプロタイプの中で、唯一上記骨'関節疾患マ 一力一多型の非感受性アレルである 117496Aアレルを含む。従って、本ハプロタイプ は、 OA (特に一次性 OA)をはじめとして、軟骨基質の変性 ·消失 ·産生能低下、軟骨 細胞分化能低下が関連する骨 ·関節疾患、例えば、骨粗鬆症、慢性関節リウマチ、 関節炎、滑膜炎、代謝性関節症、スポーツによる関節障害、先天性骨系統疾患 [例 えば、軟骨形成の低下している骨系統疾患 ·変形性関節症を合併する先天性骨系 統疾患 (例:軟骨無形成症、多発性骨端異形成症、脊椎骨端異形成症、骨幹端異 形成症、 Stickler症候群、偽性軟骨無形成症等）]などの疾患に対して保護的であり 得る。 As shown in Example 3 below, as a result of the polymorphism discovery, the NCBL SNP database (http: 〃 www.ncbi.nlm) contains all the CALM2 gene etason and their flanking region, promoter region, and intron 1. In addition to the eight known polymorphisms (rsl353644, rs878665, rsl723484, rs815815, rs815816, rsl7036325, rsl027478 and rs2454084) registered in .nih.gov / SNP /), the above two bone and joint disease marker polymorphisms (11496G 〉 A and 1 17117A〉 G) together with the following eight new polymorphisms [GenBank accession No. AC073283.8 represented by the base sequence (complementary strand sequence) SNP (T / C) at the base shown by base number 120456 ), SNP (C / G) at the base represented by base number 118756, SNP (C / T) at the base represented by base number 115958, SNP (G / A) at the base represented by base number 115084, base number 114307 SNP (G / A) at the base indicated by SNP (G / A) in the base represented by 106372, deletion (TG /-) in the base sequence represented by base numbers 115810 to 115809, and insertion in the base sequence represented by base numbers 115303 to 115302 (GA / G [ C] A)] was found (see Table 2). Of these known and novel polymorphisms, 8 polymorphisms with relatively high minor allele frequency (MAF) [5 known polymorphisms [ReiSNP ID: rsl3536 44 (122702C> T), rsl723484 (116513A> G) ), Rs815815 (115602A> G), rs815816 (1149 32T> C) and rsl7036325 (106596A> G)] and three new polymorphs (118756G> C, 1174 960 Hachijobi 115810-115809-> below 0) As a result of estimating the haplotype structure using, as shown in Example 7, the five common haplotypes (See Table 6), of which haplotype III (122702C, 11875 6G, 117496A, 116513A, 115810-115809—, 115602A, 114932T, 106596A) is significantly correlated with HOA without AD The frequency was low compared to the control (non-HOA group). Haplotype III includes the 117496A allele, which is the only insensitive allele of the above-mentioned bone 'joint disease, among the five haplotypes. Therefore, this haplotype is not only OA (particularly primary OA), but also bone and joint diseases related to degeneration / disappearance / decreased ability to produce cartilage and decreased ability to differentiate chondrocytes, such as osteoporosis, rheumatoid arthritis, arthritis. , Synovitis, metabolic arthropathy, sports joint disorders, congenital bone system diseases [eg bone system diseases with reduced cartilage formation · congenital bone system diseases complicated with osteoarthritis (eg : Achondroplasia, multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)].
[0019] また、ハプロタイプ IIIにおける他の 7つの多型は、いずれも 117496Aアレルと完全連 鎖の状態にある。そのため、これらの多型部位においてハプロタイプ IIIと異なるァレ ルが検出されれば、ハプロタイプ III以外のハプロタイプであり、従って、本発明の感 受性アレルの 1つである 117496Gを有する蓋然性が極めて高い。例えば、 122702Tァ レルはハプロタイプ IIのみに、 118756Cアレルはハプロタイプ Vのみに、 106596Gァレ ルはハプロタイプ IVのみにそれぞれ存在し、 116513Gアレル、 115810〜115809TGァ レル、 115602Gアレルおよび 114932Cアレルはハプロタイプ IIもしくは IVに存在するこ とから、これらのアレルを検出することによつても、骨'関節疾患に対する遺伝的感受 性を高確度に判定することが可能である。  [0019] In addition, the other seven polymorphisms in haplotype III are all in a completely linked state with the 117496A allele. Therefore, if an allele that is different from haplotype III is detected in these polymorphic sites, it is a haplotype other than haplotype III, and therefore has a very high probability of having 117496G, which is one of the susceptibility alleles of the present invention. . For example, 122702T allele exists only in haplotype II, 118756C allele only in haplotype V, 106596G allele only in haplotype IV, 116513G allele, 115810-115809TG allele, 115602G allele and 114932C allele haplotype II or Since these alleles are present in IV, genetic susceptibility to bone and joint diseases can be determined with high accuracy by detecting these alleles.
本発明の診断方法において検出される多型は、上記した多型のうちのいずれか 1 つであってもよいし、 2つ以上であってもよい。後記実施例においてハプロタイプ構造 の推定に用いた 8個の多型のうち 117496G〉A以外の多型を検出する場合は、ハプロ タイプ III以外のハプロタイプであると同定されるまで順次行ってもよい。但し、ハプロ タイプ Iとハプロタイプ IIIを区別するためには、 117496G〉Aの多型を検出する必要が ある。  The polymorphism detected in the diagnostic method of the present invention may be any one of the polymorphisms described above, or may be two or more. When detecting polymorphisms other than 117496G> A among the 8 polymorphisms used for estimation of the haplotype structure in the examples described later, these may be sequentially performed until a haplotype other than haplotype III is identified. However, in order to distinguish between haplotype I and haplotype III, it is necessary to detect the polymorphism of 117496G> A.
[0020] 本発明の診断方法において、多型の検出は公知の SNP検出方法のいずれも使用 すること力 Sできる。古典的な検出方法としては、例えば、被験者の細胞等から抽出し たゲノム DNAを試料とし、 GenBankァクセッション番号 AC073283.8で表される塩基配 列の部分塩基配列であって、上記したいずれかの多型部位の塩基（好ましくは、塩 基番号 117496または 117117で示される塩基、あるいは塩基番号 122702、 118756、 11 6513、 115810〜115809、 115602、 1114932または 106596で示される塩基）を含む約 1 5〜約 500塩基の連続した塩基配列を含有してなる核酸をプローブとして用い、例え ば Wallaceら（Proc. Natl. Acad. Sci. USA, 80， 278-282 (1983))の方法に従って、ストリ ンジエンシーを正確にコントロールしながらハイブリダィゼーシヨンを行い、プローブと 完全相補的な配列のみを検出する方法や、上記核酸と上記核酸にお!/、て多型部位 の塩基が他の塩基に置換された核酸の!/、ずれか一方を標識し、他方を未標識とした ミックスプローブを用い、変性温度から徐々に反応温度を低下させながらハイブリダィ ゼーシヨンを行い、一方のプローブと完全相補的な配列を先にハイブリダィズさせ、ミ スマッチを有するプローブとの交差反応を防ぐ方法などが挙げられる。ここで標識剤 としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。 放射性同位元素としては、例えば、〔125I〕、 〔mI〕、 〔¾〕、 〔14C〕などが用いられる。上 記酵素としては、安定で比活性の大きなものが好ましぐ例えば、 /3—ガラクトシダー ゼ、 β ダルコシダーゼ、アルカリフォスファターゼ、パーォキシダーゼ、リンゴ酸脱 水素酵素などが用いられる。蛍光物質としては、例えば、フルォレスカミン、フルォレ ッセンイソチオシァネートなどが用いられる。発光物質としては、例えば、ルミノール、 ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。 [0020] In the diagnostic method of the present invention, any known SNP detection method can be used to detect a polymorphism. The power to do S. As a classic detection method, for example, a genomic DNA extracted from a subject's cells or the like is used as a sample, and a partial base sequence of the base sequence represented by GenBank accession number AC073283.8, which About 1 including the base at the polymorphic site (preferably, the base represented by the base number 117496 or 117117, or the base represented by the base numbers 122702, 118756, 11 6513, 115810 to 115809, 115602, 1114932 or 106596) A nucleic acid comprising a continuous base sequence of 5 to about 500 bases is used as a probe. For example, according to the method of Wallace et al. (Proc. Natl. Acad. Sci. USA, 80, 278-282 (1983)) Hybridization is performed while accurately controlling the nuance, and only the sequence that is completely complementary to the probe is detected, or the base of the polymorphic site in the nucleic acid and the nucleic acid is changed to another base. Replaced nucleic acid ! / Using a mixed probe with one of the labels labeled and the other unlabeled, hybridization is performed while gradually lowering the reaction temperature from the denaturation temperature, and the sequence that is completely complementary to one probe is hybridized first. And a method for preventing cross reaction with a probe having a mismatch. Here, as the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used. As the radioisotope, for example, [ 125 I], [ m I], [¾], [ 14 C] and the like are used. As the above enzyme, those which are stable and have high specific activity are preferred. For example, / 3-galactosidase, β-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
好ましくは、多型の検出は、例えば、 WO 03/023063に記載された種々の方法、例 えば、 RFLP法、 PCR-SSCP法、 ASOハイブリダィゼーシヨン、ダイレクトシークェンス法 、 ARMS法、変性剤濃度勾配ゲル電気泳動法、 RNaseA切断法、化学切断法、 DOL 法、 TaqMan PCR法、インベーダー法、 MALDI-TOF/MS法、 TDI法、モレキュラー'ビ ーコン法、ダイナミック 'アレルスぺシフィック'ノヽィブリダィゼーシヨン法、ノ ドロック'プ ローブ法、 UCAN法、 DNAチップまたは DNAマイクロアレイを用いた核酸ハイブリダィ ゼーシヨン法、および ECA法などにより実施することができる（WO 03/023063,第 17 頁第 5行〜第 28頁第 20行を参照）。以下、代表的な方法として、 TaqMan PCR法とィ ンベーダ一法について、より詳細に説明する。 Preferably, detection of polymorphism is performed by various methods described in WO 03/023063, for example, RFLP method, PCR-SSCP method, ASO hybridization, direct sequence method, ARMS method, denaturing agent Gradient gel electrophoresis, RNaseA cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic 'allele specific' neutral It can be carried out by a dialysis method, a node lock probe method, a UCAN method, a nucleic acid hybridization method using a DNA chip or a DNA microarray, an ECA method, etc. (WO 03/023063, page 17, page 5) Line to page 28, line 20). Hereinafter, TaqMan PCR method and A more detailed description will be given of the method of the vanvader.
[0022] (l) TaqMan PCR法  [0022] (l) TaqMan PCR method
TaqMan PCR法は、蛍光標識したアレル特異的オリゴヌクレオチド（TaqManプロ一 ブ）と Taq DNAポリメラーゼによる PCRとを利用した方法である。 TaqManプローブとし ては、 GenBankァクセッション番号 AC073283.8で表される塩基配列の部分塩基配列 であって、上記したいずれかの多型部位の塩基を含む約 15〜約 30塩基の連続した 塩基配列からなるオリゴヌクレオチドが用いられる。該プローブは、その 5'末端が FA Mや VICなどの蛍光色素で、 3'末端力 STAMRAなどのクェンチヤ一（消光物質）でそれ ぞれ標識されており、そのままの状態ではクェンチヤ一が蛍光エネルギーを吸収する ため蛍光は検出されない。プローブは双方のアレルについて調製し、一括検出のた めに互いに蛍光波長の異なる蛍光色素（例えば、一方のアレルを FAM、他方を VIC) で標識すること力好ましい。また、 TaqManプローブからの PCR伸長反応が起こらない ように 3'末端はリン酸化されている。 TaqManプローブとハイブリダィズする領域を含 むゲノム DNAの部分配列を増幅するように設計されたプライマーおよび Taq DNAポリ メラーゼとともに PCRを行うと、 TaqManプローブが铸型 DNAとハイブリダィズし、同時 に PCRプライマーからの伸長反応が起こる力 伸長反応が進むと Taq DNAポリメラー ゼの 5'ヌクレアーゼ活性によりハイブリダィズした TaqManプローブが切断され、蛍光 色素が遊離してクェンチヤ一の影響を受けなくなり、蛍光が検出される。铸型の増幅 により蛍光強度は指数関数的に増大する。  The TaqMan PCR method uses a fluorescently labeled allele-specific oligonucleotide (TaqMan probe) and PCR using Taq DNA polymerase. The TaqMan probe is a partial base sequence of the base sequence represented by GenBank accession number AC073283.8, which is a continuous base of about 15 to about 30 bases including the bases of any of the polymorphic sites described above. An oligonucleotide consisting of a sequence is used. The probe is labeled with a fluorescent dye such as FAM or VIC at the 5 'end and labeled with a quencher (quenching substance) such as 3' end force STAMRA. Fluorescence is not detected due to absorption. It is preferable to prepare probes for both alleles and label them with fluorescent dyes having different fluorescence wavelengths (for example, one allele is FAM and the other is VIC) for batch detection. In addition, the 3 'end is phosphorylated to prevent PCR extension from the TaqMan probe. When PCR is performed with a primer designed to amplify a partial sequence of genomic DNA containing a region that hybridizes to the TaqMan probe and Taq DNA polymerase, the TaqMan probe hybridizes to the vertical DNA and simultaneously from the PCR primer. Force of elongation reaction As the elongation reaction proceeds, the hybridized TaqMan probe is cleaved by the 5 'nuclease activity of Taq DNA polymerase, the fluorescent dye is released and is not affected by quenching, and fluorescence is detected. The fluorescence intensity increases exponentially due to the vertical amplification.
