JPWO2006098401A1 - Method for measuring the risk of developing rheumatoid arthritis - Google Patents

Abstract

The present invention provides rheumatoid arthritis susceptibility genes in Japanese and provides a method for detecting and determining the risk of developing rheumatoid arthritis. The present invention also provides rheumatoid arthritis susceptibility diagnostic markers, probes, primers, and reagent kits that can be used effectively in carrying out this detection method. The present invention provides the SEC8L1 gene as a rheumatoid arthritis susceptibility gene and SNP440, SNP441, SNP442, SNP443, SNP445, SNP447 and SNP449 (present in LD block 4) as rheumatoid arthritis sensitivity SNPs. Furthermore, as a method of detecting the risk of developing rheumatoid arthritis, at least one selected from SNP441 selected from the above blocks and other SNPs (SNP440, SNP445, SNP447 and SNP449) targeting genomic DNA obtained from a human subject. A method is provided that includes detecting and identifying one SNP.

Description

  The present invention relates to a method for measuring and determining the risk of developing rheumatoid arthritis. More specifically, the method detects SNPs (single nucleotide polymorphisms), particularly haplotypes associated with rheumatoid arthritis from a sample containing human genomic DNA, so that the sample provider (subject) is affected with rheumatoid arthritis. It is a method of detecting and determining whether or not there is a potential risk.

  Furthermore, the present invention relates to a diagnostic marker for rheumatoid arthritis disease, a probe, a primer, and a reagent kit that can be used effectively in carrying out such a detection method.

  Rheumatoid arthritis (hereinafter simply referred to as “RA”) is one of the most common autoimmune diseases, and its prevalence is said to be almost uniform and 0.3% to 1% worldwide. (Non-patent Document 1). Chronic inflammation of the synovium and subsequent joint destruction are the main features. Although the detailed pathophysiology of rheumatoid arthritis has not been elucidated, it is suggested that genetic factors and environmental factors (infection, hormones, pregnancy, etc.) are involved in both family studies and twin studies (Non-Patent Documents 2 and 3). ). The correlation between the HLA region and RA has been known for some time, and in particular, it has been known that a plurality of HLA-DRB1 alleles encoding shared epitopes are involved in the risk and severity of disease (Non-patent Document 4). However, only about 1/3 of RA's genetic factors can be explained only by HLA (Non-patent Document 5), and it is expected that a plurality of genes other than HLA are involved in the development of RA. The involvement of candidate genes such as TNFα, CTLA-4, and cytokine-related genes has been investigated, but the results are not consistent (Non-patent Document 6). Genome-wide linkage analysis using RA families has been reported by at least four groups (Non-Patent Documents 7 to 12), and several regions other than HLA have been identified in multiple studies. A common region has not been identified.

  In general, in multifactorial diseases such as rheumatoid arthritis, linkage analysis alone does not give sufficient results to identify susceptibility genes, and detailed analysis of the region by linkage disequilibrium analysis using SNP is necessary. In fact, genes related to multifactorial diseases such as type 2 diabetes and Crohn's disease have begun to be identified by such a method (Non-Patent Documents 13 to 15). In rheumatoid arthritis, an association between a haplotype in the PADI4 gene and Japanese RA has been reported (Non-patent Document 16). PADI4 is located on chromosome 1 q36 and has been reported as a candidate region in two studies so far (Non-patent Documents 7 and 8). Recently, the same group has identified an association between SNP and RA in the RANX1 binding region in the SLC22A4 gene (Non-patent Document 17).

However, as mentioned above, RA is a multifactorial disease that involves both genetic and environmental factors, and it is difficult to determine the risk of morbidity based on mutations or abnormalities in a single gene. There is a need for further study.
Felson D: Epidemiology of the rheumatic diseases.In: Koopman WJ (ed) Arthritis and allied conditions: a textbook of rheumatology, 13th ed.Williams & Wilkins, Philadelphia, PA, pp3-34, 1996. Lawrence JS. Heberden Oration, 1969. Rheumatoid arthritis--nature or nurture? Ann Rheum Dis 1970; 29: 357-79. del Junco D, Luthra HS, Annegers JF, Worthington JW, Kurland LT.The familial aggregation of rheumatoid arthritis and its relationship to the HLA-DR4 association.Am J Epidemiol 1984; 119: 813-29. Wordsworth BP, Lanchbury JS, Sakkas LI, Welsh KI, Panayi GS, Bell JI.HLA-DR4 subtype frequencies in rheumatoid arthritis indicate that DRB1 is the major susceptibility locus within the HLA class II region.Proc Natl Acad Sci USA 1989; 86: 10049-53. Deighton CM, Walker DJ, Griffiths ID, Roberts DF.The contribution of HLA to rheumatoid arthritis.Clin Genet 1989; 36: 178-82. Barton A, Ollier W. Genetic approaches to the investigation of rheumatoid arthritis. Curr Opin Rheumatol 2002; 14: 260-9. Cornelis F, Faure S, Martinez M, Prud'homme JF, Fritz P, Dib C, et al. New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study.Proc Natl Acad Sci USA 1998; 95: 10746-50 . Shiozawa S, Hayashi S, Tsukamoto Y, Goko H, Kawasaki H, Wada T, et al. Identification of the gene loci that predispose to rheumatoid arthritis. Int Immunol 1998; 10: 1891-5. Jawaheer D, Seldin MF, Amos CI, Chen WV, Shigeta R, Monteiro J, et al. A genomewide screen in multiplex rheumatoid arthritis families suggests genetic overlap with other autoimmune diseases. Am J Hum Genet 2001; 68: 927-36. MacKay K, Eyre S, Myerscough A, Milicic A, Barton A, Laval S, et al. Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom. Arthritis Rheum 2002; 46: 632-9 . Jawaheer D, Seldin MF, Amos CI, Chen WV, Shigeta R, Etzel C, et al. Screening the genome for rheumatoid arthritis susceptibility genes: a replication study and combined analysis of 512 multicase families. Arthritis Rheum 2003; 48: 906-16 . Eyre S, Barton A, Shephard N, Hinks A, Brintnell W, MacKay K, et al. Investigation of susceptibility loci identified in the UK rheumatoid arthritis whole-genome scan in a further series of 217 UK affected sibling pairs. Arthritis Rheum 2004; 50: 729-35. Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, et al. Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet 2000; 26: 163-75. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.Nature 2001; 411: 599-603. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.Nature 2001; 411: 603-6. Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, Suzuki M, et al. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet 2003; 34: 395-402. Tokuhiro S, Yamada R, Chang X, Suzuki A, Kochi Y, Sawada T, et al. An intronic SNP in a RUNX1 binding site of SLC22A4, encoding an organic cation transporter, is associated with rheumatoid arthritis. Nat Genet 2003; 35: 341-8.

  An object of the present invention is to provide a method for determining the risk of developing rheumatoid arthritis for an individual subject. A further object of the present invention is to provide a reagent and a reagent kit useful for carrying out the method easily.

  For the purpose of finding RA susceptibility genes and gene polymorphisms involved in the development of rheumatoid arthritis (RA) in the Japanese population, the present inventors have previously reported repeated linkages among multiple races. Nine Japanese RA susceptibility genes areas, TIRA1-9 (Chromosome 7 long arm 31-34: 7q31-34) (Takeuchi F, Moritani M, Nabeta H, Mori M, Goto M, Hanyu T, et al. , by preliminaly genome-wide screening .: 10th Asia Pasific League of Associations for Rheumatology Congress. Bangkok, Thailand, 2002 Dec 1-6) was selected as a candidate region, and between rheumatoid arthritis patients and controls (healthy subjects) A large-scale association analysis and linkage disequilibrium analysis were performed between the controls) and the SEC8L1 gene present on the human chromosome 7 (7q31-34) was found to be a candidate disease susceptibility gene for RA. In gene intron 10 There was a specific genetic polymorphism (SNP441) that showed a significant difference in the allele frequency between case controls, and it was found that the SNP441 and haplotypes containing it were important in defining the susceptibility of rheumatoid arthritis.

  And, by detecting the SEC8L1 gene polymorphism (SNP441) and haplotypes containing this for individual subjects, it was found that the potential risk of developing rheumatoid arthritis can be detected with high probability for the subject, The present invention has been completed.

