JP2004166702A - Method for detecting Paget's disease of bone - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of detecting bone Paget's disease and an animal presenting pathological conditions of bone Paget's disease. <P>SOLUTION: The detection of bone Paget's disease correlates a mutation or an expression dose of chondroitin/chondroitin sulfate synthase gene comprising plurality of specific amino acid sequences to bone Paget's disease. A knockout animal is obtained by knockouting the above-described chondroitin/chondroitin sulfate synthase gene. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a method for detecting Paget's disease of bone, and more particularly, to a method for detecting Paget's disease of bone that associates mutation or expression level of a gene of an enzyme involved in the synthesis of chondroitin / chondroitin sulfate with bone Paget disease. .

  Bone Paget's disease is a disease in which both osteoclasts and osteoblasts increase, and bone resorption and secondary bone formation associated therewith are abnormally enhanced locally, resulting in bone deformation and thickening. The prevailing theory is that the disease is triggered by a viral infection, and reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from the patient's peripheral blood and bone marrow mononuclear cells. Non-Patent Document 1 describes that measles virus could be detected by the method described above. However, it has not been shown that the measles virus causes bone Paget's disease directly, and the cause of the pathology of bone Paget's disease is unknown. Non-Patent Document 2 states that patients with this disease are rare in Japan, but there are many patients in the United States and Europe, and the higher the elderly, the higher the incidence, and the higher the incidence in men than in women. Is described.

  In addition, a mutant locus causing bone Paget's disease is already known and described in Non-Patent Document 3, but there are many genes at this locus, and when any of the genes is mutated, bone It was not known if Paget's disease would occur. Therefore, in order to diagnose Paget's disease of bone by a simple method, it is necessary to clarify which gene is mutated to cause the disease.

J. Bone Miner Res., 11 (1996), pp.1602-1607

Endocr. Metab. Clin. North. Am., 24 (1995), pp. 437-450 Am. J. Hum. Genet., 69 (2001), pp.1055-1061

  Expectations for a simple method for detecting Paget's disease of bone have been elucidated by elucidating the causative genes that cause the pathology of Paget's disease of bone.

  The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that all of the gene mutations in Paget's disease of bone have occurred at loci of DNA involved in the synthesis of chondroitin / chondroitin sulfate. The present invention has been completed by applying it to the detection of Paget's disease of bone.

  That is, the gist of the present invention is a method for detecting Paget's disease of bone, wherein the mutation or expression level of a gene encoding chondroitin synthase is associated with Paget's disease of bone.

  According to the present invention, a novel method for detecting Paget's disease of bone and a pathological animal having Paget's disease of bone are provided.

  The detection method of the present invention is a method for detecting Paget's disease of bone, wherein the mutation or expression level of a chondroitin synthase gene is associated with Paget's disease of bone.

  The “chondroitin synthase” in the detection method of the present invention is not particularly limited as long as it is an enzyme involved in the synthesis of chondroitin or chondroitin sulfate. In other words, chondroitin and chondroitin sulfate are usually synthesized in vivo as proteoglycans, so they synthesize not only enzymes involved in elongation of the basic skeleton of chondroitin / chondroitin sulfate, but also, for example, a linking portion between proteoglycan protein and glycosaminoglycan. Enzymes and the like are also included. If such an enzyme is mutated or not expressed, chondroitin / chondroitin sulfate is not synthesized.

  The “chondroitin synthase” in the detection method of the present invention transfers “D-glucuronic acid residue or N-acetyl-D-galactosamine residue to the non-reducing terminal sugar residue of chondroitin / chondroitin sulfate. Glycosyltransferase having D-galactose bound to β- and 4-glycosidic bonds to “xylose residue linked to amino acid residue” (hereinafter also referred to as “glycosyltransferase 1” for convenience) (Hereinafter also referred to as “glycosyltransferase 2” for convenience).

  The above-mentioned “glycosyltransferase 1” refers to “D-glucuronic acid (hereinafter also simply referred to as“ GlcUA ”) residue or N-acetyl-D- residue relative to the sugar residue at the non-reducing end of chondroitin / chondroitin sulfate. There is no particular limitation as long as it has an activity of transferring a galactosamine (hereinafter, also simply referred to as “N-acetylgalactosamine” or “GalNAc”) residue. Such glycosyltransferase 1 is involved in elongation of chondroitin / chondroitin sulfate, and when mutation or suppression of expression occurs in such an enzyme, normal biosynthesis of chondroitin / chondroitin sulfate is hindered and bone formation is abnormal. Is thought to lead to Paget's disease of bone.

The “chondroitin” in the above “glycosyltransferase 1” refers to a disaccharide in which a uronic acid (hereinafter, also simply referred to as “UA”) residue and a GalNAc residue are linked by a 1,3 glycosidic bond to β1,4 glycoside. It is a kind of glycosaminoglycan having a basic skeleton (ie,-[4UA1-3GalNAcβ1-] _n : n is an integer of 2 or more) formed by bonding repeatedly. Chondroitin does not have a sulfate group attached to this basic skeleton, but chondroitin sulfate has a sulfated carbon atom at the 2-, 4-, or 6-position of a GalNAc residue, or the 2-position of a uronic acid residue. Have been.

  The “sugar residue at the non-reducing terminal of chondroitin / chondroitin sulfate” is any one of a GalNAc residue and a UA residue constituting the basic skeleton of chondroitin / chondroitin sulfate, and “glycotransferase 1” is When the residue is a GalNAc residue, it transfers UA, and when the sugar residue is a UA residue, it transfers GalNAc, but this enzyme has at least one transfer activity.

  Specific examples of such “glycosyltransferase 1” include “chondroitin synthase containing a protein having an amino acid sequence consisting of amino acids 1 to 882 of SEQ ID NO: 2”.

  The enzyme has an activity of transferring a GalNAc residue from a GalNAc donor substrate (eg, UDP-GalNAc) to a GalNAc acceptor having a structure represented by the following formula 1, and synthesizing a compound having a structure represented by the following formula 2. In addition, it has an activity of transferring a GlcUA residue from a GlcUA donor substrate (eg, UDP-GlcUA) to a GlcUA acceptor having a structure represented by the following formula 3, and synthesizing a compound having a structure represented by the following formula 4 ( Reference Example 2 in the present specification).

