WO2012063829A1 - Novel gene, and osteoporosis testing method based on single nucleotide polymorphism on fong gene locus - Google Patents

Novel gene, and osteoporosis testing method based on single nucleotide polymorphism on fong gene locus Download PDF

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WO2012063829A1
WO2012063829A1 PCT/JP2011/075744 JP2011075744W WO2012063829A1 WO 2012063829 A1 WO2012063829 A1 WO 2012063829A1 JP 2011075744 W JP2011075744 W JP 2011075744W WO 2012063829 A1 WO2012063829 A1 WO 2012063829A1
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base
seq
osteoporosis
protein
sequence
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志郎 池川
郁代 稲葉
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独立行政法人理化学研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1014—Hydroxymethyl-, formyl-transferases (2.1.2)
    • 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
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    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to a test method for predicting or determining osteoporosis and a reagent used in the test method.
  • the present invention further relates to a gene associated with osteoporosis, a protein expressed from the gene, and an antibody against the protein.
  • osteoporosis is one of the most common diseases. Osteoporosis develops after middle age and about 80% of patients are women. Currently, more than 200 million people worldwide suffer from osteoporosis, and the number of patients is expected to increase rapidly as the population ages.
  • Bone mineral density is a phenotype commonly used in the examination of osteoporosis, and is a useful tool for evaluating the risk of fracture due to osteoporosis. It is widely known that BMD is affected by genetic factors, and the heritability is estimated to be 50-90%.
  • Non-Patent Documents 1-3 Several experimental approaches can be considered to identify loci involved in osteoporosis risk. For example, attempts have been made to identify candidate genes by extensive screening of genes related to bone metabolism and pathogenic genes of rare single gene bone diseases (Non-Patent Documents 1-3). However, only a few genes are reproducible.
  • GWAS genome-wide correlation analysis
  • An object of the present invention is to provide a method for accurately examining the onset risk of osteoporosis and the presence or absence of onset, and a test reagent used in the method.
  • the present invention also provides a gene associated with osteoporosis, a protein expressed from the gene, and an antibody against the protein.
  • SNP single nucleotide polymorphism
  • the present invention is as follows. (1) A method for determining the risk of osteoporosis and / or the presence or absence of osteoporosis, comprising analyzing a single nucleotide polymorphism present in the FONG locus and examining osteoporosis based on the analysis result. (2) The single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the 61st base of the base sequence of SEQ ID NO: 1 or a base in a linkage disequilibrium relationship with the base (1 ) Method.
  • (B) hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence 389 to 832 in the nucleotide sequence shown in SEQ ID NO: 2 or a probe that can be prepared from the nucleotide sequence; DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence.
  • (C) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 3.
  • A a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
  • B A protein having the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3, comprising an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 3.
  • the present invention it is possible to accurately and easily predict the risk of developing osteoporosis that has been difficult to predict. Moreover, the onset of osteoporosis can be determined accurately and simply. Therefore, the present invention contributes to the prevention and early treatment of osteoporosis.
  • a white portion indicates an untranslated region, and a black portion indicates a code region.
  • B The figure which shows the expression level of the FONG gene in each human tissue. The vertical axis shows the relative value of the FONG mRNA amount to the ⁇ -actin (ACTB) mRNA amount (mean ⁇ standard error based on two independent tests).
  • ACTB ⁇ -actin
  • test method of the present invention comprises analyzing the SNPs contained in the human FONG locus, and testing osteoporosis based on the analysis results, and / or the risk of developing osteoporosis and / or Or it is the determination method of the presence or absence of onset. That is, in the present invention, “examination” includes an examination of the risk of developing osteoporosis and an examination of the presence or absence of osteoporosis.
  • the FONG gene locus is a region including a gene encoding the FONG gene product, which is present in the long arm 33.1 region of human chromosome 2.
  • the FONG locus is preferably GenBank Accession No. 2 on human chromosome 2. It means the area from 20062258 to 200176734 in NC_00000.11.
  • SNPs present in the FONG locus include human rs7605378.
  • the rs number indicates the registration number of the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/) of the National Center for Biotechnology Information.
  • rs7605378 is present in intron 3 of the FONG locus.
  • rs7605378 is GenBank Accession No. It means a polymorphism of adenine (A) / cytosine (C) at the 50886343rd base of NT_005403.16, and when this base is A, the possibility of osteoporosis is high.
  • rs7605378 has a high possibility of osteoporosis in the order of AA> AC> CC.
  • rs7605378 a sequence having a total length of 121 bp including the SNP base and a region of 60 bp before and after that is shown in SEQ ID NO: 1.
  • the 61st base has a polymorphism.
  • a base corresponding to the above base is analyzed.
  • a base corresponding to the above-mentioned base means a corresponding base of the human FONG gene locus. That is, “analyzing a base corresponding to the above-mentioned base” includes analyzing the corresponding base in the sequence even if the sequence is slightly changed at a position other than the SNP due to a difference in race. It is.
  • the base to be analyzed in the present invention is not limited to the above, and a polymorphism of a base in linkage disequilibrium with the above base may be analyzed.
  • the “base in linkage disequilibrium with the above-mentioned base” satisfies the relationship of r 2 > 0.5, preferably r 2 > 0.8, more preferably r 2 > 0.9 with the above-mentioned base.
  • the base in linkage disequilibrium with the above base can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja).
  • DNA collected from a plurality of people can be identified by sequence analysis using a sequencer and searching for SNPs in linkage disequilibrium.
  • bases that are in linkage disequilibrium with the above bases include rs10194676, rs57696619, rs2346662, rs75777763, rs2689763, rs769699, rs796363, rs769957, rs769958, rs7976963, rs2689766, and rs59093685, 01. Details of these SNPs are shown in Table 1. In Table 1, r 2 represents an r 2 of each SNP against Rs7605378.
  • rs10194076 is GenBank Accession No. This means a polymorphism of thymine (T) / cytosine (C) at the 50888080 position of NT_005403.16.
  • T thymine
  • C cytosine
  • rs57696619 has a high possibility of osteoporosis when thymine (T) is deleted.
  • rs59036985 has a high possibility of osteoporosis when 4 bases (AAAG) are inserted.
  • the risk of osteoporosis increases in the order of homozygote of risk allele> heterozygote of risk allele and non-risk allele> nonzygote of non-risk allele.
  • sequences having a total length of 121 bp (124 bp in the case of rs59093685) including these SNP bases and a region of 60 bp before and after that are shown in SEQ ID NOs: 4 to 17, respectively.
  • the 61st base has a polymorphism.
  • the “61st base” may be appropriately read as the 61st to 64th bases in SEQ ID NO: 16.
  • the osteoporosis can be examined by examining the base type of the SNP and associating the obtained result with osteoporosis based on the above criteria.
  • the SNP may be analyzed alone, or a plurality of SNPs including at least one of the SNPs may be collectively analyzed (haplotype analysis).
  • the sequence of the FONG gene locus may be analyzed for the sense strand or the antisense strand.
  • the sample used for the analysis of the SNP contained in the FONG gene locus is not particularly limited as long as it is a sample containing chromosomal DNA. Examples thereof include body fluid samples such as blood and urine, cells such as oral mucosa, and hair such as hair. Can be mentioned. Although these samples can be used directly for the analysis of SNP, it is preferable to isolate chromosomal DNA from these samples by a conventional method and analyze it.
  • the analysis of SNPs contained in the FONG gene locus can be performed by a normal gene polymorphism analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, invader method and the like.
  • Sequence analysis can be performed by a normal method. Specifically, a sequence reaction is performed using a primer set at a position of several tens of bases on the 5 ′ side of a base showing polymorphism, and the type of base at the corresponding position is determined from the analysis result. can do. In addition, when performing a sequence, it is preferable to amplify the fragment
  • SNP analysis can be performed by examining the presence or absence of amplification by PCR.
  • a primer having a sequence corresponding to a region containing a base showing a polymorphism and having a 3 'end corresponding to each polymorph is prepared.
  • PCR can be performed using each primer, and the type of polymorphism can be determined depending on the presence or absence of the amplification product.
  • the presence or absence of amplification may be examined by the LAMP method (Japanese Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Japanese Patent No. 2443586), ICAN method (Japanese Patent Laid-Open No. 2002-233379), and the like. it can.
  • a single-strand amplification method may be used.
  • telomere length a DNA fragment containing a SNP site
  • telomere length a DNA fragment containing a SNP site
  • PCR-SSCP single-strand conformation polymorphism method
  • Genetic. 1992 Jan 1; 12 (1): 139-146. a method for determining which type of polymorphism is based on the mobility of the amplified product in electrophoresis.
  • DNA containing the target SNP contained in the FONG locus is amplified, and the amplified DNA is dissociated into single-stranded DNA.
  • the dissociated single-stranded DNA is separated on a non-denaturing gel, and the type of polymorphism can be determined by the difference in mobility of the separated single-stranded DNA on the gel.
  • a base showing polymorphism when included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by a restriction enzyme (RFLP method).
  • RFLP method restriction enzyme
  • the DNA sample is cleaved with a restriction enzyme.
  • the DNA fragments can then be separated and the type of polymorphism determined by the size of the detected DNA fragment.
  • Test Reagent of the Present Invention also provides test reagents such as primers and probes for testing osteoporosis.
  • a probe that includes the SNP site at the FONG locus and can determine the type of base at the SNP site based on the presence or absence of hybridization.
  • a probe having a length of 10 bases or more having a sequence containing the 61st base of the base sequence selected from SEQ ID NOs: 1, 4 to 17 or a complementary sequence thereof can be mentioned.
  • the length of the probe is more preferably 15 to 35 bases, still more preferably 20 to 35 bases.
  • the primer examples include a primer that can be used for PCR for amplifying the polymorphic site at the FONG locus, or a primer that can be used for sequence analysis (sequencing) of the polymorphic site. .
  • a primer capable of amplifying or sequencing a region containing the 61st base of the base sequence selected from SEQ ID NOs: 1, 4 to 17 can be mentioned.
  • the length of such a primer is preferably 10 to 50 bases, more preferably 15 to 35 bases, and further preferably 20 to 35 bases.
  • a primer having a sequence 5 ′ side of the base preferably 30 to 100 bases upstream, or a 3 ′ side region of the base, preferably 30 to 100 bases.
  • a primer having a sequence complementary to the downstream region is exemplified.
  • a primer used to determine polymorphism based on the presence or absence of amplification by PCR it has a sequence containing the base, a primer containing the base on the 3 ′ side, a complementary sequence of the sequence containing the base, Examples include a primer containing a base complementary to the above base on the 3 ′ side.
  • the test reagent of the present invention may contain a polymerase for PCR, a buffer, a hybridization reagent and the like in addition to these primers and probes.
  • the gene of the present invention is a human FONG gene.
  • An example of the human FONG gene is DNA containing the nucleotide sequence of SEQ ID NO: 2.
  • the base sequence of SEQ ID NO: 2 is the base sequence of the FONG gene based on the transcription product from the FONG locus.
  • the gene having the above sequence is presumed to encode a protein consisting of the amino acid sequence of SEQ ID NO: 3.
  • the protein consisting of the amino acid sequence of SEQ ID NO: 3 is presumed to have formiminotransferase activity.
  • examples of the gene of the present invention include DNA containing the 389th to 832rd base sequences, which is a region presumed to encode the protein in the base sequence of SEQ ID NO: 2.
  • the gene of the present invention is not limited to the gene of the above sequence because there is a possibility that substitution or deletion is present in one or a plurality of bases due to differences in race.
  • the gene of the present invention includes an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO: 3, and is equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 3. It may be a DNA encoding a protein having the following functions.
  • the “one or several” specifically means preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
  • the gene of the present invention has an identity of 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more with respect to the entire amino acid sequence of SEQ ID NO: 3. And a DNA encoding a protein having the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3.