That is, the present invention provides the following inventions:
(1) Rheumatoid arthritis disease diagnosis marker A marker for diagnosing rheumatoid arthritis, comprising at least one oligo or polynucleotide selected from the group consisting of the following (1) to (7):
(1) an oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across G or C located at position 379258 (SNP440) in the base sequence of the human SEC8L1 gene;
(2) in the base sequence of the human SEC8L1 gene, an oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and 3 ′ side across C or T located at position 393668 (SNP441);
(3) in the base sequence of human SEC8L1 gene, an oligo or polynucleotide having a base sequence with a maximum length of 300 bases on each of the 5 'side and 3' side across C or T located at position 406991 (SNP442);
(4) In the base sequence of the human SEC8L1 gene, an oligo or polynucleotide having a base sequence with a maximum length of 300 bases on each of the 5 ′ side and 3 ′ side across A or T located at position 411544 (SNP443),
(5) an oligo or polynucleotide having a base sequence having a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across A or G located at position 476136 (SNP445) in the base sequence of the human SEC8L1 gene;
(6) an oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across A or G located at position 569133 (SNP447) in the base sequence of the human SEC8L1 gene;
(7) An oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across T or C located at position 628601 (SNP449) in the base sequence of the human SEC8L1 gene.

(2) Method for detecting the risk of developing rheumatoid arthritis (A) Targeting genomic DNA obtained from a human subject, the base described in (2) below and at least one base selected from the bases described in (1) and (5) to (7) A method for detecting a risk of developing rheumatoid arthritis for a subject comprising the step of detecting and identifying
(1) Base at position 379258 (SNP440) of the human SEC8L1 gene
(2) Base at position 393668 (SNP441) of the human SEC8L1 gene
(5) Base at position 476136 (SNP445) of human SEC8L1 gene
(6) Base at position 569133 (SNP447) of human SEC8L1 gene
(7) Base position at position 628601 (SNP449) of the human SEC8L1 gene. (B) The base (SNP441) of (2) detected and identified in the above step (A) is cytosine, and the bases described in (1) and (5) to (7) (SNP440, SNP445, SNP447, SNP449) At least one base selected from the following conditions:
Base (1) (SNP440): Guanine
Base (5) (SNP445): Guanine
Base of (6) (SNP447): Guanine
(7) Base (SNP449): The method for detecting the risk of developing rheumatoid arthritis according to Item 2, comprising a step of determining that the subject is susceptible to rheumatoid arthritis when cytosine is satisfied.

(3) Rheumatoid arthritis disease risk detection reagent or reagent kit Item 4. Hybridizes to the following oligo or polynucleotide (2) (provided that the oligo or polynucleotide is RNA, the base symbol “t” in the sequence listing shall be read as “u”; the same shall apply hereinafter). Oligonucleotide having a length of 15 to 30 bases or a label thereof, and an oligonucleotide having a length of 15 to 30 bases that hybridizes to at least one oligo or polynucleotide selected from the group consisting of (1) and (5) to (7) Primer set used to detect the risk of developing rheumatoid arthritis, comprising a combination of nucleotides or a label thereof:
(1) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 379258 (SN440),
(2) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 393668 (SN441),
(5) In the nucleotide sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more containing a nucleotide located at position 476136 (SN445),
(6) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 569133 (SN447),
(7) A continuous oligo or polynucleotide having a length of 16 bases or more containing a nucleotide located at position 628601 (SN449) in the base sequence of the human SEC8L1 gene.
Item 5. Hybridizes to the following oligo or polynucleotide (2) (provided that the oligo or polynucleotide is RNA, the base symbol “t” in the sequence listing shall be read as “u”; the same shall apply hereinafter). A 16-500 base-long oligonucleotide that hybridizes to at least one oligo or polynucleotide selected from the group consisting of (1) and (5)-(7) A probe set used for detecting the risk of developing rheumatoid arthritis, comprising an oligonucleotide or a combination thereof with a label:
(1) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases including a nucleotide located at position 379258 (SN440),
(2) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases containing a nucleotide located at position 393668 (SN441),
(5) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases including a nucleotide located at position 476136 (SN445),
(6) In the nucleotide sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases containing a nucleotide located at position 569133 (SN447),
(7) A continuous oligo or polynucleotide having a length of 16 to 500 bases comprising a nucleotide located at position 628601 (SN449) in the base sequence of the human SEC8L1 gene.
Item 6. Item 6. The probe set according to Item 5, which is a solid phase probe in which each oligonucleotide or a labeled product thereof is immobilized on an arbitrary solid phase.
Item 7. A reagent for detecting a risk of rheumatoid arthritis, or a kit containing the reagent set according to item 4 or / and the probe set according to item 5 or 6.

  According to the present invention, the relative risk of developing rheumatoid arthritis (RA) can be easily detected. The detection method of the present invention is a method that can be easily carried out in vitro and without requiring specialized knowledge of a doctor or the like. For subjects whose potential risk of developing RA is found to be relatively high by the detection method of the present invention, to that effect, in order to prevent the onset of RA or delay even a little It is possible to take appropriate measures. Therefore, the present invention is extremely useful as a test method for preventing the onset of RA or delaying the onset of RA. Furthermore, the present invention provides a reagent used in the above detection method, which makes it possible to easily carry out the above method.

1. Definition of terms used in the present invention Indications by abbreviations such as nucleotide sequences (nucleotide sequences) and nucleic acids in the present specification are defined by IUPAC-IUB [IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Patent Office) and conventional symbols in the field.

  In the present specification, unless otherwise specified, “gene” refers to double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand), and single-stranded DNA having a sequence complementary to the sense strand ( Antisense strands) and fragments thereof are included, and the term “gene” in the present specification indicates the regulatory region, coding region, exon, and intron without distinction unless otherwise specified. And

  Further, in this specification, “nucleotide”, “oligonucleotide” and “polynucleotide” are synonymous with nucleic acid and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.

  As used herein, “gene polymorphism” refers to alleles when there are two or more genetically determined alleles. Specifically, in a human population, one or more nucleotide substitutions, deletions, insertions, translocations, reverses at specific sites in one or more other individual genomes based on the genome sequence of one individual. When there is a mutation such as a position, it is statistically certain that the mutation is not a mutation that has occurred in the individual or individuals, or within the population at a frequency of 1% or more, not a specific mutation within the individual. If it is pedigree that it is present in the family, the mutation is referred to as a “gene polymorphism”. As used herein, the term “gene polymorphism” refers to a single nucleotide polymorphism (SNP (or SNPs)), which is a polymorphism caused by a single nucleotide change, and extends over a plurality of nucleotides in succession. Both polymorphisms are included. A “single nucleotide polymorphism” is a polymorphism caused by a single nucleic acid change. Polymorphisms are present at a frequency greater than 1%, preferably greater than 10% of the selected population. The “haplotype” refers to a combination of gene mutations existing in one allele (haploid).

  In the present specification, the “rheumatoid arthritis susceptibility gene” (RA susceptibility gene) refers to a gene that determines a predisposition to develop a polygenic disease rheumatoid arthritis (RA).

  In the present specification, “allele frequency” refers to the ratio of alleles to the number of all genes present in a population at one gene locus.

  As used herein, “linkage disequilibrium” refers to a relationship in which the frequency of an arbitrary allele combination in a population appears more frequently with a nearby specific allele than expected by chance. For example, if locus X has alleles a and b (which are present with equal frequency) and neighboring locus Y has alleles c and d (which are present with equal frequency), then another The haplotype ac, which is a combination of genetic polymorphisms, is expected to exist at a frequency of 0.25 in the population. An allele ac is said to be in linkage disequilibrium if the haplotype ac is larger than these expected values, that is, if a specific genotype ac is frequently encountered. Linkage disequilibrium is caused by the natural selection of a particular combination of alleles or by the evolutionary recent introduction of a population, resulting from the fact that linked alleles have not reached equilibrium. obtain. Therefore, the form of linkage disequilibrium is different in another group, such as ethnicity and race, and even if ac is in linkage disequilibrium in one group, ad is in linkage disequilibrium in another group. In some cases. Detection of genetic polymorphism in linkage disequilibrium is effective in detecting susceptibility to a disease even though the polymorphism itself does not directly cause the disease. For example, an allele a at a locus X may not be a causative gene element of the disease, but may be disease susceptibility due to linkage disequilibrium with the allele c at the locus Y.

  As used herein, “linkage disequilibrium analysis” refers to analyzing the degree of linkage disequilibrium in the genomic region. For example, the linkage disequilibrium analysis can be performed by calculating a linkage disequilibrium coefficient | D ′ | indicating the strength of linkage disequilibrium between two markers from typing data of a healthy person (control person).

  In the present specification, “minor allele” refers to an allele having a low gene frequency when two alleles exist at a locus of one gene.

