JP2003061677A - Sensitive gene of systemic lupus erythematosus and its employment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensitive gene of systemic lupus erythematosus, to provide a method for predicting the risk of the systemic erythematodes crisis with the sensitive gene, and a device for predicting the risk of the systemic erythematodes crises with the sensitive gene. SOLUTION: A new polymorphic gene selected from the following group: (1) c. 705G>T in human CD19 gane; (2) IVS14-30C>T in human CD19 gene; (3) c. *132(GT)12-18 in human CD19 gene; and (4) c. 695T>C in human FCGR2B.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a susceptibility gene for rheumatic diseases. In particular, it relates to a susceptibility gene associated with the onset of systemic lupus erythematosus (hereinafter referred to as SLE) in Japanese. The present invention also relates to a method of predicting the risk of developing SLE using the susceptibility gene.

[0002]

2. Description of the Related Art Rheumatoid arthritis (hereinafter referred to as RA)
Rheumatoid diseases represented by erythematosus and systemic lupus erythematosus (hereinafter referred to as SLE) are autoimmune diseases that cause intractable chronic inflammation in major organs of the whole body.

RA is a disease that frequently causes joint failure throughout the body. Its pathological condition is abnormal growth of synovium consisting of several layers of cells lining the joint capsule and covering the joint space and chronic inflammation, and accompanying bone and / or cartilage absorption. In addition, the prevalence in the entire population is 0.5 to 1.0%
(See Reference 1). It often occurs first in women in their 30s and 40s and the ratio of affected males and females is approximately 1: 4.
When a first-degree relative has a patient with rheumatism, that person is said to be four to eight times more likely to be affected in the future (see Reference 2). Furthermore, the coincidence rate of onset in twins is 12% monozygotic and 4% dizygotic (see References 3-5), suggesting that genetic factors are strongly involved.

The frequency of SLE is lower than that of RA, and its prevalence is about 0.01 to 0.10%. It is a disease that often first appears in women in their 10s to 30s, and the ratio of males and females is 1: 9. Due to the systemic immune reaction with the appearance of autoantibodies against the nucleus and cytoplasmic components in the cells of self,
Each organ and blood cell shape of the whole body is affected. So far, SLE
It has been reported that the prevalence of siblings in these patients is about 20 to 400 times higher than in general, and twin studies have revealed that the risk of monozygotic twins is approximately 12 times higher than that of dizygotic cases (Reference). 6), SLE is suspected to have a stronger genetic effect than RA.

As described above, there is no doubt that some genetic factors are involved in the onset of rheumatic diseases, but the coincidence rate in monozygotic twins is considerably lower than 100%, and cases of family clustering. In the development of rheumatic diseases, it is suggested that the interaction of multiple genetic factors and environmental factors as well as immunologically acquired factors such as gene rearrangement is involved in the development of rheumatic diseases. To be done. That is, the rheumatic disease is considered to be a multifactorial disease. However, the genetic factors involved in its development have not yet been fully elucidated.

On the other hand, CD19 is a molecule that regulates B cell differentiation, activation and proliferation. Deficiency and overexpression of CD19 have been demonstrated in mice to cause hypogammaglobulinemia and autoantibody production, respectively. The human CD19 gene is located at the 16p11.2. Locus, and rheumatoid arthritis (hereinafter referred to as RA)
And one of the candidate loci for Crohn's disease (hereinafter referred to as CD)
Has been suggested.

The CD19 gene is a 95 kDa gene specific to B lymphocytes, and belongs to the immunoglobulin gene superfamily expressed by pre-B cells in the early stage from H chain rearrangement to plasma cell differentiation (Reference 7). -9
reference). In addition, the CD19 molecule is CD2 on the surface of B cells
1, binds to CD18 and Leu13 to form the B cell co-receptor complex and regulates B cell receptor (BCR) signaling.