GlcUAβ1- (3GalNAcβ1-4GlcUAβ1) _k- (3GalNAc) _l (1)
GalNAcβ1-4GlcUAβ1- (3GalNAcβ1-4GlcUAβ1) _k − (3GalNAc) _l (2)
GalNAcβ1- (4GlcUAβ1-3GalNAcβ1) _m- (4GlcUA) _n (3)
GlcUAβ1-3GalNAcβ1- (4GlcUAβ1-3GalNAcβ1) _m − (4GlcUA) _n (4)
In the formulas 1 to 4, “-” indicates a glycosidic bond, and a number indicates a carbon position of the sugar ring where the glycosidic bond is present. “Α” and “β” denote the anomers of the glycosidic bond at the 1-position of the sugar ring, wherein the trans-position relative to the 5-position CH ₂ OH or COOH is “α”, and the cis-position is “β”. Show. k and m each represent an integer of 1 or more, and l and n each represent 1 or 0.

  Examination of the locus of the enzyme gene consisting of the nucleotide sequence (SEQ ID NO: 1) using OMIM (Online Mendelian Inheritance in Man) revealed that it was present at 5q31.1 (see Reference Example 1).

  Another example of “glycosyltransferase 1” is “chondroitin synthase containing a protein consisting of an amino acid sequence consisting of amino acids 1 to 755 of SEQ ID NO: 4”.

  The enzyme has an activity of transferring a GalNAc residue from a GalNAc donor (for example, UDP-GalNAc) to a GalNAc acceptor having the structure represented by the above formula 1, and synthesizing a compound having the structure represented by the above formula 2 and Has the activity of transferring a GlcUA residue from a GlcUA donor (eg, UDP-GlcUA) to a GlcUA acceptor having a structure represented by the above formula 3, and synthesizing a compound having a structure represented by the above formula 4 (this specification). Reference Example 4 in the book).

  Examination of the gene locus using OMIM revealed that the enzyme gene consisting of the base sequence (SEQ ID NO: 3) was present at 2q36.3 (see Reference Example 3 in this specification). .

  The `` activity of transferring a GlcUA residue or a GalNAc residue to a sugar residue at the non-reducing end of chondroitin '' is, for example, a GlcUA donor substrate or a GalNAc donor substrate labeled with radioactivity, and the like. After an enzymatic reaction using an unlabeled chondroitin / chondroitin sulfate (including low-molecular-weight chondroitin / low-molecular-weight chondroitin sulfate (including sugar chains of 20 sugars or less) obtained by reducing the molecular weight thereof) with an acceptor substrate The reaction product can be easily detected by using gel filtration and an autoradiograph or a scintillation counter. The GlcUA donor substrate is preferably a sugar nucleotide having GlcUA, for example, adenosine diphosphate-D-glucuronic acid (ADP-GlcUA), uridine diphosphate-D-glucuronic acid (UDP-GlcUA), Examples include guanosine diphosphate-D-glucuronic acid (GDP-GlcUA) and cytidine diphosphate-D-glucuronic acid (CDP-GlcUA), with UDP-GlcUA being most preferred. This is because UDP-GlcUA works as a GlcUA donor substrate in a living body.

  Further, the GalNAc donor substrate is preferably a sugar nucleotide having GalNAc, for example, adenosine diphosphate-N-acetyl-D-galactosamine (ADP-GalNAc), uridine diphosphate-N-acetyl-D -Galactosamine (UDP-GalNAc), guanosine diphosphate-N-acetyl-D-galactosamine (GDP-GalNAc), cytidine diphosphate-N-acetyl-D-galactosamine (CDP-GalNAc), and UDP- GalNAc is most preferred. This is because UDP-GalNAc works as a GalNAc donor substrate in the living body.

  The above “glycosyltransferase 2” has a function of transferring D-galactose (hereinafter also simply referred to as “galactose” or “Gal”) to “xylose residue linked to an amino acid residue” by β1,4 glycosidic bond. There is no particular limitation as long as it is "glycosyltransferase". Such glycosyltransferase 2 is involved in the early stage of the production of chondroitin / chondroitin sulfate, and mutation of such an enzyme prevents normal chondroitin / chondroitin sulfate biosynthesis, resulting in abnormal bone formation. Is thought to lead to Paget's disease of bone.

  The “amino acid residue” in the above “glycosyltransferase 2” is preferably an amino acid that forms a peptide (protein). In particular, in the structure of chondroitin / chondroitin sulfate proteoglycan existing in a living body, a peptide (protein ) And chondroitin / chondroitin sulfate-binding amino acid to which xylose is present at the reducing end of the characteristic tetrasaccharide structure (GlcUAβ1-3Galβ1-3Galβ1-4Xyl: hereinafter referred to as “linkage tetrasaccharide”) Is most preferred. Most preferably, L-serine is exemplified as such an amino acid.

  That is, “Glycosyltransferase 2” transfers a Gal residue from a Gal donor substrate (eg, UDP-Gal) to a Gal acceptor having a structure represented by the following formula 5, and obtains a compound having a structure represented by the following formula 6: It has an activity to synthesize (see Reference Example 5 in this specification).

Xylβ1-Ser (5)
Galβ1-4Xylβ1-Ser (6)
In the above formulas 5 and 6, “Ser” represents a serine residue (including the case in a peptide chain) in which the xylose residue represented by the above “Xyl” is bonded to its side chain, and “Xyl” is the above “Ser” "Represents a xylose residue bonded to the side chain of the serine residue, and"-"," β ", and the numbers are the same as in the above formulas 1 to 4.

  Examination of the gene locus using OMIM revealed that the enzyme gene consisting of the nucleotide sequence (SEQ ID NO: 5) was present at 5q35 (see Reference Example 5 in this specification).

  The “activity of transferring Gal to a“ xylose residue linked to an amino acid residue ”by β1,4 glycosidic bond” is, for example, a Gal donor substrate labeled with radioactivity or the like, and a xylose not labeled with radioactivity. After reacting with a binding peptide (for example, SEQ ID NO: 15: N-terminal sequence of bikunin in which xylose is bonded as a side chain to a serine residue at the ninth amino acid residue: manufactured by Peptide Institute) The reaction product can be easily detected by using gel filtration and an autoradiograph or a scintillation counter. The Gal donor substrate is preferably a sugar nucleotide having Gal, for example, adenosine diphosphate-D-galactose (ADP-Gal), uridine diphosphate-D-galactose (UDP-Gal), Examples include guanosine diphosphate-D-galactose (GDP-Gal) and cytidine diphosphate-D-galactose (CDP-Gal), with UDP-Gal being most preferred. This is because UDP-Gal works as a Gal donor substrate in vivo.

