JP4870976B2 - Test method for autoimmune disease using whole blood - Google Patents

Description

  The present invention relates to a method for examining an autoimmune disease, particularly juvenile idiopathic arthritis, using a sample derived from a subject, particularly whole blood, and a marker gene and kit therefor.

  Autoimmune disease is a phenomenon in which the immune system, which is a biological defense mechanism, attacks self cells. Antibodies and lymphocytes that react with self-antigens are induced, resulting in tissue damage and lesions. Autoimmune diseases are roughly classified into two types: diseases without organ specificity and diseases with specificity. There are many cases where the cause of autoimmunity is suggested but the cause is not clear, and there are many problems to be solved, such as diagnostic methods, including their onset mechanism. One of the autoimmune diseases whose cause has not been clarified is juvenile idiopathic arthritis (JIA).

  Juvenile idiopathic arthritis (JIA) is a type of rheumatic disease and is the most common rheumatic disease in children. Juvenile idiopathic arthritis is classified into several subtypes, with severe differences in severity and prognosis. Roughly classified, it can be classified into those in which systemic symptoms such as fever, skin rash and pericarditis appear intermittently (systemic type) and those in which some joints have symptoms (small joint type and multi-joint type). In particular, systemic juvenile idiopathic arthritis has a strong inflammatory reaction throughout the body and a poor prognosis. On the other hand, polyarticular juvenile idiopathic arthritis does not directly affect life, but it causes polyarticular deformation and lowers QOL. Both have arthritis, but the prognosis is very different, so the distinction is very important.

  However, all conventional JIA diagnoses have been performed based on clinical findings (Non-Patent Documents 1 to 4), and an effective method for discriminating both is not known. Therefore, a method for examining juvenile idiopathic arthritis and a method for determining its subtype have been desired.

  In recent years, molecular biological research on immune diseases has been actively promoted, and studies on cytokines, their receptors, and intracellular signal transduction mechanisms after receiving signals are progressing. However, just focusing on the individual genes that work here is not enough to express the characteristics of immune diseases. For this reason, attempts have been made to obtain expression information of a large number of genes at once by using a DNA microarray or the like.

  A method for deriving disease information by statistically processing a large amount of gene expression data obtained by DNA microarray measurement has been studied.

  Golub et al. Have shown that acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) can be distinguished by the expression pattern of the “predictor” gene cluster (Non-patent Document 5). Although it is not so difficult to distinguish morphologically between ALL and AML using current pathological diagnosis, this paper is the first report showing the possibility of blood cell classification by expression profile. Yes, it has the greatest impact on similar papers since then, and is the basis for that idea.

  Alizadeh et al. Measured the B lymphocytes collected from the peripheral blood of patients with diffuse large B-cell lymphoma using a DNA microarray as a sample, and performed hierarchical clustering of the obtained gene expression data. The patient's peripheral blood B lymphocytes show two gene expression patterns similar to those of B cells located in the germinal center of the lymphoid tissue, and cases of gene expression patterns similar to B cells activated in vitro. It was found that there is a case (Non-Patent Document 6). As a result of investigating the survival rate of both, Kaplan-Meier plots revealed that patients with B cells with the latter expression pattern had a worse prognosis than patients with B cells with the former expression pattern. . In addition, it is clear that the results obtained by clustering gene expression information performed by the authors are more highly correlated with the prognosis than following the prognosis prediction that follows the conventional pathological diagnosis. It was.

  The results of Alizadeh et al. Are significant in that they can derive clinically available laws from gene expression information. However, the results have been verified only for a limited number of B-cell lymphomas, and it has not been verified whether the law can be applied to completely new clinical cases.

J. Rheumatol., Vol. 25, 1991-1994, 1998 Arthritis. Rheum., Vol. 29, 274-281, 1986 Bull. Rheum. Dis., Vol. 38, 1-7, 1989 EULAR Publishers, 47-50, 1978 Science, vol. 286, 531-537, 1999 Nature, vol. 403, 503-511, 2000

  An object of this invention is to provide the new parameter | index and test method for the diagnosis of an autoimmune disease, and pathological condition prediction.

  Conventionally, in the examination of autoimmune diseases, findings relating to blood cell images from whole blood and measurement of immune-related active substances such as cell extracts prepared by separating leukocytes such as lymphocytes and cytokines in serum blood have been measured. It has been mainly used. However, the present inventors can examine autoimmune diseases by measuring the expression level of genes expressed in peripheral blood cells using total RNA extracted from whole blood from which none of the components are separated. I found. In addition, for this reason, the present inventors have found that genes whose expression levels are remarkably varied between samples derived from patients suffering from autoimmune diseases and samples derived from healthy individuals, and patients suffering from autoimmune diseases. We found genes whose expression levels fluctuate significantly depending on their pathological conditions.

That is, the present invention includes the following inventions.
(1) A method for examining an autoimmune disease by detecting the expression of a gene expressed in peripheral blood cells,
Extracting the total RNA from the whole blood of the subject, and measuring the expression level of the gene in the total RNA,
Said method.
(2) The method according to (1), wherein the autoimmune disease is juvenile idiopathic arthritis.
(3) A method for examining an autoimmune disease, comprising detecting expression of a gene contained in a gene set determined by the following steps (i) to (v) in a sample derived from a subject:
(I) normalizing the expression data of genes that are specifically expressed in autoimmune diseases;
(Ii) ranking high discrimination genes and creating a high discrimination gene list;
(Iii) selecting a gene set from the high discrimination gene list;
(Iv) performing a discrimination ability evaluation of the gene set in step (iii);
(V) A gene set having a discrimination ability is determined.
(4) In a sample derived from a subject, a gene selected from a highly discriminating gene list composed of genes identified by the nucleotide sequences represented by SEQ ID NOs: 1 to 100 and ranked in the order of SEQ ID NOs. A method for examining an autoimmune disease, comprising detecting the expression of a gene included in the gene set, wherein the gene set has a discrimination rate of 50% or more for the autoimmune disease.
(5) The method according to (4), wherein the autoimmune disease is juvenile idiopathic arthritis.

(6) The method according to (4) or (5), wherein the gene set includes at least the first to fifth genes in the high discrimination gene list.
(7) The method according to (4) or (5), wherein the gene set comprises at least 5 genes selected from the 1st gene to the 20th gene in the high discrimination gene list.
(8) systemic juvenile idiopathic arthritis and polymorphism, including detecting expression of a gene included in a gene set determined by the following steps (i) to (v) in a sample derived from a subject: To determine articular juvenile idiopathic arthritis:
(I) standardize expression data of genes specifically expressed between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis;
(Ii) ranking high discrimination genes and creating a high discrimination gene list;
(Iii) selecting a gene set from the high discrimination gene list;
(Iv) performing a discrimination ability evaluation of the gene set in step (iii);
(V) A gene set having a discrimination ability is determined.
(9) In a sample derived from a subject, a gene selected from a highly discriminating gene list composed of genes specified by the nucleotide sequences represented by SEQ ID NOs: 101 to 200 and ranked in the order of SEQ ID NOs. Detecting the expression of a gene included in the gene set, wherein the discrimination rate between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis of the gene set is 50% or more, A method for discriminating between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis.
(10) The method according to (9), wherein the gene set includes at least the first to fifth genes in the high discrimination gene list.