  • the gene of the present invention includes a probe that can be prepared from the above base sequence, for example, a complementary sequence of the base sequence shown in SEQ ID NO: 2 or a complementary sequence of the 389th to 832th base sequences of the base sequence shown in SEQ ID NO: 2 and a string It may be a DNA that hybridizes under a gentle condition and encodes a protein having a function equivalent to that of the protein consisting of the amino acid sequence of SEQ ID NO: 3.
  • stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • highly homologous DNAs for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more DNAs having homology.
  • DNAs with lower homology are not hybridized with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 ⁇ SSC, 0.1% SDS, preferably 60 ° C. 0.1 ⁇ SSC, 0.1% SDS, more preferably 68 ° C., 0.1 ⁇ SSC, 0.1% SDS at a salt concentration and temperature of 1 time, more preferably 2 to 3 times. The conditions to do are mentioned.
  • the probe that can be prepared from the base sequence may be a part of the complementary sequence of the DNA.
  • a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template.
  • hybridization washing conditions include 50 ° C., 2 ⁇ SSC, 0.1% SDS.
  • Protein of the present invention is a protein encoded by the gene of the present invention.
  • Examples of the protein of the present invention include a protein containing the amino acid sequence of SEQ ID NO: 3.
  • a protein comprising the amino acid sequence of SEQ ID NO: 3 is presumed to have formiminotransferase activity.
  • the protein of the present invention includes an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 3, and has the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3. It may be a protein having The “one or several” specifically means preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
  • the protein of the present invention has an identity of 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more with respect to the entire amino acid sequence of SEQ ID NO: 3. And a protein having the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3.
  • Antibody of the present invention is an antibody against the protein of the present invention. As long as it specifically recognizes the protein of the present invention, it may be a polyclonal antibody or a monoclonal antibody.
  • a polyclonal antibody can be obtained, for example, by immunizing a non-human mammal such as a mouse or rabbit with an immunogen containing the protein of the present invention, and collecting an antibody that specifically recognizes the protein of the present invention from the antiserum. Can do. It may be used for immunization by binding to a carrier protein such as BSA or KLH. The antibody can be purified by protein A or the like.
  • the monoclonal antibody can be obtained, for example, by immunizing a non-human mammal such as a mouse with an immunogen containing the protein of the present invention and fusing lymphocytes isolated from the mammal with mouse myeloma cells to produce a hybridoma. It can be obtained by selecting an antibody that specifically recognizes the protein of the present invention from the antibodies produced by the hybridoma.
  • Monoclonal antibodies also include fragments of monoclonal antibodies such as F (ab ′) 2 antibodies, F (ab ′) antibodies, single chain antibodies (scFv), diabodies, and minibodies. .
  • the antibody of the present invention can be used for detection of fusion protein containing the protein of the present invention, immunoprecipitation, ELISA and the like.
  • Example 1 Identification of SNPs correlated with osteoporosis
  • GWAS genome-wide correlation analysis
  • the osteoporosis subjects (cases) and non-osteoporosis subjects (controls; controls) used for the primary and secondary screening were BioBank Japan (BBJ) (Nakamura, Y. The BioBank Japan Project. Clin Adv). Hematol Oncol 5, 696-7 (2007)).
  • the case group of the first follow-up was obtained from BBJ.
  • Samples of outpatient postmenopausal female volunteers who were not related to each other were used for the secondary follow-up case group and the primary and secondary follow-up control groups.
  • the control group has various diseases other than osteoporosis.
  • Osteoporosis was determined according to the criteria of the Japan Osteoporosis Society, and osteoporosis was determined when both the lumbar spine and femoral neck were less than 70% of the BMD of young adult men.
  • the standard corresponds to a World Health Period (WHO) standard T-score of less than -2.5.
  • WHO World Health Period
  • Genomic DNA extracted from peripheral blood leukocytes by a conventional method was used as a sample, and high density oligonucleotide array (Perlegen Science) was used for analysis.
  • QQ Quantile-quantile
  • RAF risk allele frequency (risk allele frequency)
  • a P value of Pearson's ⁇ 2 test (allele model)
  • b odds ratio (odds ratio; OR) of risk allele from 2 ⁇ 2 frequency table
  • c Results of Breslow-Day test
  • d Meta-analysis of all 4 trials (primary and secondary screening and primary and secondary follow-up) using Japanese subjects
  • e All using Japanese subjects Meta-analysis of 4 trials and trials using Han population
  • haplotype analysis was performed on the 14 tag SNPs selected from the linkage disequilibrium (LD) block in the vicinity of s7605378. Compared to analyzing rs7605378 alone, no haplotypes that were more significantly correlated with osteoporosis were found (Table 5).
  • rs7605378 is the SNP that is most strongly correlated with osteoporosis.
  • LOC348751 is composed of five exons, the predicted exon 1 was not confirmed by RT-PCR in this experiment, and only a part of exon 2 and exons 3-5 were confirmed.
  • the main splicing variant was composed of four exons, and contained exons 3 and 4 of LOC348751 in common.
  • exon 5 is rich in diversity among splicing variants.
  • the predicted gene structure of LOC348751 and the gene structure of FONG are shown in FIG.
  • a cDNA fragment corresponding to the nucleotide sequence of 413-731 of FONG was cloned into the pCR2.1TOPO vector (Invitrogen).
  • the DIG-labeled probe was synthesized using DIG RNA Labeling Kit (Roche) based on the cloned vector.
  • Extraction of mRNA from kidney, liver, skeletal muscle, and bone was performed by FastTrack 2.0 mRNA Isolation kit (Invitrogen). 2 ⁇ g of mRNA extracted from each tissue was subjected to electrophoresis, and mRNA was detected using DIG Easy Hyb and DIG Wash and Block Buffer set (Roche) according to the manufacturer's instructions.
  • a band corresponding to the predicted length of the transcript was detected in all tissues (FIG. 6 (a)).
  • FONG expression was examined in various human tissues by real-time PCR, it was revealed that FONG is highly expressed in liver and skeletal muscle, and is also expressed moderately in bone. (FIG. 6B).
  • the 147 amino acid residue protein comprises a signal peptide and a formiminotransferase domain-N-terminal subdomain (FTCD- N domain).
  • the FTCD-N domain is underlined in FIG.
  • FTCD is a mammalian metabolic enzyme involved in the conversion of histidine to glutamic acid, and its N-terminal domain transfers the formimino group from N-formimino-L-glutamic acid to tetrahydrofolic acid, resulting in L-glutamic acid and 5-formiminotetrahydro Has activity to produce folic acid.
  • Glutamate signaling is important for bone homeostasis, for example, glutamate is secreted by osteoclasts, and glutamate transporter 1 KO mice are known to develop osteoporosis. That is, FONG may control bone metabolism.
  • rs7605378 and 12 SNPs that are completely linked to the SNP are present in intron 3 of the FONG locus or in the 3 'flanking region, and do not affect the amino acid sequence of the protein expressed by FONG. Therefore, these SNPs may be related to osteoporosis by affecting the expression of FONG.
  • FONG was newly identified as a candidate for an osteoporosis susceptibility gene.
  • SNPs related to osteoporosis were found. These SNPs are useful for the examination of osteoporosis.

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Abstract

Provided is an osteoporosis testing method. A single nucleotide polymorphism on a FONG gene locus is analyzed, and the occurrence of osteoporosis is detected on the basis of the results of the analysis.

Description

新規遺伝子およびＦＯＮＧ遺伝子座の一塩基多型に基づく骨粗鬆症の検査方法Test method for osteoporosis based on novel nucleotide and single nucleotide polymorphism of FONG locus
　本発明は骨粗鬆症を予測または判定するための検査方法及び該検査方法に用いられる試薬に関する。本発明は、さらに、骨粗鬆症に関連する遺伝子、当該遺伝子より発現されるタンパク質、および当該タンパク質に対する抗体に関する。 The present invention relates to a test method for predicting or determining osteoporosis and a reagent used in the test method. The present invention further relates to a gene associated with osteoporosis, a protein expressed from the gene, and an antibody against the protein.
　骨や関節の病気は世界中で深刻な問題であり、その中でも骨粗鬆症（ｏｓｔｅｏｐｏｒｏｓｉｓ）は最も多い疾患の一つである。骨粗鬆症は、中年以降に発症し、患者の約８割は女性である。現在、全世界で２億人以上が骨粗鬆症に罹患しており、人口の高齢化に伴い患者は急激に増大するものと予想される。 病 気 Bone and joint diseases are a serious problem all over the world, and among them osteoporosis is one of the most common diseases. Osteoporosis develops after middle age and about 80% of patients are women. Currently, more than 200 million people worldwide suffer from osteoporosis, and the number of patients is expected to increase rapidly as the population ages.
　骨粗鬆症は、臨床上は、骨量の低下、骨強度の減少、および骨折傾向の増大等を特徴とする。骨密度（ｂｏｎｅ　ｍｉｎｅｒａｌ　ｄｅｎｓｉｔｙ；　ＢＭＤ）は骨粗鬆症の検査に一般的に用いられる表現型であり、骨粗鬆症による骨折リスクを評価するのに有用なツールである。ＢＭＤが遺伝的要因により影響を受けることは広く知られており、遺伝率は５０～９０％であると推定されている。 Osteoporosis is clinically characterized by decreased bone mass, decreased bone strength, increased fracture tendency, and the like. Bone mineral density (BMD) is a phenotype commonly used in the examination of osteoporosis, and is a useful tool for evaluating the risk of fracture due to osteoporosis. It is widely known that BMD is affected by genetic factors, and the heritability is estimated to be 50-90%.
　骨粗鬆症リスクに関与する遺伝子座を同定するためにはいくつかの実験的アプローチが考えられる。例えば、骨代謝に関連する遺伝子や、まれな単一遺伝子骨疾患の病原遺伝子を幅広くスクリーニングすることで候補となる遺伝子を同定する試みがなされている（非特許文献１－３）。しかしながら、再現性が得られているのは、ほんのわずかな遺伝子のみである。 Several experimental approaches can be considered to identify loci involved in osteoporosis risk. For example, attempts have been made to identify candidate genes by extensive screening of genes related to bone metabolism and pathogenic genes of rare single gene bone diseases (Non-Patent Documents 1-3). However, only a few genes are reproducible.
　また、ゲノムワイド相関解析（ＧＷＡＳ）によって骨粗鬆症の感受性遺伝子を同定する試みがなされている。ＧＷＡＳによれば、表現型にほとんど影響しない遺伝子バリアントの検出も可能であり、未知の感受性遺伝子を見出すことができると期待される。最近、いくつかのグループがＧＷＡＳを行い、骨粗鬆症感受性に関連する複数の遺伝子座を同定している（非特許文献４－９、特許文献１、２）。例えば、アジア人種について骨粗鬆症のＧＷＡＳを行い、ＪＡＧ１やＡＬＤＨ７Ａ１等のいくつかの遺伝子が同定されている（非特許文献９，１０）。しかしながら、骨粗鬆症に対するこれら遺伝子の遺伝的な寄与は完全には明らかになっていない。 In addition, attempts have been made to identify susceptibility genes for osteoporosis by genome-wide correlation analysis (GWAS). According to GWAS, it is possible to detect gene variants that hardly affect the phenotype, and it is expected that unknown susceptibility genes can be found. Recently, several groups have performed GWAS and identified multiple loci associated with osteoporosis susceptibility (Non-patent Documents 4-9, Patent Documents 1 and 2). For example, osteoporosis GWAS is performed on Asian races, and several genes such as JAG1 and ALDH7A1 have been identified (Non-Patent Documents 9 and 10). However, the genetic contribution of these genes to osteoporosis is not completely clear.
　そして、ＦＯＮＧ遺伝子座の多型と骨粗鬆症との関係は報告されていない。 And the relationship between the polymorphism of the FONG locus and osteoporosis has not been reported.