  In addition, each base number described in this specification is based on positional information of The National Center for Biotechnology Information data base. In this specification, “xxxx position” in the case of “xxxx position in the base sequence of the human SEC8L1 gene” means position information when the first base at position 132395078 of the human SEC8L1 gene is 1. To do.

2. It is difficult to identify genetic factors and environmental factors of rheumatoid arthritis (RA), which is a rheumatoid arthritis susceptibility gene multifactorial disease, because individual factors are less involved in the pathogenesis and pathology. An approach to perform case-control-related analysis over a wide range of areas using uniformly distributed high-frequency SNPs (area-wide case-control-related analysis) is used to identify disease susceptibility genes for RA, which is a multifactor disease. It is an effective and powerful approach.

Therefore, the present inventors selected the region of chromosome 31 q31-34 (7q31-34) as an RA susceptibility candidate region in order to search for RA susceptibility genes in Japanese, and gene-centric evenly-spaced common Constituent components of the exorcist complex by performing region-wide case-control-related analysis and linkage disequilibrium (LD) mapping using the SNP “single nucleotide polymorphism (SNP) that is frequently arranged in the gene region” It was found that the human SEC8L1 gene encoding SEC8 (protein) is a gene that regulates RA sensitivity. In yeast, this Sec8 complex is required for exocytosis and provides a guide to the secretion of secretory vesicles from the Golgi apparatus (35). Mammalian exocysts are also thought to be involved in the transport of vesicles from the Golgi apparatus to the cell membrane, like yeast (36). The human SEC8L1 gene is the genome sequence of human chromosome 7 (Genbank Sequence Accession IDs: NC 000007), an 812691 bp gene located at nucleotide numbers 132395078 to 133207769 (Gene Name: Homo sapiens gene exocyst complex component 4).

  In addition, the identification of the RA susceptibility gene of the present invention was specifically performed by the method described below, as described in Examples.

  As the RA susceptibility candidate region, select chromosome 7 q31-34 (7q31-34) and use 728 SNPs with a minor allele frequency of 15% or more arranged at equal intervals in the region in two steps. Case-control-related analyzes were performed (380 patients with rheumatoid arthritis x 2, 380 healthy controls x 2). Of the 728 SNPs in the first-stage association analysis, 48 SNPs showed significant association with RA, and in the second-stage case-control association analysis, the four SNPs in the SEC8L1 gene (SNP441, SNP442, SNP443, SNP445) showed significant differences with reproducibility (P <0.05). Among them, SNP441 showed the strongest correlation with rheumatoid arthritis at P = 0.000059.

  These SNPs in the SEC8L1 gene were all located in intron 10 in the SEC8L1 gene, and were present in the conserved linkage disequilibrium block 4 (LD block 4). In addition, almost all SNPs (SNP441-461) located within the block were found to correlate with RA. This reveals that the RA-sensitive locus is present in linkage disequilibrium block 4 in the 300 kb region spanning introns 10 to 13 of the SEC8L1 gene. Here, linkage disequilibrium block 4 (LD block 4) is located at base numbers 132747619 to 133035115 in the genome sequence of human chromosome 7 (corresponding to positions 393668 to 681164 in the base sequence of the SEC8L1 gene). It is an area. In the LD block 4, 21 SNPs exist as shown in FIG. 1B. There was no gene other than the SEC8L1 gene in the block, and the observed correlation with RA seemed to originate from the SEC8L1 gene itself.

  Next, in order to clarify the correlation of SNPs present in linkage disequilibrium block 4 (LD block 4), SNPs (SNP440, SNP441, SNP445, SNP447, SNP449) that showed a significant difference from the block were selected, The distribution of genotype combinations between SNPs was compared between case controls. As a result, an RA-sensitive haplotype (P = 0.0015) and an RA-resistant haplotype (P = 0.0000062) were identified.

  As explained above, SNP441 present in linkage disequilibrium block 4 on the nucleotide sequence of the human SEC8L1 gene located on the human chromosome 7 (7q31) and the haplotype containing it showed a strong correlation with RA. This human SEC8L1 gene was thought to contribute to RA sensitivity (RA susceptibility gene).

  As shown in Experimental Examples 2 and 3, the human SEC8L1 gene is ubiquitously expressed in human tissues including synovial fibroblasts, and all lymphocyte cells (T cells, trees that are immunocompetent cells). SEC8L1 was expressed in dendritic cells, macrophages, B cells, NK cells, etc.). These support the findings of the present invention that the SEC8L1 gene is a potent rheumatoid arthritis (RA) disease susceptibility gene.

3. Rheumatoid arthritis diagnosis marker The present invention is an oligo or polynucleotide comprising a rheumatoid arthritis sensitive SNP (RA sensitive SNP) present in the linkage disequilibrium block 4 (LD block 4) of the human SEC8L1 gene which is the RA sensitive gene. Thus, an oligo or polynucleotide that is specifically recognized on the human genome is provided as a diagnostic marker for rheumatoid arthritis (RA diagnosis marker).

  Here, as the RA-sensitive SNP, the following seven SNPs present in the LD block 4 can be exemplified.

(1) SNP440; SNP located at position 379258 in the nucleotide sequence of the human SEC8L1 gene
(2) SNP441; SNP located at position 393668 in the nucleotide sequence of the human SEC8L1 gene
(3) SNP442; SNP located at position 406991 in the nucleotide sequence of the human SEC8L1 gene
(4) SNP443; SNP located at position 411544 in the nucleotide sequence of the human SEC8L1 gene
(5) SNP445; SNP located at position 476136 in the nucleotide sequence of the human SEC8L1 gene
(6) SNP447; SNP located at position 569133 in the nucleotide sequence of the human SEC8L1 gene
(7) SNP449: SNP located at position 628601 in the nucleotide sequence of the human SEC8L1 gene.

  The length (base length) of the oligo or polynucleotide containing each of these SNPs is not particularly limited as long as it is a length specifically recognized on the human genome. The length is usually 10 bases or more, preferably 20 bases or more. Therefore, the RA marker for diagnosis of RA of the present invention comprises 51 nucleotides (of each SNP above) centered on each RA-sensitive SNP ((1) to (7)) present in the LD block in the nucleotide sequence of the human SEC8L1 gene. 5'-side and 3'-side 25 bases each), 201 bases (each 5'-side and 3'-side 100 bases each), 601 bases (each 5'-side and 3'-side 300 bases each) Each) or the like.

Specific examples of the diagnostic marker for RA disease of the present invention include oligos or polynucleotides containing the following base sequences:
(1) An oligo or polynucleotide having a base length in the range of 51 to 601 containing at least the base sequence set forth in SEQ ID NO: 1 and the base sequence set forth in SEQ ID NO: 2 across SNP440 (base G or C),
(2) an oligo or polynucleotide having a base length in the range of 51 to 601 containing at least the base sequence set forth in SEQ ID NO: 3 and the base sequence set forth in SEQ ID NO: 4 across SNP441 (base C or T),
(3) an oligo or polynucleotide having a base length in the range of 51 to 601, comprising at least the base sequence set forth in SEQ ID NO: 5 and the base sequence set forth in SEQ ID NO: 6 across SNP442 (base C or T);
(4) an oligo or polynucleotide having a base length of 51 to 601 containing at least the base sequence set forth in SEQ ID NO: 7 and the base sequence set forth in SEQ ID NO: 8 across SNP443 (base A or T),
(5) an oligo or polynucleotide having a base length of 51 to 601 containing at least the base sequence set forth in SEQ ID NO: 9 and the base sequence set forth in SEQ ID NO: 10 across SNP445 (base A or G),
(6) an oligo or polynucleotide having a base length in the range of 51 to 601 containing at least the base sequence set forth in SEQ ID NO: 11 and the base sequence set forth in SEQ ID NO: 12 across SNP447 (base A or G),
(7) An oligo or polynucleotide having a base length in the range of 51 to 601, comprising at least the base sequence set forth in SEQ ID NO: 13 and the base sequence set forth in SEQ ID NO: 14 across SNP449 (base T or C).

  Oligos or polynucleotides (DNA fragments) specified by these nucleotide sequences are genetic predisposition markers that define susceptibility (susceptibility, resistance) to rheumatoid arthritis. Therefore, by detecting a gene containing the marker, a genetic predisposition for rheumatoid arthritis in a subject can be examined and diagnosed. In this sense, the oligo or polynucleotide can be defined and used as a diagnostic marker for RA disease.