例えば、 GenBankァクセッション番号 AC073283.8で表される塩基配列中、塩基番号 117496で示される塩基における多型の検出において、当該塩基を含むアレル特異 的オリゴヌクレオチド（約 15〜約 30塩基長； Aアレルは FAMで、 Gアレルは VICでそれ ぞれ 5'末端標識し、 3'末端は!/、ずれも TAMRAで標識）を TaqManプローブとして用 いた場合、被験者のジエノタイプが AA、あるいは GGであれば、それぞれ FAMあるい は VICの強い蛍光強度を認め、他方の蛍光はほとんど認められない。一方、被験者 のジエノタイプ力 SAGであれば、 FAMおよび VIC両方の蛍光が検出される。  For example, in the detection of a polymorphism at the base represented by base number 117496 in the base sequence represented by GenBank accession number AC073283.8, an allele-specific oligonucleotide (about 15 to about 30 bases in length; If the A allele is FAM, the G allele is labeled 5 'with VIC, and the 3' end is! /, And both are labeled with TAMRA), the subject's dienotype is AA or GG. If present, the strong fluorescence intensity of FAM or VIC is recognized, and the other fluorescence is hardly recognized. On the other hand, if the subject's dienotypic force SAG, both FAM and VIC fluorescence are detected.
[0023] (2)インベーダー法 [0023] (2) Invader method
インベーダー法では、 TaqMan PCR法と異なり、アレル特異的オリゴヌクレオチド（ァ レルプローブ）自体は標識されず、多型部位の塩基の 5 '側に铸型 DNAと相補性のな い配列（フラップ)を有し、 3 '側には铸型に特異的な相補配列を有する。インべーダ 一法では、さらに铸型の多型部位の 3 '側に特異的な相補配列を有するオリゴヌタレ ォチド (インベーダープローブ；該プローブの 5 '末端である多型部位に相当する塩基 は任意である）と、 5 '側がヘアピン構造をとり得る配列を有し、ヘアピン構造を形成し た際に 5 '末端の塩基と対をなす塩基から 3 '側に連続する配列がアレルプローブのフ ラップと相補的な配列であることを特徴とする FRET (fluorescence resonance energy t ransfer)プローブとが用いられる。 FRETプローブの 5 '末端は蛍光標識（例えば、 FAM や VICなど）され、その近傍にはクェンチヤ一（例えば、 TAMRAなど）が結合しており 、そのままの状態 (ヘアピン構造)では蛍光は検出されなレ、。 Unlike the TaqMan PCR method, the invader method uses an allele-specific oligonucleotide (a (Rel probe) itself is not labeled, has a non-complementary sequence (flap) on the 5 'side of the base of the polymorphic site, and has a complementary sequence specific to the 3' side on the 3 'side . In the Invader method, an oligonucleotide having a specific complementary sequence on the 3 'side of the polymorphic site of the saddle type (Invader probe; any base corresponding to the polymorphic site at the 5' end of the probe is optional. The 5 'side has a sequence that can take a hairpin structure, and when the hairpin structure is formed, the sequence that is 3' side continuous from the base paired with the 5 'end base is the flap of the allele probe. And a FRET (fluorescence resonance energy transfer) probe characterized in that it is a complementary sequence. The 5 'end of the FRET probe is fluorescently labeled (for example, FAM, VIC, etc.), and a quencher (for example, TAMRA, etc.) is bound in the vicinity thereof, and fluorescence is not detected as it is (hairpin structure). Les.
铸型であるゲノム DNAにアレルプローブおよびインベーダープローブを反応させる と、三者が相補結合した際に多型部位にインベーダープローブの 3 '末端が侵入する 。この多型部位の構造を認識する酵素（cleavase)を用いてアレルプローブの一本鎖 部分（即ち、多型部位の塩基から 5 '側のフラップ部分）を切り出すと、フラップは FRE Tプローブと相補的に結合し、フラップの多型部位が FRETプローブのヘアピン構造 に侵入する。この構造を cleavaseが認識して切断することにより、 FRETプローブの末 端標識された蛍光色素が遊離してクェンチヤ一の影響を受けなくなって蛍光が検出 される。多型部位の塩基が铸型とマッチしないアレルプローブは cleavaseによって切 断されなレ、が、切断されなレ、アレルプローブも FRETプローブとハイブリダィズすること ができるので、同様に蛍光が検出される。但し、反応効率が異なるため、多型部位の 塩基がマッチするアレルプローブでは、マッチしな!/、アレルプローブに比べて蛍光強 度が顕著に強い。  When an allele probe and an invader probe are reacted with the genomic DNA, which is a cage type, the 3 'end of the invader probe enters the polymorphic site when the three partners bind complementarily. Using the enzyme that recognizes the structure of this polymorphic site (cleavase), the single-stranded part of the allele probe (ie, the 5 'side flap part from the base of the polymorphic site) is excised, and the flap is complementary to the FRET probe. Binds and the flap polymorphic site enters the hairpin structure of the FRET probe. When this structure is recognized and cleaved by cleavase, the end-labeled fluorescent dye of the FRET probe is released and is not affected by quenching, and fluorescence is detected. Allele probes whose bases at the polymorphic site do not match the saddle type are not cleaved by cleavase, but those that are not cleaved can also be hybridized with the FRET probe, so fluorescence is detected in the same manner. However, since the reaction efficiencies are different, the allele probe that matches the base of the polymorphic site does not match! /, And the fluorescence intensity is significantly stronger than the allele probe.
通常、 3種のプローブおよび cleavaseと反応させる前に、铸型 DNAはアレルプロ一 ブおよびインベーダープローブがハイブリダィズする部分を含む領域を増幅し得るプ ライマーを用いて PCRにより増幅しておくことが好まし!/、。  In general, before reacting with the three probes and cleavase, it is preferable to amplify the vertical DNA by PCR using a primer that can amplify the region containing the hybridized portion of the allele probe and invader probe. ! /
上記のようにして多型を調べた結果、任意の骨 ·関節疾患非罹患者集団におけるよ りも任意の骨 ·関節疾患患者集団にぉレヽて有意に高!、アレルを保有して!/、ると判定 された場合、特に該アレルについてホモ接合体であると判定された場合には、被験 者は当該骨 ·関節疾患に対する遺伝的感受性が高いと診断することができる。例え ば、 GenBankァクセッション番号 AC073283.8で表される塩基配列中、 As a result of examining the polymorphism as described above, it was significantly higher in any bone / joint disease patient population than in any bone / joint disease non-affected population, and possessed alleles! / , Especially if the allele is determined to be a homozygote. The person can be diagnosed with a high genetic susceptibility to the bone and joint disease. For example, in the base sequence represented by GenBank accession number AC073283.8,
(1)塩基番号 117496で示される塩基が Gのアレルを保有する場合、および/または (1) When the base represented by the base number 117496 has an allele of G, and / or
(2)塩基番号 117117で示される塩基が Gのアレルを保有する場合、 (2) When the base represented by base number 117117 has an allele of G,
被験者は、軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能低下が関連する 骨 ·関節疾患、例えば、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎、滑膜 炎、代謝性関節症、スポーツによる関節障害、先天性骨系統疾患 [例えば、軟骨形 成の低下して!/、る骨系統疾患 ·変形性関節症を合併する先天性骨系統疾患 (例：軟 骨無形成症、多発性骨端異形成症、脊椎骨端異形成症、骨幹端異形成症、 Stickler 症候群、偽性軟骨無形成症等) ]に罹患しやすレ、 (感受性である）と診断することがで きる。その一方で、該被験者は、軟骨基質の産生能 '軟骨細胞分化能の異常亢進が 関連する疾患、例えば、先天性骨系統疾患 [例えば、軟骨形成の亢進している骨系 統疾患 (例：多発性外骨腫、片側肥大、オリエール病、マフツチ症候群等）]、骨軟骨 腫、骨腫瘍、軟骨腫瘍などの疾患に対しては非感受性であり得る。  Subjects are osteoarthritis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy related to bone / joint diseases associated with degeneration / disappearance of cartilage matrix, decreased production ability, and reduced chondrocyte differentiation ability , Sports joint disorders, congenital bone system diseases [eg, reduced cartilage formation! /, Bone system diseases · congenital bone system diseases complicated by osteoarthritis (eg, soft bone aplasia, Multiple epiphyseal dysplasia, vertebral epidysodysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)] can be diagnosed . On the other hand, the subject may have a disease associated with abnormal production of chondrocytes, such as a congenital bone disease [for example, a bone disease with increased cartilage formation (eg, Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor, and other diseases.
[0025] 一方、 GenBankァクセッション番号 AC073283.8で表される塩基配列中、 [0025] On the other hand, in the base sequence represented by GenBank accession number AC073283.8,
(1)塩基番号 117496で示される塩基が Aのアレルを保有する場合、および/または (1) When the base represented by the base number 117496 has an allele of A, and / or
(2)塩基番号 117117で示される塩基が Aのアレルを保有する場合、 (2) When the base represented by base number 117117 has an allele of A,
被験者は、軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能低下が関連する 骨 ·関節疾患、例えば、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎、滑膜 炎、代謝性関節症、スポーツによる関節障害、先天性骨系統疾患 [例えば、軟骨形 成の低下して!/、る骨系統疾患 ·変形性関節症を合併する先天性骨系統疾患 (例：軟 骨無形成症、多発性骨端異形成症、脊椎骨端異形成症、骨幹端異形成症、 Stickler 症候群、偽性軟骨無形成症等）]に感受性ではないと診断することができる。その一 方で、該被験者は、軟骨基質の産生能 ·軟骨細胞分化能の異常亢進が関連する疾 患、例えば、先天性骨系統疾患 [例えば、軟骨形成の亢進している骨系統疾患（例： 多発性外骨腫、片側肥大、オリエール病、マフツチ症候群等）]、骨軟骨腫、骨腫瘍、 軟骨腫瘍などの疾患に感受性であり得る。  Subjects are osteoarthritis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy related to bone / joint diseases associated with degeneration / disappearance of cartilage matrix, decreased production ability, and reduced chondrocyte differentiation ability , Sports joint disorders, congenital bone system diseases [eg, reduced cartilage formation! /, Bone system diseases · congenital bone system diseases complicated by osteoarthritis (eg, soft bone aplasia, Multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)]. On the other hand, the subject may have a disease associated with abnormal enhancement of cartilage matrix production ability / chondrocyte differentiation ability, such as congenital bone disease [eg, bone disease with increased cartilage formation (eg, : Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor, and other diseases.
[0026] また、 GenBankァクセッション番号 AC073283.8で表される塩基配列中、 (3a)塩基番号 116513で示される塩基が Gのアレルを保有する場合、および/または (3b)塩基番号 115810〜115809で示される塩基が TGのアレルを保有する場合、およ び/または [0026] In addition, in the base sequence represented by GenBank accession number AC073283.8, (3a) When the base represented by base number 116513 possesses an allele of G, and / or (3b) When the base represented by base numbers 115810 to 115809 possesses an allele of TG, and / or
(3c)塩基番号 115602で示される塩基が Gのアレルを保有する場合、および/または (3d)塩基番号 114932で示される塩基が Cのアレルを保有する場合、および/または (3e)塩基番号 122702で示される塩基が Tのアレルを保有する場合もしくは塩基番号 1 06596で示される塩基が Gのアレルを保有する場合、ある!/、は  (3c) when the base represented by base number 115602 carries the G allele, and / or (3d) when the base represented by base number 114932 carries the allele of C, and / or (3e) base number 122702 When the base indicated by T has an allele of T or when the base indicated by base number 1 06596 has an allele of G!
(4)塩基番号 118756で示される塩基が Cのアレルを保有する場合、  (4) When the base represented by the base number 118756 has an allele of C,
被験者は、軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能低下が関連する 骨 ·関節疾患、例えば、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎、滑膜 炎、代謝性関節症、スポーツによる関節障害、先天性骨系統疾患 [例えば、軟骨形 成の低下して!/、る骨系統疾患 ·変形性関節症を合併する先天性骨系統疾患 (例：軟 骨無形成症、多発性骨端異形成症、脊椎骨端異形成症、骨幹端異形成症、 Stickler 症候群、偽性軟骨無形成症等) ]に罹患しやすレ、 (感受性である）と診断することがで きる。その一方で、該被験者は、軟骨基質の産生能 '軟骨細胞分化能の異常亢進が 関連する疾患、例えば、先天性骨系統疾患 [例えば、軟骨形成の亢進している骨系 統疾患 (例：多発性外骨腫、片側肥大、オリエール病、マフツチ症候群等）]、骨軟骨 腫、骨腫瘍、軟骨腫瘍などの疾患に対しては非感受性であり得る。  Subjects are osteoarthritis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy related to bone / joint diseases associated with degeneration / disappearance of cartilage matrix, decreased production ability, and reduced chondrocyte differentiation ability , Sports joint disorders, congenital bone system diseases [eg, reduced cartilage formation! /, Bone system diseases · congenital bone system diseases complicated by osteoarthritis (eg, soft bone aplasia, Multiple epiphyseal dysplasia, vertebral epidysodysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)] can be diagnosed . On the other hand, the subject may have a disease associated with abnormal production of chondrocytes, such as a congenital bone disease [for example, a bone disease with increased cartilage formation (eg, Multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.]], osteochondroma, bone tumor, cartilage tumor, and other diseases.