特開２０１０－００００６６号公報JP 2010-000066 A 特開２００５－００６５３８号公報JP 2005-006538 A
　本発明は、骨粗鬆症の発症リスクや発症の有無を正確に検査する方法、及び該方法に用いられる検査試薬を提供することを課題とする。また、本発明は、骨粗鬆症に関連する遺伝子、当該遺伝子より発現されるタンパク質、および当該タンパク質に対する抗体を提供する。 An object of the present invention is to provide a method for accurately examining the onset risk of osteoporosis and the presence or absence of onset, and a test reagent used in the method. The present invention also provides a gene associated with osteoporosis, a protein expressed from the gene, and an antibody against the protein.
　本発明者らは上記課題の解決のために鋭意検討した結果、ＦＯＮＧ遺伝子座に存在する一塩基多型（ＳＮＰ）が骨粗鬆症と相関することを同定した。そして、これらの多型を調べることにより骨粗鬆症の発症リスクや発症の推定を正確に実施できることを見出し、本発明を完成するに至った。 As a result of intensive studies for solving the above problems, the present inventors have identified that a single nucleotide polymorphism (SNP) present at the FONG locus is correlated with osteoporosis. And by investigating these polymorphisms, it discovered that the onset risk of osteoporosis and estimation of onset could be implemented correctly, and came to complete this invention.
　すなわち、本発明は以下の通りである。
（１）ＦＯＮＧ遺伝子座に存在する一塩基多型を分析し、該分析結果に基づいて骨粗鬆症を検査することを特徴とする、骨粗鬆症の発症リスクおよび／または発症の有無の判定方法。
（２）前記一塩基多型が、配列番号１の塩基配列の塩基番号６１番目の塩基に相当する塩基、または該塩基と連鎖不平衡の関係にある塩基における一塩基多型である、（１）に記載の方法。
（３）前記連鎖不平衡の関係にある塩基が、配列番号４～１７から選ばれる塩基配列の塩基番号６１番目の塩基に相当する塩基である、（２）に記載の方法。
（４）配列番号１、４～１７から選ばれる塩基配列において、塩基番号６１番目の塩基を含む１０塩基以上の配列、又はその相補配列を有する骨粗鬆症検査用プローブ。
（５）配列番号１、４～１７から選ばれる塩基配列において、塩基番号６１番目の塩基を含む領域を増幅することのできる骨粗鬆症検査用プライマー。
（６）以下の（Ａ）～（Ｄ）から選択されるＤＮＡ。
（Ａ）配列番号２に示す塩基配列における３８９～８３２番目の塩基配列を含むＤＮＡ。
（Ｂ）配列番号２に示す塩基配列における３８９～８３２番目の塩基配列と相補的な塩基配列又は該塩基配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質をコードするＤＮＡ。
（Ｃ）配列番号３に示すアミノ酸配列を含むタンパク質をコードするＤＮＡ。
（Ｄ）配列番号３のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入又は付加を含むアミノ酸配列を含み、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質をコードするＤＮＡ。
（７）以下の（ａ）または（ｂ）のタンパク質。
（ａ）配列番号３に示すアミノ酸配列を含むタンパク質。
（ｂ）配列番号３のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入又は付加を含むアミノ酸配列を含み、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質。
（８）前記タンパク質に対する抗体。 That is, the present invention is as follows.
(1) A method for determining the risk of osteoporosis and / or the presence or absence of osteoporosis, comprising analyzing a single nucleotide polymorphism present in the FONG locus and examining osteoporosis based on the analysis result.
(2) The single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the 61st base of the base sequence of SEQ ID NO: 1 or a base in a linkage disequilibrium relationship with the base (1 ) Method.
(3) The method according to (2), wherein the base in the linkage disequilibrium relationship is a base corresponding to the 61st base of the base sequence selected from SEQ ID NOs: 4 to 17.
(4) A probe for testing osteoporosis having a sequence of 10 bases or more including the 61st base in the base sequence selected from SEQ ID NOs: 1, 4 to 17, or a complementary sequence thereof.
(5) A primer for testing osteoporosis capable of amplifying a region containing the base at position 61 in the base sequence selected from SEQ ID NOs: 1, 4 to 17.
(6) DNA selected from the following (A) to (D).
(A) DNA containing the 389th to 832rd base sequences in the base sequence shown in SEQ ID NO: 2.
(B) hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence 389 to 832 in the nucleotide sequence shown in SEQ ID NO: 2 or a probe that can be prepared from the nucleotide sequence; DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence.
(C) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 3.
(D) a protein having an equivalent function to the protein consisting of the amino acid sequence of SEQ ID NO: 3, comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 3 DNA to encode.
(7) The following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(B) A protein having the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3, comprising an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 3.
(8) An antibody against the protein.
　本発明によれば、これまで予測が困難であった骨粗鬆症の発症リスクを正確かつ簡便に予測することができる。また、骨粗鬆症の発症を正確かつ簡便に判定することができる。したがって、本発明は骨粗鬆症の予防や早期治療に貢献するものである。 According to the present invention, it is possible to accurately and easily predict the risk of developing osteoporosis that has been difficult to predict. Moreover, the onset of osteoporosis can be determined accurately and simply. Therefore, the present invention contributes to the prevention and early treatment of osteoporosis.
１次スクリーニングにおけるＱ－Ｑプロットを示す図。斜め線からの偏差は、いずれのＳＮＰｓも骨粗鬆症と相関しないという帰無仮説からの偏差に相当する。The figure which shows the QQ plot in a primary screening. Deviations from diagonal lines correspond to deviations from the null hypothesis that none of the SNPs correlate with osteoporosis. ２次スクリーニングにおけるＱ－Ｑプロットを示す図。斜め線からの偏差は、いずれのＳＮＰｓも骨粗鬆症と相関しないという帰無仮説からの偏差に相当する。The figure which shows the QQ plot in a secondary screening. Deviations from diagonal lines correspond to deviations from the null hypothesis that none of the SNPs correlate with osteoporosis. ヒト第２染色体ＦＯＮＧ遺伝子座領域の連鎖不平衡（ＬＤ）マップを示す図。The figure which shows the linkage disequilibrium (LD) map of a human 2nd chromosome FONG locus region. ＦＯＮＧ遺伝子からの転写産物に基づく遺伝子の塩基配列と推定アミノ酸配列。図中、ＦＴＣＤ－Ｎドメインと推定される部分を下線で示す。The base sequence and deduced amino acid sequence of the gene based on the transcription product from the FONG gene. In the figure, the part presumed to be the FTCD-N domain is indicated by an underline. 予測されていたＬＯＣ３４８７５１の遺伝子構造と、ＦＯＮＧの遺伝子構造を示す図。図中、白抜き部分は非翻訳領域を、黒塗り部分はコード領域を示す。The figure which shows the gene structure of LOC3488751 and the gene structure of FONG which were estimated. In the figure, a white portion indicates an untranslated region, and a black portion indicates a code region. （ａ）ノーザンブロッティングによるＦＯＮＧ遺伝子の発現解析の結果を示す写真。レーン１：腎臓、レーン２：骨格筋、レーン３：肝臓、レーン４：骨。（ｂ）各ヒト組織でのＦＯＮＧ遺伝子の発現レベルを示す図。縦軸は、βアクチン（ＡＣＴＢ）のｍＲＮＡ量に対するＦＯＮＧのｍＲＮＡ量の相対値（２回の独立した試験に基づく平均±標準誤差）を示す。(A) A photograph showing the results of FONG gene expression analysis by Northern blotting. Lane 1: kidney, lane 2: skeletal muscle, lane 3: liver, lane 4: bone. (B) The figure which shows the expression level of the FONG gene in each human tissue. The vertical axis shows the relative value of the FONG mRNA amount to the β-actin (ACTB) mRNA amount (mean ± standard error based on two independent tests).
＜１＞本発明の検査方法
　本発明の検査方法は、ヒトのＦＯＮＧ遺伝子座に含まれるＳＮＰを分析し、該分析結果に基づいて骨粗鬆症を検査することを特徴とする、骨粗鬆症の発症リスクおよび／または発症の有無の判定方法である。すなわち、本発明において、「検査」とは骨粗鬆症の発症リスクの検査及び骨粗鬆症の発症の有無の検査を含む。 <1> Test Method of the Present Invention The test method of the present invention comprises analyzing the SNPs contained in the human FONG locus, and testing osteoporosis based on the analysis results, and / or the risk of developing osteoporosis and / or Or it is the determination method of the presence or absence of onset. That is, in the present invention, “examination” includes an examination of the risk of developing osteoporosis and an examination of the presence or absence of osteoporosis.
　ＦＯＮＧ遺伝子座は、ヒト第２染色体長腕３３．１領域に存在する、ＦＯＮＧ遺伝子産物をコードする遺伝子を包含する領域である。ＦＯＮＧ遺伝子座は、好ましくは、ヒト第２染色体上のＧｅｎＢａｎｋ　Ａｃｃｅｓｓｉｏｎ　Ｎｏ．ＮＣ＿０００００２．１１の２００６２５２５８～２００７１６７３４の領域を意味する。 The FONG gene locus is a region including a gene encoding the FONG gene product, which is present in the long arm 33.1 region of human chromosome 2. The FONG locus is preferably GenBank Accession No. 2 on human chromosome 2. It means the area from 20062258 to 200176734 in NC_00000.11.
　ＦＯＮＧ遺伝子座に存在する具体的なＳＮＰとしては、ヒトｒｓ７６０５３７８を挙げることができる。ここで、ｒｓ番号はＮａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　ＩｎｆｏｒｍａｔｉｏｎのｄｂＳＮＰデータベース（ｈｔｔｐ／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｐｒｏｊｅｃｔｓ／ＳＮＰ／）の登録番号を示す。ｒｓ７６０５３７８はＦＯＮＧ遺伝子座のイントロン３に存在する。ｒｓ７６０５３７８はＧｅｎＢａｎｋ　Ａｃｃｅｓｓｉｏｎ　Ｎｏ．　ＮＴ＿００５４０３．１６の５０８８６３４３番目の塩基におけるアデニン（Ａ）／シトシン（Ｃ）の多型を意味し、この塩基がＡである場合は骨粗鬆症の可能性が高い。また、アレルを考慮して解析した場合は、ｒｓ７６０５３７８がＡＡ＞ＡＣ＞ＣＣの順で骨粗鬆症の可能性が高い。 Specific examples of SNPs present in the FONG locus include human rs7605378. Here, the rs number indicates the registration number of the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/) of the National Center for Biotechnology Information. rs7605378 is present in intron 3 of the FONG locus. rs7605378 is GenBank Accession No. It means a polymorphism of adenine (A) / cytosine (C) at the 50886343rd base of NT_005403.16, and when this base is A, the possibility of osteoporosis is high. Moreover, when analyzing in consideration of alleles, rs7605378 has a high possibility of osteoporosis in the order of AA> AC> CC.
　なお、ｒｓ７６０５３７８について、ＳＮＰ塩基及びその前後６０ｂｐの領域を含む合計１２１ｂｐの長さの配列を、配列番号１に示した。６１番目の塩基が多型を有する。 For rs7605378, a sequence having a total length of 121 bp including the SNP base and a region of 60 bp before and after that is shown in SEQ ID NO: 1. The 61st base has a polymorphism.
　上記塩基に相当する塩基を本発明においては解析する。ここで、「上記塩基に相当する塩基」とは、ヒトＦＯＮＧ遺伝子座の該当塩基を意味する。すなわち、「上記塩基に相当する塩基を解析する」ことには、仮に、人種の違いなどによって上記配列がＳＮＰ以外の位置で若干変化したとしても、その中の該当塩基を解析することが含まれる。 In the present invention, a base corresponding to the above base is analyzed. Here, “a base corresponding to the above-mentioned base” means a corresponding base of the human FONG gene locus. That is, “analyzing a base corresponding to the above-mentioned base” includes analyzing the corresponding base in the sequence even if the sequence is slightly changed at a position other than the SNP due to a difference in race. It is.