4). Method for detecting the risk of developing rheumatoid arthritis The present invention also provides a method for detecting the risk of developing rheumatoid arthritis (RA) in a subject (whether or not it is likely to develop rheumatoid arthritis). The method for detecting a risk of RA according to the present invention is selected from the bases described in (2) below and the bases described in (1) and (5) to (7) for genomic DNA obtained from a subject. And detecting and identifying at least one base.

(1) Base at position 379258 (SNP440) of the human SEC8L1 gene
(2) Base at position 393668 (SNP441) of the human SEC8L1 gene
(5) Base at position 476136 (SNP445) of human SEC8L1 gene
(6) Base at position 569133 (SNP447) of human SEC8L1 gene
(7) Base at position 628601 (SNP449) of the human SEC8L1 gene.

  These are a set of RA disease-susceptible SNPs present in LD block 4 (positions 393668 to 681164 in the base sequence of the human SEC8L1 gene).

  As a combination of the base (SNP) detected and identified here, the base (SNP441) of (2) and any one base selected from (1) and (5) to (7) (SNP440, SNP445, SNP447 and SNP449), for example, a combination of base (2) (SNP441) and base (1) (SNP440), base (2) (SNP441) and base (5) (SNP445) ), (2) base (SNP441) and (6) base (SNP447), (2) base (SNP441) and (7) base (SNP449). it can. However, the SNP to be combined with the base (SNP441) in (2) is not necessarily one, and may be any two or three selected from (1) and (5) to (7) Or all four of (1) and (5)-(7).

  The detection method of the present invention is preferably a genomic DNA obtained from a human subject, wherein (2) the base (SNP441) and at least one base selected from (1) and (5) to (7) ( SNP440, SNP445, SNP447, SNP449) is detected and identified (step (A)). In the method, the base (SNP441) detected in step (A) and identified (SNP441) and at least one base selected from (1) and (5) to (7) satisfy the following conditions: When the condition is satisfied, the subject can have a step [step (B)] that determines that the subject is likely to suffer from rheumatoid arthritis (RA susceptibility).

Base (2) (SNP441): cytosine
Base (1) (SNP440): Guanine
Base (5) (SNP445): Guanine
Base of (6) (SNP447): Guanine
Base (7) (SNP449): cytosine.

  According to this method, it is possible to determine and diagnose the RA susceptibility and RA difficulty, that is, the risk of RA morbidity. The determination (diagnosis) of the RA morbidity risk can be performed mechanically without requiring the determination of a person having specialized knowledge such as a doctor using the above-mentioned base classification as a determination criterion (determination index). For this reason, it can be said that the method of the present invention is a method for detecting the risk of suffering from rheumatoid arthritis.

  The genomic DNA is extracted and the target base is detected by a known method [for example, Bruce, et al., Geneme Analysis / A laboratory Manual (vol.4), Cold Spring Harbor Laboratory, NY., (1999)]. Can be used.

  The sample from which genomic DNA is extracted is any cell (including cultured cells, excluding germ cells), tissue (including cultured tissue), organ, or body fluid (eg, saliva) isolated from subjects and clinical samples. Lymphatic fluid, airway mucosa, semen, sweat, urine, etc.). As the material, leukocytes or mononuclear cells separated from peripheral blood are preferable, and leukocytes are particularly preferable. These materials can be isolated according to methods commonly used in clinical laboratory tests.

For example, when leukocytes are used as a material, leukocytes are first separated from peripheral blood isolated from a subject according to a conventional method. Subsequently, proteinase K and sodium dodecyl sulfate (SDS) are added to the obtained leukocytes to degrade and denature proteins, and then phenol / chloroform extraction is performed to obtain genomic DNA (including RNA). RNA can be removed by RNase as necessary. Incidentally, extraction of the genomic DNA is not limited to the above method, methods well known in the art (e.g., Sambrook J. et al,.. "Molecular Cloning:. A Laboratry Manual (2 nd Ed)" Cold Spring Harbor Laboratory, NY) and commercially available DNA extraction kits can be used. Furthermore, if necessary, DNA containing the SEC8L1 gene on human chromosome 7 or its LD block 4 may be isolated. The DNA can be isolated by PCR or the like using genomic DNA as a template, using a primer that hybridizes to the SEV8L1 gene or its LD block 4.

  In the step of detecting the target base, an RA-sensitive SNP that is highly related to rheumatoid arthritis is detected from the extract containing human genomic DNA obtained as described above. The base is detected by directly determining the SEC8L1 gene on human chromosome 7 further isolated from a sample containing human genomic DNA, preferably the base sequence in LD block 4, and various SNPs located in each block. A method for examining the mutation of the base may be used.

  For example, as a method for detecting the target base, in addition to the method for directly determining the gene sequence of the corresponding region as described above, when the polymorphic sequence is a restriction enzyme recognition site, the difference in restriction enzyme cleavage pattern is used. Genotype determination method (hereinafter referred to as RFLP), polymorphism-specific probe-based hybridization method (for example, sticking a specific probe on a chip, glass slide, nylon membrane, etc. Determine the type of polymorphism by detecting the difference in hybridization intensity for different probes, or the efficiency of specific probe hybridization, and the amount of probe that polymerase degrades during template duplex amplification To identify the genotype, and to change the fluorescence emitted by certain double-strand-specific fluorescent dyes By detecting the temperature difference of double-stranded melting, a method for identifying the polymorphism by this, a complementary sequence is attached to both ends of the polymorphic site-specific oligo probe, and the probe in the self molecule depends on the temperature. And a method for specifying a genotype by using a difference between whether a secondary structure is formed or hybridized to a target region. In addition, a method in which a base-extension reaction is performed by polymerase from a template-specific primer and a base incorporated into a polymorphic site at that time is identified (by using dideoxynucleotide, each is fluorescently labeled, and each fluorescence is Detection method, method of detecting incorporated dideoxynucleotide by mass spectrometry), and further using template-specific primers followed by the presence or absence of base pairs complementary to the mutation site or non-complementary base pairs. There is a method to make it recognize.

  Hereinafter, conventionally known representative methods for detecting gene polymorphisms are listed, but the present invention is not limited to these.

  (a) RFLP (restriction fragment length polymorphism) method, (b) PCR-SSCP method (single-stranded DNA higher-order structure polymorphism analysis) [Biotechniques, 16, 296-297 (1994), and Biotechniques, 21 , 510-514 (1996)], (c) ASO (Allele Specific Oligonucleotide) hybridization method [Clin. Chim. Acta, 189, 153-157 (1990)], (d) Direct sequencing method [Biotechniques, 11, 246 -249 (1991)], (e) ARMS (Amplification Refracting Mutation System) method (Nuc. Acids. Res., 19, 3561-3567 (1991); Nuc. Acids. Res., 20, 4831-4837 (1992) (F) Denaturing Gradient Gel Electrophoresis (DGGE) method [Biotechniques, 27, 1016-1018 (1999)], (g) RNase A cleavage method [DNA Cell. Biol., 14, 87 -94 (1995)], (h) chemical cleavage method [Biotechniques, 21, 216-218 (1996)], (i) DOL (Dye-labeled Oligonucleotide Ligation) method [Genome Res., 8, 549-556 (1998) )], (J) T qMan-PCR method [Genet. Anal., 14, 143-149 (1999); J. Clin. Microbiol., 34, 2933-2936 (1996)], (k) Invader method [Science, 5109, 778-783 ( 1993); J. Biol. Chem., 30, 21387-21394 (1999); Nat. Biotechnol., 17, 292-296 (1999)], (l) MALDI-TOF / MS method (Matrix Assisted Laser Desorption-time of Flight / Mass Spectrometry) [Genome Res., 7, 378-388 (1997); Eur. J. Clin. Chem. Clin. Biochem., 35, 545-548 (1997)], (m) TDI (Template -directed Dye-terminator Incorporation method [Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)], (n) Molecular Beacons method [Nat. Biotechnol., 1, p49- 53 (1998); gene medicine, 4, p46-48 (2000)], (o) Dynamic Allele-Specific Hybridization (DASH) method [Nat. Biotechnol., 1.p.87 -88 (1999); gene medicine, 4, 47-48 (2000)], (p) padlock Probe (Padlock Probe) method [Nat. Genet., 3, p225-232 (1998); Gene medicine, 4, p50-51 (2000)], (q) UCAN method [Takara Shuzo Co., Ltd. home page (http: http://www.takara.co.jp)], (r) DNA chip or DNA microarray ("SNP gene polymorphism strategy" Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p128-135), (s) ECA method [ Anal. Chem., 72, 1334-1341, (2000)].