[0027] 尚、後記実施例においてハプロタイプ構造の推定に用いた 8個の多型のうち 2個以 上の多型を調べた際に、 1以上の結果が他の結果と相反する場合には、 1174960A の多型における結果が優先される。  [0027] It should be noted that when two or more polymorphisms among the eight polymorphisms used for estimation of the haplotype structure in the examples described later are examined, if one or more results conflict with other results. 1174960A polymorphic results are preferred.
[0028] 上述のように、 GenBankァクセッション番号 AC073283.8で表される塩基配歹 IJ (相補 鎖配列）中、塩基番号 117496および 117117で示される塩基における多型は、本発明 において初めて見出された新規多型（117496Aおよび 117117Gが新規アレル）であり 、且つ骨 ·関節疾患に対する遺伝的感受性の診断に利用できるマーカー多型である [0028] As described above, the polymorphisms at the bases indicated by base numbers 117496 and 117117 in the base arrangement IJ (complementary strand sequence) represented by GenBank accession number AC073283.8 are first seen in the present invention. New polymorphisms released (117496A and 117117G are new alleles) and marker polymorphisms that can be used to diagnose genetic susceptibility to bone and joint diseases
〇 Yes
従って、本発明はまた、 GenBankァクセッション番号 AC073283.8で表される塩基配 列 (相補鎖配列）の部分塩基配列であって、 (a)塩基番号 117496で示される塩基（但し、該塩基は Aである）、または Therefore, the present invention also provides a partial base sequence of the base sequence (complementary strand sequence) represented by GenBank accession number AC073283.8, (a) a base represented by base number 117496 (provided that the base is A), or
(b)塩基番号 117117で示される塩基（但し、該塩基は Gである）  (b) a base represented by base number 117117 (provided that the base is G)
を含む、約 15〜約 500塩基（好ましくは約 15〜約 200塩基、より好ましくは約 15〜約 50 塩基）の連続した塩基配列を含有してなる核酸を提供する。力、かる SNP部位を含む新 規核酸は、上記した本発明の診断方法において、当該塩基における多型を検出す るのに好ましく用いることができる。そのような核酸は、配列番号： 1で表される塩基配 列情報に基づいて、 DNA/RNA自動合成機を用いて合成することができる。 A nucleic acid comprising a continuous base sequence of about 15 to about 500 bases (preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases). A novel nucleic acid containing a strong SNP site can be preferably used to detect a polymorphism in the base in the above-described diagnostic method of the present invention. Such a nucleic acid can be synthesized using a DNA / RNA automatic synthesizer based on the nucleotide sequence information represented by SEQ ID NO: 1.
また、 GenBankァクセッション番号 AC073283.8で表される塩基配列（相補鎖配列） 中、塩基番号 122702、 118756、 117496、 116513、 115810〜115809、 115602、 114932 および 106596で示される塩基もしくは塩基配列における多型を用いて同定された 5つ のコモンハプロタイプ（表 6参照）は、いずれも新規ハプロタイプである。このうちハプ 口タイプ IIIは、 OA (特に ADを伴わない HOA)をはじめとした種々の骨 ·関節疾患に対 する非感受性アレル（ 117496A)を有する。  In addition, in the base sequence (complementary strand sequence) represented by GenBank accession number AC073283.8, in the base or base sequence represented by base numbers 122702, 118756, 117496, 116513, 115810 to 115809, 115602, 114932 and 106596 The five common haplotypes identified using the polymorphism (see Table 6) are all new haplotypes. Of these, haptic type III has an insensitive allele (117496A) for various bone and joint diseases, including OA (particularly HOA without AD).
従って、本発明はまた、 GenBank accession No. AC073283.8で表される塩基配列（ 相補鎖配列）中、塩基番号 122702、 118756、 117496、 116513、 115602、 114932およ び 106596で示される塩基もしくは塩基配列が、それぞれ G、 A、 A、 A、 Tおよび Aで あり、塩基番号 115810〜115809で示される塩基が欠失しているハプロタイプの塩基 配列を含む核酸を提供する。  Accordingly, the present invention also provides a base or base represented by base numbers 122702, 118756, 117496, 116513, 115602, 114932 and 106596 in the base sequence (complementary strand sequence) represented by GenBank accession No. AC073283.8. Provided is a nucleic acid comprising a haplotype base sequence in which the sequences are G, A, A, A, T, and A, respectively, and the bases represented by base numbers 115810 to 115809 are deleted.
かかる核酸もまた、本発明の診断方法において、 OA (特に ADを伴わない HOA)等 の骨 ·関節疾患に対する非感受性ハプロタイプを検出するためのプローブ等として用 いること力 Sできる。そのような核酸は、当該ハプロタイプ構造を有する CALM2遺伝子 を保有するヒトの細胞 ·組織から単離したゲノム DNAより、配列番号： 1で表される塩基 配列の全部または一部を含む核酸をプローブ等として用いることにより単離すること 力できる。あるいは、該核酸は、任意のハプロタイプ構造を有する CALM2遺伝子を保 有するヒトの細胞 ·組織から単離したゲノム DNAより、同様の方法によって対応する部 分の核酸を単離した後、常法に従ってその塩基配列を決定し、所望のハプロタイプと は異なるハプロタイプであった場合に、部位特異的変異誘発などの自体公知の手法 を用いて、所望のハプロタイプ構造に改変することによつても得ることができる。 [0029] 本発明はまた、上記本発明の診断方法に用いるためのキットを提供する。即ち、本 発明の診断用キットは、 117496G〉Aもしくは該多型と連鎖不平衡係数 D 'が 0.9以上の 連鎖不平衡状態にある、好ましくは完全連鎖状態にある（即ち、 D ' =l)、 CALM2遺伝 子内および/またはその周辺領域内に存在する多型、並びに/あるいは 117117A〉 Gもしくは該多型と連鎖不平衡係数 D 'が 0.9以上の連鎖不平衡状態にある、好ましく は完全連鎖状態にある（即ち、 D ' =l)、 CALM2遺伝子内および/またはその周辺領 域内に存在する多型であって、一方のアレル頻度力 任意の骨'関節疾患非罹患者 集団におけるよりも任意の骨 ·関節疾患患者集団において高い多型からなる群より選 択される 1以上の多型の各々を検出し得る 1組以上の核酸プローブおよび/または プライマーを含むことを特徴とする。 Such a nucleic acid can also be used as a probe for detecting an insensitive haplotype for bone / joint diseases such as OA (particularly HOA without AD) in the diagnostic method of the present invention. Such a nucleic acid is probed with a nucleic acid containing all or part of the base sequence represented by SEQ ID NO: 1 from genomic DNA isolated from a human cell or tissue carrying the CALM2 gene having the haplotype structure. It can be isolated by using as Alternatively, the nucleic acid can be isolated from a genomic DNA isolated from a human cell or tissue having a CALM2 gene having an arbitrary haplotype structure by isolating the corresponding part of the nucleic acid according to the same method. When the base sequence is determined and the haplotype is different from the desired haplotype, it can also be obtained by modifying to the desired haplotype structure using a method known per se such as site-directed mutagenesis. . [0029] The present invention also provides a kit for use in the diagnostic method of the present invention. That is, the diagnostic kit of the present invention is in a linkage disequilibrium state with 117496G> A or the polymorphism and a linkage disequilibrium coefficient D ′ of 0.9 or more, preferably in a complete linkage state (ie, D ′ = l). A polymorphism present in the CALM2 gene and / or its surrounding region, and / or 117117A> G or a linkage disequilibrium coefficient with the polymorphism D ′ of 0.9 or more, preferably a complete linkage Polymorphism in the CALM2 gene and / or its surrounding region that is in a state (ie, D ′ = l), and one allele frequency power is more arbitrary than in the population without any bone 'joint disease It comprises one or more nucleic acid probes and / or primers capable of detecting each of one or more polymorphisms selected from the group consisting of high polymorphisms in the bone / joint disease patient population.
[0030] 具体的には、本発明の診断用キットに用いられる核酸プローブは、上記本発明の 診断方法において検出すべき多型部位の塩基を含む領域でゲノム DNAとハイブリダ ィズする核酸であり、標的部位に対して特異的であり且つ多型性を容易に検出し得 る限りその長さ（ゲノム DNAとハイブリダィズする部分の塩基長）に特に制限はなぐ 例えば約 15塩基以上、好ましくは約 15〜約 500塩基、より好ましくは約 15〜約 200塩 基、いっそう好ましくは約 15〜約 50塩基である。  [0030] Specifically, the nucleic acid probe used in the diagnostic kit of the present invention is a nucleic acid that hybridizes with genomic DNA in a region containing the base of the polymorphic site to be detected in the diagnostic method of the present invention. As long as it is specific to the target site and the polymorphism can be easily detected, its length (base length of the portion that hybridizes with genomic DNA) is not particularly limited. For example, about 15 bases or more, preferably about 15 to about 500 bases, more preferably about 15 to about 200 bases, and even more preferably about 15 to about 50 bases.
該プローブは、多型性の検出に適した付加的配列（ゲノム DNAと相補的でな!/、配 歹 IJ)を含んでいてもよい。例えば、上記インベーダー法に用いられるアレルプローブ は、多型部位の塩基の 5 '末端にフラップと呼ばれる付加的配列を有する。  The probe may contain additional sequences suitable for detection of polymorphisms (not complementary to genomic DNA! /, IJ). For example, the allele probe used in the invader method has an additional sequence called a flap at the 5 ′ end of the base at the polymorphic site.
また、該プローブは、適当な標識剤、例えば、放射性同位元素 (例: 125I、 mI、 3H、 14 C等）、酵素（例： 0 ガラクトシダーゼ、 0 ダルコシダーゼ、アルカリフォスファタ一 ゼ、パーォキシダーゼ、リンゴ酸脱水素酵素等）、蛍光物質（例：フルォレスカミン、フ ノレオレツセンイソチオシァネート等）、発光物質 (例:ルミノール、ルミノール誘導体、 ルシフェリン、ルシゲニン等）などで標識されていてもよい。あるいは、蛍光物質（例： F AM VIC等）の近傍に該蛍光物質の発する蛍光エネルギーを吸収するクェンチヤ一 (消光物質）がさらに結合されていてもよい。力、かる実施態様においては、検出反応 の際に蛍光物質とクェンチヤ一とが分離して蛍光が検出される。 The probe may be a suitable labeling agent such as a radioisotope (eg, 125 I, m I, 3 H, 14 C, etc.), an enzyme (eg, 0 galactosidase, 0 dalcosidase, alkaline phosphatase, Oxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluoreoretsen isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) Also good. Alternatively, a quencher (quenching substance) that absorbs fluorescence energy emitted by the fluorescent substance may be further bound in the vicinity of the fluorescent substance (eg, FAM VIC). In this embodiment, in the detection reaction, the fluorescent substance and the quencher are separated and the fluorescence is detected.
[0031] 好ましくは、本発明の診断用キットに用いられる核酸プローブは、 GenBank accessio n No. AC073283.8で表される塩基配列（相補鎖配列）中、塩基番号 117496および/ または 117117で示される多型部位、あるいは塩基番号 122702、 118756、 116513、 115 810〜115809、 115602、 114932および 106596からなる群より選択される塩基番号で示 される多型部位、より好ましくは 117496および/または 117117で示される多型部位の 塩基を含む、約 15〜約 500塩基、好ましくは約 15〜約 200塩基、より好ましくは約 15〜 約 50塩基の連続した塩基配列を含有してなる核酸である。ここで、 [0031] Preferably, the nucleic acid probe used in the diagnostic kit of the present invention is GenBank accessio. n No. In the base sequence (complementary strand sequence) represented by AC073283.8, the polymorphic site represented by base numbers 117496 and / or 117117, or base numbers 122702, 118756, 116513, 115 810 to 115809, 115602, 114932 And a polymorphic site represented by a base number selected from the group consisting of 106596, more preferably a base of a polymorphic site represented by 117496 and / or 117117, preferably about 15 to about 500 bases, preferably about 15 to A nucleic acid comprising a continuous base sequence of about 200 bases, more preferably about 15 to about 50 bases. here,
(1)塩基番号 117496で示される塩基は Gまたは A、  (1) The base represented by base number 117496 is G or A,
(2)塩基番号 117117で示される塩基は Aまたは G、  (2) The base represented by base number 117117 is A or G,
(3)塩基番号 122702で示される塩基は Cまたは T、  (3) The base represented by base number 122702 is C or T,
(4)塩基番号 118756で示される塩基は Gまたは C、  (4) The base represented by base number 118756 is G or C,
(5)塩基番号 116513で示される塩基は Aまたは G、  (5) The base represented by base number 116513 is A or G,
(6)塩基番号 115810〜115809で示される塩基は欠失している力、、または TG、  (6) The base represented by base numbers 115810 to 115809 is a deleted force, or TG,
(7)塩基番号 115602で示される塩基は Aまたは G、  (7) The base represented by base number 115602 is A or G,
(8)塩基番号 114932で示される塩基は Tまたは C、  (8) The base represented by the base number 114932 is T or C,
(9)塩基番号 106596で示される塩基は Aまたは G、  (9) The base represented by base number 106596 is A or G,
であり、使用する多型検出法に応じて、各多型部位についていずれか一方の塩基を 有する核酸を用いることもできるし、各アレルに対応する塩基を有する 2種類の核酸 を用いることもできる。尚、上記インベーダー法に使用されるインベーダープローブに ついては、多型部位の塩基（即ち、 3 '末端の塩基）は任意の塩基でよい。 Depending on the polymorphism detection method to be used, a nucleic acid having one of the bases for each polymorphic site can be used, or two types of nucleic acids having a base corresponding to each allele can be used. . For the invader probe used in the invader method, the base at the polymorphic site (ie, the 3 ′ terminal base) may be any base.