(11) The method according to (9), wherein the gene set comprises at least 5 genes selected from the 1st to 15th genes in the high discrimination gene list.
(12) detecting the expression of the gene,
Extracting total RNA from a sample derived from a subject, and measuring the expression level of the gene in the total RNA.
(3) The method in any one of (11).
(13) In a gene set including genes selected from a list of highly discriminating genes composed of genes specified by the nucleotide sequences represented by SEQ ID NOs: 1 to 100 and ranked in the order of SEQ ID NOs, each gene is specifically 15 to 30 base length continuous primers for amplification or 20 to 1500 base length continuous probes for detecting each gene, and the autoimmune disease discrimination rate of the gene set is 50% or more A kit for identifying an autoimmune disease.
(14) The kit according to (13), wherein the autoimmune disease is juvenile idiopathic arthritis.
(15) The kit according to (13) or (14), wherein the gene set includes at least the first to fifth genes in the high discrimination gene list.

(16) The kit according to (13) or (14), wherein the gene set comprises at least 5 genes selected from the first to twentieth genes in the high discrimination gene list.
(17) In a gene set including genes selected from a list of highly discriminating genes consisting of genes specified by the nucleotide sequences represented by SEQ ID NOs: 101 to 200 and ranked in the order of SEQ ID NOs, each gene is specifically A continuous primer 15 to 30 bases long to amplify, or a continuous probe 20 to 1500 bases long to detect each gene, and systemic juvenile idiopathic arthritis and polyarticular type of the gene set A kit for discriminating between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis, with a discrimination rate of juvenile idiopathic arthritis of 50% or more.
(18) The kit according to (17), wherein the gene set comprises at least the first to fifth genes in the high discrimination gene list.
(19) The kit according to (17), wherein the gene set comprises at least 5 genes selected from the 1st to 15th genes in the high discrimination gene list.
(20) A gene set comprising genes selected from a list of highly discriminating genes consisting of genes identified by the nucleotide sequences represented by SEQ ID NOs: 1 to 100 and ranked in the order of SEQ ID NOs. A marker gene set for testing autoimmune diseases, wherein the discrimination rate of autoimmune diseases is 50% or more.
(21) A gene set comprising genes selected from a high discriminating gene list consisting of genes specified by the nucleotide sequences represented by SEQ ID NOs: 101 to 200 and ranked in the order of SEQ ID NOs. Gene for discriminating between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis, with a discrimination rate of 50% or more between systemic juvenile idiopathic arthritis and arthritic juvenile idiopathic arthritis set.

  INDUSTRIAL APPLICABILITY According to the present invention, a new index and test method for diagnosis and pathophysiological prediction of autoimmune diseases, particularly juvenile idiopathic arthritis, are provided.

  The present inventors measure the expression level of a gene expressed in peripheral blood cells in total RNA obtained by extracting total RNA from intact whole blood from which a subject's lymphocytes and the like have not been separated. It was found that autoimmune diseases can be examined by

  That is, in one embodiment, the present invention is a method for examining an autoimmune disease by measuring the expression level of a gene expressed in peripheral blood cells, the step of extracting total RNA from the whole blood of a subject And measuring the expression level of the gene in the total RNA. In the present specification, the test includes a test for the presence or absence of a disease, a test for the degree of a disease, and a test for a disease state, for example, a test for a subtype of a disease, and a determination of a prognosis of a disease.

  Conventionally, in a diagnosis used as a sample derived from a subject for diagnosis of an autoimmune disease, whole blood has not been used as it is as a subject of a molecular biological test. This is because whole blood contains various cells, and it was thought that data for determining the disease would not be obtained even if it was used as it was. However, the present inventors performed extraction of total RNA using whole blood as it is, and detected the expression of genes expressed in peripheral blood cells in the total RNA, thereby sufficiently effectively treating autoimmune diseases. It was found that the test can be performed as in the case of using a sample separated from.

  Whole blood refers to blood that has been collected from a subject. Whole blood is usually collected with a strict aseptic procedure. Anticoagulants may include citrate ions or heparin.

  Extraction of total RNA from whole blood can be performed by methods known to those skilled in the art, and is not particularly limited. For example, guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N., Anal. Biochem., 162 , 156-159 (1987)). Of these, the guanidine acid thiocyanate / phenol / chloroform method is preferred.

  Measurement of the expression level of a gene in total RNA extracted from whole blood includes measuring the mRNA level of the target gene in the sample. For this purpose, it is common to prepare cRNA, cDNA, aRNA or the like corresponding to the base sequence from mRNA of the target gene and measure it using a DNA microarray (DNA chip) or the like. In addition, as described later, mRNA or a modified method thereof, mass spectrometry, SAGE method, competitive PCR method or the like can be used for mRNA measurement.

  Autoimmune diseases are diseases caused by autoantibodies or autoantigen-sensitized lymphocytes, and also include immune complex diseases, which are diseases caused by a conjugate of an autoantibody and a corresponding antigen. Specific examples of autoimmune diseases include collagen disease, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile idiopathic arthritis (JIA), malignant rheumatoid arthritis, systemic lupus erythematosus, scleroderma (systemic sclerosis), multiple Myositis, dermatomyositis, Sjogren's syndrome (dry syndrome), psoriatic arthritis, Wegener's granulomatosis, mixed connective tissue disease (MCTD), rheumatic polymyalgia, gout, pseudogout, polyarteritis, vasculitis, Examples include adult-onset Still's disease, Behcet's disease, osteoarthritis, antiphospholipid antibody syndrome, Reiter's syndrome, ankylosing spondylitis, joint disease associated with enteritis, rheumatic fever, osteoporosis and the like. The method of the present invention is suitable for examination of rheumatic diseases, particularly juvenile idiopathic arthritis (JIA).

  Juvenile idiopathic arthritis (JIA) is a chronic disease characterized by persistent joint inflammation, and typical symptoms of arthritis include pain, swelling, and restricted movement, and it occurs in young people. Usually, arthritis persists for more than 6 weeks, and all other diseases that can cause arthritis can be ruled out, and if the cause is unknown, the doctor is diagnosed with juvenile idiopathic arthritis.

  There are several different subtypes of juvenile idiopathic arthritis. Roughly speaking, systemic symptoms such as fever, rash and pericarditis appear intermittently (systemic type), and symptoms occur in some joints. It can be divided into two types (small-joint type and multi-joint type). Systemic juvenile idiopathic arthritis is characterized by the appearance of systemic symptoms along with arthritis, and the main features are high spike fever, repeated antipyretic fever, and a salmon-like skin rash accompanying heat. Other symptoms include muscle pain, hepatosplenomegaly, lymphadenopathy, pericarditis, pleurisy. Articulated juvenile idiopathic arthritis has few systemic symptoms in the first 6 months of onset, and has more than 5 joint symptoms. Rheumatoid factor-positive types and rheumatoid factor-negative types are classified according to the presence or absence of blood autoantibodies called rheumatoid factors.

  When measuring the expression level, a gene or gene set whose expression level varies significantly between a sample from a patient suffering from an autoimmune disease and a sample from a healthy subject, or a patient suffering from an autoimmune disease Therefore, it is desirable to measure the expression level of a gene or gene contained in a gene set whose expression level varies significantly depending on the pathological condition. For this purpose, a highly discriminating gene list is created, a gene set including a plurality of genes selected from the list is selected, and the expression of the genes included in the gene set is detected. The creation of a highly discriminating gene list, selection of a gene set, and discrimination method are as described below.