  The above are typical gene polymorphism detection methods. However, the determination of the risk of RA disease of the present invention is not limited to these, and other known or future developed gene polymorphism detection methods can be widely used. . Moreover, when detecting the gene polymorphism of the present invention, these gene polymorphism detection methods may be used singly or in combination of two or more.

  By the above method, for subjects who have been found to have a relatively high potential risk of developing rheumatoid arthritis (RA), this is notified and appropriate measures to prevent the onset of RA in advance. Can be taken. Therefore, the present invention is extremely useful as a test method for preventing the onset of RA, and further as a test method for preventing the occurrence of other diseases and symptoms associated with RA.

5. Reagent for detecting the risk of developing rheumatoid arthritis, kit containing the same (1) Probe The target nucleotide (RA-sensitive SNP) and the nucleotide containing the nucleotide are detected using the RA-sensitive SNP on the SEC8L1 gene of human chromosome 7. Oligonucleotides that can specifically hybridize to the included oligo or polynucleotide and detect the SNP are used. Such an oligonucleotide has a length of 16 to 500 bases, preferably 20 to 200 bases, more preferably 20 to 50 bases each including individual SNPs (SNP440, SNP441, SNP445, SNP447, SNP449) on the SEC8L1 gene. It is usually designed as an oligo or polynucleotide having a length of 16 to 500 bases so as to specifically hybridize with a continuous gene region.

  Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the second edition, conditions described in 1989), it means that cross-hybridization with other DNA does not occur significantly. Preferably, the oligo or polynucleotide preferably has a base sequence complementary to the base sequence of the gene region containing the SNP to be detected, but if such specific hybridization is possible, the oligo or polynucleotide is completely There is no need to be complementary.

  The oligo or polynucleotide is at least one oligo or polynucleotide selected from the group consisting of the following (1), (2) and (5) to (7) (provided that the oligo or polynucleotide is RNA) In the case, the base symbol “t” in the sequence listing shall be read as “u”), and a continuous oligo or polynucleotide having a length of 16 to 500 bases may be mentioned. Corresponding to the oligo or polynucleotide to be hybridized ((1), (2) and (5)-(7)), the nucleotides are (1 ′), (2 ′) and (5 ′)-( 7 ').

(1) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases including a nucleotide located at position 379258 (SNP440),
(2) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases containing a nucleotide located at position 393668 (SNP441),
(5) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases containing a nucleotide located at position 476136 (SNP445),
(6) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases including a nucleotide located at position 569133 (SNP447),
(7) A continuous oligo or polynucleotide having a length of 16 to 500 bases comprising a nucleotide located at position 628601 (SNP449) in the base sequence of the human SEC8L1 gene.

  The oligo or polynucleotide comprises an RA-receptive SNP (SNP440, SNP441, SNP445, SNP447, SNP449) located on the SEC8L1 gene on human chromosome 7 to determine susceptibility to rheumatoid arthritis for a subject. Alternatively, it is designed as an oligo or polynucleotide “probe” that specifically hybridizes to the polynucleotide. These oligos or polynucleotides can be prepared based on the base sequence of the SEC8L1 gene, for example, using a commercially available nucleotide synthesizer according to a conventional method.

  Suitable probes for use in the detection of the risk of developing rheumatoid arthritis in the present invention are the probes comprising the oligo or polynucleotide of (2 ′) above, and (1 ′) and (5 ′) to (7 ′) above. A combination with at least one selected oligo or polynucleotide probe. Here, the combination of the probes may be a combination of the probe (2 ′) and any one probe selected from (1 ′) and (5 ′) to (7 ′). For example, (2 A combination of a probe of ') and a probe of (1'), a combination of a probe of (2 ') and a probe of (5'), a combination of a probe of (2 ') and a probe of (6'), ( A combination of the probe 2 ′) and the probe (7 ′) can be mentioned. However, the probe to be combined with the probe (2 ′) is not necessarily one, and any two or three selected from (1 ′) and (5 ′) to (7 ′) may be used. Or all four of (1 ′) and (5 ′) to (7 ′).

  More preferably, the probe is labeled with a radioactive substance, a fluorescent substance, a chemiluminescent substance, an enzyme, or the like so that an oligo or polynucleotide containing each of the above SNPs (SNP440, SNP441, SNP445, SNP447, SNP449) can be detected. (Described later).

  The probe (oligo or polynucleotide) can be used by immobilizing on an arbitrary solid phase. Therefore, the present invention also provides the probe (oligo or polynucleotide) as an immobilized probe (for example, a gene chip, a cDNA microarray, an oligo DNA array, a membrane filter, etc. on which the probe is immobilized). The probe can be preferably used as a DNA chip for detecting an RA-sensitive gene.

  The solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to. The immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good. The immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics).

  For example, the TaqMan PCR method (Livak KJ. Gene Anal 14, 143 (1999), Morris T et al., J Clin Microbiol 34, 2933 (1996)), which is an example of the ASO method, includes various RA-sensitive SNPs. An oligonucleotide having a length of about 20 bases complementary to the region is designed as a probe. The probe is labeled with a fluorescent dye at the 5 'end and a quenching substance at the 3' end and specifically hybridizes with the sample DNA, but does not emit light as it is, but is an extension reaction from the upstream of a separately added PCR primer. By this, the 5 'fluorescent dye bond is cleaved and detected by the released fluorescent dye.

  In the Invade method [Lyamichev V. et al., Nat Biotechnol 17, 292 (1999)], which is another example of the ASO method, the sequences adjacent to the polymorphic site (2 'on the 3' side and 2 'on the 5' side) Complementary oligonucleotides are designed as probes. Detection is achieved by these two probes and a third probe that is independent of the analyte.

(2) Primer The present invention also provides an oligonucleotide used as a primer for specifically amplifying a sequence region containing RA-sensitive SNP on the SEC8L1 gene of human chromosome 7.

  As such an oligonucleotide, at least one oligo or polynucleotide selected from the group consisting of the following (1), (2) and (5) to (7) (provided that the oligo or polynucleotide is RNA, (The base symbol “t” in the sequence listing shall be read as “u”), which may be oligonucleotides 15 to 30 bases in length [hereinafter these oligonucleotides are to be hybridized. May correspond to (1 "), (2") and (5 ") to (7") corresponding to oligo or polynucleotide ((1), (2) and (5)-(7)) ].

(1) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 379258 (SNP440),
(2) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more, including a nucleotide located at position 393668 (SNP441),
(5) In the nucleotide sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more containing a nucleotide located at position 476136 (SNP445),
(6) In the nucleotide sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more containing a nucleotide located at position 569133 (SNP447),
(7) A continuous oligo or polynucleotide having a length of 16 bases or more containing a nucleotide located at position 628601 (SNP449) in the base sequence of the human SEC8L1 gene.

  Such an oligonucleotide specifically hybridizes to a part of a continuous oligo or polynucleotide containing a base (nucleotide) of RA-sensitive SNP in the SEC8L1 gene, and specifically amplifies the oligo or polynucleotide. It is designed as an oligonucleotide having a length of 15-30 bases, preferably about 18-25 bases. The length of the oligo or polynucleotide to be amplified is appropriately set according to the detection method used, but is generally 15 to 1000 bases, preferably 20 to 500 bases, more preferably 20 to 200 bases It is.

  In the present invention, a suitable primer for use in the detection of the risk of developing rheumatoid arthritis is selected from the above-mentioned (2 ″) oligonucleotide primer and the above (1 ″) and (5 ″) to (7 ″). It is a combination with a primer comprising at least one oligonucleotide. Here, the primer combination may be a combination of the primer (2 ") and any one primer selected from (1") and (5 ") to (7"). For example, (2 ”) Primer and (1”) primer combination, (2 ”) primer and (5”) primer combination, (2 ”) primer and (6”) primer combination, ( Mention may be made of combinations of 2 ") primer and (7") primer. However, the primer to be combined with the primer of (2 ") is not necessarily one, and any two or three selected from (1") and (5 ") to (7") may be used. Or all four of (1 ") and (5") to (7 ").

  The above-mentioned various oligonucleotides having a base sequence that specifically hybridizes to a base sequence of 15 bases or more in the vicinity of various RA-sensitive SNPs (SNP440, SNP441, SNP445, SNP447, SNP449) on the SEC8L1 gene of human chromosome 7. Can be used as a primer in the present invention. These oligonucleotides can be prepared based on the base sequence of the SEC8L1 gene, for example, using a commercially available nucleotide synthesizer according to a conventional method.