本発明の診断用キットに用いられる核酸プライマーは、上記本発明の診断方法に おいて検出すべき多型部位の塩基を含むゲノム DNAの領域を特異的に増幅し得る ように設計されたものであればいかなるものであってもよい。例えば、 GenBank access ion No. AC073283.8で表される塩基配列（相補鎖配列）の部分塩基配列であって、 検出すべき多型部位の塩基より 5 '側の相補鎖配列の一部にハイブリダィズする、約 1 5〜約 50塩基、好ましくは約 15〜約 30塩基の塩基配列を含む核酸と、該多型部位の 塩基より 3 '側の配列の一部にハイブリダィズする、約 15〜約 50塩基、好ましくは約 15 〜約 30塩基の塩基配列を含む核酸との組み合わせであり、それらによって増幅され る核酸の断片長が約 50〜約 1,000塩基、好ましくは約 50〜約 500塩基、より好ましくは 約 50〜約 200塩基である、一対の核酸が挙げられる。 該プライマーは、多型性の検 出に適した付加的配列（ゲノム DNAと相補的でな!/、配列）、例えばリンカ一配列を含 んでいてもよい。 The nucleic acid primer used in the diagnostic kit of the present invention is designed so as to specifically amplify a genomic DNA region containing the base of the polymorphic site to be detected in the diagnostic method of the present invention. Anything is acceptable. For example, a partial base sequence of the base sequence (complementary strand sequence) represented by GenBank access ion No. AC073283.8, which hybridizes to a part of the complementary strand sequence 5 'to the base of the polymorphic site to be detected. About 15 to about 50 bases, preferably about 15 to about 30 bases, and hybridizes to a part of the sequence 3 ′ from the base of the polymorphic site with about 15 to about 50 bases. A base, preferably a combination with a nucleic acid containing a base sequence of about 15 to about 30 bases, and the fragment length of the nucleic acid amplified by them is about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably Is A pair of nucleic acids having about 50 to about 200 bases is included. The primer may contain additional sequences suitable for polymorphism detection (non-complementary to genomic DNA! /, Sequences), such as a linker sequence.
また、該プライマーは、適当な標識剤、例えば、例えば、放射性同位元素 (例：125ι、 131I、 3H、 14C等）、酵素（例： β—ガラタトシダーゼ、 β—ダルコシダーゼ、アルカリフォ スファターゼ、パーォキシダーゼ、リンゴ酸脱水素酵素等）、蛍光物質（例：フルォレス カミン、フルォレツセンイソチオシァネート等）、発光物質（例：ルミノール、ルミノール 誘導体、ルシフェリン、ルシゲニン等）などで標識されていてもよい。 The primer may be a suitable labeling agent such as a radioisotope (eg, 125 ι, 1 31 I, 3 H, 14 C, etc.), an enzyme (eg, β-galatatosidase, β-darcosidase, alkaline phosphatase). Phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescene isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) It may be labeled.
[0033] 好ましくは、本発明の診断用キットに用いられる核酸プライマーは、 GenBankァクセ ッシヨン番号 AC073283.8で表される塩基配列の部分塩基配列であって、塩基番号 11 7496および/または 117117で示される多型部位、あるいは塩基番号 122702、 118756 、 116513、 115810〜115809、 115602、 114932および 106596からなる群より選択される 塩基番号で示される多型部位、より好ましくは 117496および/または 117117で示され る多型部位の塩基を含む、約 50〜約 1,000塩基、好ましくは約 50〜約 500塩基、より 好ましくは約 50〜約 200塩基の連続した塩基配列を増幅し得る一対の核酸である。  [0033] Preferably, the nucleic acid primer used in the diagnostic kit of the present invention is a partial base sequence of the base sequence represented by GenBank Accession No. AC073283.8 and represented by Base Nos. 11 7496 and / or 117117. Selected from the group consisting of base numbers 122702, 118756, 116513, 115810 to 115809, 115602, 114932 and 106596, more preferably 117496 and / or 117117. A pair of nucleic acids that can amplify a continuous base sequence of about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably about 50 to about 200 bases, including the bases of the polymorphic site.
[0034] 本発明の診断用キットに用いられる核酸プローブまたはプライマーは、 DNAであつ ても RNAであってもよく、また、一本鎖であっても二本鎖であってもよい。二本鎖の場 合は二本鎖 DNA、二本鎖 RNA、 DNA/RNAノヽイブリツドのいずれであってもよい。従 つて、本明細書においてある塩基配列を有する核酸について記載する場合、特に断 らない限り、該塩基配列を有する一本鎖核酸、該塩基配列と相補的な配列を有する 一本鎖核酸、それらのハイブリッドである二本鎖核酸をすベて包含する意味で用いら れてレヽると理角早されるべきである。  [0034] The nucleic acid probe or primer used in the diagnostic kit of the present invention may be DNA or RNA, and may be single-stranded or double-stranded. In the case of a double strand, any of a double-stranded DNA, a double-stranded RNA, and a DNA / RNA nobled may be used. Therefore, when describing a nucleic acid having a certain base sequence in this specification, unless otherwise specified, a single-stranded nucleic acid having the base sequence, a single-stranded nucleic acid having a sequence complementary to the base sequence, and those If it is used to include all double-stranded nucleic acids that are hybrids, it should be rationalized.
上記核酸プローブまたはプライマーは、例えば、 AC073283.8で表される塩基配列 の情報に基づ!/、て、 DNA/RNA自動合成機を用いて常法に従って合成することがで きる。  The nucleic acid probe or primer can be synthesized according to a conventional method using a DNA / RNA automatic synthesizer based on the information of the base sequence represented by AC073283.8, for example.
[0035] 上記核酸プローブおよび/またはプライマーは、各々別個に（あるいは可能であれ ば混合した状態で)水もしくは適当な緩衝液 (例： TEバッファーなど）中に適当な濃度 (例： 2 X〜20 X濃度で 1〜50 Mなど）となるように溶解し、約- 20°Cで保存することが できる。 [0035] The nucleic acid probe and / or primer are each separately (or mixed if possible) in water or in an appropriate buffer (eg, TE buffer) at an appropriate concentration (eg, 2 X to Can be dissolved at a concentration of 20 X at 1-50 M) and stored at about -20 ° C. it can.
本発明の診断用キットは、多型検出法に応じて、当該方法の実施に必要な他の成 分を構成としてさらに含んでいてもよい。例えば、該キットが TaqMan PCR法による多 型検出用である場合には、該キットは、 10 X PCR反応緩衝液、 lO X MgCl水溶液、 10 X dNTPs水溶液、 Taq DNAポリメラーゼ（5U/ L)等をさらに含むことができる。  The diagnostic kit of the present invention may further include other components necessary for carrying out the method as a component depending on the polymorphism detection method. For example, when the kit is for detecting a polymorphism by TaqMan PCR, the kit contains 10 X PCR reaction buffer, lO X MgCl aqueous solution, 10 X dNTPs aqueous solution, Taq DNA polymerase (5 U / L) and the like. Further can be included.
[0036] 本発明の診断用キットは、軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能 低下が関連する骨 ·関節疾患、例えば、骨粗鬆症、変形性関節症、慢性関節リウマ チ、関節炎、滑膜炎、代謝性関節症、スポーツによる関節障害、先天性骨系統疾患 [ 例えば、軟骨形成の低下している骨系統疾患 ·変形性関節症を合併する先天性骨 系統疾患 (例:軟骨無形成症、多発性骨端異形成症、脊椎骨端異形成症、骨幹端 異形成症、 Stickler症候群、偽性軟骨無形成症等）]に対する遺伝的感受性の診断 に使用すること力できる。また、本発明の診断用キットは、軟骨基質の産生能'軟骨 細胞分化能の異常亢進が関連する疾患、例えば、先天性骨系統疾患 [例えば、軟 骨形成の亢進している骨系統疾患（例：多発性外骨腫、片側肥大、オリエール病、マ フツチ症候群等）]、骨軟骨腫、骨腫瘍、軟骨腫瘍などの疾患に対する遺伝的感受 性の診断にも使用することができる。  [0036] The diagnostic kit of the present invention is a bone / joint disease associated with degeneration / disappearance / decreased production ability of cartilage matrix, or reduced ability to differentiate chondrocytes, such as osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis. , Synovitis, metabolic arthropathy, joint disorders due to sports, congenital bone disease [eg, bone disease with reduced cartilage formation · congenital bone disease with osteoarthritis (eg, cartilage) Aplasia, multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.)]. In addition, the diagnostic kit of the present invention can be used for diseases associated with abnormal enhancement of cartilage matrix production ability 'chondrocyte differentiation ability, such as congenital bone disease (eg, bone disease with enhanced soft bone formation ( For example, multiple exostoses, unilateral hypertrophy, Oriel's disease, Maftucci syndrome, etc.)], osteochondroma, bone tumor, cartilage tumor, and other genetic susceptibility diagnosis.
[0037] 上述のように、本発明者らの、 CALM1遺伝子における多型と OAとの関連に関する 研究成果は、カルシウム/カルモジュリン（Ca/CaM)経路の異常が OAの発症や進展 に深く関与していることを強く示唆している。従って、本発明の診断方法によって、骨 •関節疾患、特に、軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能低下が関 連する骨 ·関節疾患〔例えば、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎 、滑膜炎、代謝性関節症、スポーツによる関節障害、先天性骨系統疾患 [例えば、軟 骨形成の低下している骨系統疾患 ·変形性関節症を合併する先天性骨系統疾患（ 例:軟骨無形成症、多発性骨端異形成症、脊椎骨端異形成症、骨幹端異形成症、 S tickler症候群、偽性軟骨無形成症等）]〕に対して感受性 (易罹患性)であると判定さ れた被験者は、軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能低下が関連 する骨 ·関節疾患の発症および/または進展の過程において、 Ca/CaM経路を介し た抑制作用の発現が不十分であることが予測される。 従って、本発明の診断方法によって、軟骨基質の変性 ·消失 ·産生能低下、軟骨細 胞分化能低下が関連する骨 ·関節疾患に対して感受性 (易罹患性)であると判定され た被験者であって、現に該骨 ·関節疾患に罹患している力、、現に発症の危険性が高 いと認められるヒトに対して、 Ca/CaM経路の活性化を促進する処置を施すことは、該 骨 ·関節疾患の予防および/または治療に有効である。 [0037] As described above, the present inventors' research results on the association between polymorphisms in the CALM1 gene and OA indicate that abnormalities in the calcium / calmodulin (Ca / CaM) pathway are deeply involved in the onset and progression of OA. It strongly suggests that Therefore, according to the diagnostic method of the present invention, bone / joint diseases, in particular, bone / joint diseases related to degeneration / disappearance / decreased production ability of cartilage matrix and reduced ability to differentiate chondrocytes [for example, osteoporosis, osteoarthritis, Rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders, congenital bone system diseases [eg, bone system diseases with reduced soft bone formation · congenital bones with osteoarthritis Susceptibility to systematic diseases (eg, achondroplasia, multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia)]] The subject who is determined to be susceptible) has a Ca / CaM pathway in the process of onset and / or progression of bone / joint diseases associated with degeneration / disappearance of the cartilage matrix, decreased production capacity, and decreased chondrocyte differentiation capacity. Insufficient expression of inhibitory action via It is. Therefore, subjects who have been determined to be susceptible (susceptible) to bone / joint diseases related to degeneration / disappearance / decreased production ability of cartilage matrix and reduced ability to differentiate cartilage cells by the diagnostic method of the present invention. In addition, the treatment of promoting the activation of the Ca / CaM pathway to a human who is actually recognized as having a high risk of developing the bone / joint disease is · Effective for prevention and / or treatment of joint diseases.
従って、本発明はまた、 GenBank accession No. AC073283.8で表される塩基配列（ 相補鎖配列）中、塩基番号 117496で示される塩基が G、および/または塩基番号 117 117で示される塩基が Gであるアレルを保有するヒトに投与することを特徴とする、 Ca/ CaM経路を活性化する物質を含有してなる骨'関節疾患予防'治療剤を提供する。こ こで「骨 ·関節疾患」としては、軟骨基質の変性 ·消失 ·産生能低下、軟骨細胞分化能 低下が関連する骨 ·関節疾患、例えば、骨粗鬆症、変形性関節症、慢性関節リウマ チ、関節炎、滑膜炎、代謝性関節症、スポーツによる関節障害、先天性骨系統疾患 [ 例えば、軟骨形成の低下している骨系統疾患 ·変形性関節症を合併する先天性骨 系統疾患 (例:軟骨無形成症、多発性骨端異形成症、脊椎骨端異形成症、骨幹端 異形成症、 Stickler症候群、偽性軟骨無形成症等）]などが挙げられる。また、 rCa/C aM経路」とは、例えば上記非特許文献 2 (第 694頁、 Scheme 1)に示される一連のシグ ナル伝達経路を意味し、 CaMの他、カルシニューリン、 CaM依存性キナーゼ II (CaMK II)などにより構成されている。  Therefore, the present invention also provides that the base represented by base number 117496 is G and / or the base represented by base number 117 117 is G in the base sequence represented by GenBank accession No. AC073283.8 (complementary strand sequence). A bone 'joint disease prevention' therapeutic agent comprising a substance that activates the Ca / CaM pathway, characterized by being administered to a human having the allele of Here, “bone / joint diseases” include bone / joint diseases related to degeneration / disappearance / decreased production ability of cartilage matrix and decreased ability to differentiate chondrocytes, such as osteoporosis, osteoarthritis, rheumatoid arthritis, Arthritis, synovitis, metabolic arthropathy, sports joint disorders, congenital bone system diseases [eg bone system diseases with reduced cartilage formation · congenital bone system diseases complicated with osteoarthritis (eg: Achondroplasia, multiple epiphyseal dysplasia, vertebral epiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudochondral dysplasia, etc.). The `` rCa / CaM pathway '' means a series of signal transduction pathways shown in, for example, Non-Patent Document 2 (page 694, Scheme 1), and in addition to CaM, calcineurin, CaM-dependent kinase II ( CaMK II) etc.