In one embodiment, the present invention relates to a method for examining an autoimmune disease, which comprises detecting the expression of a gene contained in a gene set determined by the following steps (i) to (v): :
(I) normalizing the expression data of genes that are specifically expressed in autoimmune diseases;
(Ii) ranking high discrimination genes and creating a high discrimination gene list;
(Iii) selecting a gene set from the high discrimination gene list;
(Iv) performing a discrimination ability evaluation of the gene set in step (iii);
(V) A gene set having a discrimination ability is determined.

  Here, the gene expressed specifically in the autoimmune disease refers to the expression level of each gene measured in the sample derived from the subject compared with the control, and the gene expression level is significantly increased and significantly decreased. It includes both genes.

  Hereinafter, an embodiment of a method for determining a gene set for discriminating an autoimmune disease by a gene discrimination analysis method will be described.

  Gene expression can be detected by measuring the mRNA level of the target gene in the sample. For this purpose, a test polynucleotide such as cDNA or aRNA corresponding to the nucleotide sequence of the target gene is usually prepared and measured using a DNA microarray (DNA chip) or the like.

  These are labeled in order to detect hybridization between the test polynucleotide and a probe for a specific gene on the microarray. For example, it can be labeled with a radioisotope, chemiluminescent compound, heavy metal atom, spectroscopic marker, magnetic marker, related enzyme, fluorescent label, and the like. A suitable labeling method corresponding to each labeling target molecule can be appropriately selected by those skilled in the art. Fluorescent labels are most preferred, and various fluorescent labels such as fluorescein, dichloro-fluorescein, BODITY (trademark), ROX, rhodamine, Cy2, Cy3, Cy5, IRD40 are known, and any of them may be used. However, Cy3 and Cy5 are particularly preferable. For labeling with Cy3 or Cy5, aminoallyl dUTP is incorporated during synthesis in both cDNA and aRNA.

  The cDNA or aRNA sample thus obtained is hybridized on a microarray on which probes that specifically hybridize with the test polynucleotide are immobilized (Brown, PO et al., Nature genet. 21, suppliment, 33-37 (1999)).

  As the DNA chip, a commercially available product (for example, AceGene (registered trademark) Human Oligo Chip 30K manufactured by Hitachi Software Engineering Co., Ltd.) may be used, or a known method (Lipshutz, RJ etal., Nature genet. 21, suppliment , 20-24 (1999)). The DNA to be immobilized includes those cloned by performing reverse transcriptase reaction and PCR with primers prepared based on sequence information such as human EST database, and oligos chemically synthesized using gene sequence information. Nucleotides are used.

  Hybridization conditions and methods are well known to those skilled in the art. For example, the hybridization buffer may be 5 × SSC, 0.5% SDS, 4 × Denhardt's solution, or the like. Hybridization is performed at 35 to 60 ° C., preferably 42 to 50 ° C., more preferably 45 ° C., for 2 hours or longer, preferably overnight or longer. Hybridization conditions can be appropriately set by those skilled in the art according to various factors including the amount of labeled molecules or detection sensitivity. After hybridization, the array is washed, the labeled molecules are read, the signal is visualized and quantified.

  Any method known in the art can be used to measure the signal intensity. For example, when a radioisotope is used as a label, the radioactivity can be measured by, for example, a liquid scintillation counter or a γ-counter. When a fluorescent substance is used as a label, the fluorescence can be detected using a fluorescent microscope, a fluorescent plate reader, a fluorescent scanner, or the like.

  For all the quantified probes, the signal intensity derived from the labeled molecule is obtained as numerical data in text format. The part with low signal intensity is greatly affected by the background, so by setting a cutoff such as the fluorescence intensity of Cy3 and Cy5 exceeding 1000, only the data whose fluorescence intensity exceeds the cutoff remains, Reject data with low fluorescence intensity. For example, for each of Cy3 and Cy5, data on a probe whose fluorescence intensity value is larger than the value obtained by adding two standard deviations to the background value is used. The ratio of the fluorescence intensity values of Cy3 and Cy5 of each probe is calculated, and standardized numerical data obtained by correcting the detection sensitivity is obtained.

  Missing values present in the data may be interpolated by some method depending on the analysis method and used for the subsequent analysis. Various interpolation methods can be applied.For example, the average value of all data for the case including the missing value to be interpolated is the average value of the data for all cases of the gene including the missing value. There is a method of interpolating with the value obtained by subtracting the average value of the data of all genes for all cases from the added value. Besides, in the report of Troyanskaya et al. (Bioinformatics, vol. 17, 520-525, 2001), there are three interpolation methods: K-Nearest Neighbors (KNN) method, Singular Value Decomposition (SVD) based method and row average method. Interpolation. By applying any of these methods, it is possible to interpolate all missing values. In some cases, missing value interpolation may not be performed.

The standardized numerical data thus selected (hereinafter also referred to as “standardized gene expression data”) is not influenced by the background and is derived from an autoimmune disease patient in comparison with a sample derived from a healthy subject. It has expression information of genes whose gene expression fluctuation width in the sample exceeds the gene expression fluctuation width caused by individual differences, and the reliability of subsequent statistical analysis can be ensured.
By performing multivariate analysis on the standardized gene expression data obtained as described above, statistical processing is performed and a gene set is selected.

Hereinafter, an outline of a method for determining a gene set will be described with reference to FIG.
Step 101:
A standardized data matrix comprising a list of probe numbers and a list of case numbers on the DNA microarray corresponding to each numerical value in the standardized gene expression data is prepared.
Step 102:
One sample is removed from the standardized data matrix as test data.
Step 103:
A list (high discriminating gene list) in which genes are arranged in an order having a high discriminating ability is generated by a ranking process for high discriminating genes described later.
Step 104:
A gene set is selected by sequentially extracting genes from the top of the high discriminating ability gene list created by the procedure 103, and it is determined whether or not the test sample removed in the procedure 102 is an autoimmune disease. The determination method follows procedures 301 to 306 described later.

Step 105:
Replace the sample that was removed for testing.
Step 106:
During the analysis, the sample is removed only once. If there is a sample that has never been removed, the process returns to step 102, where the sample is removed as a test sample and the analysis proceeds.
Step 107:
If all the analyzed results are combined and the discrimination rate of the gene set is equal to or higher than the evaluation criterion, the obtained gene set is adopted, and if the discrimination rate is equal to or lower than the evaluation criterion, the gene set is not adopted. The process is terminated. The gene set is determined by repeating the above procedure.

  The ranking process of the highly discriminating genes in the procedure 103 in FIG. 1 is performed according to the procedures 201 to 205 (FIG. 2). This treatment can be carried out according to the method of Golub et al. (Science. Vol. 286, 531-537, 1999). In the processing, the genes included in the standardized data matrix are processed one by one.

Procedure 201:
When a certain gene N is treated, the sample is labeled as a sample from a patient with an autoimmune disease or a sample from a healthy person. Based on each label, the following formula 1 is used to calculate a Signal To Noise Ratio value as an index indicating how useful the gene is for discriminant analysis of autoimmune diseases.

In Formula 1, μ _I is an average value of gene expression ratio of autoimmune disease sample, μ _II is an average value of gene expression ratio of healthy sample, σ _I is a standard deviation of gene expression ratio of autoimmune disease sample, σ _II Represents the standard deviation of the gene expression ratio of the healthy sample.