  As a gene mutation in Paget's disease of bone, for example, it is known that a mutation occurs in 18q22.1 in PDB2, 5q35 in PDB3, and 5q31 in PDB4 (OMIM # 602080). It is known that mutations occur in 2q36, 10q13, and 5q35 as genes in familial bone Paget's disease (Am. J. Hum. Genet. 69 (2001), pp. 1055-1061). Several to a dozen or so genes are encoded in such known mutation sites, and the location of each of the above-mentioned chondroitin synthase genes is 5q31.1 in SEQ ID NO: 1 (K3) and 3 K11) is 2q36.3, and SEQ ID NO: 5 (β4GalT7) is 5q35, which is completely identical to the position where the above mutation exists. As described above, since the genes of these enzymes involved in the synthesis of chondroitin / chondroitin sulfate all overlap with the gene mutation sites in Paget's disease of bone, mutations in these genes cause disorders in the biosynthesis of chondroitin / chondroitin sulfate. The probability of causing Paget's disease of bone is extremely high.

  As used herein, the term "associating the mutation or expression level of the chondroitin synthase gene with the Paget's disease of the bone" means that a mutation in the chondroitin synthase gene is detected, or that the expression level of the gene is healthy. It means that it is determined to be a Paget's disease of bone when it is lower than that of.

  As used herein, the term “gene mutation” refers to a structural change in a gene, and the “mutation” includes point mutation, inversion, deletion, insertion, duplication, translocation and the like. Further, the “mutant gene” refers to a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69, in which one or more nucleotides are substituted or It means a nucleotide sequence that has been deleted, or one or more nucleotides have been inserted or added to the nucleotide sequence. Here, the “plurality” is preferably 2 or more, more preferably 4 or more, still more preferably 6 or more, and most preferably 10 or more.

  Aspects of the mutation of the chondroitin synthase gene are preferably point mutation, deletion, insertion, inversion, duplication, translocation, more preferably point mutation, deletion, insertion, inversion, translocation, and still more preferably. Are point mutations, deletions, insertions, translocations, most preferably point mutations, deletions, insertions.

  As used herein, the term "gene expression" typically refers to the transcription of DNA or a portion thereof into RNA, or the translation of the RNA or a portion thereof into a protein. Therefore, the term “gene expression level” refers to the amount of RNA transcribed from the gene or the amount of protein translated from the RNA.

  The expression level of RNA can be quantified using, for example, a PCR method using the primers in Table 1 described in Example 1 described below. In the PCR method, the use of a quantitative PCR method is preferable, and an RT-PCR method for kinetic analysis and a quantitative real-time PCR method are exemplified. According to the present invention, the quantification of RNA is not limited to these, and Northern blots, dot blots, DNA microarrays and the like can also be used. On the other hand, nucleic acids of genes widely present in the same tissue or the like, for example, nucleic acids encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin can be used as controls. The expression level of the protein can be quantified using, for example, an ELISA method or a Western blot.

  The detection method of the present invention can be specifically performed as follows.

  That is, for example, from a sample containing a gene (e.g., epithelial tissue, connective tissue, etc., preferably connective tissue is mentioned, and it is very easy to collect, and blood is particularly preferable because the obtained sample is fresh). For example, total RNA can be extracted and purified using an existing RNA preparation kit (for example, RNA Extraction Kit: manufactured by Amersham Bioscience). Using the extracted and purified total RNA, the expression levels of K3, K11, and β4Gal-T7 were determined by RT-PCR using an existing kit (eg, SUPERSCRIPT First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen)). It can be quantified by a method such as a method. Examples of the combination of the 5 ′ primer, 3 ′ primer, and probe used include SEQ ID NO: 56 when quantifying the expression level of K3, SEQ ID NO: 57, SEQ ID NO: 58, and sequence when quantifying the expression level of K11. When quantifying the expression level of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, β4Gal-T7, a combination of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 can be mentioned. It is possible to select and prepare as appropriate.

  Then, for example, as a result of these quantifications, a patient in which the expression of any of K3, K11 and β4GalT7 gene is partially or completely suppressed can be regarded as a patient at high risk of bone Paget disease. In addition, the quantification can be performed by quantifying the amount of transcription of the K3, K11 and β4Gal-T7 genes from the RNA amount as exemplified above, for example, K3, K11 or β4Gal-T7 prepared according to a conventional method. It is also possible to directly determine the amount of K3, K11 or β4Gal-T7 protein using an antibody or the like. It is also possible to associate with.

  More specifically, as described in Example 2 below, the ratio of the expression level of the chondroitin synthase gene in peripheral blood mononuclear cells of a patient with bone Paget disease and normal peripheral blood mononuclear cells was determined. Bone Paget disease can be determined by measuring by quantitative PCR. According to the present invention, the ratio of the expression level that can be determined to be bone Paget's disease is 1/2 or less, preferably 1/5 or less, and still more preferably 1/10 or less. The term “measurement” as used herein includes any of detection, amplification, quantification, and semi-quantification.

  According to the present invention, Paget's disease of bone can be detected by confirming the presence of a mutation in the chondroitin synthase gene. That is, the nucleotide sequence of the chondroitin synthase gene extracted from peripheral blood mononuclear cells of a patient with bone Paget disease is compared with the nucleotide sequence of the chondroitin synthase gene extracted from normal peripheral blood mononuclear cells, and the presence of the mutation is determined. By detecting, it can be determined that there is bone Paget disease. More specifically, as described in Example 1 described below, the genome of the genome was prepared using primers appropriately prepared based on the base sequences of K3, K11 and β4Gal-T7 described in the sequence listing of the present specification. The K3, K11 or β4Gal-T7 gene is directly amplified from DNA using a method such as PCR, and the nucleotide sequence is analyzed according to a known nucleotide sequencing method. It is possible to conclude that

  The number of mutated nucleotides that can be determined to be a Paget disease of bone is 1 or more, preferably 4 or more, still more preferably 6 or more, and most preferably 10 or more.