Ca/CaM経路を活性化する物質としては、例えば、 CaM,カルシニューリン、 CaMKII のうちの 1以上の発現および/または活性を促進する物質が挙げられる。このような 物質としては、具体的には、例えば、 CaMもしくはその部分ペプチド、カルシニューリ ンもしくはその部分ペプチド、 CaMKIIもしくはその部分ペプチド、上記いずれかのタ ンパク質もしくはペプチドをコードする核酸等が挙げられるが、それらに限定されず、 例えば、以下に示すスクリーニング法によって得られる化合物なども包含され得る。 C aMおよびその部分ペプチド、並びにそれらをコードする核酸については、上記特許 文献 1に記載されている。カルシニューリン、 CaMKIIおよびそれらの部分ペプチド、 並びにそれらをコードする核酸についても、各サブユニットの公知のアミノ酸配列およ び塩基配列に基づいて、同様に調製することができる。 [0039] CaMの発現および/または活性を促進する物質のスクリーニング方法については、 上記特許文献 1に記載されている。カルシニューリン、 CaMKIIの発現を促進する物 質のスクリーニングについても CaMと同様にして行うことができる。 Examples of the substance that activates the Ca / CaM pathway include a substance that promotes the expression and / or activity of one or more of CaM, calcineurin, and CaMKII. Specific examples of such substances include CaM or a partial peptide thereof, calcineurin or a partial peptide thereof, CaMKII or a partial peptide thereof, and a nucleic acid encoding any one of the above proteins or peptides. However, it is not limited thereto, and for example, compounds obtained by the screening methods shown below can be included. C aM and partial peptides thereof, and nucleic acids encoding them are described in Patent Document 1 above. Calcineurin, CaMKII and their partial peptides, and nucleic acids encoding them can be similarly prepared based on the known amino acid sequence and base sequence of each subunit. [0039] A screening method for a substance that promotes CaM expression and / or activity is described in Patent Document 1 above. Screening for substances that promote the expression of calcineurin and CaMKII can be performed in the same manner as CaM.
カルシニューリンは軟骨細胞分化への関与が指摘される転写因子（例： NFATcl)の 脱リン酸化酵素であり、その活性は、例えば、該転写因子により発現制御されるシス エレメント（例： NFAT応答エレメント）を含むプロモーターの制御下にあるレポーター 遺伝子の発現を指標にして測定することができる。あるいは、該酵素の基質アナログ を合成し、 32Pラベルして脱リン酸化を直接測定することもできる。被験物質の存在下 で脱リン酸化が促進されれば、該物質をカルシニューリンの活性促進物質として選択 すること力 Sでさる。 Calcineurin is a dephosphorylation enzyme of a transcription factor (eg, NFATcl) that is indicated to be involved in chondrocyte differentiation, and its activity is, for example, a cis element (eg, NFAT response element) whose expression is controlled by the transcription factor. It can be measured using the expression of a reporter gene under the control of a promoter containing Alternatively, a substrate analog of the enzyme can be synthesized and labeled with 32 P to directly measure dephosphorylation. If dephosphorylation is promoted in the presence of the test substance, the force S can be selected to select the substance as a calcineurin activity promoter.
CaMKIIは、 cAMP応答エレメント結合性の転写因子を活性化（リン酸化）して軟骨基 質遺伝子の産生を促進すると考えられており、その活性は、例えば、該軟骨基質遺 伝子のプロモーターの制御下にあるレポーター遺伝子の発現を指標にして測定する こと力 Sできる。あるいは、該酵素の基質アナログを合成し、 32Pラベルした ATPを用いて 基質のリン酸化を直接測定することもできる。被験物質の存在下でリン酸化が促進さ れれば、該物質を CaMKIIの活性促進物質として選択することができる。 CaMKII is thought to activate (phosphorylate) cAMP response element-binding transcription factors to promote the production of cartilage substrate genes, and the activity is controlled, for example, by the promoter of the cartilage matrix gene. The ability to measure using the underlying reporter gene expression as an indicator. Alternatively, a substrate analog of the enzyme can be synthesized and the phosphorylation of the substrate can be directly measured using 32 P-labeled ATP. If phosphorylation is promoted in the presence of the test substance, the substance can be selected as a CaMKII activity promoter.
被験物質としては、例えば、タンパク質、ペプチド、非ペプチド性化合物、合成化合 物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液などが挙げられ、これ らの物質は新規なものであってもよ!/、し、公知のものであってもよ!/、。  Examples of test substances include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel. It may be! /, And it may be a publicly known one! /.
[0040] Ca/CaM経路を活性化する物質を、上記予防'治療剤として使用する場合は、常套 手段に従って製剤化することができる。該物質が CaM、カルシニューリンもしくは CaM ΚΠまたはその部分ペプチドをコードする核酸の場合、該核酸を単独あるいはレトロゥ イノレスベクター、アデノウイノレスベクター、アデノウイノレスァソシエーテッドウイノレスベタ ターなどの適当なベクターに揷入した後、常套手段に従って製剤化することができる 。該核酸は、そのままで、あるいは摂取促進のための補助剤とともに、遺伝子銃ゃハ イド口ゲルカテーテルのようなカテーテルによって投与することができる。  [0040] When a substance that activates the Ca / CaM pathway is used as the prophylactic / therapeutic agent, it can be formulated according to conventional means. When the substance is a nucleic acid encoding CaM, calcineurin or CaM or a partial peptide thereof, the nucleic acid can be used alone or in a suitable vector such as a retroinoleless vector, an adenowinores vector, or an adenowinore associated betater. Then, it can be formulated according to conventional means. The nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hard mouth gel catheter.
[0041] 例えば、 Ca/CaM経路を活性化する物質は、必要に応じて糖衣を施した錠剤、カブ セル剤、エリキシル剤、マイクロカプセル剤などとして経口的に、あるいは水もしくはそ れ以外の薬学的に許容し得る液との無菌性溶液、または懸濁液剤などの注射剤の 形で非経口的に使用できる。例えば、 Ca/CaM経路を活性化する物質を、生理学的 に認められる公知の担体、香味剤、賦形剤、べヒクル、防腐剤、安定剤、結合剤など とともに、一般に認められた製剤実施に要求される単位用量形態で混和することによ つて製造すること力できる。これら製剤における有効成分量は指示された範囲の適当 な容量が得られるようにするものである。 [0041] For example, a substance that activates the Ca / CaM pathway may be administered orally as a tablet, capsule, elixir, microcapsule, or the like with sugar coating as needed. It can be used parenterally in the form of injections such as sterile solutions with other pharmaceutically acceptable liquids or suspensions. For example, a substance that activates the Ca / CaM pathway, together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be produced by mixing in the required unit dosage form. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
[0042] 錠剤、カプセル剤などに混和することができる添加剤としては、例えば、ゼラチン， コーンスターチ，トラガント，アラビアゴムのような結合剤、結晶性セルロースのような 賦形剤、コーンスターチ，ゼラチン，アルギン酸などのような膨化剤、ステアリン酸マグ ネシゥムのような潤滑剤、ショ糖，乳糖またはサッカリンのような甘味剤、ペパーミント， ァカモノ油またはチェリーのような香味剤などが用いられる。調剤単位形態がカプセ ルである場合には、上記タイプの材料にさらに油脂のような液状担体を含有すること 力できる。注射のための無菌組成物は注射用水のようなべヒクル中に活性物質、胡 麻油，椰子油などのような天然産出植物油などを溶解または懸濁させるなどの通常 の製剤実施に従って処方することができる。注射用の水性液としては、例えば、生理 食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、 D-ソルビトール， D-マン 二トール，塩化ナトリウムなど）などが用いられ、適当な溶解補助剤、例えば、アルコ ール（例：エタノール)、ポリアルコール（例：プロピレングリコール，ポリエチレングリコ 一ル)、非イオン性界面活性剤（例：ポリソルベート 80™, HCO-50)などと併用しても よい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、溶解補助剤である 安息香酸ベンジル、ベンジルアルコールなどと併用してもよレ、。 [0042] Examples of additives that can be mixed with tablets and capsules include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. And the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, coconut oil or cherry. When the dispensing unit form is capsule, it is possible to further contain a liquid carrier such as fats and oils in the above type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice, such as dissolving or suspending active substances, naturally occurring vegetable oils such as sesame oil, coconut oil etc. in a vehicle such as water for injection. . As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used. In combination with adjuvants such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80 ™, HCO-50) Also good. As the oily liquid, for example, sesame oil, soybean oil and the like are used, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
[0043] また、上記予防 ·治療剤は、例えば、緩衝剤（例えば、リン酸塩緩衝液，酢酸ナトリ ゥム緩衝液)、無痛化剤 (例えば、塩化ベンザルコユウム，塩酸プロ力インなど)、安定 剤（例えば、ヒト血清アルブミン，ポリエチレングリコールなど)、保存剤 (例えば、ベン ジルアルコール，フエノールなど)、酸化防止剤などと配合してもよい。調製された注 射液は通常、適当なアンプルに充填される。  [0043] The prophylactic / therapeutic agent includes, for example, a buffer (for example, phosphate buffer solution, sodium acetate buffer solution), a soothing agent (for example, benzalkonium chloride, pro-in hydrochloride, etc.), a stable agent. You may mix | blend with an agent (for example, human serum albumin, polyethyleneglycol, etc.), a preservative (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection liquid is usually filled into a suitable ampoule.
[0044] このようにして得られる製剤は安全で低毒性であるので、例えば、ヒトゃ他の温血動 物 (例えば、ラット，マウス，ノヽムスター，ゥサギ，ヒッジ，ャギ，ブタ，ゥシ，ゥマ，ネコ，ィ ヌ，サル，チンパンジー，トリなど）に対して投与することができる。 [0044] Since the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rat, mouse, nomster, usagi, hidge, goat, pig, ushiki) 、 Uma 、 cat 、 i , Monkeys, chimpanzees, birds, etc.).
[0045] Ca/CaM経路を活性化する物質の投与量は、投与対象、対象臓器、症状、投与方 法などにより差異はあるが、経口投与の場合、一般的に例えば、変形性関節症患者 (60 kgとして)においては、一日にっき約 0· 1〜100 mg、好ましくは約 1.0〜50mg、より 好ましくは約 1.0〜20 mgである。非経口的に投与する場合は、その 1回投与量は投 与対象、対象臓器、症状、投与方法などによっても異なるが、例えば注射剤の形で は、通常、例えば変形性関節症患者（60 kgとして）においては、一日にっき約 0.01 〜30 mg程度、好ましくは約 0.1〜20 mg程度、より好ましくは約 0.1〜10 mg程度を投 与するのが好都合である。他の動物の場合も、 60 kg当たりに換算した量を投与する こと力 Sでさる。 [0045] Although the dose of the substance that activates the Ca / CaM pathway varies depending on the administration subject, target organ, symptom, administration method, etc., in the case of oral administration, generally, for example, patients with osteoarthritis (As 60 kg) is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of injection, it is usually treated with osteoarthritis patients (60 In terms of kg), it is convenient to apply about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, the amount converted per 60 kg is measured with the force S.
[0046] 本明細書において、塩基やアミノ酸などを略号で表示する場合、 IUPAC-IUB Com missionon Biochemical Nomenclatureによる略号あるいは当該分野における慣用略 号に基づくものであり、その例を下記する。またアミノ酸に関し光学異性体があり得る 場合は、特に明示しなければ L体を示すものとする。  [0046] In the present specification, bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there are optical isomers for amino acids, L form is shown unless otherwise specified.