Step 202:
The label attached to each sample is replaced at random, and the value of Signal to Noise Ratio is calculated again using Equation 1.
Step 203:
The process of step 202 is repeated a predetermined number of times. Golub et al. Performed 400 times, but in the example of the present invention, procedure 202 was repeated 1000 times.
Step 204:
Based on the numerical values calculated in steps 201 and 202, the pValue of the Signal To Noise Ratio value calculated in step 201 is calculated, and genes whose pValue is equal to or greater than a certain value (p> = 0.01) are removed.
Step 205:
The standardized data matrix is rearranged in the order of Signal to Noise Ratio calculated in step 202.

  The discrimination ability evaluation of the gene set in the procedure 104 in FIG. 1 is performed on the gene set selected from the ranking-processed high discrimination ability gene list created in the procedure 103. This process can be performed according to the method of Golub et al. (Science, supra) (FIG. 3). A certain number of high discriminating gene lists created by the procedure 103 are extracted from the top to select a gene set, and it is determined whether or not the sample removed in the procedure 102 is an autoimmune disease.

Step 301:
In step 102, a gene set from which the first to Nth genes in the ranked gene list are extracted is selected.
Step 302:
In order to determine whether one sample removed by the procedure 102 is an autoimmune disease or a healthy person using the gene set extracted by the procedure 301, the weight is calculated using the following equation (2). The attached vote value V is calculated.

Regarding gene i, μ _{I in} Equation 2 is the average value of the gene expression ratio of the autoimmune disease sample, μ _II is the average value of the gene expression ratio of the healthy sample, and x _i is the gene contained in the sample to be discriminated. The expression rate value of i, p (g, c), represents the value of Signal To Noise Ratio.
The direction of voting is determined depending on whether the value of x _i is close to μ _I or μ _II, and V _i is used as the voting value.

Step 303:
Voting is performed for all the genes included in the gene set extracted by the procedure 301 using the above formula 2, and the one with the larger absolute value of the vote value is finally determined by the gene set. Use as a discriminant value
Step 304:
It should be noted that, using the vote value obtained in step 302, the degree of certainty of the prediction is used as a Prediction Score, and calculation is performed using Equation 3 below.
Prediction Score:

V _win is the total number of votes voted for the winner, and V _loose is the total number of votes voted for the loser.

Step 305:
Since it is already known whether or not the sample removed in the procedure 102 is an autoimmune disease, it is examined whether or not the analysis has been performed in comparison with the result predicted this time.
Step 306:
In accordance with the method of Golub et al. (Science, supra), if the Prediction Score calculated in step 304 is less than or equal to the root half of the number of genes used, the determination result is undecidable.

  In the procedure 107 of FIG. 1, the discrimination rate (number of correct answers / (number of correct answers + number of incorrect answers)) of gene sets extracted in order from the top of the high discriminating ability gene list ranked in the processing up to the procedure 106 is calculated. To evaluate the discrimination ability of the gene set. Usually, a gene set having a discrimination rate (discrimination ability) of 50% or more, preferably 70% or more, more preferably 85% or more is determined as a high discrimination ability gene set (FIG. 4).

  And the expression of the gene contained in the determined gene set in an unknown sample can be detected, the obtained expression information can be statistically processed according to procedures 301 to 306, and autoimmune diseases can be examined ( 3 and 5).

In another embodiment, the present invention relates to systemic juvenile idiopathic arthritis and polyarticular juvenile comprising detecting the expression of a gene included in a gene set determined by the following steps (i) to (v): On how to determine idiopathic idiopathic arthritis:
(I) standardize expression data of genes specifically expressed between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis;
(Ii) ranking high discrimination genes and creating a high discrimination gene list;
(Iii) selecting a gene set from the high discrimination gene list;
(Iv) performing a discrimination ability evaluation of the gene set in step (iii);
(V) A gene set having a discrimination ability is determined.

  Here, the gene specifically expressed between systemic juvenile idiopathic arthritis and arthritic juvenile idiopathic arthritis is measured in systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis. In addition, it refers to genes with significantly different expression levels.

  The method for determining the gene list is the same as above, using samples from patients with systemic juvenile idiopathic arthritis, samples from patients with systemic juvenile idiopathic arthritis, or labeled samples. Thus, a high discriminating ability gene list can be created, a gene set can be selected, and discrimination ability evaluation and discrimination can be performed.

Gene List In one embodiment, the high discriminating ability gene list for examining autoimmune diseases is preferably composed of genes identified by the nucleotide sequences represented by SEQ ID NOs: 1 to 100 and ranked in the order of SEQ ID NOs. This is a high discriminating gene list (Table 1).

  In Table 1, the DNA chip ID number refers to the ID number in AceGene (registered trademark) Human Oligo Chip 30K manufactured by Hitachi Software Engineering Co., Ltd. http://bio.hitachi-sk.co.jp/acegene/ By inputting the ID number in human30k / search_30k / search_human30k.html, the gene targeted by the probe on the DNA chip can also be searched. In addition, “+” indicates a gene whose expression level is increased in patients with autoimmune disease, particularly juvenile idiopathic arthritis, and “−” indicates that in patients with autoimmune disease, particularly juvenile idiopathic arthritis. A gene whose expression level is decreased.

  In one embodiment, the high discriminating ability gene list for discriminating between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis is derived from genes identified by the nucleotide sequences represented by SEQ ID NOs: 101 to 200. It is a high discriminating ability gene list ranked in the order of the sequence number (Table 2).

In Table 2, “+” indicates a gene whose expression level is increased in patients with systemic juvenile idiopathic arthritis, and “−” indicates its expression in patients with systemic juvenile idiopathic arthritis. A gene whose amount is decreasing.
In the gene lists in Tables 1 and 2 above, the ID number indicates the accession number registered in GenBank.

  The gene specified by each base sequence included in each gene list includes a gene composed of each base sequence and a gene functionally equivalent to the gene. Here, “functionally equivalent” means that the polypeptide encoded by the gene of interest has the same biological and biochemical functions as the polypeptide encoded by the gene consisting of each nucleotide sequence. Point.

  Methods well known to those skilled in the art for preparing genes encoding polypeptides that are functionally equivalent to a polypeptide include hybridization techniques (Sambrook, J. et al., Molecular Cloning 2nd ed., 9.47-9.58). , Cold Spring Harbor Lab. Press, 1989). In the present specification, the term “gene” includes DNA and RNA, and includes not only genomic DNA but also mRNA, cDNA, aRNA and cRNA. Moreover, not only full-length genes but also ESTs are included.

  The gene specified by each base sequence includes a gene that hybridizes under stringent conditions with a gene consisting of a base sequence complementary to each base sequence. The stringent condition means a condition in which a specific hybrid is formed and a non-specific hybrid is not formed, that is, a high homology (homology or identity of 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more). More specifically, such conditions can be determined by conventional techniques well known in the art, such as colony hybridization, plaque hybridization, microarray, or Southern blot hybridization. After hybridization at 65 ° C in the presence of 0.7 to 1.0 M NaCl, 0.1 to 2 times the concentration of SSC (Saline Sodium Citrate; 150 mM sodium chloride, 15 mM sodium citrate) solution And can be achieved by washing the membrane at 65 ° C.