  A method in which MALDI-TOF / MS (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry) is applied to the Mass Array method [Haff LA et al. Genome Res 7, 378 (1997), Little DP et al., Nature Medicine vol.3, No.12, 1413-1416, (1997)] will be used as an example to explain the specific usage of primers. In this case, the primer is hybridized to the sample DNA fixed on the silicon chip, and ddNTP is added to extend only one base. The single base extension product is then separated and the polymorphism is detected by mass spectrometry. In this method, it is desirable to design the primer as short as possible, usually 15 bases or more in length.

(3) Labeled substance The probe or primer of the present invention is added with an appropriate labeling substance for detecting a gene polymorphism, such as a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin or the like. Is included.

  As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ′ , 7'-hexachloro-6-carboxylfluorescein, green fluorescent dye, fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems) , Yellow-green fluorescent dye), rhodamine or a derivative thereof [eg, tetramethylrhodamine (TMR)], but is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, Amersham Pharmacia, oligonucleotide ECL 3'-oligo labeling system, etc.).

  In addition, the primer of the present invention includes those to which a linker sequence for detecting a polymorphism is added at the end. Examples of such a linker sequence include a flap (a sequence completely unrelated to the sequence near the polymorphism) added to the 5 ′ end of the oligonucleotide used in the above-described invader method.

  The above probes or primers (which may be labeled) can be used as a reagent for detecting the risk of developing RA.

(4) Rheumatoid Arthritis Disease Risk Detection Reagent Kit The present invention also provides the above-described RA disease risk detection reagent as a kit. The kit includes at least one oligo or polynucleotide used as the probe or primer (which may be labeled or immobilized on a solid phase). In addition to the probe or primer described above, the kit of the present invention includes other reagents and instruments necessary for carrying out the method of the present invention, such as a hybridization reagent, a probe label, a label detection agent, and a buffer as necessary. Etc. may be included as appropriate.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Object of Analysis The analysis shown in the following examples was conducted on a total of 1566 Japanese subjects. Of the 1566, 760 were rheumatoid arthritis patients (RA patients) (156 men and 603 women), and 806 were non-rheumatoid arthritis patients (healthy controls) (189 men and 617 people) Female). All RA patients have ACR diagnostic criteria (Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31 : 315-24) and at least 3 years after onset. The study was approved by the University of Tokushima Ethics Committee, and blood was collected after obtaining written informed consent from all participants, and peripheral blood lymphocytes or Genomic DNA was extracted from immortalized lymphoblasts using EB virus and used as a test sample. Synovial tissue was collected from three RA patients who had undergone artificial joint surgery, and the literature (Mishiro T, Nakano S, Takahara S, Miki M, Nakamura Y, Yasuoka S, et al. Relationship between cathepsin B and synovial fibroblasts were cultured according to the method described in thrombin in rheumatoid arthritis. J Rheumatol 2004; 31: 1265-73). RA types are classified according to Ochi T et al. (Ochi T, Iwase R, Yonemasu K, Matsukawa M, Yoneda M, Yukioka M, et al. Natural course of joint destruction and fluctuation of serum C1q levels in patients with rheumatoid arthritis. According to Arthritis Rheum 1988; 31: 37-43).

  Table 1 summarizes the clinical features of RA patients and healthy controls.

  The age ± SD at the time of sampling was 61.9 ± 12.2 years for RA patients and 38.8 ± 15.4 years for healthy controls.

In the following examples, the association test regarding the allele frequency of SNP was performed by creating a 2 × 2 contingency table and performing a chi-square test. Chi-square test of haplotype frequency using permutation method was performed using SNPAlyze ver.3.2 Pro (DYNACOM, Japan). The degree of pairwise linkage disequilibrium was evaluated by calculating the linkage disequilibrium coefficient (| D ′ |) and the correlation coefficient (r ² ).

Example 1
(1) SNP search and typing in RA sensitive region
A genome-wide affected sib pair analysis using 53 families yielded a LOD score of 1.41 with marker D7S2560 in the 7q31-34 region (Nyholt DR. All LODs are not created equal.Am J Hum Genet 2000; 67: 282-8). Therefore, we thought that genes related to the sensitivity and severity of RA exist in the region (25.3 Mb) from D7S486 to D7S2513, which is the 1-LOD interval from D7S2560. NCBI Map View Build 34 Version2 contains 179 genes in this region. Therefore, in order to create useful marker sets for these candidate genes, SNPs were searched from public databases (dbSNP, TSC) and Celera databases. Among a huge number of SNPs, 728 SNPs that satisfy the following three criteria were extracted.

(i) SNPs present in a gene region from 10 kb upstream from the transcription start point to 10 kb downstream of the stop codon, and arranged at a uniform interval (10 to 20 kb) as much as possible.
(ii) Have a sequence suitable for the design of the probe used in the TaqMan assay.
(iii) A frequency analysis using 46 healthy control samples, and the frequency of minor alleles is 15% or higher.

  Next, a TaqMan assay system was created for the extracted 728SNP. A TaqMan probe and primer set was prepared using Primer Express from Applied Biosystems. Genotyping was performed in a reaction system (4 μl) using TaqMan Universal Master Mix or QuantiTect Prove PCR Kit, 5 ng DNA, 450 nM primer, and 100 nM probe. PCR was performed using a thermal cycler (ABI GeneAmp PCR System 9700) by repeating 40 cycles of enzyme activation at 95 ° C. for 10 minutes, then at 92 ° C. or 94 ° C. for 15 seconds and at 60 ° C. for 1 minute. 380 samples with unknown genotype were injected into a 384-well plate, and the remaining 4 wells were used as controls. After the PCR reaction, fluorescence signals based on VIC and FAM of the PCR amplification products were detected using ABI Prism 7900HT Sequence Detector System and SDS Software.

(2) Case control related analysis
In order to reduce the time and cost for experimental typing, 1520 cases were divided into two groups (1 group: 380 RA patients, 380 healthy controls), and a two-stage analysis was performed. Gender differences, age at onset, and RA subtypes were adjusted to be uniform at each stage. In the first stage of analysis, all SNPs were typed, and reproducibility of SNPs that showed an association with RA (P <0.05) was confirmed using different sample sets. At each stage, the allele frequencies of patients and healthy controls were subjected to chi-square test using a 2 × 2 contingency table. SNPs that showed a significant difference were further examined using genotype frequency, dominant model, and recessive model. Hardy-Weinberg equilibria at each SNP were also examined by chi-square test (Nielsen DM, Ehm MG, Weir BS. Detecting marker-disease association by testing for Hardy-Weinberg disequilibrium at a marker locus. Am J Hum Genet 1998; 63: 1531-40). SNPs with minor allele frequencies of less than 15%, typing success rates of less than 97%, and Hardy-Weinberg equilibrium in control samples were excluded from the analysis. Linkage disequilibrium and phase estimation in the candidate region used the genotype of the first stage 380 healthy control samples. Pairwise linkage disequilibrium was estimated as D = x ₁₁ -p ₁ q ₁ where the haplotype frequency A ₁ B ₁ was x _{11 and} the allele frequencies of A and B were p ₁ and q ₁ , respectively. The r is A, when B in the other allele frequencies were as p _2, q ₂ respectively estimated at _{r = D / (p 1 p} 2 q 1 q 2) 1/2 (Hill WG RA. Linkage disequilibrium in finite populations. Theor Appl Genet 1968; 38: 226-31). D '= D _max = minimum (p ₁ q ₂ , p ₂ q ₁ ) when D <0, and D _max = minimum (p ₁ q ₁ , p ₂ q ₂ ) when D> 0, D' = D / D _max . (Lewontin RC. The interaction of selection and linkage. I. General considerations: heterotic models. Genetics 1964; 49: 49-67). The estimation of the haplotype frequency at multiple points was performed by the maximum likelihood estimation method.

Statistics including permutation tests used SNP Alyze Pro or Fujitsu Disease Susceptibility Gene Discovery System. The structure of linkage disequilibrium was illustrated using GOLD (Abecasis GR, Cookson WO. GOLD--graphical overview of linkage disequilibrium. Bioinformatics 2000; 16: 182-3). Population attributional risk is estimated by assuming that the prevalence of RA is 0.01, f is the frequency of risk alleles, and r ₁ and r ₂ are genotype risk ratios, X = (1-f) ² + 2f ( 1-f) r ₁ + f ² r ₂ (X-1) / X (Esterbauer H, Schneitler C, Oberkofler H, Ebenbichler C, Paulweber B, Sandhofer F, et al. A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans. Nat Genet 2001; 28: 178-83).

result
The power using the Genetic Power Calculator is 380 RA patients and healthy controls, with genotype relative risks of f ₁ / f ₀ and f ₂ / f ₀ of 1.2 and 1.5, respectively, and the frequency of marker alleles 0.45 and α being 0.05, it was 0.5125 (Purcell S, Cherny SS, Sham PC. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 2003; 19: 149-50).