DNA ：デォキシリボ核酸  DNA: Deoxyribonucleic acid
cDNA ：相補的デォキシリボ核酸  cDNA: complementary deoxyribonucleic acid
A ：アデニン  A: Adenine
T ：チミン  T: Thymine
G ：グァニン  G: Guanine
C ：シトシン  C: cytosine
RNA ：リボ核酸  RNA: Ribonucleic acid
mRNA ：メッセンジャーリボ核酸  mRNA: Messenger ribonucleic acid
dATP ：デォキシアデノシン三リン酸  dATP: Deoxyadenosine triphosphate
dTTP ：デォキシチミジン三リン酸  dTTP: Deoxythymidine triphosphate
dGTP ：デォキシグアノシン三リン酸  dGTP: Deoxyguanosine triphosphate
dCTP ：デォキシシチジン三リン酸  dCTP: Deoxycytidine triphosphate
ATP ：アデノシン三リン酸  ATP: Adenosine triphosphate
EDTA ：エチレンジァミン四酢酸 SDS ：ドデシル硫酸ナ 1 EDTA: ethylenediamine tetraacetic acid SDS: Dodecyl sulfate 1
Gly ：グリシン  Gly: Glycine
Ala ： ァラニン  Ala: Alanine
Val ： パリン  Val: Palin
Leu ： ロイシン  Leu: Leucine
lie ：イソロイシン  lie: Isoleucine
Ser ：セリン  Ser: Serine
Thr ：スレ才ニン  Thr: Thre Nin
Cys ：システィン  Cys: Sistine
Met ：メチォニン  Met: Methionine
Glu ：グルタミン酸  Glu: Glutamic acid
Asp ： ァスパラギン酸  Asp: Aspartic acid
Lys ：リジン  Lys: Lysine
Arg ：ァノレギニン  Arg: Anoleginin
His ： ヒスチジン  His ： Histidine
Phe ： フエニノレアラニン  Phe: Phenylenolanine
Tyr ：チロシン  Tyr: Tyrosine
Trp ：トリブトファン  Trp: Tribute fan
Pro ：プロリン  Pro: Proline
Asn ： ァスパラギン  Asn: Asparagine
Gin ：グノレタミン Gin: Gnoretamine
Glu ：ピログルタミン酸  Glu: pyroglutamic acid
Sec •セレノ、 スティン (  Sec • Seleno, Sting (
[0047] 配列番号： 1において、 SNP部位は「塩基配列またはアミノ酸配列を含む明細書等 の作成のためのガイドライン」（平成 14年 7月）の付属書 2、表 1に示される択一的記号 により表記している。 [0047] In SEQ ID NO: 1, the SNP site is an alternative shown in Appendix 2, Table 1 of “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (July 2002). This is indicated by the symbol.
実施例  Example
[0048] 以下に実施例を挙げて本発明をより具体的に説明する力 これらは単なる例示で あって本発明を何ら限定するものではない。 [0048] The ability to explain the present invention more specifically with reference to the following examples. Therefore, the present invention is not limited at all.
[0049] 実施例 1 定量的 PCRによる、軟骨細胞におけるカルモジュリン遺伝子発現解析 正常ヒト軟骨細胞 (nHAC)は Cambrex社より購入した。 nHACを chondrocyte growth medium (Cambrex社)で約 90%の密度になるまで培養した後、細胞をアルジネートビー ズに包埋し、 chondrocytedifferentiation medium (Cambrex社）で培養した。培地は 3も しくは 4日間おきに新鮮な chondrocytedifferentiation mediumに置換した。アルジネー トビーズへの包埋から 3週間後、 Hauselmannら（J. Cell Sci. 107 (Ptl): 17-27 (1994)) の方法に従い、軟骨細胞を単離した。その後、単離した細胞から Isogen (二ツボンジ ーン）を用いて total RNAを回収した。回収した total RNA 500 ngに対して、 Multiscrib e reverse transcriptase (Applied biosystems)および random hexamerを用レヽ飞逆 与 を行い、 cDNAを得た。カルモジュリン遺伝子 (CALM 1， CALM2および CALM3)の定 量は、 QuantiTect STBR Green PCR (Qiagen)を用いて、 ABI PRISM 7700 sequence detection system (Applied biosystems)上で、添付文書の方法に従つ飞ィ丁った。退伝 子定量に用いたプライマーを、表 1に記す。各遺伝子のコピー数は、検量線を用いて 算出した。また、各遺伝子量は、 GAPDH遺伝子を指標として定量した total RNA量で 割り込み、標準化した。  Example 1 Analysis of Calmodulin Gene Expression in Chondrocytes by Quantitative PCR Normal human chondrocytes (nHAC) were purchased from Cambrex. After culturing nHAC with chondrocyte growth medium (Cambrex) to a density of about 90%, the cells were embedded in alginate beads and cultured with chondrocyte differentiation medium (Cambrex). The medium was replaced with fresh chondrocyte differentiation medium every 3 or 4 days. Three weeks after embedding in alginate beads, chondrocytes were isolated according to the method of Hauselmann et al. (J. Cell Sci. 107 (Ptl): 17-27 (1994)). Thereafter, total RNA was recovered from the isolated cells using Isogen (Nibonbon Gene). Multicycle reverse transcriptase (Applied biosystems) and random hexamer were used for 500 ng of the recovered total RNA, and cDNA was obtained. Calmodulin genes (CALM 1, CALM2 and CALM3) were quantified using the QuantiTect STBR Green PCR (Qiagen) on the ABI PRISM 7700 sequence detection system (Applied biosystems) according to the method in the package insert. It was. Table 1 shows the primers used for quantification of remission. The copy number of each gene was calculated using a calibration curve. The amount of each gene was standardized by interrupting the total RNA amount quantified using the GAPDH gene as an index.
その結果、軟骨細胞においては、 CALM2遺伝子発現量が最も高力、つた（図 1)。そ の量は、 CALM1遺伝子の約 3倍であった。一方、 CALM3遺伝子の発現量は最も低く 、 CALM1遺伝子の 1/3程度であった。この結果は、 CALM2遺伝子がタンパク質レべ ルでのカルモジュリン総量に対して、最も寄与してレ、る可能性を示唆する。  As a result, CALM2 gene expression level was the highest in chondrocytes (Fig. 1). The amount was about 3 times that of the CALM1 gene. On the other hand, the expression level of the CALM3 gene was the lowest, about 1/3 that of the CALM1 gene. This result suggests that the CALM2 gene may contribute most to the total amount of calmodulin at the protein level.
[0050] [表 1コ カルモジュリン遺伝子の定量に用いたプライマー  [0050] [Table 1 Primers used for quantification of cocalmodulin gene
標的遺伝子 酸!?列  Target gene Acid !?
フォワー £ドプτ 2ライマー リバースプライマー  Forward £ Dop τ 2 Laimer Reverse Primer
CALM! GGACAAGTCAACTATGAAGAATTCG CCACCAACCAATACATGCAG CALM! GGACAAGTCAACTATGAAGAATTCG CCACCAACCAATACATGCAG
CALM2 GATGGTCAAGTAAACTATGAAGAGTTTG CCAGAGTAAGCCACATGCAACALM2 GATGGTCAAGTAAACTATGAAGAGTTTG CCAGAGTAAGCCACATGCAA
CALM3 TGGCCAGGTCAATTATGAAGAGT GGCATGGAGAGAGACTCAGG CALM3 TGGCCAGGTCAATTATGAAGAGT GGCATGGAGAGAGACTCAGG
[0051] 実施例 2 オリゴヌクレオチドマイクロアレイ解析による正常および OA関節軟骨にお けるカルモジュリン遺伝子の発現量の比較 CALM1, CALM2,および CALM3の発現解析は約 6万種類のターゲット配列に対す るオリゴヌクレオチドプローブから構成されるマイクロアレイセット（GeneChip U95およ び U133, Affymetrix)を用いて行った。ビォチン化 cRNAの調製とアレイハイブリダィゼ ーシヨンなどの一連の操作 (ま Affymetrix GeneChip expression analysis manualに準し て行った。変形性関節症軟骨および正常関節軟骨の RNAは社内倫理委員会の承認 の下に Direct Clinical Access社より購入した。まず Total RNA 5-lOmgを铸型とし、 T7 -poly Ί primerと: superscript II (Invitrogen)を用い飞 rirst strand cDNAを合成しに。次 に、 b.coli DNA polymerase i (Invitrogenノと ligase (Invitrogen)を用いて secona strand cDNAを合成した。得られた二本鎖 cDNAを用いてビォチン化 UTPと CTP (Enzo Diagn ostics)の子在 Cin vitro transcription (Ambioruをネ丁つた。ビォテン化 cRNAを Tris(p H8.1), lOOmM酢酸カリウム， 30mM酢酸マグネシウムを含むバッファ一中で 94°Cで 3 0分間インキュベートすることにより断片化し、アレイハイブリダィゼーシヨンを行った。 洗浄および染色は専用の Fluidics Station (Affymetrix)を用いて行った。シグナルは 専用の Confocal Scanner (Molecular Dynamics)を用いて検出した。アレイハイブリダィ セーシヨンシグアノレは GeneChip analysis softwareによって resence と半 IJ疋 れた全 遺伝子のシグナル値の中央値を 1として標準化した。上記の方法により、正常膝関節 軟骨 (n=l)、変形性股関節症患者由来関節軟骨 (n=5)、正常股関節軟骨 (n=2)、お よび変形性膝関節症患者（n=4)より抽出した total RNAから作製したビォチン化 cRNA を用いて CALMl, CALM2および CALM3のマイクロアレイ解析を行った結果、変形性 股関節症と変形性膝関節症患者由来関節軟骨の両方において各々の関節由来正 常軟骨と比べて CALM2 mRNA発現量が増加していることがわかった（図 2)。すなわ ち、 CALM2がヒト OA関節軟骨で発現量が上昇する遺伝子であることが明らかとなつ た。 [0051] Example 2 Comparison of expression levels of calmodulin gene in normal and OA articular cartilage by oligonucleotide microarray analysis CALM1, CALM2, and CALM3 expression analyzes were performed using a microarray set (GeneChip U95 and U133, Affymetrix) consisting of oligonucleotide probes for approximately 60,000 target sequences. A series of operations such as biotinylated cRNA preparation and array hybridization (also performed according to the Affymetrix GeneChip expression analysis manual. RNA for osteoarthritic cartilage and normal articular cartilage was approved by the in-house ethics committee. Purchased from Direct Clinical Access Co., Ltd. First, synthesize total RNA 5-lOmg in 铸 shape, synthesize 飞 rst strand cDNA using T7-poly Ί primer and: superscript II (Invitrogen), then b.coli Secona strand cDNA was synthesized using DNA polymerase i (Invitrogen and ligase (Invitrogen). Using the resulting double-stranded cDNA, biotinylated UTP and CTP (Enzo Diagn ostics) were born. Cin vitro transcription (Ambioru Biotinylated cRNA was fragmented by incubating at 94 ° C for 30 minutes in a buffer containing Tris (pH 8.1), lOOmM potassium acetate, 30 mM magnesium acetate, and array hybridization was performed. Cleaning and cleaning Staining was performed using a dedicated Fluidics Station (Affymetrix), signals were detected using a dedicated Confocal Scanner (Molecular Dynamics), and array hybridization assay was performed using GeneChip analysis software. The median signal value of all genes was normalized as 1. Normal knee joint cartilage (n = l), articular cartilage from osteoarthritis patients (n = 5), normal hip joint cartilage (n = 2) and microarray analysis of CALMl, CALM2 and CALM3 using biotinylated cRNA prepared from total RNA extracted from knee osteoarthritis patients (n = 4). It was found that CALM2 mRNA expression was increased in both articular cartilage from patients with knee arthritis compared to normal cartilage from each joint (Fig. 2), that is, CALM2 was expressed in human OA articular cartilage. Genetics that increase in quantity It became clear that she was a child.
実施例 3 CALM2遺伝子領域における、遺伝子多型のディスカバリー Example 3 Discovery of genetic polymorphism in the CALM2 gene region
CALM2遺伝子上の多型と変形性股関節症の相関解析を行うため、飯田ら [Hda A, Ozaki , Ί anaKa Γ and Nakamura ί (2005) rine-scaie SNP map of an 丄丄一Kb genomi c region at 22ql3.1 containing the galectin-1 gene. J Hum Genet 50: 42-45]の万法 に従い、 CALM2遺伝子領域における多型ディスカバリーを行った。方法を簡単に記 す。 Genbankデータベースに報告のあるゲノム配列（ァクセッション番号: AC073283.8 )を元に、 CALM2遺伝子のプロモータ領域、イントロン 1、および全てのェクソンとその 周辺領域を増幅するプライマーを作成した。これらのプライマーを用いて、 3個人の混 合ゲノム DNA 20 ngに対して PCRを行った。ゲノム DNAは 48人から抽出した（すなわ ち、混合ゲノムの数は 48/3 = 16となる)。 PCRは、 GeneAmp PCRシステム（Applied bio systems)を用いて、以下の条件で行った。すなわち、初期変性： 94度で 2分間、サイク リングステップ（35回サイクル）：94度 30秒間（熱変性）→60度で 30秒間（アニーリング） →72度で 2分間（伸長反応）、最終伸長反応：72度で 7分間、の条件で行った。配列 決定は、蛍光 dyeターミネータ一を用いたサイクルシークェンス法により、 PCR反応産 物を铸型として行った。全ての遺伝子多型はポリフレッドコンピュータープログラムを 用いて決定した。その結果、 CALM2領域には、 16個の一塩基多型と 2個の挿入/欠 失多型があることが明らかになった（図 3、表 2)。そのうち、 10個の多型は NCBIの公 開している SNPデータベースへの登録がなぐ新規のものであった（表 2)。 To analyze the correlation between polymorphisms on the CALM2 gene and hip osteoarthritis [Hda A, Ozaki, ΊanaKa Γ and Nakamura ί (2005) rine-scaie SNP map of an 丄 丄 一 Kb genomi c region at The polymorphism discovery in the CALM2 gene region was performed according to the universal method of 22ql3.1 containing the galectin-1 gene. J Hum Genet 50: 42-45]. Briefly describe how The Based on the genome sequence reported in the Genbank database (accession number: AC073283.8), primers were prepared to amplify the promoter region of CALM2 gene, intron 1, and all exons and their surrounding regions. Using these primers, PCR was performed on 20 ng of mixed genomic DNA of 3 individuals. Genomic DNA was extracted from 48 people (ie, the number of mixed genomes was 48/3 = 16). PCR was performed using a GeneAmp PCR system (Applied bio systems) under the following conditions. That is, initial denaturation: 94 ° C for 2 minutes, cycling step (35 cycles): 94 ° 30 seconds (thermal denaturation) → 60 ° C for 30 seconds (annealing) → 72 ° C for 2 minutes (extension reaction), final extension Reaction: The reaction was performed at 72 ° C. for 7 minutes. Sequencing was carried out using the PCR reaction product in a vertical pattern by the cycle sequence method using a fluorescent dye terminator. All genetic polymorphisms were determined using the Polyfred computer program. As a result, it was revealed that there are 16 single nucleotide polymorphisms and 2 insertion / deletion polymorphisms in the CALM2 region (Figure 3, Table 2). Of these, 10 polymorphisms were new and were not registered in the SNP database published by NCBI (Table 2).