  Aspects of the mutation of the chondroitin synthase gene are preferably point mutation, deletion, insertion, inversion, duplication, translocation, more preferably point mutation, deletion, insertion, inversion, translocation, and still more preferably. Are point mutations, deletions, insertions, translocations, most preferably point mutations, deletions, insertions.

  In addition, the present invention has revealed that K3, K11 and β4Gal-T7 are related to bone Paget's disease. It is also possible to prepare knockout animals that exhibit the pathology of the disease. As the knockout animal, for example, mammals such as mice, rats, rabbits, dogs, cats, and monkeys are preferable, and mice or rats are most preferable since knockout animal preparation techniques have been established to some extent. Is not done.

  For example, knockout mice (pathological mice) showing the pathology of Paget's disease of bone can be performed according to the description of, for example, the latest technology of gene targeting (Ken Yagi edited by Yodosha) and gene targeting (translated by Tetsuo Noda, Medical Science International). it can.

  For example, exon (in the case of mK3) containing the activation domain of the mouse gene (K3 (mK3: SEQ ID NO: 65), K11 (mK11: SEQ ID NO: 67) or β4Gal-T7 (mβ4Gal-T7: SEQ ID NO: 69)) to be knocked out The fragment containing the third exon (1560 bp) of the ORF portion, the fragment containing 4598 bp corresponding to the full length of the ORF in the case of mK11, and the fragment containing the second to sixth exon of the ORF in the case of mβ4Gal-T7 ) Into the ES cells (derived from E14 / 129Sv mouse) by a known method such as electroporation, and select G418-resistant colonies. Then, the G418-resistant colony can be transferred to a 24-well plate and cultured. After a portion of the cells is cryopreserved, DNA is extracted from the remaining ES cells, and about 120 colonies in which recombination has occurred are selected by a known nucleotide sequence analysis method. Furthermore, it was confirmed that the recombination had occurred as planned using a method such as Southern blotting, and finally about 10 recombinants were selected, and about 2 clones of the ES cells were transferred to C57BL / 6 mouse embryos. Inject into the blastocyst. In this way, the mouse embryo into which the ES cells have been injected can be transplanted into the uterus of a foster parent mouse to generate a chimeric mouse. Hetero knockout mice can be obtained from chimeric mice by germ transmission.

  Knockout mice can also be prepared by a “method of suppressing gene expression” such as the small interfering RNA method (T.R. Brummelkamp et al., Science, 296, 550-553 (2002)).

  More specifically, as described in Example 3 below, a knockout mouse was prepared by introducing a chondroitin synthase gene into an ES cell, and using a clone in which the desired homologous recombination had occurred to obtain a germ line. Can be produced by establishing a mouse line.

Hereinafter, the present invention will be described more specifically with reference to examples.
Reference Example 1: Preparation of chondroitin sulfate synthase (K3) (1) Cloning of cDNA and construction of expression vector Amino acid sequence of β1,4 galactose transferase (β4Gal-T) (amino acid sequence encoded by GenBank accession No. D29805) Was used as a query to perform a BLAST search. As a result, Expression Sequence Tag (EST: GenBank accession No. AC0004219) was found. However, since this sequence was incomplete, the ORF was examined from the genome database by GenScan (Stanford University, USA). As a result, the nucleotide sequence described in SEQ ID NO: 1 (the encoded amino acid sequence is SEQ ID NO: 2) was discovered. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 was confirmed to be expressed in at least human brain by RT-PCR using Marathon-Ready cDNA (Clontech) as a template. No. 9 and SEQ ID No. 10, and combinations of 5 ′ and 3 ′ primers of SEQ ID Nos. 11 and 12 were used). In order to clone a soluble region excluding a region containing a transmembrane region of this gene (a region consisting of amino acid numbers 1 to 129 of SEQ ID NO: 2), two primers of SEQ ID NO: 7 and SEQ ID NO: 8 are used in accordance with a conventional method. PCR was performed. As the template cDNA used, Marathon-Ready cDNA human brain (Clontech) was used. The amplified band of about 2.3 kb is digested with HindIII and XbaI according to a conventional method, and inserted into a HindIII and XbaI site of a mammalian cell expression vector pFLAG-CMV-1 (manufactured by Sigma) according to a conventional method. (K3-FLAG-CMV1) was obtained. When the base sequence of the obtained expression vector was confirmed, it was confirmed that a DNA fragment consisting of the base sequence of base numbers 388 to 2649 of the base sequence described in SEQ ID NO: 1 was inserted. When the position of the DNA having this nucleotide sequence on the genome was confirmed using OMIM, it was revealed that the DNA was present at 5q31.1.
(2) Preparation of K3
15 μg of K3-FLAG-CMV1 was transfected into COS7 cells cultured in a 100-mm culture dish so as to be 70% confluent using TransFast (promega) according to the protocol. The supernatant cultured for 3 days was collected, filtered through a 0.22 μm filter, and 10 μl of the supernatant was added with 100 μl of Anti-FLAG M2-Agarose Affinity Gel (manufactured by Sigma) and mixed by inversion at 4 ° C. overnight. After the reaction, the gel was washed three times with 50 mM Tris-HCl, pH 7.4 / 20% glycerol, and an excess washing solution was removed using a syringe fitted with a 27G injection needle. This gel was suspended in 50 mmol / L Tris-HCl, pH 7.4 (20% glycerol, 10 mmol / L phenylmethylsulfonyl fluoride, 1 μg / ml leupeptin, 1 μg / ml pepstatin) so as to be 50% (v / v). The suspension was turbid, and after centrifugation, the supernatant was removed to obtain an enzyme-adsorbed gel suspension.
Reference Example 2
Elongation of chondroitin skeleton using K3 (1) Preparation of chondroitin / chondroitin sulfate odd-numbered sugar Chondroitin (chemically desulfated chondroitin sulfate derived from shark cartilage: manufactured by Seikagaku Corporation) and chondroitin sulfate (derived from shark cartilage: After limited digestion of bovine testicular hyaluronidase (manufactured by Seikagaku Corporation; also known as chondroitin sulfate C), the reaction solution was kept at 100 ° C. for 10 minutes to inactivate the enzyme by heating. The reaction solution is applied to a Superdex 30 column (60 × 1.6 cm, manufactured by Amersham Bioscience, chromatography conditions; mobile phase: 0.2 mol / L NH ₄ HCO ₃ , flow rate: 2 ml / min), and the eluate is subjected to absorbance at 225 nm. While monitoring with, fractionation was performed every 2 ml, and fractions corresponding to 10 sugars were pooled. The fraction was desalted with a PD10 column (manufactured by Amersham Bioscience), and uronic acid was quantified by a carbazole sulfate method according to a conventional method, followed by freeze-drying. The lyophilized product was dissolved in distilled water so as to have a concentration of 1 mM, and used as an even-numbered oligosaccharide sample (a decasaccharide derived from chondroitin was described as “CH10”, and a decasaccharide derived from chondroitin sulfate was described as “CS10”).

