WO2003087820A1

WO2003087820A1 - Method of examining comp failure

Info

Publication number: WO2003087820A1
Application number: PCT/JP2003/004768
Authority: WO
Inventors: Shiro Ikegawa; Akihiko Mabuchi
Original assignee: Riken
Priority date: 2002-04-16
Filing date: 2003-04-15
Publication date: 2003-10-23
Also published as: AU2003235161A1; JP2003307517A

Abstract

It is intended to provide a method of conveniently examining COMP failure. Namely, a method of examining COMP failure which comprises quantifying COMP protein per unit volume of a body fluid.

Description

Specification

Testing methods for COMP abnormalities

The present invention relates to a method for examining abnormalities of C0MP (cartilage oligomeric matrix protein). More specifically, the present invention relates to a method for examining C0MP abnormality by measuring the amount of C0MP per unit volume of body fluid of a subject, and also relates to a kit for performing the examination method. Background art

Pseudoachondroplasia (PSACH) is a dwarfism with short-limbs with normal intelligence and normal facial features. It shows a characteristic growth pattern and normal birth height, but height growth declines rapidly after one year of age, with final height of less than 13 to 4 SD in mild cases and less than 16 SD in severe cases. Short stature (1 SD is about 5 cm for adults; 16 SD is more than 30 cm below average). Many adults are less than 1 m in height. In addition, the limb has limited range of motion and deformity of large joints, premature osteoarthritis and flaccidity of whole body ligaments. In childhood, there is a generalized squamous vertebrae, characterized by an anterior tongue-like protrusion of the vertebral body in a lateral view. However, this spinal dysplasia almost normalizes in adult cases, making diagnosis difficult. The long and short tube bones are shortened, and the epiphysis and metaphysis are markedly dysplastic.

Multiple epiphyseal dysplasia (MED) is a disease that causes multiple tubular epiphyseal dysplasias, resulting in short stature and early-onset osteoarthritis. Intelligence and facial features are normal. Short stature occurs after infancy, is relatively mild, and is often in the normal range. It is not a single disease but a syndrome with considerable clinical and genetic heterogeneity. C0MP, 9 type collagen gene _{(C0L9A1 C0L9A2, C0L9A3) s matrillin} 3 (MTN3), mutations in genes DSTDT have been reported.

The mechanism by which these diseases occur is that C0MP forms pentamers. Therefore, it is thought that the mutation acts dominant-negative, but there are some facts suggesting that the interaction with other components of the cartilage extracellular matrix such as collagen is impaired. Type 9 collagen is known to bind to C0MP.

As described above, PSACH and MED are intractable hereditary diseases that cause marked skeletal abnormalities such as severe short stature and early-onset osteoarthritis. Therefore, early diagnosis and definitive diagnosis are needed for early treatment, prevention of complications, prediction of prognosis, and genetic counseling. Currently, the diagnosis of PSACH and MED is based on clinical and radiological findings. However, 1) the image of the disease is complicated, and the differences in age and cases are large in findings. There are many problems, such as experience and skills required.

In addition, in C0MP abnormalities including PSACH and MED, diagnosis at the DNA / gene level is also performed. For example, in the case of PSACH, almost all cases of PSACH examined in the past have C0MP mutations. Also, 36 C0MP mutations have been found in 60 PSACH patients. All mutations were in only one allele, and no abnormality was found in both alleles. The position of the mutation is mostly in the calmodulin-like repeat, but it is also found in the C-terminal globular domai. Exon leveled at Norre (or, or have one force S in exon 9 force ^¾ et al exon 18.

In addition, in the case of MED, C0MP mutations were found in about one-third of the cases examined. This is due to the genetic heterogeneity of MED and the presence of five causative genes other than C0MP such as type 9 collagen gene (C0L9A1 C0L9A2.C0L9A3), MTN3 and DSTDT. The mutations found in MED also range widely from exon 9 to exon 18.

COMP (cartilage oligomeric matrix protein; abnormal ¾E!) NA · In the diagnosis of erythropodia, the gene structure of the causal gene C0MP is complex, the concentration of mutations is small, and it is necessary to examine a wide range of regions. However, there is a problem that it takes much time and cost. Therefore, development of a quick and simple screening diagnostic system has been awaited. Disclosure of the invention An object of the present invention is to provide a method for easily diagnosing a COMP abnormality.