[表 2] [Table 2]
日本人サンプルを用いた多型ディスカバリーによって同定された、 CALM2領域の多型情報 Polymorphism information of CALM2 region identified by polymorphism discovery using Japanese samples
多型番号 存在領域の種類 場所3 5 ' フランキング配歹 ϋ 多型 3 ' フランキング配列 dbSNPでの報告 i- -CALM2- -1 5，フランキング領域 -2514 gatacatacatctctgagca [T/C] gcacctgtatttcagactt rs l353644Polymorphism number Type of existing region Location 3 5 'flanking distribution ϋ polymorphism 3' flanking sequence Reported by dbSNP i- -CALM2- -1 5, flanking region -2514 gatacatacatctctgagca [T / C] gcacctgtatttcagactt rs l353644
-CALM2- -2 5'フランキング領域 -268 tcgccgccacgatgctcctc [T/C] gcgccgcaaagtagcgccc -CALM2- -2 5 'flanking region -268 tcgccgccacgatgctcctc [T / C] gcgccgcaaagtagcgccc
i- -CALM2- -3 イン卜ロン 1 451 cgcctttgtccgcttgcggc [A/C] gccatgcgcccagggggcg rs 878665 i- -CALM2- -4 イン卜ロン 1 1362 gaaaaaaaaaaaaaaaaaca [C/G] ctaatgagggcggtgatct i- -CALM2- -3 Inron 1 451 cgcctttgtccgcttgcggc [A / C] gccatgcgcccagggggcg rs 878665 i- -CALM2- -4 Inron 1 1362 gaaaaaaaaaaaaaaaaaca [C / G] ctaatgagggcggtgatct
-CALM2- -5 イン卜ロン 1 2622 tta attgcttttgatacaa [G/A] taaaacaaatatcctagct  -CALM2- -5 Yin Ron 1 2622 tta attgcttttgatacaa [G / A] taaaacaaatatcctagct
i- -CALM2- -6 イン卜ロン 1 3001 ctgagagtatactaatgcct [G/A] ggaactttctctggagtgg i- -CALM2- -6 In 卜 ron 1 3001 ctgagagtatactaatgcct [G / A] ggaactttctctggagtgg
i- -CALM2- -7 イン卜ロン 1 3605 tatatagtagccttcccaaa [G/A] tcccatttcccaccccccc rs l723484 i- -CALM2- - 8 イン卜ロン 1 4160 aacttgatgctttcccccca [C/T] gccacgcatgccaggttgc i- -CALM2- -7 IN 卜 RON 1 3605 tatatagtagccttcccaaa [G / A] tcccatttcccaccccccc rs l723484 i- -CALM2--8 IN 卜 RON 1 4160 aacttgatgctttcccccccc [C / T] gccacgcatgccaggttgc
i- -CALM2- - 9 イン卜ロン 1 4516 ttgagcaga aaaataaact [G/A] ctttctggattccttcttg rs 815815 i- -CALM2- - 10 イン卜ロン 1 5034 a tgaataaggataaaattt [G/A] taatgaattttagcaaatg i- -CALM2--9 in 卜 ron 1 4516 ttgagcaga aaaataaact [G / A] ctttctggattccttcttg rs 815815 i- -CALM2--10 in 卜 ron 1 5034 a tgaataaggataaaattt [G / A] taatgaattttagcaaatg
i- -CALM2- - 11 イン卜ロン 1 5186 atttggtaaatattatag a [T/C] tgtagttcagagctctgga rs 815816 i- -CALM2- 12 イン卜ロン 2 104 aataattgactttggtatat [G/A] tcatggatactaacatatg i- -CALM2--11 In 卜 ron 1 5186 atttggtaaatattatag a [T / C] tgtagttcagagctctgga rs 815816 i- -CALM2- 12 In 卜 ron 2 104 aataattgactttggtatat [G / A] tcatggatactaacatatg
i- -CALM2- 13 イントロン 2 7815 gatatatttgaaatattatc [ G/A] taaggtatcataaagattg rs l7036325 i- -CALM2- ■14 イン卜ロン 2 フ 84フ aaagattggttaatctttag [A/G] agtcaaagtcacatttcaa rs l027478 i- -CALM2- ■15 イントロン 2 8039 ttacattttattattaatct t G/A] tgtatttggattgtggtct i- -CALM2- 13 intron 2 7815 gatatatttgaaatattatc [G / A] taaggtatcataaagattg rs l7036325 i- -CALM2- ■ 14 in 卜 ron 2 f 84 aaagattggttaatctttag [A / G] agtcaaagtcacatttcaa rs l027478 i- -CALM2- ■ 15 intron 2 8039 ttacattttattattaatct t G / A] tgtatttggattgtggtct
i- -CALM2- •16 イントロン 5 97 aaattagtgacccttcccct [ C/G] aaattaggttgctaaaaca rs2454084 i- -CALM2- ■17 イン卜ロン 1 4308-4309 ttccttgggtaaacaatctg [TG/欠失] actgacttcacaccaaata i- -CALM2- • 16 intron 5 97 aaattagtgacccttcccct [C / G] aaattaggttgctaaaaca rs2454084 i- -CALM2- ■ 17 in 卜 ron 1 4308-4309 ttccttgggtaaacaatctg [TG / deletion] actgacttcacaccaaata
i- -CALM2- 18 イン卜ロン 5 97-98 aattagtgacccttcccctg [ C/揷入] aaattaggttgctaaaaca i- -CALM2- 18 In 卜 ron 5 97-98 aattagtgacccttcccctg [C / Iritsu] aaattaggttgctaaaaca
'場所番号の定義は den Dunnen and Antonarakis ( 2000 )らの方法に従った 'Definition of place numbers follows the method of den Dunnen and Antonarakis (2000)
[0054] 実施例 4 CALM2遺伝子上の多型と変形性股関節症の相関解析 [0054] Example 4 Correlation analysis between polymorphism on CALM2 gene and osteoarthritis of the hip
CALM2遺伝子上の多型について、変形性股関節症患者群と、対照群についてジ エノタイピングを行い、その結果を用いて相関解析を行った。変形性股関節症患者 群 (ケース）としては、 138人の臼蓋形成不全症併発患者を含む、 357人を用いた。対 照群（コントロール）としては 375人を用いた。実施例 3で得られた CALM2遺伝子上の 多型について、 Assays-by-Designサービス（Applied biosystems)によってジエノタイピ ング用 TaqManプローブを作成した。この TaqManプローブを用いて、各個人から採取 したゲノム DNAを試料として 384マルチウエルプレート上でジエノタイピング反応を行 つた。検出は、 ABI Prism 7900 (Applied biosystems)を用いて行い、 Sequence Detecti on Software (Applied biosystems)を用いて解析した。相関角早析、ノヽーディ'ワインべ ノレグ平衡の計算は、山田ら（Yamada R， Tanaka T， Unoki Μ， Nagai Τ， Sawada Τ， Oh nishi Y, Tsunoda T， Yukioka M, Maeda A, Suzuki , Tateishi H, Ochi T， Nakamura Y and Yamamoto (2001) Association between a single-nucleotide polymorphism in the promoter of the human interleuKin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelihood estimation of combinatorial effect that two geneti c loci have on susceptibility to the disease. Am J Hum Genet 68: 674-685)の方法 を参考にして行った。結果を表 3にまとめる。 CALM2遺伝子領域における多型は、い ずれの多型についても、全ての解析モデル（アレル、ジエノタイプ、優性および劣性 モデル)で変形性股関節症との相関は検出されな力、つた。  The polymorphisms in the CALM2 gene were genotyped for the hip osteoarthritis patient group and the control group, and the correlation analysis was performed using the results. As the hip osteoarthritis patient group (case), 357 people including 138 acetabular dysplasia patients were used. As a control group (control), 375 people were used. For the polymorphism on the CALM2 gene obtained in Example 3, a TaqMan probe for dienotyping was prepared by Assays-by-Design service (Applied biosystems). Using this TaqMan probe, a genomic DNA collected from each individual was used as a sample to perform a dienotyping reaction on a 384 multiwell plate. Detection was performed using ABI Prism 7900 (Applied biosystems) and analyzed using Sequence Detecti on Software (Applied biosystems). Correlation angle pre-analysis and Noody's wine benoleg equilibria are calculated by Yamada et al. (Yamada R, Tanaka T, Unoki Μ, Nagai Τ, Sawada Τ, Ohnishi Y, Tsunoda T, Yukioka M, Maeda A, Suzuki, Tateishi H, Ochi T, Nakamura Y and Yamamoto (2001) Association between a single-nucleotide polymorphism in the promoter of the human interleuKin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelihood estimation of combinatorial effect that two geneti c loci have On susceptibility to the disease. Am J Hum Genet 68: 674-685). The results are summarized in Table 3. The polymorphisms in the CALM2 gene region were strong enough to detect no correlation with osteoarthritis in all analysis models (alleles, dienotypes, dominant and recessive models).
[0055] [表 3] [0055] [Table 3]
CALM2領域の多型と変形性股関節症の相関解析 Correlation analysis between polymorphism of CALM2 region and osteoarthritis of the hip
ケース（変形性股閲節症） _ コン卜 α-ル 相 （P『S) ジエノタイプの分布 ジエノタイプの分布  Case (degenerative crotch ganglia) _ kong α-ru phase (P'S) dienotype distribution dienotype distribution
HWEC test HWE c test HWE C test HWE c test
多型番号 11 12 22 総和 MA 11 12 22 総和 アレル ジエノタイプ 優性モデル劣性モデル  Polymorphism number 11 12 22 Sum MA 11 12 22 Sum Allele Dienotype Dominant model Recessive model
(P) (P (P) (P
-CALM2-1 2 59 295 356 0.09 0 .87 2 63 310 3フ 5 0 .09 0 .82 0.95 1.00  -CALM2-1 2 59 295 356 0.09 0 .87 2 63 310 3F 5 0 .09 0 .82 0.95 1.00
1-CALM2-2 0 1 356 357 0.001 1 .00 0 0 375 375 0 .00 0.31 0.31 i-CALM2-4 1 17 339 357 0 • 31 0 21 354 375 0 .03 0 • 86 0.31 i-CALM2-5 310 45 2 357 0.07 0 • 97 304 61 3 368 0 .09 1 • 00 0.12 0.11 - CALM2 - 6 0 13 344 357 0.02 0 ,94 0 8 36フ 375 0 • 01 0 .98  1-CALM2-2 0 1 356 357 0.001 1 .00 0 0 375 375 0 .00 0.31 0.31 i-CALM2-4 1 17 339 357 0 • 31 0 21 354 375 0 .03 0 • 86 0.31 i-CALM2-5 310 45 2 357 0.07 0 • 97 304 61 3 368 0 .09 1 • 00 0.12 0.11-CALM2-6 0 13 344 357 0.02 0, 94 0 8 36 F 375 0 • 01 0 .98
CALM2- 7 5 83 262 350 0 o.13 0 .86 フ 91 274 372 0 •14 0 .98 0.86 CALM2- 7 5 83 262 350 0 o.13 0 .86 F 91 274 372 0 • 14 0 .98 0.86
-CALM2-9 oマ  -CALM2-9 o Ma
5 87 262 354 0 .76 7 92 269 368 0 •14 0 .96  5 87 262 354 0 .76 7 92 269 368 0 • 14 0 .96
i-CALM2-10 0 3 354 357 0.004 1 .00 0 5 364 369 0 .01 0 .99 0.51 0.51 i一 CALM2- 11 264 87 5 356 0 • 7フ 2Ί2 94 9 375 0 .15 0 • 9フ 0.62 ΐ- CALM2-12 353 3 0 356 0.004 1, .00 371 1 0 372 0. 001 1 • 00 0.30 i-CALM2-10 0 3 354 357 0.004 1 .00 0 5 364 369 0 .01 0 .99 0.51 0.51 i 1 CALM2-11 264 87 5 356 0 • 7F 2Ί2 94 9 375 0 .15 0 • 9F 0.62 ΐ- CALM2-12 353 3 0 356 0.004 1, .00 371 1 0 372 0. 001 1 • 00 0.30
i-CALM2-13 2 42 313 357 0.06 0 .90 2 44 324 370 0, .06 0 .93 0,9フ 1.00 0.97 一 CALM2-17 8 94 254 356 0, .98 9 98 268 375 0< .15 1 ■ 00 0.99 0.97 数字の 1と 2はそれぞれ、表 2の多型のカラムの左および右の塩基に対応する i-CALM2-13 2 42 313 357 0.06 0 .90 2 44 324 370 0, .06 0 .93 0,9 1.00 0.97 1 CALM2-17 8 94 254 356 0, .98 9 98 268 375 0 <.15 1 ■ 00 0.99 0.97 The numbers 1 and 2 correspond to the left and right bases of the polymorphic column in Table 2, respectively.