  It is also possible to isolate genes functionally equivalent to each gene using gene amplification methods using primers synthesized based on nucleotide sequence information, such as polymerase chain reaction (PCR) method, LAMP method, etc. Is possible.

  In addition, functionally equivalent genes usually have high homology or identity at the amino acid sequence level. High homology or identity generally refers to homology or identity of at least 50% or more, preferably 75% or more, more preferably 85% or more, more preferably 95% or more at the amino acid level. The homology or identity of amino acid sequences and base sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993)). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)). Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

Gene set A preferred gene set for discriminating autoimmune diseases in the present invention is a high discriminating gene list consisting of genes specified by the nucleotide sequences represented by SEQ ID NOs: 1 to 100 and ranked in the order of SEQ ID NOs. A gene set comprising genes selected from the above, wherein the autoimmune disease discrimination rate is 50% or more, preferably 70% or more, more preferably 85% or more. Here, the autoimmune disease discrimination rate is determined by the above method. The gene set includes at least 5 genes selected from the gene list of Table 1, preferably 5 to 50 genes, more preferably 10 to 40 genes, and even more preferably 10 to 30 genes.

  More specifically, a suitable gene set for discriminating an autoimmune disease in the present invention consists of genes identified by the nucleotide sequences represented by SEQ ID NOs: 1 to 100 and ranked in the order of SEQ ID NOs. At least 1st to 5th gene in the discriminating gene list, preferably at least 1st to 10th gene, more preferably at least 1st to 15th gene, and more preferably at least 1st to 20th gene Including genes.

  Here, for example, “a gene set including at least the first to fifth genes in the gene list” means that genes other than the first to fifth genes may be included, preferably a sequence It includes other genes included in the above gene list consisting of genes specified by the base sequences represented by any of numbers 1 to 100.

  Another specific example of a suitable gene set is a gene set including at least 5 genes, preferably at least 10 genes selected from the 1st to 20th genes in the gene list.

  Here, for example, “a gene set including at least 5 genes selected from the 1st to 20th genes in the above gene list” refers to genes other than the 1st to 20th genes as described above. The other gene included in the said gene list which consists of a gene specified with the base sequence represented by preferably any of sequence number 1-100 preferably.

  Of the genes included in the gene set, 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, most preferably 95% or more of the genes of SEQ ID NOs: 1 to 100 It is preferably a gene included in a gene list consisting of genes specified by any of the nucleotide sequences represented.

  By detecting the expression of genes contained in these gene sets in unknown samples, it is possible to reliably discriminate autoimmune diseases with a high discrimination rate.

  A preferred gene set for discriminating systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis according to the present invention comprises a gene identified by the nucleotide sequence represented by SEQ ID NOs: 101 to 200. A gene set comprising genes selected from a list of highly discriminating genes that are ranked in the order of, wherein the discrimination rate between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis is 50% or more, preferably Is a gene set of 70% or more, more preferably 85% or more. Here, the discrimination rate between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis is determined by the above method. The gene set includes at least 5 genes selected from the gene list of Table 2, preferably 5 to 50 genes, more preferably 10 to 40 genes, and even more preferably 10 to 30 genes.

  More specifically, a suitable gene set for discriminating between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis according to the present invention is specified by the nucleotide sequences represented by SEQ ID NOs: 101 to 200. At least 1st to 5th gene, preferably at least 1st to 10th gene, more preferably at least 1st to 15th gene in the high discriminating gene list. Including genes.

  Here, for example, “a gene set including at least the first to fifth genes in the gene list” means that genes other than the first to fifth genes may be included, preferably a sequence It includes other genes included in the above gene list consisting of genes specified by the base sequences represented by any of numbers 101 to 200.

  Another specific example of a suitable gene set is a gene set including at least 5 genes, preferably at least 10 genes, selected from the 1st to 15th genes in the gene list.

  Here, for example, “a gene set including at least 5 genes selected from the 1st to 15th genes in the above gene list” refers to genes other than the 1st to 15th genes as described above. The other gene included in the said gene list which consists of a gene specified by the base sequence represented by either of the sequence numbers 101-200 preferably.

  Of the genes included in the gene set, 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, most preferably 95% or more of the genes of SEQ ID NOs: 101 to 200 It is preferably a gene included in a gene list consisting of genes specified by any of the nucleotide sequences represented.

  By detecting the expression of genes contained in these gene sets in an unknown sample, systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis can be reliably discriminated at a high discrimination rate.

Measurement of gene expression level A method for detecting the expression of each gene in a sample derived from a subject, more specifically, a method for measuring the expression level of each gene is based on each nucleotide sequence in the sample derived from the subject. A method for measuring mRNA of a specified gene can be mentioned.

  Hereinafter, a method for measuring mRNA of a gene specified by each base sequence in a sample derived from a subject will be described. Here, measurement of mRNA of each gene includes measurement of cRNA, cDNA or aRNA prepared from the mRNA.

  The method includes a step of extracting total RNA from a sample derived from a subject, particularly whole blood, and a step of measuring the expression level of a target gene in the total RNA, that is, a gene specified by each base sequence.

  The method for extracting total RNA from the sample derived from the subject is not particularly limited. For example, guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine thiocyanate / phenol, The chloroform method (Chomczynski, P. and Sacchi, N., (1987) Anal. Biochem., 162, 156-159) and the like can be employed. Of these, the guanidine acid thiocyanate / phenol / chloroform method is preferred.

  Total RNA derived from a sample of a healthy subject not suffering from an autoimmune disease used as a control can be extracted and purified in the same manner as described above, but a commercially available product may be used.

Subsequently, a test polynucleotide such as cRNA, cDNA or aRNA is prepared from the total RNA obtained above and labeled with an appropriate label such as a radioisotope, a fluorescent substance, a luminescent substance, etc. Detect intensity. As the radioisotope, ³² P, ¹²⁵ I, ³⁵ S and the like can be used. As the fluorescent substance, for example, CyDye, fluorescein (FITC), sulforhodamine (SR), tetramethylrhodamine (TRITC) and the like can be used. As the luminescent substance, luciferin or the like can be used.

  For the detection of the signal intensity, any method known in the art for detecting a label can be used. For example, when a radioisotope is used as a label, the radioactivity can be measured by, for example, a liquid scintillation counter or a γ-counter. When a fluorescent substance is used as a label, the fluorescence can be detected using a fluorescent microscope, a fluorescent plate reader, a fluorescent scanner, or the like.

  Specifically, it can be measured by hybridizing the test polynucleotide to a carrier on which a probe that specifically hybridizes to the test polynucleotide is immobilized (Brown, PO et al., Nature genet. 21, suppliment, 33-37 (1999)). For example, cDNA is prepared from the extracted total RNA by reverse transcriptase reaction, and aminoallyl-UTP or the like is added when aRNA is prepared from the cDNA by T7 enzyme reaction, and indirectly labeled with a fluorescent label (eg, Cy3, Cy5, etc.) Then, hybridization is performed with the immobilized probe, and a fluorescent signal derived from the label is detected and analyzed.

  As the carrier on which the probe is immobilized, for example, a DNA microarray (DNA chip) can be used. The DNA chip is preferably one in which DNA synthesized based on the EST sequence on the database or the total RNA sequence is immobilized. As the DNA chip, a commercially available product (for example, AceGene (registered trademark) Human Oligo Chip 30K manufactured by Hitachi Software Engineering Co., Ltd.) may be used, or a known method (Lipshutz, RJ etal., Nature genet. 21, suppliment, 20-24 (1999)).