10 μl of enzyme-adsorbed gel suspension in 50 mmol / L MES buffer (pH 6.5) containing 10 nmol / L MnCl ₂ and 171 μmol / L ATP sodium salt, test substances (chondroitin (CHEL), chondroitin sulfate (CSEL)) , CH10 or CS10) and 0.036 nmol of [ ³ H] UDP-GalNAc to make a total volume of 30 μl. The enzyme reaction was performed at 37 ° C. for 1 hour, and then the reaction solution was kept at 100 ° C. for 1 minute to inactivate the enzyme and stop the reaction.

  After filtering each reaction solution with a micro filter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 1.0 cm: manufactured by Amersham Biosciences Inc., chromatography conditions; mobile phase: 0.2 mol / L NaCl, (The flow rate was 1.0 ml / min), and the eluate was collected for each 0.5 ml fraction, and the radioactivity was measured with a scintillation counter (FIG. 1). As a result, strong GalNAc transfer activity was observed when CHEL (fractions 17 to 18), CH10 (fraction 23) and CS10 (fraction 23) were used as the GalNAc receptor substrate, and GalNAc transfer activity against CSEL was observed. Was not observed (16 fractions). 23 fractions of the reaction product obtained from CH10 and CS10 were fractions showing the molecular weight at which 11 sugars eluted. The 11 saccharide obtained from CH10 is referred to as “CH11 (K3)”, and the 11 saccharide obtained from CS10 is referred to as “CS11 (K3)”.

  Fractions 21-25 of CS11 (K3) were collected, pooled and desalted on a PD10 column. The sample thus obtained was bisected and freeze-dried. One half was dissolved in 100 μl of 0.1 mol / L TrisHCl buffer (pH 7.4) containing 30 mM sodium acetate (CS11 (K3) A), and the other was digested with chondroitinase ACII (100 mU chondroitinase). ACII (manufactured by Seikagaku Corporation) was dissolved in 100 μl of the CS11 (K3) fraction, digested with enzyme at 37 ° C. for 10 hours, and heated to inactivate the enzyme: CS11 (K3) B).

After CS11 (K3) A and CS11 (K3) B were filtered through a 0.22 μm pore size microfilter (manufactured by Millipore), a Superdex peptide column (30 × 10 mm: manufactured by Amersham Biosciences, chromatography conditions; mobile phase) : 0.2 mol / L NaCl, flow rate 0.5 ml / min), and the eluate was collected for each 0.5 ml fraction, and the radioactivity was measured with a scintillation counter. As a result, the monosaccharide was detected in CS11 (K3) B. The radioactivity peak shifted to the fraction (FIG. 2). From these results, it was revealed that K3 can transfer GalNAc to GlcUA at the non-reducing terminal of chondroitin sulfate-derived decasaccharide by β1,4 bond to prepare 11-sugar.
(2) Preparation of chondroitin / chondroitin sulfate even sugar Chondroitin (chemically desulfating chondroitin sulfate derived from shark cartilage: manufactured by Seikagaku Corporation) and chondroitin sulfate (derived from shark cartilage: manufactured by Seikagaku Corporation: chondroitin sulfate C) Was digested with bovine testicular hyaluronidase (manufactured by Sigma), and the reaction solution was kept at 100 ° C. for 10 minutes to inactivate the enzyme. The reaction solution was centrifuged at 10,000 × g for 10 minutes, and the supernatant was recovered and further digested with bovine liver-derived β-glucuronidase (manufactured by Sigma). The enzyme reaction was stopped by keeping the reaction solution at 100 ° C. for 10 minutes. This reaction solution is applied to a Superdex 30 column (60 × 1.6 cm, manufactured by Amersham Biosciences, chromatography conditions; mobile phase: 0.2 mol / L NH ₄ HCO ₃ , flow rate: 2 ml / min), and the eluate is measured at an absorbance of 225 nm. While monitoring, fractionation was performed every 2 ml, and fractions corresponding to 11 sugars were pooled. Each fraction was desalted using a PD10 column (manufactured by Amersham Bioscience), and uronic acid was quantified by a carbazole sulfate method according to a conventional method, followed by freeze-drying. The lyophilized product was dissolved in distilled water so as to have a concentration of 1 mmol / L, and used as an odd-numbered oligosaccharide sample (11-saccharide derived from chondroitin: “CH11”, 11-saccharide derived from chondroitin sulfate: “CS11”).

  Also, chondroitin (chemically desulfating chondroitin sulfate derived from shark cartilage: manufactured by Seikagaku Corporation) and chondroitin sulfate (derived from shark cartilage: manufactured by Seikagaku Corporation: also called chondroitin sulfate C) are derived from bovine liver. Samples were digested with β-glucuronidase (manufactured by Sigma) (described as “CHOL” and “CSOL”, respectively).

10 μl of the enzyme-adsorbed gel suspension in 10 mmol / L MnCl ₂ , 50 mmol / L acetate buffer (pH 5.6), 1 nmol of the test substance (CHOL, CSOL, CH11 or CS11), and [ ¹⁴ C] UDP-GlcUA 0.432 nmol was added to make the total volume 30 μl. The enzyme reaction was performed at 37 ° C. for 1 hour, and then the reaction solution was kept at 100 ° C. for 1 minute to inactivate the enzyme and stop the reaction.