b; マイナ一ァレノレ頻度 (minor allele frequency)b; minor allele frequency
; メヽーディワインべノレグ平衡 （Hardy— Weinberg Equilibrium)  ; Medieval Wine Benoreg Equilibrium (Hardy— Weinberg Equilibrium)
d; 計算不可 d; cannot be calculated
o o
o o  o o
o ο  o ο
ο o ο ο  ο o ο ο
ο  ο
ο ο [0056] 実施例 5 CALM2遺伝子上の多型と、臼蓋形成不全症を伴わない変形性股関節症 の相関解析 ο ο Example 5 Correlation analysis of polymorphism on CALM2 gene and hip osteoarthritis without acetabular dysplasia
変形性股関節症群を、臼蓋形成不全症を伴う患者群と伴わない患者群に分け、実 施例 4の方法に従い相関解析を行った。その結果、 i-CALM2-5および i_CALM2-6が 臼蓋形成不全症を伴わない患者群で有意な相関を示した (Pく 0.05を有意水準とした 場合)。 i-CALM2-5についてはメジャーアレルの頻度力 i-CALM2-6についてはマイ ナーアレルの頻度力 ケース群で高力、つた（表 4)。この結果は、 CALM2遺伝子が臼 蓋形成不全症を伴わない変形性股関節症の感受性遺伝子であることを示唆する。  The hip osteoarthritis group was divided into a patient group with and without acetabular dysplasia, and a correlation analysis was performed according to the method of Example 4. As a result, i-CALM2-5 and i_CALM2-6 showed a significant correlation in patients without acetabular dysplasia (when P was 0.05). For i-CALM2-5, frequency power of major alleles For i-CALM2-6, frequency power of minor alleles was high in the case group (Table 4). This result suggests that the CALM2 gene is a susceptibility gene for osteoarthritis of the hip without acetabular dysplasia.
[0057] [表 4] [0057] [Table 4]
Figure imgf000033_0001
実施例 6 臼蓋形成不全症を伴わなレ、変形性股関節症患者群と対照群における連 鎖不平衡マップの作成
Figure imgf000033_0001
Example 6 Creation of linkage disequilibrium maps in patients with acetabular dysplasia, hip osteoarthritis patients and controls
ハプロタイプ相関解析を行うために、臼蓋形成不全症を伴わな!/、変形性股関節症 患者群（219人、ケース）、および対照群（375人、コントロール）について、それぞれ 2 多型間について連鎖不平衡定数 D 'の計算を行った。多型は、マイナーアレルの頻 度が 0.はり大きいものを選択して使用した。計算方法は、実施例 4で記載した山田ら の方法に従った。その結果、ケースおよびコントロールともに、各 2多型間の D 'は 0.6 以上となった（表 5)。この結果は、用いた多型によって決定される CALM2遺伝子領 域力 ひとつの連鎖不平衡ブロックにあることを示唆する。 To perform haplotype correlation analysis, do not accompany acetabular dysplasia! /, Osteoarthritis of the hip For the patient group (219 cases) and the control group (375 controls), the linkage disequilibrium constant D ′ was calculated between the two polymorphisms. The polymorphism was selected with a minor allele frequency of 0. larger. The calculation method followed the method of Yamada et al. Described in Example 4. As a result, in both cases and controls, the D 'between the two polymorphisms was 0.6 or more (Table 5). This result suggests that the CALM2 gene region force determined by the polymorphism used is in a single linkage disequilibrium block.
[表 5] [Table 5]
Figure imgf000035_0001
Figure imgf000035_0001
[0060] 実施例 7 臼蓋形成不全症を伴わない変形性股関節症患者群を用いたハプロタイプ 角早析 [0060] Example 7 Haplotype angular segregation using osteoarthritis patient group without acetabular dysplasia
実施例 6で使用した多型を用いて、それぞれの集団（ケースおよびコントロール）に おけるハプロタイプ構造を推定した。ハプロタイプ構造の推定には、 Excoffierらの方 法 [Excoffier し and ^latkin M (1995) Maximum-likelihood estimation of molecular ha plotypefrequencies in a diploid population. Mol Biol Evol 12: 921-927]を参考とした 。その結果、この領域には、主に 5種類のハプロタイプが存在することが明らかとなつ た。また、それらのハプロタイプ頻度の合計は、全体の 95%を占めていた。これらのデ ータを用いてハプロタイプ相関解析を行ったところ、ハプロタイプ IIIが臼蓋形成不全 症を伴わない変形性股関節症と有意な相関を示した（有意水準を Pく 0.05とした場合) 。ハプロタイプ IIIの頻度は、コントロール群で頻度が高かった（表 6)。この結果は、実 施例 5とともに、 CALM2遺伝子が臼蓋形成不全症を伴わない変形性股関節症の感 受性遺伝子であることを示唆する。  Using the polymorphism used in Example 6, the haplotype structure in each population (case and control) was estimated. For estimation of the haplotype structure, the method of Excoffier et al. [Excoffier and ^ latkin M (1995) Maximum-likelihood estimation of molecular haplotype frequency in a diploid population. Mol Biol Evol 12: 921-927] was used as a reference. As a result, it has become clear that there are mainly five types of haplotypes in this area. The total frequency of those haplotypes accounted for 95% of the total. When haplotype correlation analysis was performed using these data, haplotype III showed a significant correlation with hip osteoarthritis without acetabular dysplasia (when the significance level was set to P 0.05). The frequency of haplotype III was higher in the control group (Table 6). This result, together with Example 5, suggests that the CALM2 gene is a susceptibility gene for osteoarthritis of the hip without acetabular dysplasia.
[0061] [表 6] [0061] [Table 6]
臼盖形成不全症を伴^ ίし、変形性股関節症を用いたハプロタイプ相関解析 Haplotype correlation analysis using osteoarthritis associated with acetabular dysplasia
ノ、 \づノ u！々 ,ィつ "ノ^ /、プロタイプ頻度 多型 （ -CALM2 No, \ Zuno u! , Itsu "no ^ /, Protype frequency polymorphism (-CALM2
/ P値  / P value
ケース コントロール 1 4 5 7 17 9 11 13  Case control 1 4 5 7 17 9 11 13
I 0.76 2 2 1 2 2 2 1 2
Figure imgf000037_0001
I 0.76 2 2 1 2 2 2 1 2
Figure imgf000037_0001
ο  ο
o o 産業上の利用可能性 oo Industrial applicability
[0062] 本発明によれば、骨 ·関節疾患（例えば OA)に対する遺伝的感受性を容易に判定 すること力 Sできる。また、本発明により該骨 ·関節疾患に感受性であると判定された被 験者を投与対象とする本発明の骨'関節疾患予防'治療剤は、 Ca/CaM経路を介し た骨 ·関節疾患に対する抑制作用が不十分であり得るヒトのみを対象とするので、予 防 ·治療効果が顕著に得られうる。  [0062] According to the present invention, it is possible to easily determine genetic susceptibility to a bone / joint disease (for example, OA). In addition, the bone 'joint disease prevention' therapeutic agent of the present invention to be administered to a subject who has been determined to be susceptible to the bone / joint disease according to the present invention is an agent for bone / joint disease via the Ca / CaM pathway. Since only humans that may have insufficient inhibitory effects are targeted, prophylactic / therapeutic effects can be obtained significantly.
[0063] 本発明を好まし!/ヽ態様を強調して説明してきたが、好まし!/、態様が変更され得るこ とは当業者にとって自明であろう。本発明は、本発明が本明細書に詳細に記載され た以外の方法で実施され得ることを意図する。したがって、本発明は添付の「請求の 範囲」の精神および範囲に包含されるすべての変更を含むものである。  [0063] While the present invention has been described with emphasis on the preferred embodiment, it will be apparent to those skilled in the art that the preferred embodiment can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.
本出願は、 日本で出願された特願 2006-293216 (出願日：平成 18年 10月 27日）を基 礎としており、そこに開示される内容は本明細書にすべて包含されるものである。また 、ここで述べられた特許および特許出願明細書を含む全ての刊行物に記載された内 容は、ここに引用されたことによって、その全てが明示されたと同程度に本明細書に 組み込まれるものである。  This application is based on Japanese Patent Application No. 2006-293216 filed in Japan (filing date: October 27, 2006), and all the contents disclosed therein are included in this specification. . In addition, the contents described in all publications including the patents and patent application specifications mentioned herein are incorporated herein by reference to the same extent as if all were specified. Is.

Claims

請求の範囲 The scope of the claims
[1] 配列番号： 1で表される塩基配列中、塩基番号 5227で示される塩基（但し、該塩基 はアデニンである）もしくは塩基番号 5606で示される塩基（但し、該塩基はグァニン である）を含む該塩基配列の部分配列であって、約 15塩基以上の連続した塩基配列 を含む核酸。  [1] In the base sequence represented by SEQ ID NO: 1, the base represented by base number 5227 (where the base is adenine) or the base represented by base number 5606 (however, the base is guanine) A nucleic acid comprising a partial sequence of the base sequence comprising a continuous base sequence of about 15 bases or more.
[2] 配列番号： 1で表される塩基配列において、塩基番号 21、 3967、 5227、 6210、 7121 [2] In the base sequence represented by SEQ ID NO: 1, base numbers 21, 3967, 5227, 6210, 7121
、 7791および 16127で示される塩基がそれぞれシトシン、グァニン、アデニン、アデ二 ン、アデニン、チミンおよびアデニンであり、塩基番号 6913〜6914で示される塩基が 欠失してレ、るハプロタイプの塩基配列を含む核酸。 , 7791 and 16127 are cytosine, guanine, adenine, adenine, adenine, thymine and adenine, respectively, and the nucleotide sequence of base numbers 6913 to 6914 is deleted. Nucleic acid containing.
[3] 配列番号： 1で表される塩基配列において、塩基番号 5227で示される塩基および[3] In the base sequence represented by SEQ ID NO: 1, the base represented by base number 5227 and
/または塩基番号 5606で示される塩基における多型を検出することを含む、骨 ·関節 疾患に対する遺伝的感受性の診断方法。 A method for diagnosing genetic susceptibility to a bone / joint disease, comprising detecting a polymorphism in the base represented by the nucleotide number 5606.
[4] 配列番号： 1で表される塩基配列において、塩基番号 21、 3967、 6210、 6913〜6914[4] In the base sequence represented by SEQ ID NO: 1, base numbers 21, 3967, 6210, 6913-6914
、 7121、 7791および 16127で示される塩基もしくは塩基配列からなる群より選択される, 7121, 7791 and 16127, or selected from the group consisting of base sequences
1以上の塩基もしくは塩基配列における多型を検出することを含む、骨 ·関節疾患に 対する遺伝的感受性の診断方法。 A method for diagnosing genetic susceptibility to bone and joint diseases, comprising detecting a polymorphism in one or more bases or base sequences.
[5] 骨 ·関節疾患が、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎、滑膜炎、 代謝性関節症、スポーツによる関節障害、骨軟骨腫、骨腫瘍、軟骨腫瘍および先天 性骨系統疾患からなる群より選択される請求項 3または 4記載の方法。 [5] Osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders, osteochondroma, bone tumor, cartilage tumor and congenital bone system The method according to claim 3 or 4, which is selected from the group consisting of diseases.
[6] 配列番号： 1で表される CALM2遺伝子の塩基配列中、塩基番号 5227で示される塩 基がグァニンである、および/または塩基番号 5606で示される塩基がグァニンである アレルを保有するヒトに投与することを特徴とする、カルシウム/カルモジュリン経路 を活性化する物質を含有してなる骨 ·関節疾患予防 ·治療剤。 [6] A human having an allele in which the base represented by base number 5227 is guanine and / or the base represented by base number 5606 is guanine in the base sequence of CALM2 gene represented by SEQ ID NO: 1. An agent for the prevention / treatment of bone / joint diseases comprising a substance that activates the calcium / calmodulin pathway, wherein
[7] 物質がカルモジュリン、カルシニューリンおよびカルモジュリン依存性キナーゼ IIか らなる群より選択される 1以上のタンパク質の発現および/または活性を促進するも のである、請求項 6記載の剤。 [7] The agent according to claim 6, wherein the substance promotes the expression and / or activity of one or more proteins selected from the group consisting of calmodulin, calcineurin and calmodulin-dependent kinase II.
[8] 骨 ·関節疾患が、骨粗鬆症、変形性関節症、慢性関節リウマチ、関節炎、滑膜炎、 代謝性関節症、スポーツによる関節障害および先天性骨系統疾患からなる群より選 択される、請求項 6記載の剤。 [8] The bone / joint disease is selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports joint disorders and congenital bone system diseases The agent according to claim 6, which is selected.
PCT/JP2007/070893 2006-10-27 2007-10-26 Gene sensitive to bone/joint disease and use thereof WO2008050855A1 (en)

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