The present inventors have conducted intensive studies to solve the above problems, and as a result, the blood C0MP level in patients with C0MP abnormality has been confirmed to be normal, and for multiple epiphyseal dysplasia confirmed to have no C0MP abnormality. It was found to be significantly lower than in patients. The present invention has been completed based on this finding. The blood C0MP protein was measured in rheumatoid arthritis, osteoarthritis, etc. (Clark AG et al., Arthritis Rheum 42 (11): 2356-64, 1999), but it was not possible to measure CMP containing PSACH and MED. No measurements were reported for abnormalities.

That is, according to the present invention, there is provided a method for examining C0MP abnormality, which comprises measuring the amount of C0MP (cartilage oligomer substrate protein) per unit amount of body fluid.

Preferably, the C0MP abnormality is pseudochondrodysplasia or multiple epiphyseal dysplasia caused by mutation of the C0MP gene.

Preferably, the bodily fluid is blood.

Preferably, the amount of C0MP per unit volume of body fluid of the subject is measured, and if the obtained measurement value is lower than the amount of C0MP per unit volume of body fluid of a normal subject, it is C0MP disorder or C0MP disorder. Judge that there is a possibility of onset.

Preferably, the amount of C0MP per unit body fluid is measured by an antigen-antibody reaction using an anti-C0MP antibody.

According to another aspect of the present invention, there is provided a kit for performing the above-described detection method of the present invention, which comprises at least an anti-C0MP antibody. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the mean and range of the results of measuring the plasma C0MP concentration in each subject for each test group.

FIG. 2 shows the results of measuring the plasma C0MP concentration in each subject, together with the age of the subject. In the figure, · indicates PSACH (with COMP mutation), ▲ indicates MED (with C0MP mutation), △ indicates MED (no COMP mutation), X indicates control. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

The test method for C0MP abnormality according to the present invention includes measuring the C0MP amount per unit body fluid (for example, the C0MP concentration in a body fluid). According to the test of the present invention, it is possible to determine whether or not C0MP abnormality has developed and / or the likelihood of developing it.

The C0MP abnormality in the present invention includes all bone disorders caused by mutation of the C0MP gene, and includes, for example, PSACH or MED caused by mutation of the C0MP gene.

In the present invention, the amount of C0MP per unit body fluid is measured. C0MP is a glycoprotein belonging to the thrombospondin gene family. The C0MP gene is located on chromosome 19 at 19ql2-13.1 and is approximately 30 kb in size. Consists of 19 exons. Encodes a protein consisting of 757 amino acids. The C0MP peptide (COMP monomer) has a globular domain at the N- and -C terminus, with four EGF-like domains arranged in tandem and eight calmodulin-like repeats arranged in tandem immediately following it. It is a feature of. C0MPs bind to each other at the N-terminal disulfide bridge to form a pentamer structure. The expression of C0MP is tissue specific and is localized in the extracellular matrix of cartilage, especially in the territorial matrix around chondrocytes. Although the details of the function of C0MP are not clear at present, it is thought to interact with other components of the extracellular matrix of cartilage, such as collagen and proteoglycan, in a calcium-dependent manner.

In the present specification, the body fluid includes blood, synovial fluid, urine, saliva, etc., but is preferably blood or joint fluid, and more preferably blood, because it contains C0MP in a relatively large amount.

In the present invention, the amount of C0MP per unit body fluid of a subject was measured, and the obtained measurement value was lower than the C0MP amount per unit body fluid of a normal subject corrected for age. In this case, it can be determined that the patient has a COMP disorder or a possibility of developing the COMP disorder.

The method for measuring the amount of C0MP is not particularly limited, and any method known to those skilled in the art can be used as a method for measuring the amount of protein. In the case of quantifying the protein, the following methods can be used: スタンプ Estlot blot method, enzyme immunoassay (EIA) including enzyme-linked immunosorbent assay, immunocytochemistry, and immunocytochemistry. Preferably, the amount of C0MP per unit body fluid can be measured by an antigen-antibody reaction using an anti-C0MP antibody. When measuring the amount of C0MP per unit amount of body fluid by an antigen-antibody reaction using an anti-C0MP antibody, enzyme immunoassay (RIA), EIA, enzyme-linked immunosorbent assay (ELISA), It can be used, and it is particularly preferable to carry out by ELISA.

The anti-C0MP antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody, and can be produced by a conventional method. Alternatively, a commercially available human C0MP antibody can be used.