  After filtering each reaction solution with a micro filter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 1.0 cm: manufactured by Amersham Biosciences Inc., chromatography conditions; mobile phase: 0.2 mol / L NaCl, The eluate was collected for each 0.5 ml fraction, and the radioactivity was measured with a scintillation counter (FIG. 3). As a result, strong GlcUA transfer activity was observed when CHOL (fractions 17 to 18), CH11 (fraction 23) and CS11 (fraction 23) were used as GlcUA receptor substrates, and GlcUA transfer activity against CSOL was observed. Was not observed (16 fractions). The 22 to 23 fractions of the reaction product obtained from CH11 and CS11 were fractions showing the molecular weight at which 12 sugars eluted. The dodecasaccharide obtained from CH11 is referred to as “CH12 (K3)”, and the dodecasaccharide obtained from CS11 is referred to as “CS12 (K3)”.

  Fractions 21 to 25 of CS12 (K3) were collected, pooled, and desalted on a PD10 column. The sample thus obtained was bisected and freeze-dried. One half was dissolved in 100 μl of 0.1 mol / L TrisHCl buffer (pH 7.4) containing 30 mmol / L sodium acetate (CS12 (K3) A), and the other was digested with chondroitinase ACII (100 m units). Chondroitinase ACII (manufactured by Seikagaku Corporation) was dissolved in 100 μl of the CS11 (K3) fraction, digested with enzyme at 37 ° C. for 10 hours, and heated to inactivate the enzyme: CS12 (K3) B). .

After filtering CS12 (K3) A and CS12 (K3) B through a micro filter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 1.0 cm: manufactured by Amersham Biosciences, chromatography conditions; migration) Phase: 0.2 mol / L NaCl, flow rate 0.5 ml / min). The eluate was fractionated into 0.5 ml fractions, and the radioactivity was measured with a scintillation counter. The radioactivity peak shifted to the sugar fraction (FIG. 4). From these results, it was revealed that K3 can transfer GlcUA to 11 sugars derived from chondroitin sulfate by β1,3 bond to prepare dodecasaccharide.
Reference Example 3
Preparation of chondroitin sulfate synthase (K11)
(1) Cloning of cDNA and Construction of Expression Vector A BLAST search was performed using the amino acid sequence of chondroitin sulfate glucuronyltransferase (CSGlcA-T) (the amino acid sequence encoded by GenBank accession No. AB037823) as a query. As a result, EST (GenBank accession No. NM_018590) was found. However, since this sequence was incomplete, the ORF was examined from the genome database by GenScan (Stanford University, USA). As a result, the nucleotide sequence described in SEQ ID NO: 3 (the encoded amino acid sequence was SEQ ID NO: 4) was discovered. The gene consisting of the nucleotide sequence of SEQ ID NO: 3 was confirmed to be expressed in at least human brain by RT-PCR using Marathon-Ready cDNA (Clontech) as a template. In order to clone a soluble region excluding a region containing a transmembrane region of this gene (a region consisting of amino acids 1 to 96 of SEQ ID NO: 4), two types of primers of SEQ ID NO: 13 and SEQ ID NO: 14 are used in accordance with a conventional method. PCR was performed. As the template cDNA used, Marathon-Ready cDNA human brain (Clontech) was used. The amplified band of about 2 kb was digested with EcoRI and BamHI according to a conventional method, and inserted into the EcoRI and BamHI sites of an expression vector for mammalian cells pFLAG-CMV-1 (manufactured by Sigma) according to a conventional method to obtain K11-FLAG-. CMV1 was obtained. When the base sequence of the obtained vector was confirmed, it was confirmed that a DNA fragment consisting of the base sequence of base numbers 287 to 2328 of the base sequence described in SEQ ID NO: 3 was inserted. The position of the DNA having this nucleotide sequence on the genome was confirmed using OMIM, and it was revealed that the DNA was present at 2q36.3.
(2) Preparation of K11
Using TransFast (promega), 15 mg of K11-FLAG-CMV1 was used to introduce the gene into COS7 cells cultured in a 100 mm culture dish so as to be 70% confluent according to the protocol. The supernatant cultured for 3 days was collected and filtered through a 0.22 μm filter, and 100 μl of Anti-FLAG M2-Agarose Affinity Gel (manufactured by Sigma) was added to 10 ml of the supernatant, and the mixture was inverted and mixed at 4 ° C. overnight. After the reaction, the gel was washed three times with 50 mmol / L Tris-HCl, pH 7.4 (20% glycerol), and excess washing solution was removed using a syringe fitted with a 27G injection needle. This gel was adjusted to 50% (v / v) in 50 mmol / L TrisHCl buffer, pH 7.4, (20% glycerol, 10 mmol / L phenylmethylsulfonyl fluoride, 1 μg / ml leupeptin, 1 μg / ml pepstatin). After suspending and centrifuging the suspension, the supernatant was removed to obtain an enzyme-adsorbed gel suspension.
Reference example 4
Extension of chondroitin skeleton using K11
(1) Preparation of chondroitin / chondroitin sulfate odd-numbered sugar
10 μl of enzyme-adsorbed gel suspension in 50 mmol / L MES buffer (pH 6.5) containing 10 nmol / L MnCl ₂ and 171 μmol / L ATP sodium salt, 1 nmol of test substance (CHEL, CSEL, CH10 or CS10) Then, 0.036 nmol of [ ³ H] UDP-GalNAc was added to make the total amount 30 μl. The enzyme reaction was performed at 37 ° C. for 1 hour, and then the reaction solution was kept at 100 ° C. for 1 minute to inactivate the enzyme and stop the reaction.

  After filtering each reaction solution with a micro filter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 1.0 cm: manufactured by Amersham Biosciences Inc., chromatography conditions; mobile phase: 0.2 mol / L NaCl, The eluate was collected for each 0.5 ml fraction, and the radioactivity was measured with a scintillation counter (FIG. 5). As a result, strong GalNAc transfer activity was observed when CHEL (fraction 18), CH10 (fraction 23) and CS10 (fraction 23) were used as GalNAc receptor substrates, and weak GalNAc transfer activity was observed for CSEL. Observed (16 fractions). The 22 to 23 fractions of the reaction product obtained from CH10 and CS10 were fractions showing the molecular weight at which 11 sugars eluted. The 11 saccharide obtained from CH10 is referred to as “CH11 (K11)”, and the 11 saccharide obtained from CS10 is referred to as “CS11 (K11)”.