  Moreover, the expression level of the gene in the sample derived from the subject can also be measured by performing an amplification reaction using the test polynucleotide as a template using a primer and detecting the specific amplification reaction.

  The amplification method is not particularly limited, and examples include known methods using the principle of the polymerase chain reaction (PCR) method, such as PCR method, RT-PCR method, real-time PCR method, ATAC-PCR method and the like. Examples of other amplification methods include LAMP method, ICAN method, RCA method, LCR method, and SDA method. Amplification is performed until the amplification product is at a detectable level.

  In order to detect whether or not a specific amplification reaction has occurred after the amplification reaction, a known means capable of specifically recognizing an amplification product obtained by the amplification reaction can be used. For example, a specific amplification reaction can be detected by confirming whether an amplified fragment of a specific size is amplified using agarose gel electrophoresis or the like.

  Alternatively, a label such as a radioisotope, a fluorescent substance, or a luminescent substance can be allowed to act on dNTP or dUTP incorporated during the amplification reaction to detect this label. There are no particular restrictions on the type of label, the method for introducing the label, and the like, and various conventionally known means can be used. As described above, the expression level of each gene can be measured in the sample derived from the subject by specifically increasing or decreasing the amplification reaction.

  Other methods for measuring the gene expression level include, for example, the subtraction method (Sive, HL and John, T. St., Nucleic Acids Research 16, 10937 (1988), Wang, Z., and Brown, DD, Proc. Natl. Acad. Sci. USA 88, 11505-11509 (1991)), differential display method (Liang, P., and Pardee, AB, Science 257, 967-971 (1992), Liang, P., Averboukh, L. , Keyomarsi, K., Sager, R., and Pardee, AB, Cancer Research 52, 6966-6968 (1992)), differential hybridization (John, T. St., and Davis, RW Cell, 16, 443) -452 (1979)), and cross-hybridization using appropriate probes (Molecular Cloning, A Laboratory Manual, Maniatis, T., Fritsch, EF, Sambrook, J., Cold Spring Harbor Laboratory Press (1982)) Etc.

  In the above method, a sample derived from a subject means a sample such as blood, body fluid, tissue or excrement isolated from a subject or a clinical donation, but a sample derived from blood, for example, all Blood, blood cell extract (for example, an extract of leukocytes such as lymphocytes) and serum are preferably used, and whole blood is particularly preferably used.

The present invention also relates to a gene set comprising genes selected from a list of highly discriminating genes consisting of genes identified by the nucleotide sequences represented by SEQ ID NOs: 1 to 100 and ranked in the order of SEQ ID NOs: Including a continuous primer of 15 to 30 bases in length for specifically amplifying each gene, or (b) a continuous probe of 20 to 1500 bases in length for detecting each gene. The present invention relates to a kit for discriminating an autoimmune disease, wherein the disease discrimination rate is 50% or more.

  The present invention also relates to a gene set comprising genes selected from a high discriminating gene list consisting of genes identified by the nucleotide sequences represented by SEQ ID NOs: 101 to 200 and ranked in the order of SEQ ID NOs: (a) Including a continuous primer of 15 to 30 bases in length for specifically amplifying each gene, or (b) a continuous probe of 20 to 1500 bases in length for detecting each gene, The present invention relates to a kit for discriminating between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis, which has a discrimination rate of 50% or more between juvenile idiopathic arthritis and arthritic juvenile idiopathic arthritis.

The primer described in (a) can be easily designed and amplified by a conventional method such as using commercially available primer design software based on each base sequence. Examples of usable software include Oligo ^™ (National Bioscience Inc.) and GENETYX (Software Development Co., Ltd.). The length having a substantial function as a primer is usually 15 to 30 bases, more preferably 18 to 20 bases.

  The probe described in (b) above is a polynucleotide that specifically hybridizes with a test polynucleotide such as cDNA, cRNA, or aRNA prepared from mRNA of a gene specified by each base sequence, and is 20 to 1500. Those having a base length are preferred. Specifically, in the Northern hybridization method, a single-stranded oligonucleotide or double-stranded DNA having a length of about 20 bases is preferably used. In the case of a DNA chip, a double-stranded DNA having a length of about 100 to 1500 bases or a single-stranded oligonucleotide having a length of about 20 to 100 bases is preferably used.

  The probe may be fixed to a carrier such as a glass plate, a plastic substrate, a nylon membrane, a microbead, or a silicon chip. Such an immobilized carrier and a method for producing the same have already been described. A chip can be mentioned.

  These primers and probes may be labeled with an appropriate label (for example, enzyme label, radioactive label, fluorescent label, etc.), and may be modified with biotin, phosphoric acid, amine or the like. In designing the primer and probe, it is preferable to consider the melting temperature (Tm), GC content, and the like.

  In addition to the above-described components, the kit of the present invention may optionally contain reagents necessary for the examination of autoimmune diseases according to the present invention, such as hybridization, probe labeling, and label detection. Good.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, the scope of the present invention is not limited to the range of an Example.

  Using a DNA chip (AceGene (registered trademark) Human Oligo Chip Human 30K) equipped with oligo DNA designed from about 30,000 humans, the genes expressed in peripheral blood cells are extracted from the peripheral blood of the subject. Using the total RNA as a control, the gene expression level was measured.

  Specifically, aminoally amplified aRNA was prepared using total RNA extracted from peripheral blood cells, the aRNA was indirectly labeled (post-labeled) with fluorescent dyes Cy3 and Cy5, respectively, and a DNA chip (AceGene (registered trademark)) Human Oligo Chip Human 30K) was used for hybridization, and changes in the expression level of genes expressed in peripheral blood cells were measured with a fluorescence scanner.

The following were used as measurement samples:
Total RNA extracted from peripheral blood cells of patients with systemic juvenile idiopathic arthritis (soJIA): 51 cases Total RNA extracted from peripheral blood cells of patients with polyarticular juvenile idiopathic arthritis (polyJIA): 5 cases Healthy children Total RNA extracted from peripheral blood cells: 8 cases Total RNA extracted from peripheral blood cells of healthy adults (for reference): 45 cases Measurement samples should be stored at room temperature, and after extraction of total RNA until the end of measurement The period was stored at -60 ° C or lower in an ultra-low temperature bath (manufactured by SANYO).

Example 1 Extraction of total RNA
Total RNA was extracted from peripheral blood collected using a PAXgene vacuum blood collection tube (PreAnalytiX) using PAXgene RNA Blood RNA Kit (PreAnalytiX). Details are as follows.

Blood collection tubes stored at 4 ° C. (more than a day) were allowed to stand at room temperature for 30 minutes before RNA extraction. Centrifuged at 4000G for 10 minutes at room temperature. The supernatant was removed by decanting, and 5 ml of RNAse free H ₂ O was added to the pellet and vortexed. Centrifuged at 4000G for 10 minutes at room temperature. The supernatant was removed by decanting, 360 μl of Buffer BR1 was added, and vortexed and transferred to a 1.5 ml tube by pipetting. Buffer BR2 (300 μl) and proteinase K (40 μl) were added and vortexed. Incubated at 55 ° C. for 10 minutes, vortexed 2-3 times in the middle. After centrifugation at 15000 G for 3 minutes at room temperature, the supernatant was pipetted into a 1.5 ml tube. 350 μl of 99.5% EtOH was added and vortexed. 700 μl of sample was added to the PAXgene column and centrifuged at 15000 G for 1 minute at room temperature. The remaining sample was added to the PAXgene column and centrifuged at 15000 G for 1 minute at room temperature.