For example, a polyclonal antibody that recognizes C0MP can be obtained by immunizing a mammal with C0MP as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. The antigen can be used for immunization by dissolving it in a buffer containing an appropriate adjuvant, if desired. After breeding the immunized mammal for a certain period of time, a small amount of serum from the mammal is sampled, and the antibody titer is measured. If the antibody titer increases, the antigen is administered an appropriate number of times according to the situation. One to two months after the last administration, blood is collected from the immunized mammal by a conventional method, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography. A polyclonal antibody can be obtained by separation and purification by a conventional method such as chromatography, such as ion exchange chromatography and affinity chromatography.

A monoclonal antibody recognizing C0MP can be obtained, for example, by preparing a hybridoma by cell fusion between an antibody-producing cell and a myeloma cell line, and culturing the hybridoma. The production of hybridomas is performed by a known method (G. Kohler et al.). al., Nature, 256 495 (1975)). The hybridoma obtained by cell fusion is cloned by limiting dilution, etc., and further screened by enzyme immunoassay using C0MP to obtain a monoclonal / reactive antibody that specifically recognizes C0MP. Producing cell lines can be obtained. In order to produce the desired monoclonal antibody from the thus obtained hybridoma, the hybridoma is cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody is isolated from the culture supernatant or ascites. It may be purified. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in appropriate combination.

The test method of the present invention can also be performed using a commercially available human C0MP protein quantification kit. For example, (1) Product number: COMP 200; Wieslab, Lund, Sweden, and (2) Kamiya Bimedical Co. (Seattle, WA, USA), Catalog No. BP-003 can be used.

According to the present invention, a kit for performing the above-described test method of the present invention, which contains at least an anti-C0MP antibody, is also provided.

The test method of the present invention has the following significance in addition to the simple and objective diagnosis using a blood marker.

(1) About PSACH

Problems in the diagnosis of conventional PSACH include early diagnosis and diagnosis in adult cases. PSACH has normal intelligence and facial features with no extraskeletal complications. The only apparent features are dwarfism with short limbs and deformed joints. Differential diagnosis from many diseases such as achondroplasia, Morquio's disease, spine and epiphyses * It shows a characteristic growth pattern, height growth decreases rapidly after 1 year of age, and the final height is as low as 13 to 4 SD in mild cases and less than 16 SD in severe cases. However, birth height is normal and, by 2-3 years, in most cases is within the normal range. This often delays diagnosis. PSACH has generalized flat vertebrae in childhood and childhood, Since anterior tongue-like protrusions of various vertebral bodies are observed, diagnosis is relatively easy if radiographic examination is performed properly. However, this spinal dysplasia almost normalizes in adult cases. After closure of the epiphyseal line, it is very difficult to evaluate the dysplasia of the epiphysis and metaphysis of the long and short canal. Thus, there are many difficulties in clinical diagnosis of PSACH. On the other hand, early, definitive diagnosis is a great gospel for patients. In this disease, deformation of the scoliosis and limbs progresses rapidly unless proper care is taken during childhood and childhood, resulting in major disabilities and sequelae. Above all, serious irreversible complications, such as abnormalities of the upper cervical vertebra (dislocation of the atlanto-axial joint), need to be aware of the risks and to prevent them. In addition, long-term preventive measures are needed for osteoarthritis that is associated with a high rate. Furthermore, since the disease is a hereditary disease, confirmation of the diagnosis is the basis for genetic counseling to the individual and his / her family (eg, estimating the risk of developing a second child). As mentioned above, early diagnosis and definitive diagnosis in treatment and management are particularly important in this disease, but this method makes it possible.

(2) MED

MED has genetic heterogeneity, and mutations of genes other than C0MP such as type 9 collagen gene (C0L9A1 C0L9A2.C0L9A3), MTN3, and DSTDT have been reported. Conventionally, the only way to distinguish them is to analyze mutations in each gene and identify one of the mutations. By this method, MED caused by C0MP mutation can be easily distinguished. In addition, it was difficult to distinguish mild cases of MED from general osteoarthritis (OA) ゃ generalized OA (generalized OA) .However, this method makes it possible to easily distinguish MED due to C0MP mutation. Became.