  Fractions 21-25 of CS11 (K11) were collected, pooled and desalted on a PD10 column. The sample thus obtained was bisected and freeze-dried. One half was dissolved in 100 μl of 0.1 mol / L TrisHCl buffer (pH 7.4) containing 30 mM sodium acetate (CS11 (K11) A), and the other was digested with chondroitinase ACII (100 m chondroitin). Nase ACII (manufactured by Seikagaku Corporation) was dissolved in 100 μl of the CS11 fraction, subjected to enzyme digestion at 37 ° C. for 10 hours, and heated to inactivate the enzyme: CS11 (K11) B).

After CS11 (K11) A and CS11 (K11) B were filtered through a microfilter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 10 mm: manufactured by Amersham Biosciences, chromatography conditions; mobile phase) : 0.2 mol / L NaCl, flow rate 0.5 ml / min). The eluate was fractionated for each 0.5 ml fraction, and the radioactivity was measured with a scintillation counter. The radioactivity peak shifted to the fraction (FIG. 6). These results suggest that K11 can transfer GalNAc to GlcUA at the non-reducing end of chondroitin sulfate-derived decasaccharide by β1,4 bond to prepare the decasaccharide.
(2) Preparation of chondroitin / chondroitin sulfate even sugar
10 nmol / L MnCl ₂ , 10 μl of the enzyme-adsorbed gel suspension in 50 mmol / L acetate buffer (pH 5.6), 1 nmol of the test substance (CHOL, CSOL, CH11 or CS11), and [ ¹⁴ C] UDP-GlcUA 0.432 nmol was added to make the total volume 30 μl. The enzyme reaction was performed at 37 ° C. for 1 hour, and then the reaction solution was kept at 100 ° C. for 1 minute to inactivate the enzyme and stop the reaction.

  After filtering each reaction solution with a micro filter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 1.0 cm: manufactured by Amersham Biosciences Inc., chromatography conditions; mobile phase: 0.2 mol / L NaCl, The eluate was collected for each 0.5 ml fraction, and the radioactivity was measured with a scintillation counter (FIG. 3). As a result, strong GlcUA transfer activity was observed when CHOL (fraction 18), CH11 (fraction 23) and CS11 (fraction 22) were used as GlcUA receptor substrates, and GlcUA transfer activity was observed for CSOL. Was not done. The 22 to 23 fractions of the reaction products obtained from CH11 and CS11 were fractions showing the molecular weight at which 12 sugars eluted. The dodecasaccharide obtained from CH11 is referred to as “CH12 (K11)”, and the dodecasaccharide obtained from CS11 is referred to as “CS12 (K11)”.

  Fractions 21 to 25 of CS12 (K11) were collected, pooled, and desalted on a PD10 column. The sample thus obtained was bisected and freeze-dried. One half was dissolved in 100 μl of 0.1 mol / L TrisHCl buffer (pH 7.4) containing 30 mmol / L sodium acetate (CS12 (K11) A), and the other was digested with chondroitinase ACII (100 m units). Chondroitinase ACII (manufactured by Seikagaku Corporation) was dissolved in 100 μl of the CS11 (K11) fraction, digested with enzyme at 37 ° C. for 10 hours, and heated to inactivate the enzyme: CS12 (K11) B). .

After filtering CS12 (K11) A and CS12 (K11) B through a micro filter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 1.0 cm: manufactured by Amersham Biosciences, chromatography conditions; migration) Phase: 0.2 mol / L NaCl, flow rate 0.5 ml / min). The eluate was fractionated into 0.5 ml fractions, and the radioactivity was measured with a scintillation counter. The radioactivity peak shifted to the sugar fraction (FIG. 4). From these results, it was revealed that the enzyme of the present invention can transfer GlcUA to 11 sugars derived from chondroitin sulfate by β1,3 bond to prepare 12 sugars.
Reference example 5
Preparation of chondroitin sulfate synthase (β4Gal-T7) and confirmation of its activity
Β4Gal-T7 was cloned according to the method of Almeida et al. (Described in EXPERIMENTAL PROCEDURES in J. Biol. Chem., 274, 37, 26165-26171 (1999)). When the nucleotide sequence of the obtained clone was confirmed according to a conventional method, it was revealed that the clone consisted of the nucleotide sequence of SEQ ID NO: 5. The location of the DNA having this nucleotide sequence on the genome was confirmed using OMIM, and it was revealed that the DNA was present at 5q35.

Further, expression of β4Gal-T7 was performed according to the method of Gotoh et al. (J. Biol. Chem., 277, 41, 38189-38196 (2002) to obtain a suspension of an affinity carrier to which β4Gal-T7 was bound. Gel 10 μl, receptor substrate (xylose-binding peptide (SEQ ID NO: 15: N-terminal sequence of bikunin in which xylose is bound to the ninth amino acid residue at serine as a side chain): manufactured by Peptide Research Institute) 6.25 mg 171 μmol of adenosine triphosphate (ATP) sodium salt, 0.036 mol of [ ³ H] UDP-Gal were added to 50 mmol of MES buffer (pH 6.5) containing 10 nmol / L of MnCl ₂ to make a total volume of 30 μl, The reaction was performed for 1 hour at 37 ° C. Thereafter, the reaction solution was kept at 100 ° C. for 1 minute to inactivate the enzyme and stop the reaction.

After the reaction solution was filtered through a microfilter (manufactured by Millipore) having a pore size of 0.22 μm, a Superdex peptide column (30 × 1.0 cm; manufactured by Amersham Biosciences, Inc., chromatography conditions; mobile phase: 0.2 mol / L NaCl, flow rate 1.0) The eluate was collected for each 0.5 ml fraction, and the radioactivity was measured with a scintillation counter. As a result, strong radioactivity was observed in a fraction of a polymer different from the fraction eluted with [ ³ H] UDP-Gal. This confirmed that β4Gal-T7 binds galactose to the receptor substrate.

Example 1 DNA extraction from blood sample and nucleotide sequence analysis
DNA was extracted from 1 ml of blood using a GFX Genomic Blood DNA Purification Kit (manufactured by Amersham Bioscience). The exon of the chondroitin sulfate synthase (K3, K11, β4Gal-T7) gene was amplified by various primers (see Table 1 below) using 250 ng of the obtained 14 to 16 μg of DNA as a template. Using the fragment obtained in this way as a template, the nucleotide sequence is analyzed by a conventional method, and the nucleotide sequence of each healthy chondroitin synthase gene is compared with the nucleotide sequence to easily determine the presence or absence of a mutation or single nucleotide polymorphism. You can find out.