　また、本発明において解析する塩基は上記のものに限定されず、上記の塩基と連鎖不平衡にある塩基の多型を分析してもよい。ここで「上記の塩基と連鎖不平衡にある塩基」とは、上記の塩基とｒ2＞０．５、好ましくはｒ2＞０．８、さらに好ましくはｒ2＞０．９の関係を満たす塩基をいう。上記の塩基と連鎖不平衡にある塩基は、例えば、HapMapデータベース(http://www.hapmap.org/index.html.ja)等を用いて同定することができる。もしくは、複数人（通常は２０～４０人程度）から採取したＤＮＡをシークエンサーにて配列解析し、連鎖不平衡にあるＳＮＰを探索することにより同定することもできる。上記の塩基と連鎖不平衡にある塩基としては、例えば、ｒｓ１０１９４０７６、ｒｓ５７６９６６１９、ｒｓ２３４６６６２、ｒｓ７５７７７６３、ｒｓ２６８９７６３、ｒｓ７６９９５４、ｒｓ７９６３６３、ｒｓ７６９９５７、ｒｓ７６９９５８、ｒｓ７９７１６３、ｒｓ２６８９７６６、ｒｓ５９０３６９８５、およびｒｓ５８３１９９０１が挙げられる。これらＳＮＰｓの詳細について表１に示す。表１中、ｒ2はｒｓ７６０５３７８に対する各ＳＮＰのｒ2を示す。また、表１中、各ＳＮＰについて、メジャーアレルがリスクアレルである。すなわち、例えば、ｒｓ１０１９４０７６は、ＧｅｎＢａｎｋ　Ａｃｃｅｓｓｉｏｎ　Ｎｏ．　ＮＴ＿００５４０３．１６の５０８８７０８０番目の塩基におけるチミン（Ｔ）／シトシン（Ｃ）の多型を意味し、この塩基がＴである場合は骨粗鬆症の可能性が高い。なお、ｒｓ５７６９６６１９は、チミン（Ｔ）が欠失している場合に骨粗鬆症の可能性が高い。なお、ｒｓ５９０３６９８５は、４塩基（ＡＡＡＧ）が挿入されている場合に骨粗鬆症の可能性が高い。いずれも、リスクアレルのホモ接合体＞リスクアレルと非リスクアレルのヘテロ接合体＞非リスクアレルのホモ接合体の順で骨粗鬆症の可能性が高い。また、これらＳＮＰ塩基及びその前後６０ｂｐの領域を含む合計１２１ｂｐ（ｒｓ５９０３６９８５の場合は１２４ｂｐ）の長さの配列を、それぞれ配列番号４～１７に示した。６１番目の塩基が多型を有する。なお、「６１番目の塩基」とは、ｒｓ５９０３６９８５の場合は、配列番号１６における６１～６４番目の塩基として適宜読み替えてよい。 In addition, the base to be analyzed in the present invention is not limited to the above, and a polymorphism of a base in linkage disequilibrium with the above base may be analyzed. Here, the “base in linkage disequilibrium with the above-mentioned base” satisfies the relationship of r 2 > 0.5, preferably r 2 > 0.8, more preferably r 2 > 0.9 with the above-mentioned base. Say base. The base in linkage disequilibrium with the above base can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja). Alternatively, DNA collected from a plurality of people (usually about 20 to 40 people) can be identified by sequence analysis using a sequencer and searching for SNPs in linkage disequilibrium. Examples of bases that are in linkage disequilibrium with the above bases include rs10194676, rs57696619, rs2346662, rs75777763, rs2689763, rs769699, rs796363, rs769957, rs769958, rs7976963, rs2689766, and rs59093685, 01. Details of these SNPs are shown in Table 1. In Table 1, r 2 represents an r 2 of each SNP against Rs7605378. In Table 1, for each SNP, the major allele is a risk allele. That is, for example, rs10194076 is GenBank Accession No. This means a polymorphism of thymine (T) / cytosine (C) at the 50888080 position of NT_005403.16. When this base is T, the possibility of osteoporosis is high. In addition, rs57696619 has a high possibility of osteoporosis when thymine (T) is deleted. Note that rs59036985 has a high possibility of osteoporosis when 4 bases (AAAG) are inserted. In any case, the risk of osteoporosis increases in the order of homozygote of risk allele> heterozygote of risk allele and non-risk allele> nonzygote of non-risk allele. In addition, sequences having a total length of 121 bp (124 bp in the case of rs59093685) including these SNP bases and a region of 60 bp before and after that are shown in SEQ ID NOs: 4 to 17, respectively. The 61st base has a polymorphism. In the case of rs59036985, the “61st base” may be appropriately read as the 61st to 64th bases in SEQ ID NO: 16.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　上記ＳＮＰの塩基の種類を調べ、得られた結果を上記のような基準に基づいて骨粗鬆症と関連付けることによって、骨粗鬆症を検査することができる。上記ＳＮＰは単独で解析されてもよいし、上記ＳＮＰの少なくとも１つを含む複数のＳＮＰｓをまとめて解析（ハプロタイプ解析）してもよい。なお、ＦＯＮＧ遺伝子座の配列はセンス鎖を解析してもよいし、アンチセンス鎖を解析してもよい。 The osteoporosis can be examined by examining the base type of the SNP and associating the obtained result with osteoporosis based on the above criteria. The SNP may be analyzed alone, or a plurality of SNPs including at least one of the SNPs may be collectively analyzed (haplotype analysis). The sequence of the FONG gene locus may be analyzed for the sense strand or the antisense strand.
　ＦＯＮＧ遺伝子座に含まれるＳＮＰの解析に用いる試料としては、染色体ＤＮＡを含む試料であれば特に制限されないが、例えば、血液、尿等の体液サンプル、口腔粘膜などの細胞、毛髪等の体毛などが挙げられる。ＳＮＰの解析にはこれらの試料を直接使用することもできるが、これらの試料から染色体ＤＮＡを常法により単離し、これを用いて解析することが好ましい。 The sample used for the analysis of the SNP contained in the FONG gene locus is not particularly limited as long as it is a sample containing chromosomal DNA. Examples thereof include body fluid samples such as blood and urine, cells such as oral mucosa, and hair such as hair. Can be mentioned. Although these samples can be used directly for the analysis of SNP, it is preferable to isolate chromosomal DNA from these samples by a conventional method and analyze it.
　ＦＯＮＧ遺伝子座に含まれるＳＮＰの解析は、通常の遺伝子多型解析方法によって行うことができる。例えば、シークエンス解析、ＰＣＲ、ハイブリダイゼーション、インベーダー法などが挙げられるが、これらに限定されない。 The analysis of SNPs contained in the FONG gene locus can be performed by a normal gene polymorphism analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, invader method and the like.
　シークエンス解析は通常の方法により行うことができる。具体的には、多型を示す塩基の５’側　数十塩基の位置に設定したプライマーを使用してシークエンス反応を行い、その解析結果から、該当する位置がどの種類の塩基であるかを決定することができる。なお、シークエンスを行う場合、あらかじめＳＮＰ部位を含む断片をＰＣＲなどによって増幅しておくことが好ましい。 Sequence analysis can be performed by a normal method. Specifically, a sequence reaction is performed using a primer set at a position of several tens of bases on the 5 ′ side of a base showing polymorphism, and the type of base at the corresponding position is determined from the analysis result. can do. In addition, when performing a sequence, it is preferable to amplify the fragment | piece containing a SNP site | part beforehand by PCR etc.
　また、ＳＮＰの解析は、ＰＣＲによる増幅の有無を調べることによって行うことができる。例えば、多型を示す塩基を含む領域に対応する配列を有し、かつ、３’末端が各多型に対応するプライマーをそれぞれ用意する。それぞれのプライマーを使用してＰＣＲを行い、増幅産物の有無によってどのタイプの多型であるかを決定することができる。また、ＬＡＭＰ法（特許第３３１３３５８号明細書）、ＮＡＳＢＡ法（Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｓｅｑｕｅｎｃｅ－Ｂａｓｅｄ　Ａｍｐｌｉｆｉｃａｔｉｏｎ；特許２８４３５８６号明細書）、ＩＣＡＮ法（特開２００２－２３３３７９号公報）などによって増幅の有無を調べることもできる。その他、単鎖増幅法を用いてもよい。 Also, SNP analysis can be performed by examining the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing a base showing a polymorphism and having a 3 'end corresponding to each polymorph is prepared. PCR can be performed using each primer, and the type of polymorphism can be determined depending on the presence or absence of the amplification product. Further, the presence or absence of amplification may be examined by the LAMP method (Japanese Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Japanese Patent No. 2443586), ICAN method (Japanese Patent Laid-Open No. 2002-233379), and the like. it can. In addition, a single-strand amplification method may be used.
　また、ＳＮＰ部位を含むＤＮＡ断片を増幅し、増幅産物の電気泳動における移動度の違いによってどのタイプの多型であるかを決定することもできる。このような方法としては、例えば、ＰＣＲ－ＳＳＣＰ（ｓｉｎｇｌｅ－ｓｔｒａｎｄ　ｃｏｎｆｏｒｍａｔｉｏｎ　ｐｏｌｙｍｏｒｐｈｉｓｍ）法（Genomics. 1992 Jan 1; 12(1): 139-146.）が挙げられる。具体的には、まず、ＦＯＮＧ遺伝子座に含まれる目的のＳＮＰを含むＤＮＡを増幅し、増幅したＤＮＡを一本鎖ＤＮＡに解離させる。次いで、解離させた一本鎖ＤＮＡを非変性ゲル上で分離し、分離した一本鎖ＤＮＡのゲル上での移動度の違いによってどのタイプの多型であるかを決定することができる。 It is also possible to amplify a DNA fragment containing a SNP site and determine which type of polymorphism is based on the mobility of the amplified product in electrophoresis. Examples of such a method include a PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1; 12 (1): 139-146.). Specifically, first, DNA containing the target SNP contained in the FONG locus is amplified, and the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing gel, and the type of polymorphism can be determined by the difference in mobility of the separated single-stranded DNA on the gel.
　さらに、多型を示す塩基が制限酵素認識配列に含まれる場合は、制限酵素による切断の有無によって解析することもできる（ＲＦＬＰ法）。この場合、まず、ＤＮＡ試料を制限酵素により切断する。次いで、ＤＮＡ断片を分離し、検出されたＤＮＡ断片の大きさによってどのタイプの多型であるかを決定することができる。 Furthermore, when a base showing polymorphism is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by a restriction enzyme (RFLP method). In this case, first, the DNA sample is cleaved with a restriction enzyme. The DNA fragments can then be separated and the type of polymorphism determined by the size of the detected DNA fragment.
　ハイブリダイゼーションの有無を調べることによって多型の種類を解析することも可能である。すなわち、各塩基に対応するプローブを用意し、いずれのプローブにハイブリダイズするかを調べることによってＳＮＰがいずれの塩基であるかを調べることもできる。 It is also possible to analyze the type of polymorphism by examining the presence or absence of hybridization. That is, a probe corresponding to each base is prepared, and it is also possible to examine which base the SNP is by examining which probe hybridizes.
　このようにしてＳＮＰがいずれの塩基であるかを決定することで、骨粗鬆症を検査するためのデータを得ることができる。 Thus, it is possible to obtain data for examining osteoporosis by determining which base the SNP is.