  Of the 728 SNPs typed in the first stage, 69 SNPs were excluded from the analysis because the minor allele frequency was less than 15% or Hardy-Weinberg equilibrium was not established in the control sample. Of the 695 SNPs analyzed, 48 SNPs present in 13 gene regions showed significant correlation with RA (P <0.05). Therefore, a second-stage analysis of these 48 SNPs was conducted for the second group (380 RA patients, 380 healthy controls).

  Among 48 SNPs, 3SNPs were also excluded from the analysis because they did not meet the regulations. Of the remaining 45 SNPs, 5 SNPs (SNP411, 441, 442, 443, and 445: 0.69% of the original) showed a significant difference with reproducibility. When the results of each stage were integrated, SNP441, 442, 443, and 445 showed smaller P values, while the significant difference of SNP411 disappeared. This SNP411 was confirmed to have a significant difference at each analysis stage, but the allele frequency was reversed between the first stage and the second stage, so it is considered that the significant difference disappeared in the integrated result.

  Among these SNPs, SNP441 showed the strongest significant difference (correlation with RA) (P = 0.000059, odds ratio 1.34, 95% confidence interval 1.16-1.55) (see Table 2). As in this experiment, it is important to eliminate the appearance of false positives associated with multiple tests in association analysis using a large number of SNPs. SNP441 remained significant even after Bonferroni correction, which corrects false positives by multiple testing (P = 0.039).

Population attributable risk of SNP441 is 40% when a 0.57 to allele frequency, genotype risk ratio, when the C / T for T / T to the r _1, C / C and r _2, were respectively 1.075 and 1.708 . The SNP441 was only 3.9 Mb from the marker D7S2560, which peaked in the affected sibling pair analysis. This means that there was no contradiction between the results of the linkage disequilibrium analysis using the sporadic sample and the results of the affected sibling pair analysis using the family sample.

  Table 2 shows the results of association analysis for SNPs present in the SEC8L1 gene.

  As can be seen from the above, SNP441, 442, 443, and 445, which showed significant differences in RA patients as described above, were all present in the 82 kb region of intron 10 of the SEC8L1 gene, which is a component of the exotox complex. The SNP449-SNP455 and SNP457-SNP461 12SNPs in the SEC8L1 gene were not significantly different in the second stage analysis, but were significantly different in the analysis integrating the results of the first stage and the second stage. SNP444, 446 and 456 showed a significant difference in the first stage, so the second stage analysis was performed, but since the Hardy-Weinberg equilibrium in the integrated result was not established, only the result of the first stage was described. Excluded from linkage disequilibrium analysis.

  As a result of the integration, 6SNPs in 4 genes showed a strong correlation with RA in at least one trait (P <0.01) (Table 3).

(3) Linkage disequilibrium analysis, haplotype analysis
The SEC8L1 gene is an 800 kb gene composed of 18 exons. It is said that the linkage disequilibrium between SNPs ranges from 10 to 60 kb and may reach several hundred kb in polymorphisms frequently observed in previous studies. In order to prove that this correlation is due to the SEC8L1 gene itself, the linkage disequilibrium pattern of the region including the two genes before and after the SEC8L1 gene (CHCHD3 gene, FLJ32786 gene) was evaluated. As a result, six linkage disequilibrium blocks were identified based on | D '|> 0.9 (FIG. 1).

  Of these, almost all SNPs (SNP441-461) present in block 4 were correlated with RA. This block is an approximately 300 kb region spanning introns 10-13 within the SEC8L1 gene (FIGS. 1B and C). There were no other genes in this linkage disequilibrium block, and this revealed that the observed correlation with RA was derived from the SEC8L1 gene itself.

A haplotype analysis was performed considering the possibility that the SNPs in these blocks 4 could be combined to contribute to the risk of RA. There were 22 SNPs in Block 4, but 3 SNPs (SNP444, 446, 456) were excluded from the analysis for the above reasons. Of the 19 SNPs present in the remaining block 4, those in linkage disequilibrium with r ² > 0.9 or more were classified into 5 groups. A haplotype was constructed by selecting 1 SNP from each group (SNP440, 441, 445, 447, 449). SNP440 and 447 were not subjected to the second stage typing, but the second stage analysis was performed for haplotype analysis. As a result, as shown in Table 4, four frequent haplotypes (haplotypes 1 to 4) were identified.

  Haplotype 1 (G, C, G, G, C) had the highest frequency, showed a positive correlation with RA (P = 0.0015), and was recognized as an RA-sensitive haplotype. On the other hand, haplotype 2 showed a negative correlation with RA (P = 0.0000062) and was recognized as an RA resistant haplotype. The P value was obtained by permutation test for correction of multiple tests (haplotype 1: P = 0.001, haplotype 2: P <0.0001). From this, although single point SNP441 and the haplotype containing SNP441 showed the strong significance with respect to RA, it is thought that it is important in prescribing the sensitivity of RA similarly.

Experimental Example 2 Expression of SEC8L1 in Human Tissue For real-time PCR, extraction of total RNA from synovial fibroblasts (FLS) was performed using RNeasy KIT. RNA from other tissues (brain, bone marrow, lung, heart, thymus, spleen, kidney, skeletal muscle) was prepared using Human Total TNA Master Panel II Kit. The prepared RNA was reverse transcribed using oligo dT primer and SuperScript II, and quantified by real-time PCR using SEC8L1 gene and β-actin TaqMan Assays on Demand as a control. Based on the expression level of SEC8L1 in bone marrow as a reference (1), the expression level of other tissues was calculated by the standard curve method.
The results are shown in FIG. As shown in FIG. 2, SEC8L1 was ubiquitously expressed in normal tissues of healthy controls, and particularly high expression was observed in skeletal muscle. In RA patients, SEC8L1 was moderately expressed in cultured synovial fibroblasts. Synovial fibroblasts are known to release MMP (an enzyme that degrades articular cartilage) by inflammatory cytokines such as TNF-α released from macrophages and are thought to be deeply involved in the pathogenesis. Cells.

Experimental Example 3 Expression of SEC8 gene in mouse immune cells
Since the expression of Sec8L1 was specifically observed in the brain and spleen, in order to elucidate the localization in a more limited living body, which tissue or cell was highly expressed was examined. Since SEC8L1 might be involved in inflammatory pathology in rheumatoid arthritis, we focused on immunocompetent cells.

After excising the spleen of a mouse (strain; BALB / c), react with an antibody to which a magnetic bead is bound to an antibody against a molecular marker specific for each lymphocyte (B cell: B220, NK cell: DX5α, Macrophage: Mac1) After that, lymphocytes were separated using the autoMACS ^™ (automatic magnetic separation) system. T cells were separated using a pan T cell isolation kit (a kit for excluding Tcells, manufactured by Miltenyi). The purity of each obtained lymphocyte population was evaluated by FACS analysis. As a result, all of the cell populations had a purity of 80% or more. Dendritic cells were cultured for 7 days in a GM-CSF (20 ng / ml) environment using a bone marrow cell population derived from the arch of the extremity of a mouse (strain; BALB / c) as a starting material. After culturing, the cells were similarly separated by autoMACS ^™ using a magnetic bead-bound antibody against CD11c, which is a specific marker molecule for dendritic cells.

  RNA was extracted from various lymphocytes separated and purified by the above method using RNeasy micro kit (manufactured by Qiagen), and the expression of mouse SEC8L1 gene was analyzed by RT-PCR. The cDNA was prepared by RT-PCR using “superscript first-strand synthesis for RT-PCR” (Miltenyi) and the primers shown below.

(1) primer-1 (designed to detect introns 3, 4 and 5 of mouse SEC8L1 gene)
(i) Forward primer: 5'-CGACATGCTGGTGTCAGCAGT-3 '(SEQ ID NO: 15)
(ii) Reverse primer: 5′-TCATCTCCCTCACACTTGAGCCTC-3 ′ (SEQ ID NO: 16).

