WO2008050855A1 - Gène sensible à une maladie des os/articulations et utilisation de celui-ci - Google Patents

Gène sensible à une maladie des os/articulations et utilisation de celui-ci 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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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

L'invention concerne un procédé destiné au diagnostic de la sensibilité génétique à une maladie des os/articulations, en particulier une maladie choisie dans le groupe constitué par l'ostéoporose, l'ostéoarthrite, l'arthrite rhumatoïde, l'arthrite, la synovite, l'arthropathie métabolique, un trouble des articulations induit par une activité sportive, l'ostéochondrome, la tumeur des os, la tumeur du cartilage et un trouble constitutionnel congénital de l'os. Ce procédé comprend la détection du polymorphisme se présentant dans un nucléotide au niveau du nucléotide n° 5227 et/ou dans un nucléotide au niveau du nucléotide n° 5606 dans la séquence nucléotidique représentée dans SEQ ID NO : 1. L'invention concerne également un agent prophylactique/thérapeutique pour une maladie des os/articulations comprenant une substance capable d'activer la voie du calcium-calmoduline, en particulier une substance capable d'exprimer et/ou de stimuler l'activité d'au moins une protéine choisie dans le groupe constitué par la calmoduline, la calcineurine et la kinase II dépendante de la calmoduline, qui peut être administrée à un patient humain dont a été déterminée la présence d'un allèle sensible à la maladie grâce au procédé de diagnostic mentionné ci-dessus.
PCT/JP2007/070893 2006-10-27 2007-10-26 Gène sensible à une maladie des os/articulations et utilisation de celui-ci WO2008050855A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HUANG Q.-Y. ET AL.: "The association of common Polymorphisms in the QPCT gene with bone mineral density in the Chinese population", J. HUM. GENET., vol. 52, no. 9, September 2007 (2007-09-01), pages 757 - 762, XP019520281 *
IKEGAWA S.: "Genome kara Mita Undoki Shikkan, (Henkeisei Kansetsusho ya Tsuikanban Hernia ni Okeru Genome Kaiseki)", THE BONE, vol. 20, no. 3, May 2006 (2006-05-01), pages 89 - 93, XP003021893 *
IKEGAWA S.: "Henkeisei Kansetsusho to Sono Gen'in Idenshi eno Approach", SAISHIN IGAKU, vol. 60, 2005, pages 153 - 160, XP003021891 *
IKEGAWA S.: "Taikeiteki Idenshi Tagata Kaiseki ni yoru Hone . Kansetsu no common disease Kanren Idenshi no Dotei to Sono Kino Kaiseki -Henkeisei Kansetsusho o Rei ni", MOLECULAR MEDICINE, vol. 42, no. 11, 2005, pages 1197 - 1201, XP003021892 *
MIYAMOTO Y.: "Henkeisei Kansetsusho ni Okeru Idenshi Tagata Kaiseki", THE JOURNAL OF THE JAPANESE ORTHOPAEDIC ASSOCIATION, vol. 80, no. 8, August 2006 (2006-08-01), pages S989 + ABSTR. NO. 2-1-S7-2, XP003021894 *

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