＜２＞本発明の検査用試薬
　本発明はまた、骨粗鬆症を検査するためのプライマーやプローブなどの検査試薬を提供する。このようなプローブとしては、ＦＯＮＧ遺伝子座における上記ＳＮＰ部位を含み、ハイブリダイズの有無によってＳＮＰ部位の塩基の種類を判定できるプローブが挙げられる。具体的には、配列番号１、４～１７から選ばれる塩基配列の６１番目の塩基を含む配列、又はその相補配列を有する１０塩基以上の長さのプローブが挙げられる。プローブの長さはより好ましくは、１５～３５塩基であり、さらに好ましくは２０～３５塩基である。 <2> Test Reagent of the Present Invention The present invention also provides test reagents such as primers and probes for testing osteoporosis. Examples of such a probe include a probe that includes the SNP site at the FONG locus and can determine the type of base at the SNP site based on the presence or absence of hybridization. Specifically, a probe having a length of 10 bases or more having a sequence containing the 61st base of the base sequence selected from SEQ ID NOs: 1, 4 to 17 or a complementary sequence thereof can be mentioned. The length of the probe is more preferably 15 to 35 bases, still more preferably 20 to 35 bases.
　また、プライマーとしては、ＦＯＮＧ遺伝子座における上記多型部位を増幅するためのＰＣＲに用いることのできるプライマー、又は上記多型部位を配列解析（シークエンシング）するために用いることのできるプライマーが挙げられる。具体的には、配列番号１、４～１７から選ばれる塩基配列の６１番目の塩基を含む領域を増幅したりシークエンシングしたりすることのできるプライマーが挙げられる。このようなプライマーの長さは１０～５０塩基が好ましく、１５～３５塩基がより好ましく、２０～３５塩基がさらに好ましい。 Examples of the primer include a primer that can be used for PCR for amplifying the polymorphic site at the FONG locus, or a primer that can be used for sequence analysis (sequencing) of the polymorphic site. . Specifically, a primer capable of amplifying or sequencing a region containing the 61st base of the base sequence selected from SEQ ID NOs: 1, 4 to 17 can be mentioned. The length of such a primer is preferably 10 to 50 bases, more preferably 15 to 35 bases, and further preferably 20 to 35 bases.
　上記多型部位をシークエンシングするためのプライマーとしては、上記塩基の５’側領域、好ましくは３０～１００塩基上流の配列を有するプライマーや、上記塩基の３’側領域、好ましくは３０～１００塩基下流の領域に相補的な配列を有するプライマーが例示される。ＰＣＲによる増幅の有無で多型を判定するために用いるプライマーとしては、上記塩基を含む配列を有し、上記塩基を３’側に含むプライマーや、上記塩基を含む配列の相補配列を有し、上記塩基の相補塩基を３’側に含むプライマーなどが例示される。 As a primer for sequencing the polymorphic site, a primer having a sequence 5 ′ side of the base, preferably 30 to 100 bases upstream, or a 3 ′ side region of the base, preferably 30 to 100 bases. A primer having a sequence complementary to the downstream region is exemplified. As a primer used to determine polymorphism based on the presence or absence of amplification by PCR, it has a sequence containing the base, a primer containing the base on the 3 ′ side, a complementary sequence of the sequence containing the base, Examples include a primer containing a base complementary to the above base on the 3 ′ side.
　なお、本発明の検査用試薬はこれらのプライマーやプローブに加えて、ＰＣＲ用のポリメラーゼやバッファー、ハイブリダイゼーション用試薬などを含むものであってもよい。 The test reagent of the present invention may contain a polymerase for PCR, a buffer, a hybridization reagent and the like in addition to these primers and probes.
＜３＞本発明の遺伝子
　本発明の遺伝子は、ヒトのＦＯＮＧ遺伝子である。ヒトのＦＯＮＧ遺伝子としては、例えば、配列番号２の塩基配列を含むＤＮＡを挙げることができる。配列番号２の塩基配列は、ＦＯＮＧ遺伝子座からの転写産物に基づくＦＯＮＧ遺伝子の塩基配列である。上記配列の遺伝子は、配列番号３のアミノ酸配列からなるタンパク質をコードしていると推定される。配列番号３のアミノ酸配列からなるタンパク質は、ホルムイミノトランスフェラーゼ活性を有すると推定される。また、本発明の遺伝子としては、特に、配列番号２の塩基配列中、前記タンパク質をコードすると推定される領域である３８９～８３２番目の塩基配列を含むＤＮＡが挙げられる。 <3> Gene of the Present Invention The gene of the present invention is a human FONG gene. An example of the human FONG gene is DNA containing the nucleotide sequence of SEQ ID NO: 2. The base sequence of SEQ ID NO: 2 is the base sequence of the FONG gene based on the transcription product from the FONG locus. The gene having the above sequence is presumed to encode a protein consisting of the amino acid sequence of SEQ ID NO: 3. The protein consisting of the amino acid sequence of SEQ ID NO: 3 is presumed to have formiminotransferase activity. In addition, examples of the gene of the present invention include DNA containing the 389th to 832rd base sequences, which is a region presumed to encode the protein in the base sequence of SEQ ID NO: 2.
　本発明の遺伝子は、人種の違いなどによって１又は複数の塩基に置換や欠失等が存在する可能性があるため、上記配列の遺伝子に限定されない。 The gene of the present invention is not limited to the gene of the above sequence because there is a possibility that substitution or deletion is present in one or a plurality of bases due to differences in race.
　すなわち、本発明の遺伝子は、配列番号３のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入又は付加等を含むアミノ酸配列を含み、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質をコードするＤＮＡであってもよい。前記「１若しくは数個」とは、具体的には好ましくは１～２０個、より好ましくは１～１０個、さらに好ましくは１～５個を意味する。 That is, the gene of the present invention includes an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO: 3, and is equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 3. It may be a DNA encoding a protein having the following functions. The “one or several” specifically means preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
　本発明の遺伝子は、配列番号３のアミノ酸配列全体に対して、８０％以上、好ましくは９０％以上、より好ましくは９５％以上、より好ましくは９７％以上、特に好ましくは９９％以上の同一性を有し、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質をコードするＤＮＡであってもよい。 The gene of the present invention has an identity of 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more with respect to the entire amino acid sequence of SEQ ID NO: 3. And a DNA encoding a protein having the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3.
　また、本発明の遺伝子は、上記塩基配列から調製され得るプローブ、例えば、配列番号２に示す塩基配列の相補配列または配列番号２に示す塩基配列の３８９～８３２番目の塩基配列の相補配列とストリンジェントな条件下でハイブリダイズし、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質をコードするＤＮＡであってもよい。ここで、「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。一例を示せば、相同性が高いＤＮＡ同士、例えば８０％以上、好ましくは９０％以上、より好ましくは９５％以上、より好ましくは９７％以上、特に好ましくは９９％以上の相同性を有するＤＮＡ同士がハイブリダイズし、それより相同性が低いＤＮＡ同士がハイブリダイズしない条件、あるいは通常のサザンハイブリダイゼーションの洗いの条件である６０℃、１×ＳＳＣ、０．１％　ＳＤＳ、好ましくは、６０℃、０．１×ＳＳＣ、０．１％　ＳＤＳ、さらに好ましくは、６８℃、０．１×ＳＳＣ、０．１％　ＳＤＳに相当する塩濃度、温度で、１回、より好ましくは２～３回洗浄する条件が挙げられる。 Further, the gene of the present invention includes a probe that can be prepared from the above base sequence, for example, a complementary sequence of the base sequence shown in SEQ ID NO: 2 or a complementary sequence of the 389th to 832th base sequences of the base sequence shown in SEQ ID NO: 2 and a string It may be a DNA that hybridizes under a gentle condition and encodes a protein having a function equivalent to that of the protein consisting of the amino acid sequence of SEQ ID NO: 3. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more DNAs having homology. Are hybridized and DNAs with lower homology are not hybridized with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C. 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS at a salt concentration and temperature of 1 time, more preferably 2 to 3 times. The conditions to do are mentioned.
　上記塩基配列から調製され得るプローブは、上記ＤＮＡの相補配列の一部であってもよい。そのようなプローブは、公知の遺伝子配列に基づいて作製したオリゴヌクレオチドをプライマーとし、これらの塩基配列を含むＤＮＡ断片を鋳型とするＰＣＲによって作製することができる。例えば、プローブとして、３００ｂｐ程度の長さのＤＮＡ断片を用いる場合には、ハイブリダイゼーションの洗いの条件としては、５０℃、２×ＳＳＣ、０．１％　ＳＤＳが挙げられる。 The probe that can be prepared from the base sequence may be a part of the complementary sequence of the DNA. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, 0.1% SDS.
＜４＞本発明のタンパク質
　本発明のタンパク質は、本発明の遺伝子によりコードされるタンパク質である。 <4> Protein of the present invention The protein of the present invention is a protein encoded by the gene of the present invention.
　本発明のタンパク質としては、例えば配列番号３のアミノ酸配列を含むタンパク質が挙げられる。配列番号３のアミノ酸配列を含むタンパク質は、ホルムイミノトランスフェラーゼ活性を有すると推定される。 Examples of the protein of the present invention include a protein containing the amino acid sequence of SEQ ID NO: 3. A protein comprising the amino acid sequence of SEQ ID NO: 3 is presumed to have formiminotransferase activity.
　本発明のタンパク質は、配列番号３のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入又は付加等を含むアミノ酸配列を含み、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質であってもよい。前記「１若しくは数個」とは、具体的には好ましくは１～２０個、より好ましくは１～１０個、さらに好ましくは１～５個を意味する。 The protein of the present invention includes an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 3, and has the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3. It may be a protein having The “one or several” specifically means preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
　本発明のタンパク質は、配列番号３のアミノ酸配列全体に対して、８０％以上、好ましくは９０％以上、より好ましくは９５％以上、より好ましくは９７％以上、特に好ましくは９９％以上の同一性を有し、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質であってもよい。 The protein of the present invention has an identity of 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more with respect to the entire amino acid sequence of SEQ ID NO: 3. And a protein having the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3.
＜５＞本発明の抗体
　本発明の抗体は本発明のタンパク質に対する抗体である。本発明のタンパク質を特異的に認識するものである限り、ポリクローナル抗体であってもよいし、モノクローナル抗体でもよい。 <5> Antibody of the present invention The antibody of the present invention is an antibody against the protein of the present invention. As long as it specifically recognizes the protein of the present invention, it may be a polyclonal antibody or a monoclonal antibody.
　ポリクローナル抗体は、例えば、本発明のタンパク質を含む免疫原でマウスやウサギなどの非ヒト哺乳動物を免疫し、その抗血清から本発明のタンパク質を特異的に認識する抗体を回収することによって得ることができる。ＢＳＡやＫＬＨなどのキャリアタンパク質に結合させて免疫に使用してもよい。抗体はプロテインＡなどによって精製することができる。 A polyclonal antibody can be obtained, for example, by immunizing a non-human mammal such as a mouse or rabbit with an immunogen containing the protein of the present invention, and collecting an antibody that specifically recognizes the protein of the present invention from the antiserum. Can do. It may be used for immunization by binding to a carrier protein such as BSA or KLH. The antibody can be purified by protein A or the like.
　モノクローナル抗体は、例えば、本発明のタンパク質を含む免疫原でマウスなどの非ヒト哺乳動物を免疫し、該哺乳動物から単離したリンパ球をマウスミエローマ細胞と融合させてハイブリドーマを作製し、得られたハイブリドーマが産生する抗体の中から、本発明のタンパク質を特異的に認識する抗体を選択することによって得ることができる。モノクローナル抗体には、Ｆ（ａｂ’）2化抗体、Ｆ（ａｂ’）化抗体、単鎖抗体（ｓｃＦｖ）、ダイアボディ（Ｄｉａｂｏｄｉｅｓ）、およびミニボディ（Ｍｉｎｉｂｏｄｉｅｓ）等のモノクローナル抗体のフラグメントも含まれる。 The monoclonal antibody can be obtained, for example, by immunizing a non-human mammal such as a mouse with an immunogen containing the protein of the present invention and fusing lymphocytes isolated from the mammal with mouse myeloma cells to produce a hybridoma. It can be obtained by selecting an antibody that specifically recognizes the protein of the present invention from the antibodies produced by the hybridoma. Monoclonal antibodies also include fragments of monoclonal antibodies such as F (ab ′) 2 antibodies, F (ab ′) antibodies, single chain antibodies (scFv), diabodies, and minibodies. .