(2) primer-2 (designed to detect intron 14 of mouse SEC8L1 gene)
(i) Forward primer: 5'-CTTCATAAGGGCAGCCTTTG-3 '(SEQ ID NO: 17)
(ii) Reverse primer: 5′-GGGGAAAGCATCTGGGATGT-3 ′ (SEQ ID NO: 18).

As a result of RT-PCR using the above primer-1 set and primer-2 set, as shown in FIG. 3, any lymphoid cells (B cells, macrophages, T cells, NK) corresponding to immunocompetent cells were obtained. SEC8L1 expression was confirmed in cells and dendritic cells (DC). In addition, as a result of detection by Primer-1, the expression of T cells was particularly higher than that of other lymphoid cells, followed by high expression of dendritic cells (DC), macrophages and B cells. In the case of Primer-2, a high level of SEC8L1 expression was observed in T cells, albeit slightly. (Figure 3)
As shown above, SEC8L1 gene expression was observed in all lymphocytes, and particularly high expression was observed in T cells. Infiltration of numerous T cells has been confirmed in the synovium of rheumatoid arthritis patients, and T cells are said to play a very important role in rheumatoid arthritis (Smeets, TJ et al. Poor expression of T cell-derived). cytokines and activation and proliferation markers in early rheumatoid synovial tissue. Clin. Immunol. Immunopathol. 88: 84-90 (1998)). T cells are CD4 positive cells and are often differentiated into Th1 cells that induce cellular immunity. The cytokines released by these are IFN-γ and IL-17, which are suggested to act on macrophages and synovial fibroblasts. It has also been reported that inflammatory cytokines such as TNF-α and IL-1 are released, causing cartilage destruction and synovial inflammation (Lubberts, E. et al. IL-17 promotes bone erosion in murine collagen- induced arthritis through loss of the receptor activator of NF-kB ligand / osteoprotegerin balance. J. Immunol. 170: 2655-2662 (2003)). Furthermore, T cells and macrophages also produce and secrete RANKL (Recptor Activator of NF-kB Ligand), an osteoclast activator, and induce bone destruction (Nakae, S. et al. IL-17). Production from activated T cell is required for the spontaneous development of destructive arthritis in mice deficient IL-1 receptor antagonist. PNAS.100: 5986-5990 (2003)). SEC8L1 is a subunit of Exocyst Complex, and is mainly involved in vesicle transport. When vesicle and cell membrane are bound, they are tethered (the vesicle is temporarily connected to the cell membrane) and bound to the membrane. (Grindstaff, KK et al. Sec6 / 8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cell. Cell. 93: 731-740 (1998)) . In patients with rheumatoid arthritis, this Exocyst Complex dysfunction results in impaired expression of certain cytokines and their transport to the SEC8L1 membrane, suggesting the possibility of inducing and promoting rheumatoid arthritis.

FIG. A is a diagram showing pairwise linkage disequilibrium among SNPs of 380 healthy Japanese controls using | D ′ |. FIG. B shows a comparison between -log ₁₀ (P value) and physical position in case control-related analysis. The horizontal line indicates P = 0.05. Only 21 SNPs typed from 1520 (2 groups: 380 patients in each group and 380 healthy controls) are listed. The arrow indicates the physical position of SNP441 that showed the strongest significant difference. FIG. C shows the genomic structure around the SEC8L1 gene and the positional relationship of six linkage disequilibrium blocks. The vertical line indicates an exon of the SEC8L1 gene. It is a figure which shows the expression pattern with respect to the expression in the bone marrow of SEC8L1 gene in a human tissue. It is the result of normalizing the expression level of SEC8L1 with the expression level of β-actin. In the figure, “FLS” means synovial fibroblasts. The expression of SEC8L1 in living cell-derived lymphocytes (Bsaibou, macrophages, T cells, NK cells, dendritic cells (DC)) collected from mouse spleen and bone marrow is shown. Expression was confirmed in all cells. As a result of detection by Primer-1, the expression of T cells was higher than that of other lymphocytes, followed by dendritic cells, macrophages and B cells. Primer-2 also showed high SEC8L1 expression in T cells, albeit slightly.

Claims (7)

A marker for diagnosing rheumatoid arthritis, comprising at least one oligo or polynucleotide selected from the group consisting of the following (1) to (7):
(1) an oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across G or C located at position 379258 (SNP440) in the base sequence of the human SEC8L1 gene;
(2) in the base sequence of the human SEC8L1 gene, an oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and 3 ′ side across C or T located at position 393668 (SNP441);
(3) in the base sequence of human SEC8L1 gene, an oligo or polynucleotide having a base sequence with a maximum length of 300 bases on each of the 5 'side and 3' side across C or T located at position 406991 (SNP442);
(4) In the base sequence of the human SEC8L1 gene, an oligo or polynucleotide having a base sequence with a maximum length of 300 bases on each of the 5 ′ side and 3 ′ side across A or T located at position 411544 (SNP443),
(5) an oligo or polynucleotide having a base sequence having a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across A or G located at position 476136 (SNP445) in the base sequence of the human SEC8L1 gene;
(6) an oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across A or G located at position 569133 (SNP447) in the base sequence of the human SEC8L1 gene;
(7) An oligo or polynucleotide having a base sequence of a maximum length of 300 bases on each of the 5 ′ side and the 3 ′ side across T or C located at position 628601 (SNP449) in the base sequence of the human SEC8L1 gene. (A) Targeting genomic DNA obtained from a human subject, the base described in (2) below and at least one base selected from the bases described in (1) and (5) to (7) A method for detecting a risk of developing rheumatoid arthritis for a subject comprising the step of detecting and identifying
(1) Base at position 379258 (SNP440) of the human SEC8L1 gene
(2) Base at position 393668 (SNP441) of the human SEC8L1 gene
(5) Base at position 476136 (SNP445) of human SEC8L1 gene
(6) Base at position 569133 (SNP447) of human SEC8L1 gene
(7) Base at position 628601 (SNP449) of the human SEC8L1 gene (B) The base (SNP441) of (2) detected and identified in the above step (A) is cytosine, and the bases described in (1) and (5) to (7) (SNP440, SNP445, SNP447, SNP449) At least one base selected from the following conditions:
Base (1) (SNP440): Guanine
Base (5) (SNP445): Guanine
Base of (6) (SNP447): Guanine
The method of detecting a risk of developing rheumatoid arthritis according to claim 2, comprising a step of determining that the subject is susceptible to rheumatoid arthritis when the base (SNP449): cytosine is satisfied. Hybridizes to the following oligo or polynucleotide (2) (provided that the oligo or polynucleotide is RNA, the base symbol “t” in the sequence listing shall be read as “u”; the same shall apply hereinafter). Oligonucleotide having a length of 15 to 30 bases or a label thereof, and an oligonucleotide having a length of 15 to 30 bases that hybridizes to at least one oligo or polynucleotide selected from the group consisting of (1) and (5) to (7) Primer set used to detect the risk of developing rheumatoid arthritis, comprising a combination of nucleotides or a label thereof:
(1) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 379258 (SN440),
(2) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 393668 (SN441),
(5) In the nucleotide sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more containing a nucleotide located at position 476136 (SN445),
(6) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 569133 (SN447),
(7) A continuous oligo or polynucleotide having a length of 16 bases or more containing a nucleotide located at position 628601 (SN449) in the base sequence of the human SEC8L1 gene. Hybridizes to the following oligo or polynucleotide (2) (provided that the oligo or polynucleotide is RNA, the base symbol “t” in the sequence listing shall be read as “u”; the same shall apply hereinafter). A 16-500 base-long oligonucleotide that hybridizes to at least one oligo or polynucleotide selected from the group consisting of (1) and (5)-(7) A probe set used for detecting the risk of developing rheumatoid arthritis, comprising an oligonucleotide or a combination thereof with a label:
(1) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases including a nucleotide located at position 379258 (SN440),
(2) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases containing a nucleotide located at position 393668 (SN441),
(5) In the base sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases including a nucleotide located at position 476136 (SN445),
(6) In the nucleotide sequence of the human SEC8L1 gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases containing a nucleotide located at position 569133 (SN447),
(7) A continuous oligo or polynucleotide having a length of 16 to 500 bases comprising a nucleotide located at position 628601 (SN449) in the base sequence of the human SEC8L1 gene.   6. The probe set according to claim 5, which is a solid-phased probe in which each oligonucleotide or a labeled product thereof is immobilized on an arbitrary solid phase.   A reagent for detecting a risk of developing rheumatoid arthritis, or a kit comprising the reagent set comprising the primer set according to claim 4 and / or the probe set according to claim 5.