'The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example

Blood C0MP protein was measured in 13 patients with pseudochondrodysplasia / polyphyseal dysplasia (gene mutations are shown in Table 1) whose C0MP abnormality (mutation) was confirmed at the gene level. As controls, blood C0MP proteins of a total of 47 healthy subjects and patients with multiple epiphyseal dysplasia confirmed to have no C0MP abnormality were measured and compared. C0MP gene mutation identified in cases in the examples

Case mutation (amino acid change) _ _ _

PSACH1 deletion of amino acids 290-325

Substitution of PSACH2 1 amino acid (D290N)

Substitution of PSACH3 1 amino acid (C387G)

PSACH4 1 amino acid substitution (D473G)

PSACH5 1 Amino acid substitution (D518N)

PSACH6 1 Amino acid substitution (G719D)

MED1 Substitution of 1 amino acid (C371F)

MED2 1 Substitution of amino acid (C371F)

MED3 1 Amino acid substitution (D385N)

MED4 Deletion of amino acids 501-503

MED5 C-terminal, deletion of the 743th or less amino acid

MED6 C-terminal, deletion of the 743th or less amino acid

MED7 1 Substitution of amino acid (R718W)

PSACH: pseudochondrodysplasia, MED: multiple epiphyseal dysplasia.

D290N: Substitution of 290th aspartic acid with asparagine

C387G: Substitution of cysteine at position 387 for glycine

D473G: Substitution of 473rd aspartic acid with glycine

D518N: Replacement of 518th aspartic acid with asparagine

G719D: Substitution of 719th glycine with aspartic acid

C371F: Substitution of 371st cysteine with fenilalanine

D385N: Substitution of 385th aspartic acid with asparagine

R718W: Arginine at position 718 replaced with tryptophan The samples were separated by centrifugation (3000 rpm for 10 minutes or 3500 rpm for 5 minutes), and plasma frozen and used until use was used. C0MP protein was measured using a commercially available human C0MP protein quantification kit (Product number: COMP 200; Wieslab, Lund, Sweden) according to the manufacturer's instructions. This kit quantifies intact or fragmented COMP by ELISA using a human C0MP-specific antibody. Duplicate measurement was performed using 60 ml of the sample diluted 50-fold with the dilution buffer attached to the Kit.

The results are shown in FIGS. 1 and 2. From the results of FIGS. 1 and 2, the following was found.

(1) Blood C0MP in patients with C0MP abnormalities was found to be significantly lower (p <0.0001) than in healthy individuals and patients with MED who were confirmed to have no C0MP abnormalities.

(2) MED patients without C0MP abnormalities at the DNA / gene level were at the same level as healthy subjects.

(3) Because the measured values are affected by age, the effect of age was corrected using a regression equation. As a result, the value in patients was lower than the lowest value in healthy subjects, and there was no overlap between the patient group and the control group.

From the above results, the blood C0MP of patients with C0MP abnormality was decreased, indicating that this system can detect the presence or absence of C0MP abnormality. In particular, it was shown that the presence or absence of abnormalities in C0MP of MED can be easily identified. In addition, since the C0MP abnormality is reflected in the patient's blood, the stage and disease C0MP of the C0MP abnormality patient may be able to be estimated by measuring the same person over time. Industrial applicability

Conventionally, there has been no objective diagnosis method for C0MP abnormality, and a subjective diagnosis based on clinical images and radiographic findings by a physician, and a time-consuming and costly diagnosis at the DNA * gene level Had been done. According to the present invention, it has become possible to provide an objective and simple test method using a body fluid sample such as blood. The test method of the present invention can perform a test using plasma in one step, and can perform a process from collection of a sample to determination of a result within two days.

Claims

The scope of the claims

1. A method for examining C0MP abnormalities, which comprises measuring the amount of C0MP (cartilage oligomeric protein) per unit volume of body fluid.

2. The detection method according to claim 1, wherein the COMP abnormality is pseudochondrodysplasia or multiple epiphyseal dysplasia caused by a mutation in the C0MP gene.

3. The detection method according to claim 1, wherein the body fluid is blood.

4. Measure the amount of C0MP per unit volume of body fluid of the subject, and if the measured value is lower than the amount of C0MP per unit volume of body fluid of the normal subject, the patient has C0MP abnormality or C0MP abnormality. 4. The detection method according to claim 1, wherein it is determined that there is a possibility of onset.

5. The test method according to any one of claims 1 to 4, wherein the amount of C0MP per unit body fluid is measured by an antigen-antibody reaction using an anti-C0MP antibody.

6. A kit for performing the test method according to any one of claims 1 to 5, comprising at least an anti-C0MP antibody.