　本発明の抗体は本発明のタンパク質を含む融合タンパク質の検出や免疫沈降やＥＬＩＳＡなどに使用できる。 The antibody of the present invention can be used for detection of fusion protein containing the protein of the present invention, immunoprecipitation, ELISA and the like.
　以下、本発明を実施例によりさらに具体的に説明する。但し、本発明はこれらの実施例に限定されない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
（実施例１）骨粗鬆症と相関するＳＮＰｓの同定
　まず、骨粗鬆症感受性を決定する遺伝的変異を同定するために、日本人被検者を用いてゲノムワイド相関解析（ＧＷＡＳ）を行った。解析に用いた被検者の特徴は表２に示すとおりである。 (Example 1) Identification of SNPs correlated with osteoporosis First, genome-wide correlation analysis (GWAS) was performed using Japanese subjects in order to identify genetic mutations that determine osteoporosis sensitivity. The characteristics of the subject used for the analysis are as shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　１次および２次スクリーニングに用いた骨粗鬆症の被検者（ケース；　症例）および非骨粗鬆症の被検者（コントロール；　対照）は、ＢｉｏＢａｎｋ　Ｊａｐａｎ（ＢＢＪ）（Nakamura, Y. The BioBank Japan Project. Clin Adv Hematol Oncol 5, 696-7 (2007)）より得た。１次追試の症例群は、ＢＢＪより得た。２次追試の症例群、並びに、１次および２次追試の対照群には、互いに血縁関係を有さない外来の閉経後女性ボランティアのサンプルを用いた。対照群は、骨粗鬆症以外の種々の疾患を有する。 The osteoporosis subjects (cases) and non-osteoporosis subjects (controls; controls) used for the primary and secondary screening were BioBank Japan (BBJ) (Nakamura, Y. The BioBank Japan Project. Clin Adv). Hematol Oncol 5, 696-7 (2007)). The case group of the first follow-up was obtained from BBJ. Samples of outpatient postmenopausal female volunteers who were not related to each other were used for the secondary follow-up case group and the primary and secondary follow-up control groups. The control group has various diseases other than osteoporosis.
　骨粗鬆症の判定は日本骨粗鬆症学会の基準に準じて行い、腰椎（ｌｕｍｂａｒ　ｓｐｉｎｅ）および大腿骨頸部（ｆｅｍｏｒａｌ　ｎｅｃｋ）の両方について若年成年男性のＢＭＤの７０％未満である場合に骨粗鬆症と判定した。当該基準は、世界保健期間（ＷＨＯ）の基準であるＴスコアで－２．５未満に相当する。ＢＭＤは、標準的なプロトコルに従って二重エネルギー放射線吸収測定法を行うことで測定した。 Osteoporosis was determined according to the criteria of the Japan Osteoporosis Society, and osteoporosis was determined when both the lumbar spine and femoral neck were less than 70% of the BMD of young adult men. The standard corresponds to a World Health Period (WHO) standard T-score of less than -2.5. BMD was measured by performing a dual energy radiation absorption measurement according to standard protocols.
　１次および２次スクリーニングにおいて、統計解析にはＦｉｓｈｅｒの正確確率検定（ａｌｌｅｌｅ　ｆｒｅｑｕｅｎｃｙ　ｍｏｄｅｌ，　ｄｏｍｉｎａｎｔ－ｅｆｆｅｃｔ　ｍｏｄｅｌ，　およびｒｅｃｅｓｓｉｖｅ－ｅｆｆｅｃｔ　ｍｏｄｅｌの３モデル）を用いた。追試において、統計解析にはχ2検定（ａｌｌｅｌｅ　ｆｒｅｑｕｅｎｃｙ　ｍｏｄｅｌ，　ｄｏｍｉｎａｎｔ－ｅｆｆｅｃｔ　ｍｏｄｅｌ，　およびｒｅｃｅｓｓｉｖｅ－ｅｆｆｅｃｔ　ｍｏｄｅｌの３モデル）を用いた。オッズ比（Ｏｄｄｓ　Ｒａｔｉｏ；　ＯＲ）および信頼区間（Ｃｏｎｆｉｄｅｎｃｅ　Ｉｎｔｅｒｖａｌ；　ＣＩ）はメジャーアレルを参考に算出した。ハプロタイプの相関解析はＨａｐｌｏｖｉｅｗ　ｓｏｆｔｗａｒｅを用いて行った（Barrett, J.C et al. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263-5 (2005)）。メタ解析はＭａｎｔｅｌ－Ｈａｅｎｓｚｅｌ法により行った。 In the primary and secondary screening, Fisher's exact test (three models of all-frequency model, dominant-effect model, and recessive-effect model) was used for statistical analysis. In the follow-up test, the χ 2 test (all frequency model, dominant-effect model, and recessive-effect model) was used for statistical analysis. The odds ratio (Odds Ratio; OR) and confidence interval (Confidence Interval; CI) were calculated with reference to the major allele. Haplotype correlation analysis was performed using Haploview software (Barrett, JC et al. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263-5 (2005)). Meta-analysis was performed by the Mantel-Haenszel method.
　本研究は東京大学医科学研究所および理化学研究所ゲノム医科学研究センターの倫理委員会によって承認された。 This research was approved by the Ethics Committee of the Institute of Medical Science at the University of Tokyo and the Genomic Medical Research Center of RIKEN.
［１次スクリーニング］
　１次スクリーニングでは、ＢＢＪに登録された骨粗鬆症の症例１９０人と対照１５５７人について、常染色体に存在する２６８，０６４個のＳＮＰｓの遺伝型を解析し、２２４，５０７個のＳＮＰｓについて遺伝型のデータを取得した。上記２６８，０６４個のＳＮＰｓは、日本人集団におけるタグＳＮＰｓとしてＪＳＮＰ（Haga, H. et al. Gene-based SNP discovery as part of the Japanese Millennium Genome Project: identification of 190,562 genetic variations in the human genome. Single-nucleotide polymorphism. J Hum Genet 47, 605-10 (2002)）またはＨａｐＭａｐデータベース（Frazer, K.A. et al. A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851-61 (2007)）から選択されたものであり、日本人集団の一般的なＳＮＰｓの約５６％をカバーする。試料としては末梢血白血球から常法により抽出したゲノムＤＮＡを用い、解析にはｈｉｇｈ　ｄｅｎｓｉｔｙ　ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　ａｒｒａｙｓ（Ｐｅｒｌｅｇｅｎ　Ｓｃｉｅｎｃｅ）を用いた。Ｑｕａｎｔｉｌｅ－ｑｕａｎｔｉｌｅ（Ｑ－Ｑ）プロット（図１）により、骨粗鬆症に関連する可能性のある多くのＳＮＰｓが存在することが明らかとなった。 [Primary screening]
In the primary screening, the genotypes of 268,064 SNPs present in the autosome were analyzed for 190 osteoporosis cases and 1557 controls registered in the BBJ, and genotype data for 224,507 SNPs. Acquired. The above 268,064 SNPs are JSNP (Haga, H. et al. Gene-based SNP discovery as part of the Japanese Millennium Genome Project: identification of 190,562 genetic variations in the human genome. Single -nucleotide polymorphism. J Hum Genet 47, 605-10 (2002)) or HapMap database (Frazer, KA et al. A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851-61 (2007)) Which covers about 56% of the general SNPs in the Japanese population. Genomic DNA extracted from peripheral blood leukocytes by a conventional method was used as a sample, and high density oligonucleotide array (Perlegen Science) was used for analysis. The Quantile-quantile (QQ) plot (FIG. 1) revealed that there are many SNPs that may be associated with osteoporosis.
［２次スクリーニング］
　１次スクリーニングにおいてデータの得られた２２４，５０７個のＳＮＰｓの内、Ｐ値の低い上位３０００個のＳＮＰｓを選択し、２次スクリーニングを行った。症例５２６人と対照１５３７人について、前記３０００個のＳＮＰｓの遺伝型を解析し、１６５４個のＳＮＰｓについて遺伝型のデータを取得した。症例群の解析はｈｉｇｈ　ｄｅｎｓｉｔｙ　ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　ａｒｒａｙｓ（Ｐｅｒｌｅｇｅｎ　Ｓｃｉｅｎｃｅ）により、対照群の解析はｍｕｌｔｉｐｌｅｘ－ＰＣＲ　ｉｎｖａｄｅｒ　ａｓｓａｙ（Ohnishi, Y. et al. A high-throughput SNP typing system for genome-wide association studies. J Hum Genet 46, 471-7 (2001)）により行った。Ｑｕａｎｔｉｌｅ－ｑｕａｎｔｉｌｅ（Ｑ－Ｑ）プロットを図２に示す。 [Secondary screening]
Of the 224,507 SNPs for which data was obtained in the primary screening, the top 3000 SNPs with the lowest P values were selected, and secondary screening was performed. For 526 cases and 1537 controls, the genotypes of the 3000 SNPs were analyzed, and genotype data were obtained for 1654 SNPs. The analysis of the case group was performed by high density oligonucleotide array (Perlegen Science), and the analysis of the control group was performed by multipleplex-PCR inverter assay (Ohnishi, Y. et al. A high-throughput SNP typing system for genome-wide association studies. 46, 471-7 (2001)). A Quantile-quantile (QQ) plot is shown in FIG.
［１次追試］
　２次スクリーニングにおいて結果の得られた１６５４個のＳＮＰｓ（図２）の内、Ｐ値の低い上位３個のＳＮＰｓを選択し、追試を行った。症例１３２６人と対照１２９２人について、前記３個のＳＮＰｓの遺伝型を解析した。相関解析における有意性の閾値をＰ＜１．６７×１０-2（＝０．０５／３）と設定した。当該閾値は、３回のテストに基づくボンフェローニ補正後のＰ＜０．０５に相当する。前記３個のＳＮＰｓの内、最小のＰ値を示すｒｓ７６０５３７８のみが骨粗鬆症と有意に相関を示した（Ｐ＝２．９９×１０-3）。ｒｓ７６０５３７８について表３に示す。なお、ｒｓ７６０５３７８について、Ａが骨粗鬆症のリスクアレルである。 [First follow-up]
Of the 1654 SNPs obtained in the secondary screening (FIG. 2), the top 3 SNPs with the lowest P values were selected for additional testing. The genotypes of the three SNPs were analyzed for 1326 cases and 1292 controls. The significance threshold in the correlation analysis was set as P <1.67 × 10 −2 (= 0.05 / 3). The threshold value corresponds to P <0.05 after Bonferroni correction based on three tests. Of the three SNPs, only rs7605378 showing the smallest P value significantly correlated with osteoporosis (P = 2.99 × 10 −3 ). Table 3 shows rs7605378. For rs7605378, A is an osteoporosis risk allele.