Although the ligand for the CD19 molecule has not been identified at present, it is thought that the CD19 signal may be generated as a result of binding of the activated fragment C3d of the complement component to CD21. . It is considered that this is because CD21 is a receptor for the cleavage fragment of the third component of complement (hereinafter referred to as C3), and the antigen-C3d complex binds to B cells via CD21.

The CD19 includes CD40 (see Reference 10), CD38 (see Reference 11), CD72 (see Reference 10),
VLA4 (Reference 12) and FcγRIIB (References 13, 14)
, As a signaling partner for several other cell surface receptors. It has been shown that altering the density of CD19 on the cell surface in gene-targeted or transgenic mice has a significant effect on B cell function. CD19
B cells from deficient (CD19 ^-/- ) mice are hyporesponsive to transmembrane signals and show abnormal differentiation and activation.
(See References 15-21). In contrast, overexpression of CD19 in transgenic mice results in B cells that are hyperreactive with transmembrane signals and have an increased humoral immune response. Also, such mice show a decrease in B cell numbers in the peripheral pool, presumably caused by high negative selection in bone marrow. in addition,
Production of anti-double-stranded (ds) DNA antibody and autoantibodies including rheumatoid factor is also observed (References 15, 17-20, 22, 23).
reference). Thus, CD19 regulates an important signal threshold for proliferation of peripheral B cell pools,
It seems to function as a molecule, so to speak (see Reference 24).

The 240 amino acid cytoplasmic region of CD19 contains nine conserved tyrosine residues (see reference 8), and its phosphorylation following ligation of BCR and / or CD19 is a regulatory molecule on the cell surface. Provides a Src homology (SH2) recognition motif that supplies Lyn, Fyn and Lck protein tyrosine kinases (PTKs), and the proto-oncogene Vav are C
Interacts with D19. CD19 also interacts with phosphatidylinositol 3-kinase (PI3-kinase) (see Reference 25). PI3-kinase regulates membrane localization and activation of many effector molecules downstream of the signal.

[0011] At present, susceptibility candidate loci suggested by genome-wide screening have been mapped in many chromosomal regions according to the hypothesis that the onset of autoimmune diseases involves many genetic factors. In particular, the candidate region for RA or Crohn's disease includes 16p11.2, which also includes the gene encoding CD19 (Reference 26,
27). In addition, B cells in SLE patients are considered to be activated (see Reference 28). Interestingly, the level of CD19 expression on B cells has been shown to be approximately 20% higher in patients with systemic sclerosis, one of the systemic autoimmune diseases associated with antinuclear antibodies.
(See Reference 29).

On the other hand, genome-wide linkage analysis suggested that chromosomal region 1q23 is a candidate region for human SLE (see References 30 and 31) and its synteny region in mouse lupus lupus erythematosus (see Reference 32). Three FcγRII genes (ie FCGR2A, 2B and
2C) and two FCγRIII genes (ie FCGR3A and
3B) has been mapped to a region of about 200 kb in 1q23 (see References 33 to 36) and is considered to be a candidate gene for SLE susceptibility genes. FcγRs in different populations
The association between polymorphisms and SLE has been extensively studied and F
cγRIIA-131H / R, FcγRIIIA-17
6F / V and FcγRIIIB-NA1 / 2 polymorphisms have been shown to be associated with SLE in some human populations
(See References 38 and 40). However, the results are inconsistent in populations with different genetic backgrounds, suggesting that other genes in this chromosomal region may be predominantly associated with SLE.

FCγR2B is the only immunoreceptor tyrosine group inhibitory motif (IT) in the FcγR family.
(Also referred to as IM) and has the ability to transmit an inhibitory signal in B cells and myelomonocytic cells (see Reference 39). FcγRII in mice
Deletion of B has recently been shown to be associated with autoimmune diseases such as collagen-induced arthritis and Gutpasture syndrome (see refs. 41, 42). Furthermore,
Spontaneous development of antinuclear antibodies and glomerulonephritis
It has been reported that the FcγRIIB deletion was introduced into the background of L / 6 mice (see Reference 43). (NZB x NZ
W) Fcγ in B cells of splenic germinal centers of F1 mice (ref. 44) and other autoimmune-prone mice (ref. 45)
It was reported to be associated with a decrease in RIIB expression level and induce an abnormal enhancement of IgG antibody production. In addition, four SNPs in the translation region in linkage disequilibrium with each other
It was also shown to form a sensitive allele of FcγRIIB in the pus-prone mouse line. These findings suggest that FCGR2B may be the major susceptibility gene for human SLE in chromosomal region 1q23.