Example 2 RNA extraction from blood sample and expression level analysis
10 ml of blood is centrifuged at 3,000 rpm for 10 minutes, and the intermediate layer is separated. Furthermore, centrifugation is performed using Lympho Prep (Nycomed) to separate the intermediate layer. By this operation, 9 × 10 ⁷ mononuclear cells (PBMC) in peripheral blood are obtained. Total RNA can be prepared from the entire amount of PBMC using an RNA Extraction Kit (Amersham Bioscience). A 1st strand cDNA was synthesized from 10 ng of the total RNA by a SUPERSCRIPT First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen). Using this cDNA as a template, the expression level of each gene can be quantified by real-time PCR. The primers and probes used are shown in Table 2.

Example 3 Preparation of Disease Model Mouse Knockout mouse is an exon containing an activation domain of mouse K3 (mK3: SEQ ID NO: 65), K11 (mK11: SEQ ID NO: 67) or β4Gal-T7 (mβ4Gal-T7: SEQ ID NO: 69) (In the case of mK3, an approximately 10 kb fragment containing the third exon (1560 bp) of the ORF portion, in the case of mK11, an approximately 10 kb fragment containing 4598 bp corresponding to the full length of the ORF, and in the case of mβ4Gal-T7, the ORF 2 Transfection (electroporation) of ES cells (derived from E14 / 129Sv mice) with 80 μg of a linear targeting vector into which a chromosome fragment (about 10 kb) centered on the sixth exon containing the sixth exon was inserted. ) And G418 resistant colonies were selected. G418 resistant colonies were transferred to a 24-well plate and cultured. After a part of the cells was cryopreserved, DNA was extracted from the remaining ES cells, and about 120 clones having recombination were selected by PCR. Furthermore, it was confirmed that the recombination had occurred as expected by Southern blotting, and finally about 10 recombinants were selected. ES cells of two of the selected clones were injected into blastocysts of C57BL / 6 mice. The mouse embryo into which the ES cells were injected was implanted into the uterus of a foster parent mouse to produce a chimeric mouse. Thereafter, hetero knockout mice were obtained by germ transmission.

  INDUSTRIAL APPLICABILITY The present invention can be used for the detection of Paget's disease of bone and the production of a diseased animal of Paget's disease of bone.

It is a figure which shows the chromatography chart which shows the synthesis | combination of the odd-numbered sugar by the GalNAc transfer activity of K3. Open circles are charts showing GalNAc transfer activity to chondroitin sulfate, and black circles are diagrams showing GalNAc transfer activity to chondroitin. The open triangle is a chart showing GalNAc transfer activity to chondroitin sulfate 10-saccharide, and the closed triangle is a chart showing GalNAc transfer activity to chondroitin 10-saccharide. FIG. 4 is a view showing a chromatography chart of 11-saccharide prepared by the GalNAc transfer activity of K3 and its chondroitinase ACII digest. Open circles show charts of 11 sugars that have not been digested with chondroitinase ACII, and black circles show charts of digested products after chondroitinase ACII digestion. FIG. 3 is a view showing a chromatography chart showing the synthesis of even-numbered sugars by the GlcUA transfer activity of K3. Open circles are charts showing GlaUA transfer activity on chondroitin sulfate, and filled circles are GlcUA transfer activities on chondroitin. The open triangle is a chart showing GlcUA transfer activity on chondroitin sulfate 11 sugar, and the open triangle is a chart showing GlcUA transfer activity on chondroitin 11 sugar. FIG. 3 is a view showing a chromatography chart of a dodecasaccharide prepared by the GlcUA transfer activity of K3 and its chondroitinase ACII digest. Open circles show charts of 12 sugars that have not been digested with chondroitinase ACII, and solid circles show charts of digested products after chondroitinase ACII digestion. It is a figure which shows the chart of the chromatography which shows the synthesis | combination of the odd-number sugar by GalNAc transfer activity of K11. Open circles are charts showing GalNAc transfer activity to chondroitin sulfate, and black circles are diagrams showing GalNAc transfer activity to chondroitin. The open triangle is a chart showing GalNAc transfer activity to chondroitin sulfate 10-saccharide, and the closed triangle is a chart showing GalNAc transfer activity to chondroitin 10-saccharide. FIG. 2 is a view showing a chromatography chart of 11-saccharide prepared by Gal11 transfer activity of K11 and its chondroitinase ACII digest. Open circles show charts of 11 sugars that have not been digested with chondroitinase ACII, and black circles show charts of digested products after chondroitinase ACII digestion. FIG. 3 is a view showing a chromatography chart showing synthesis of even-numbered sugars by the GlcUA transfer activity of K11. Open circles are charts showing GlaUA transfer activity on chondroitin sulfate, and filled circles are GlcUA transfer activities on chondroitin. The open triangle is a chart showing GlcUA transfer activity on chondroitin sulfate 11 sugar, and the open triangle is a chart showing GlcUA transfer activity on chondroitin 11 sugar. FIG. 2 is a view showing a chromatography chart of a dodecasaccharide prepared by the GlcUA transfer activity of K11 and its chondroitinase ACII digest. Open circles show charts of 12 sugars that have not been digested with chondroitinase ACII, and solid circles show charts of digested products after chondroitinase ACII digestion.

Claims (5)

A method for detecting Paget's disease of bone, wherein the mutation or expression level of a chondroitin synthase gene is associated with Paget's disease of bone. The `` chondroitin synthase '' is a glycosyltransferase having an activity of transferring a D-glucuronic acid residue or an N-acetyl-D-galactosamine residue to a sugar residue at a non-reducing terminal of chondroitin. The method for detecting Paget's disease of bone according to claim 1. 3. The method according to claim 1, wherein the "chondroitin synthase" is "a glycosyltransferase that transfers D-galactose to a" xylose residue linked to an amino acid residue "through a β1,4 glycosidic bond". A method for detecting Paget's disease of bone. The "chondroitin synthase gene" is a gene comprising the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69. Item 6. The method for detecting Paget's disease of a bone according to Item 1. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 70, or a glycosyltransferase gene comprising an amino acid sequence having homology thereto A knockout animal, the expression of which is partially or completely suppressed.

Images