Figure JPOXMLDOC01-appb-T000003
 ＲＡＦ：リスクアレル頻度（ｒｉｓｋ　ａｌｌｅｌｅ　ｆｒｅｑｕｅｎｃｙ）
ａ：Ｐｅａｒｓｏｎのχ2検定（ａｌｌｅｌｅ　ｍｏｄｅｌ）のＰ値
ｂ：２×２頻度表からのリスクアレルのオッズ比（ｏｄｄｓ　ｒａｔｉｏ；　ＯＲ）
ｃ：Ｂｒｅｓｌｏｗ－Ｄａｙ検定の結果
ｄ：日本人被検者を用いた全４試験（１次および２次スクリーニング並びに１次および２次追試）のメタ解析
ｅ：日本人被検者を用いた全４試験および漢民族集団を用いた試験のメタ解析
Figure JPOXMLDOC01-appb-T000003
 RAF: risk allele frequency (risk allele frequency)
a: P value of Pearson's χ 2 test (allele model) b: odds ratio (odds ratio; OR) of risk allele from 2 × 2 frequency table
c: Results of Breslow-Day test d: Meta-analysis of all 4 trials (primary and secondary screening and primary and secondary follow-up) using Japanese subjects e: All using Japanese subjects Meta-analysis of 4 trials and trials using Han population
［２次追試］
　検証のため、独立した人的集団を被検者として２次追試を行った。症例２４０人と対照２８５人について、ｒｓ７６０５３７８の遺伝型を解析したところ、ｒｓ７６０５３７８と骨粗鬆症との有意な相関が再現された（Ｐ＝３．９７×１０-2）。なお、相関解析における有意性の閾値はＰ＜０．０５と設定した。 [Secondary follow-up]
For verification, a second follow-up test was conducted with an independent human group as the subject. When 240 genotypes and 285 controls were analyzed for rs7605378 genotype, a significant correlation between rs7605378 and osteoporosis was reproduced (P = 3.97 × 10 −2 ). The significance threshold in the correlation analysis was set to P <0.05.
　また、１次および２次スクリーニング並びに１次および２次追試からなる全４試験のメタ解析を行ったところ、ｒｓ７６０５３７８のｃｏｍｂｉｎｅｄ　Ｐ　ｖａｌｕｅは１．５１×１０-8（ＯＲ＝１．２５；　９５％　ＣＩ：１．１６－１．３５）であり（表３）、ＧＷＡＳの有意性の閾値として設定したＰ＜２．２３×１０-7（＝０．０５／２２４，５０７）をパスした。当該閾値は、２２４，５０７回のテストに基づくボンフェローニ補正後のＰ＜０．０５に相当する。よって、ｒｓ７６０５３７８が骨粗鬆症と有意に相関することが確認された。 Further, when meta-analysis of all four tests including primary and secondary screening and primary and secondary follow-up was performed, the combined P value of rs7605378 was 1.51 × 10 −8 (OR = 1.25; 95% CI: 1.16-1.35) (Table 3), which passed P <2.23 × 10 −7 (= 0.05 / 224,507) set as a threshold value for significance of GWAS. The threshold corresponds to P <0.05 after Bonferroni correction based on 224,507 tests. Therefore, it was confirmed that rs7605378 is significantly correlated with osteoporosis.
［漢民族集団を用いた追試］
　さらに、漢民族集団（ここでは香港人集団）において、ｒｓ７６０５３７８と骨粗鬆症との相関を解析した。症例３３８人と対照１２２人について、ｒｓ７６０５３７８の遺伝型を解析したところ、骨粗鬆症と相関する傾向が認められた。 [Further examination using Han ethnic group]
Furthermore, the correlation between rs7605378 and osteoporosis was analyzed in the Han ethnic group (here Hong Kong group). Analysis of the genotype of rs7605378 for 338 cases and 122 controls showed a tendency to correlate with osteoporosis.
　さらに、日本人集団を用いた４試験とこの漢民族集団を用いた試験からなる全５試験のメタ解析を行ったところ、ｒｓ７６０５３７８のｃｏｍｂｉｎｅｄ　Ｐ　ｖａｌｕｅは１．３３×１０-8（ＯＲ＝１．２４；　９５％　ＣＩ：１．１５－１．３３）であり（表３）、日本人を用いた４試験のメタ解析の結果と比較して、さらに高い有意性を示した。よって、ｒｓ７６０５３７８が骨粗鬆症と有意に相関することが再度確認された。 Furthermore, when meta-analysis of all 5 studies comprising 4 studies using a Japanese population and a study using this Han ethnic group was performed, the combined P value of rs7605378 was 1.33 × 10 −8 (OR = 1. 24; 95% CI: 1.15 to 1.33) (Table 3), which showed higher significance compared to the results of the meta-analysis of 4 trials using Japanese. Therefore, it was confirmed again that rs7605378 is significantly correlated with osteoporosis.
［骨粗鬆症と最も強く相関するＳＮＰの同定］
　ＨａｐＭａｐプロジェクトデータベース（リリース２３ａ）を参照し、ｒｓ７６０５３７８に対するＤ’＞０．８であり、かつ、マイナーアレル頻度＞０．１であるＳＮＰｓを選択し、ＦＯＮＧ領域の連鎖不平衡（ＬＤ）マップを作成した（図３）。ｒｓ７６０５３７８近傍の連鎖不平衡（ＬＤ）ブロックには、ＨａｐＭａｐプロジェクトデータベースに登録された５１個のＳＮＰｓと１個の予測遺伝子が含まれていた。当該５１個のＳＮＰｓ全てをｒ2＞０．８でカバーする、ｒｓ７６０５３７８を含む１４個のタグＳＮＰｓを選択し、症例２０３９人と対照１２９２人について、遺伝型を解析したところ、ｒｓ７６０５３７８よりも有意に骨粗鬆症と相関するＳＮＰは見出せなかった。なお、Ｄ’およびｒ2はいずれも連鎖不平衡の程度を示す尺度である。 [Identification of SNPs most strongly correlated with osteoporosis]
Refer to the HapMap project database (Release 23a), select SNPs with D '> 0.8 for rs7605378 and minor allele frequency> 0.1, and create linkage disequilibrium (LD) map of FONG region (FIG. 3). The linkage disequilibrium (LD) block near rs7605378 contained 51 SNPs and one predicted gene registered in the HapMap project database. 14 tag SNPs including rs7605378 that cover all 51 SNPs with r 2 > 0.8 were selected, and the genotypes of 2039 cases and 1292 controls were analyzed, which was significantly higher than rs7605378. SNPs that correlate with osteoporosis were not found. D ′ and r 2 are both scales indicating the degree of linkage disequilibrium.
　さらに、ｒｓ７６０５３７８に対してｒ2＞０．８を示す約２５ｋｂの領域を、症例２４人から取得したゲノムＤＮＡのダイレクトシーケンスにより検索したところ、ＨａｐＭａｐデータベースに登録された３９個の既知ＳＮＰｓに加え、２０個の新規ＳＮＰｓが見出された（表４）。当該５９個のＳＮＰｓ全てを用いてペアワイズｒ2値を算出し、ｒ2＞０．９５を示す２２個のタグＳＮＰｓを選択して遺伝型を解析したところ、ｒｓ７６０５３７８よりも有意に骨粗鬆症と相関するＳＮＰは見出されなかった。 Furthermore, when a region of about 25 kb showing r 2 > 0.8 with respect to rs7605378 was searched by direct sequencing of genomic DNA obtained from 24 cases, in addition to 39 known SNPs registered in the HapMap database, Twenty new SNPs were found (Table 4). Pair-wise r 2 values were calculated using all the 59 SNPs, and 22 tag SNPs showing r 2 > 0.95 were selected and analyzed for genotype, which was significantly more correlated with osteoporosis than rs7605378. No SNP was found.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　さらに、ｓ７６０５３７８近傍の連鎖不平衡（ＬＤ）ブロックから選択した前記１４個のタグＳＮＰｓについて、ハプロタイプ解析を行った。ｒｓ７６０５３７８を単独で解析するのに比較して、より有意に骨粗鬆症と相関したハプロタイプは見出されなかった（表５）。 Furthermore, haplotype analysis was performed on the 14 tag SNPs selected from the linkage disequilibrium (LD) block in the vicinity of s7605378. Compared to analyzing rs7605378 alone, no haplotypes that were more significantly correlated with osteoporosis were found (Table 5).
Figure JPOXMLDOC01-appb-T000005
 ＯＲ：アレル２に対するアレル１のオッズ比
ＣＩ：信頼区間（Ｃｏｎｆｉｄｅｎｃｅ　Ｉｎｔｅｒｖａｌ）
前記２次追試で用いた骨粗鬆症症例群において１％以上の頻度で出現する全てのハプロタイプを示す。１および２は、それぞれ骨粗鬆症症例群におけるマイナーアレルおよびメジャーアレルを示す。
＊１４個のＳＮＰｓのアレル（左からｒｓ１２３７３７８８、ｒｓ７５７２４７３、ｒｓ１２６１５４３５、ｒｓ１０９３１８７５、ｒｓ１２４７３６７９、ｒｓ６７５９６４４、ｒｓ１７５２９４９７、ｒｓ６７４３２７１、ｒｓ４６７３４９１、ｒｓ７６０５３７８、ｒｓ２０３０６５３、ｒｓ２１６４８８９、ｒｓ１２４７０９８６、およびｒｓ１０２０３１２２）
Figure JPOXMLDOC01-appb-T000005
 OR: odds ratio of allele 1 to allele 2 CI: confidence interval (Confidence Interval)
All haplotypes appearing at a frequency of 1% or more in the osteoporosis case group used in the second follow-up test are shown. 1 and 2 show the minor allele and major allele in the osteoporosis case group, respectively.
* Alleles of 14 SNPs (from left: rs123737788, rs7572473, rs12615435, rs10931875, rs124736779, rs67569644, rs1754297, rs6743271, rs4673491, rs7605378, rs2064869, rs12406889, rs12464889, rs124068
　以上より、ｒｓ７６０５３７８が骨粗鬆症と最も強く相関するＳＮＰであることが明らかとなった。また、ｒｓ７６０５３７８に対してｒ2＝１を示す、すなわちｒｓ７６０５３７８と完全連鎖する１２個のＳＮＰｓが検出された（表４）。よって、特にこれら１３個のＳＮＰは、骨粗鬆症の原因ＳＮＰｓであると推測され、いずれも骨粗鬆症の検査に用いることができる。 From the above, it was revealed that rs7605378 is the SNP that is most strongly correlated with osteoporosis. In addition, 12 SNPs having r 2 = 1 with respect to rs7605378, that is, completely linked to rs7605378 were detected (Table 4). Therefore, in particular, these 13 SNPs are presumed to be the cause SNPs of osteoporosis, and any of them can be used for the examination of osteoporosis.
（実施例２）ＦＯＮＧの同定と発現
［ＦＯＮＧの同定］
　ＮＣＢＩ　ｇｅｎｏｍｅ　ｄａｔａｂａｓｅ（ｂｕｉｌｄ　３６．３）において、ｒｓ７６０５３７８はＬＯＣ３４８７５１遺伝子内に存在する。前記データベースにおいて、ＬＯＣ３４８７５１にはＲｅｆｓｅｑ転写産物（９６６ｂｐ）が登録されているが、あくまでｉｎ　ｓｉｌｉｃｏ予測に基づくものであった。そこで、骨を試料として、発現した転写産物の全長配列をＲＡＣＥおよびＲＴ－ＰＣＲによりクローニングした。その結果、１４７アミノ酸残基のタンパク質の存在が予測される１９９７ｂｐの新規な転写産物が同定された（図４）。当該タンパク質のアミノ酸配列を配列番号３に、当該転写産物に基づく遺伝子の塩基配列を配列番号２に示す。この新規に見出された遺伝子をＦＯＮＧと命名した。 (Example 2) Identification and expression of FONG [Identification of FONG]
In the NCBI genome database (build 36.3), rs7605378 is present in the LOC348751 gene. In the database, Refseq transcript (966 bp) is registered in LOC348751, but it was based on in silico prediction only. Therefore, using bone as a sample, the full-length sequence of the expressed transcript was cloned by RACE and RT-PCR. As a result, a 1997-bp novel transcript predicted to have a protein of 147 amino acid residues was identified (FIG. 4). The amino acid sequence of the protein is shown in SEQ ID NO: 3, and the base sequence of the gene based on the transcript is shown in SEQ ID NO: 2. This newly found gene was named FONG.