  The column was placed in a new collection tube, 350 μl of Buffer BR3 was added, and centrifuged at 15000 G for 1 minute at room temperature. The column was placed in a new collection tube, 80 μl of DNAse I (DNAse: 10 RDD: 70 μl) was added, and the mixture was allowed to stand for 15 minutes. 350 μl μl of Buffer BR3 was added to the stationary column, and centrifuged at 15000 rpm for 1 minute at room temperature. The column was placed in a new collection tube, 500 μl of Buffer BR4 was added, and centrifuged at 15000 G for 1 minute at room temperature. The column was placed in a new collection tube, 500 μl of Buffer BR4 was added, and centrifuged at 15000 G for 3 minutes at room temperature. The column was placed in a new collection tube and centrifuged at 15000 G for 1 minute at room temperature to remove EtOH. The column was set in an elution tube, 50 μl of Buffer BR5 was added so as to wet the entire membrane, allowed to stand for 5 minutes, and then centrifuged at 15000 G for 1 minute at room temperature. The eluate was added onto the column again, allowed to stand for 5 minutes, and then centrifuged at 15000 G for 1 minute at room temperature. The obtained total RNA was stored at -60 ° C or lower.

The concentration was measured with NanoDrop (registered trademark) (NanoDrop (registered trademark) Technologies Inc.) using 1 μl of total RNA. In addition, 1 μl of total RNA was used to check the quality with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc.) (18S rRNA: 28S rRNA abundance ratio is preferably about 1: 2).
Based on the concentration calculated by NanoDrop (registered trademark), 3 pieces of each total RNA divided into 1 μg were prepared and stored together with the remaining sample at a temperature of −60 ° C. or lower.

Example 2 aRNA Amplification Peripheral blood cell-derived total RNA was each aRNA amplified using Amino Allyl Message Amp ^™ aRNA kit (# 1752: Ambion) to prepare aminoallyl-labeled aRNA. Details are as in 1) to 6) below.

1) Preparation of First Strand cDNA
(1) The following reagents were mixed and incubated for 10 minutes in a heat block at 70 ° C.
Total RNA 2μg
T7 Oligo (dT) Primer 1μl
Nuclease free DW up to 12μl
(2) A premix was prepared with the following composition (per sample).
10 _× First Strand Buffer 2μl
Ribonuclease Inhibitor 1μl
dNTP Mix 4μl
Reverse Transcriptase 1μl
(3) The premix of (2) was added to the reaction solution of (1), mixed several times by pipetting, and then incubated in a heat block at 42 ° C. for 2 hours. The sample was transferred to ice and cooled.

2) Synthesis of Second Strand cDNA The following reagents were added to the above sample, mixed by pipetting several times, and incubated at 16 ° C. for 2 hours.
Nuclease free DW 63μl
10 × second Strand Buffer 10μl
dNTP Mix 4μl
DNA Polymerase 2μl
RNAse H 1μl

3) Purification of cDNA The filter cartridge attached to the kit was set in a wash tube, and the filter was moistened with 50 μl of cDNA binding buffer. The incubation was continued for 5 minutes at room temperature. 250 μl of cDNA binding buffer was added to the synthesized cDNA sample solution, mixed by pipetting several times, and then transferred to the filter cartridge. Centrifugation was performed at 10000 G for 1 minute until the solution passed through the filter. The liquid in the wash tube was discarded and the filter cartridge was returned. 650 μl of cDNA wash buffer was added to the filter. Centrifugation was performed at 10000 G for 1 minute until the solution passed through the filter. The liquid in the wash tube was discarded, the filter cartridge was returned, and again centrifuged at 10000 G for 1 minute, and the wash buffer remaining on the filter was completely shaken off. The filter cartridge was set in a new tube, and 9 μl of nuclease free DW that had been preheated to 50 ° C. was put in the filter. At this time, I tried to put DW in the center of the filter as much as possible. After incubating at room temperature for 2 minutes, it was centrifuged at 10,000 G for 1 minute. 9 μl of the above nuclease free DW was put in a filter, incubated at room temperature for 2 minutes, and centrifuged at 10,000 G for 1 minute, and 14 μl of cDNA sample solution was collected.

4) Synthesis of aminoallyl-labeled aRNA 14 μl of the cDNA sample solution obtained by the above operations 1) to 3) was placed in a 0.2 ml centrifuge tube, and the following reagents were added.
aaUTP Solution (50 mM) 3 μl
ATP, CTP, GTP mix (25 mM) 12 μl
UTP Solution (50 mM) 3 μl
T7 10 × Reaction Buffer 4μl
T7 Enzyme Mix 4μl
The solution was mixed by pipetting several times, and then incubated for 14 hours on a 37 ° C. thermal cycler. After the incubation, the thermal cycler was automatically set to 4 ° C. 2 μl of DNAse was added, mixed by pipetting, and incubated for 30 minutes on a 37 ° C. thermal cycler.

5) Purification of aminoallyl-labeled aRNA
The aRNA collection tube and filter cartridge attached to the Amino Allyl Message Amp ^™ aRNA kit (Ambion) were set. Transfer the sample solution obtained in 4) to a 1.5 ml centrifuge tube, add 58 μl of nuclease free DW, 350 μl of aRNA binding buffer, 250 μl of 100% EtOH, mix the solution by pipetting several times, and then aRNA collection tube And centrifuged at 10000 G for 1 minute. The filter cartridge was removed, the liquid in the centrifuge tube was discarded, and the filter was returned. 650 μl of aRNA wash buffer was put in a filter and centrifuged at 10000 G for 1 minute. After discarding the liquid in the tube, the filter cartridge was returned, and again centrifuged at 1000 G for about 1 minute, and the wash buffer remaining on the filter was completely shaken off. The filter cartridge was set in a new tube, and 50 μl of nuclease free DW that had been preheated to 50 ° C. was put in the filter. At this time, I tried to put DW in the center of the filter as much as possible. After incubating at room temperature for 2 minutes, it was centrifuged at 10,000 G for 2 minutes. 50 μl of the above nuclease free DW was put in a filter, incubated at room temperature for 2 minutes, and centrifuged at 10,000 G for 2 minutes, and 100 μl of aminoallyl-labeled aRNA sample was collected.

6) Concentration measurement and storage of aminoallyl-labeled aRNA Take 1 μl from the aminoallyl-labeled aRNA sample solution obtained in 5) and use Bioanalyzer (registered trademark) (Agilent) using RNA 6000 Nano LabChip (registered trademark) kit (Agilent Technologies). Electrophoresis by Technologies). This confirmed whether aRNA amplification was normally performed. 1 μl of the aminoallyl-labeled aRNA sample solution was used, and the concentration was measured with NanoDrop (registered trademark) (NanoDrop (registered trademark) Technologies Inc.). The aRNA solution was dispensed into one reaction use (5 μg), EtOH precipitated by the following method, and stored at −20 ° C. or lower.