　なお、ＬＯＣ３４８７５１は５つのエクソンで構成されるが、本実験のＲＴ－ＰＣＲでは、予測されていたエクソン１は確認されず、エクソン２の一部およびエクソン３－５のみが確認された。上記１９９７ｂｐの転写産物を含む多数のスプライシングバリアントを同定したところ、主要なスプライシングバリアントは４つのエクソンで構成されており、ＬＯＣ３４８７５１のエクソン３および４を共通して含んでいた。一方、エクソン５についてはスプライシングバリアント間で多様性に富むことが明らかとなった。予測されていたＬＯＣ３４８７５１の遺伝子構造と、ＦＯＮＧの遺伝子構造を図５に示す。 Although LOC348751 is composed of five exons, the predicted exon 1 was not confirmed by RT-PCR in this experiment, and only a part of exon 2 and exons 3-5 were confirmed. When a large number of splicing variants including the above-described 1997 bp transcript were identified, the main splicing variant was composed of four exons, and contained exons 3 and 4 of LOC348751 in common. On the other hand, it was revealed that exon 5 is rich in diversity among splicing variants. The predicted gene structure of LOC348751 and the gene structure of FONG are shown in FIG.
［ＦＯＮＧの発現］
　ＦＯＮＧの発現と転写産物のサイズを確認するため、以下の手順でノーザンブロッティングを行い、ＲＮＡの検出を行った。 [Expression of FONG]
In order to confirm the expression of FONG and the size of the transcript, Northern blotting was performed by the following procedure to detect RNA.
　ＦＯＮＧの４１３－７３１番目の塩基配列に相当するｃＤＮＡフラグメントをｐＣＲ２．１ＴＯＰＯベクター（Ｉｎｖｉｔｒｏｇｅｎ）にクローニングした。ＤＩＧラベルしたプローブを、クローニング後のベクターを基に、ＤＩＧ　ＲＮＡ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ（Ｒｏｃｈｅ）を用いて合成した。腎臓、肝臓、骨格筋、および骨からのｍＲＮＡの抽出はＦａｓｔＴｒａｃｋ　２．０　ｍＲＮＡ　Ｉｓｏｌａｔｉｏｎ　ｋｉｔ　（Ｉｎｖｉｔｒｏｇｅｎ）により行った。各組織から抽出されたｍＲＮＡ２μｇを電気泳動に供し、ＤＩＧ　Ｅａｓｙ　ＨｙｂおよびＤＩＧ　Ｗａｓｈ　ａｎｄ　Ｂｌｏｃｋ　Ｂｕｆｆｅｒ　ｓｅｔ　（Ｒｏｃｈｅ）を製造元の指示に従って使用しｍＲＮＡの検出を行った。予測された転写産物の長さに相当するバンドが、全ての組織に共通して検出された（図６（ａ））。 A cDNA fragment corresponding to the nucleotide sequence of 413-731 of FONG was cloned into the pCR2.1TOPO vector (Invitrogen). The DIG-labeled probe was synthesized using DIG RNA Labeling Kit (Roche) based on the cloned vector. Extraction of mRNA from kidney, liver, skeletal muscle, and bone was performed by FastTrack 2.0 mRNA Isolation kit (Invitrogen). 2 μg of mRNA extracted from each tissue was subjected to electrophoresis, and mRNA was detected using DIG Easy Hyb and DIG Wash and Block Buffer set (Roche) according to the manufacturer's instructions. A band corresponding to the predicted length of the transcript was detected in all tissues (FIG. 6 (a)).
　また、リアルタイムＰＣＲにより、種々のヒト組織におけるＦＯＮＧの発現を調べたところ、ＦＯＮＧは、肝臓および骨格筋において高度に発現しており、また骨においても中程度に発現していることが明らかとなった（図６（ｂ））。 In addition, when FONG expression was examined in various human tissues by real-time PCR, it was revealed that FONG is highly expressed in liver and skeletal muscle, and is also expressed moderately in bone. (FIG. 6B).
［ＦＯＮＧの機能推定］
　Ｐｒｏｔｅｉｎ　ｍｏｔｉｆ　ａｎａｌｙｓｉｓ　ｐｒｏｇｒａｍ（http://www.ebi.ac.uk/Tools/InterProScan/）によると、前記１４７アミノ酸残基のタンパク質は、シグナルペプチド、およびホルムイミノトランスフェラーゼドメイン－Ｎ末サブドメイン（ＦＴＣＤ－Ｎドメイン）を有していると予測された。ＦＴＣＤ－Ｎドメインを図４に下線で示す。ＦＴＣＤは、ヒスチジンからグルタミン酸への変換に関与する哺乳類の代謝酵素であり、そのＮ末ドメインはＮ－ホルムイミノ－Ｌ－グルタミン酸からテトラヒドロ葉酸へホルムイミノ基を転移し、Ｌ－グルタミン酸と５－ホルムイミノテトラヒドロ葉酸を生成する活性を有する。グルタミン酸シグナル伝達は骨の恒常性に重要であり、例えば、グルタミン酸は破骨細胞により分泌され、また、グルタミン酸トランスポータ１のＫＯマウスは骨粗鬆症を発症することが知られている。すなわちＦＯＮＧは骨の代謝を制御している可能性がある。 [Function estimation of FONG]
According to the Protein motif analysis program (http://www.ebi.ac.uk/Tools/InterProScan/), the 147 amino acid residue protein comprises a signal peptide and a formiminotransferase domain-N-terminal subdomain (FTCD- N domain). The FTCD-N domain is underlined in FIG. FTCD is a mammalian metabolic enzyme involved in the conversion of histidine to glutamic acid, and its N-terminal domain transfers the formimino group from N-formimino-L-glutamic acid to tetrahydrofolic acid, resulting in L-glutamic acid and 5-formiminotetrahydro Has activity to produce folic acid. Glutamate signaling is important for bone homeostasis, for example, glutamate is secreted by osteoclasts, and glutamate transporter 1 KO mice are known to develop osteoporosis. That is, FONG may control bone metabolism.
　また、ｒｓ７６０５３７８および当該ＳＮＰと完全連鎖する１２個のＳＮＰｓは、いずれもＦＯＮＧ遺伝子座のイントロン３に、または３’フランキング領域に存在し、ＦＯＮＧより発現されるタンパク質のアミノ酸配列に影響しない。よって、これらＳＮＰｓはＦＯＮＧの発現に影響することで骨粗鬆症と関連している可能性がある。 Also, rs7605378 and 12 SNPs that are completely linked to the SNP are present in intron 3 of the FONG locus or in the 3 'flanking region, and do not affect the amino acid sequence of the protein expressed by FONG. Therefore, these SNPs may be related to osteoporosis by affecting the expression of FONG.
　以上の通り、骨粗鬆症感受性遺伝子の候補として新規にＦＯＮＧを同定した。 As described above, FONG was newly identified as a candidate for an osteoporosis susceptibility gene.
　上記の通り、骨粗鬆症と関連するＳＮＰｓが見出された。これらのＳＮＰｓは、骨粗鬆症の検査に有用である。 As described above, SNPs related to osteoporosis were found. These SNPs are useful for the examination of osteoporosis.

Claims (8)

  1. 　ＦＯＮＧ遺伝子座に存在する一塩基多型を分析し、該分析結果に基づいて骨粗鬆症を検査することを特徴とする、骨粗鬆症の発症リスクおよび／または発症の有無の判定方法。 A method for determining the onset risk of osteoporosis and / or the presence or absence of onset, comprising analyzing a single nucleotide polymorphism present in the FONG locus and examining osteoporosis based on the analysis result.
  2. 　前記一塩基多型が、配列番号１の塩基配列の塩基番号６１番目の塩基に相当する塩基、または該塩基と連鎖不平衡の関係にある塩基における一塩基多型である、請求項１に記載の方法。 The single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the base number 61 in the base sequence of SEQ ID NO: 1 or a base in linkage disequilibrium with the base. the method of.
  3. 　前記連鎖不平衡の関係にある塩基が、配列番号４～１７から選ばれる塩基配列の塩基番号６１番目の塩基に相当する塩基である、請求項２に記載の方法。 The method according to claim 2, wherein the base in the linkage disequilibrium relationship is a base corresponding to the 61st base of the base sequence selected from SEQ ID NOs: 4 to 17.
  4. 　配列番号１、４～１７から選ばれる塩基配列において、塩基番号６１番目の塩基を含む１０塩基以上の配列、又はその相補配列を有する骨粗鬆症検査用プローブ。 A probe for testing osteoporosis having a sequence of 10 bases or more including the 61st base in the base sequence selected from SEQ ID NOs: 1, 4 to 17, or a complementary sequence thereof.
  5. 　配列番号１、４～１７から選ばれる塩基配列において、塩基番号６１番目の塩基を含む領域を増幅することのできる骨粗鬆症検査用プライマー。 A primer for osteoporosis testing that can amplify a region containing the base at position 61 in the base sequence selected from SEQ ID NOs: 1, 4 to 17.
  6. 　以下の（Ａ）～（Ｄ）から選択されるＤＮＡ。
    （Ａ）配列番号２に示す塩基配列における３８９～８３２番目の塩基配列を含むＤＮＡ。
    （Ｂ）配列番号２に示す塩基配列における３８９～８３２番目の塩基配列と相補的な塩基配列又は該塩基配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、配列番号３のアミノ酸配列をからなるタンパク質と同等の機能を有するタンパク質をコードするＤＮＡ。
    （Ｃ）配列番号３に示すアミノ酸配列を含むタンパク質をコードするＤＮＡ。
    （Ｄ）配列番号３のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入又は付加を含むアミノ酸配列を含み、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質をコードするＤＮＡ。     DNA selected from the following (A) to (D).
    (A) DNA containing the 389th to 832rd base sequences in the base sequence shown in SEQ ID NO: 2.
    (B) hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence 389 to 832 in the nucleotide sequence shown in SEQ ID NO: 2 or a probe that can be prepared from the nucleotide sequence; DNA encoding a protein having a function equivalent to a protein comprising an amino acid sequence.
    (C) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 3.
    (D) a protein having an equivalent function to the protein consisting of the amino acid sequence of SEQ ID NO: 3, comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 3 DNA to encode.
  7. 　以下の（ａ）または（ｂ）のタンパク質。
    （ａ）配列番号３に示すアミノ酸配列を含むタンパク質。
    （ｂ）配列番号３のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入又は付加を含むアミノ酸配列を含み、配列番号３のアミノ酸配列からなるタンパク質と同等の機能を有するタンパク質。     The following protein (a) or (b).
    (A) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
    (B) A protein having the same function as the protein consisting of the amino acid sequence of SEQ ID NO: 3, comprising an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 3.
  8. 　請求項７に記載のタンパク質に対する抗体。 An antibody against the protein according to claim 7.
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JP2002010789A (en) * 1999-08-05 2002-01-15 Genset Corp Est and human protein to be encoded

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* Cited by examiner, † Cited by third party
Title
GUO Y. ET AL.: "Genome-wide association study identifies ALDH7A1 as a novel susceptibility gene for osteoporosis", PLOS GENET., vol. 6, no. 1, 8 January 2010 (2010-01-08), pages EL000806 *
HSU Y.H. ET AL.: "Large-scale genome-wide linkage analysis for loci linked to BMD at different skeletal sites in extreme selected sibships", J BONE MINER RES., vol. 22, no. 2, February 2007 (2007-02-01), pages 184 - 194 *
KUNG A.W. ET AL.: "Association of JAG1 with bone mineral density and osteoporotic fractures: a genome-wide association study and follow-up replication studies", AM J HUM GENET., vol. 86, no. 2, 12 February 2010 (2010-02-12), pages 229 - 239 *

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