Example 3 Coupling Reaction and Fragmentation The pellet obtained in Example 2 was dissolved in a coupling buffer and labeled by binding the fluorescent dye Cy3 or Cy5 to each aRNA. Details are as follows.
CyDye (Amersham Bioscience) dissolved in DMSO was added to the aRNA solution and reacted for 1 hour in a heat block set at 40 ° C. Unreacted CyDye was removed with a gel filtration column (Micro Bio-Spin (registered trademark) P-30). CyDye-modified aRNA was purified and concentrated with an ultrafiltration membrane (Microcon (registered trademark) YM-30).

  The resulting solution was mixed with 5 × fragmentation buffer. After heating for 15 minutes in a heat block set at 94 ° C, it was quenched with crushed ice. Fragmentation was confirmed by 3% agarose gel electrophoresis. The resulting solution was purified and concentrated with an ultrafiltration membrane (Microcon (registered trademark) YM-10).

Example 4 Hybridization hybridization solution, salmon sperm DNA, and the like were added to prepare a target solution. The hybridization solution was prepared by dissolving 109.60 g of tetramethylammonium chloride (Aldrich, T1, 952-6, 98 +%) in about 100 ml of sterile distilled water, adding sterile distilled water to make up to 200 ml, and then adding 0.2 ml. It was prepared by filtering through a mm-diameter filter, dispensing into a 50 ml plastic centrifuge tube, and storing at room temperature (15 ° C. to 27 ° C.).

  The target solution was heat-denatured for 2 minutes in a heat block set at 95 ° C., and then rapidly cooled with crushed ice. Formamide was added to prepare a hybridization solution. Hybridization was performed using an automatic hybrid device (HS400 TECAN).

  Wash in 2x SSC-0.1% SDS solution for 5 minutes (30 ° C), wash in 2x SSC-0.1% SDS solution for 5 minutes (30 ° C), and wash in 2x SSC solution for 5 minutes (30 ° C). Then, it was washed with 1 × SSC solution for 5 minutes (30 ° C.).

The fluorescence intensity of Cy3 and Cy5 of each spot on the DNA chip was measured using a reader (ScanArray (registered trademark) Lite) (Packard Biochip Technologies). Digitization of image data was performed using DNASISArray (registered trademark), and the obtained numerical data was standardized on Excel (Microsoft).
The details of the reagents used in the above examples are as follows.

  The standardized numerical data thus selected is not affected by the background, does not include errors due to differences in detection sensitivity between Cy3 and Cy5, has been obtained in the majority of cases analyzed, and is healthy. Variation of gene expression in juvenile idiopathic arthritis in comparison with, or gene expression variation in comparison between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis It has the expression information of the gene exceeding the expression fluctuation range, and was able to secure the reliability of the subsequent statistical analysis.

  Based on the above standardized gene expression data, according to the method of Golub et al. (Science, supra), the above-mentioned procedures 101 to 107, procedures 201 to 205, and procedures 301 to 306 are used to create a high discriminating gene list and determine a gene set Carried out.

  The high discriminating ability gene list for examining juvenile idiopathic arthritis prepared in this example is ranked in the order of SEQ ID NOs consisting of genes identified by any of SEQ ID NOS: 1 to 100 described in Table 1 above. It is a gene list attached. Table 4 below shows the discrimination rate of juvenile idiopathic arthritis of a gene set including the first to Nth genes (N is 1 to 100) in the gene list.

  It was shown that juvenile idiopathic arthritis can be discriminated with a discrimination rate of 0.9 or higher when any of the above gene sets including genes selected from the gene list shown in Table 1 is used.

  The high discriminating ability gene list for discriminating systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis prepared in this example is any one of SEQ ID NOS: 101 to 200 described in Table 2 above. It is a gene list ranked in the order of the sequence number which consists of a specified gene.

  Table 5 shows the discrimination rate between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis in a gene set including the first to Nth genes (N is 1 to 100) in the gene list.

  When using any of the above gene sets including genes selected from the gene list shown in Table 2, systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis can be discriminated with a discrimination rate of 0.9 or higher. It has been shown.

The outline of the determination method of a gene set is shown. An overview of the ranking process for highly discriminating genes is shown. An overview of the discrimination ability evaluation of a gene set is shown. The outline of the gene set determination method is shown. The outline of the unknown gene sample discrimination method is shown.

Claims (15)

In a sample of from a subject, see contains detecting the expression of genes of the gene set comprising a gene that is identified by the nucleotide sequence represented by SEQ ID NO: 1 to 5 nucleotide sequence represented by SEQ ID NO: 1 The expression level of the gene specified in (4) decreases and the expression level of the gene specified in the nucleotide sequences represented by SEQ ID NOs: 2 to 4 increases. This indicates juvenile idiopathic arthritis. To detect gene expression to test for osteoarthritis . The method according to claim 1, wherein the gene set further comprises at least 5 genes selected from genes specified by the nucleotide sequences represented by SEQ ID NOs: 6 to 20 . The method according to claim 1, wherein the gene set further comprises at least 5 genes selected from genes specified by the nucleotide sequences represented by SEQ ID NOs: 6 to 100 . In a sample of from a subject, see contains detecting the expression of genes of the gene set comprising a gene that is identified by the nucleotide sequence represented by SEQ ID NO: 101-105, SEQ ID NO: 101 to 105 A decrease in the expression level of the gene identified by the nucleotide sequence indicates systemic juvenile idiopathic arthritis. To discriminate between systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis , A method of detecting expression . The method according to claim 4 , wherein the gene set further comprises at least 5 genes selected from the genes specified by the nucleotide sequences represented by SEQ ID NOs: 106 to 115 . The method according to claim 4 , wherein the gene set further comprises at least 5 genes selected from genes identified by the nucleotide sequences represented by SEQ ID NOs: 106 to 200 . Detecting gene expression
Extracting total RNA from a sample derived from a subject, and measuring the expression level of the gene in the total RNA.
Any one method of claims 1-6. 20 for detecting a 15-30 bases long contiguous primer or each gene, for specifically amplifying each gene of a set of genes including the gene identified by the nucleotide sequence represented by SEQ ID NO: 1-5 1500 bases long contiguous probe including, a kit for determining the juvenile idiopathic arthritis. The kit according to claim 8 , wherein the gene set further comprises at least 5 genes selected from genes identified by the nucleotide sequences represented by SEQ ID NOs: 6 to 20 . The kit according to claim 8 , wherein the gene set further comprises at least 5 genes selected from genes specified by the nucleotide sequences represented by SEQ ID NOs: 6 to 100 . 20 for detecting a 15-30 bases long contiguous primer or each gene, for specifically amplifying each gene of a set of genes including the gene identified by the nucleotide sequence represented by SEQ ID NO: 101 to 105 1500 bases long contiguous including probes, kits for determining the total body juvenile idiopathic arthritis and articulated juvenile idiopathic arthritis. The kit according to claim 11 , wherein the gene set further comprises at least 5 genes selected from genes specified by the nucleotide sequences represented by SEQ ID NOs: 106 to 115 . The kit according to claim 11 , wherein the gene set further comprises at least 5 genes selected from the genes specified by the nucleotide sequences represented by SEQ ID NOs: 106 to 200 . Comprising a gene identified by the nucleotide sequence shown in SEQ ID NO: 1 to 5 marker gene set for testing the juvenile idiopathic arthritis. Marker gene set for discriminating SEQ ID NO: 101 to contain a gene identified by the nucleotide sequence represented by 105, systemic-onset juvenile idiopathic arthritis and articulated juvenile idiopathic arthritis.

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