So far, only partial reports on the above-mentioned various molecules related to SLE and immune function have been reported, and specific reports on genetic factors that are susceptible to SLE. It has not been done.

[0015]

In view of such a situation, the present invention provides a susceptibility gene for systemic lupus erythematosus, and a method for predicting the onset risk of systemic lupus erythematosus using the susceptibility gene. Is to provide.

[0016]

The present inventors have found that typical rheumatic diseases, particularly systemic lupus erythematosus (SL).
In order to elucidate the etiology and pathology of E), based on the importance of pathology, mRNA expression profiling of normal and diseased tissues, results of linkage analysis, information from animal models, etc. Mutation analysis was performed systematically and the relationship with disease was analyzed. As a result of such earnest research, the following solutions have been found.

That is, the present invention is as follows: At least one novel polymorphic gene selected from the following groups: (1) c. 705G>T; (2) IVS14-30C in human CD19 gene
>T; (3) c. In human CD19 gene ^* 132 (G
T) _12-18 ; and (4) c. In human FCGR2B. 695T> C.

2. A method of predicting the risk of developing systemic lupus erythematosus in a subject comprising: (1) preparing a genomic DNA sample from the subject; (2) obtaining at least one of the following polymorphisms in the genomic DNA sample: Genotyping; (a) c. In the human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. Genotype of 695; and (3) when the polymorphism selected in (2) above is the polymorphism of (d) above and the genotype is a polymorphism resulting in codon 232T / T And (e) human leukocytes HL
Determining the presence or absence of A-DRB1 ^* 1501; (4) Risk of developing systemic lupus erythematosus in a subject by determining the risk rate of developing SLE based on the genotypes determined in (2) and (3) above. Predict rates.

3. A computer-aided device for predicting the risk of developing systemic lupus erythematosus in a subject, comprising: (1) a device for purifying genomic DNA; (2) a device purified in (1) above. Regarding genomic DNA,
Device for determining the genotype of at least one polymorphism selected from the group consisting of (a) c. In human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. 695 genotypes; and (3) storage means for storing a score table in which a predetermined score is associated with the polymorphic genotype described in (2) above; and (4) determined in (2) Searching the score table stored in the storage means of (3) based on the determined genotype,
Means for extracting corresponding score data, and outputting all of the extracted score data and the total score of the scores.

4. An apparatus for predicting the risk of developing systemic lupus erythematosus in a subject using a computer, comprising: (1) a systemic lupus erythematosus susceptibility polymorphism gene selected from the group consisting of: a) c. in the human CD19 gene 705G>T; (b) IVS14-30C in human CD19 gene
>T; (c) c. ^* 132 (G
T) _12-18 ; and (d) c. In human FCGR2B. Storage means for storing a score table in which a predetermined score is associated with a genotype of 695T>C; and (2) the storage of (1) based on the genotype determined for the selected polymorphism. Means for searching the score table stored in the means, extracting corresponding score data, and outputting all of the extracted score data and the total score of the scores.

5. In a computer, (1) determining the genotype of at least one of the following polymorphisms in the obtained genomic DNA sample; (a) c. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. Genotype of 695; and (2) when the polymorphism selected in (1) above is the polymorphism of (d) above and the genotype is a polymorphism resulting in codon 232T / T And (e) human leukocytes HL
Determining the presence or absence of A-DRB1 ^* 1501; (3) Risk of developing systemic lupus erythematosus in a subject by determining the risk of developing SLE based on the genotypes determined in (1) and (2) above. Predicting rate; a program for performing.

6. (1) Purifying the genomic DNA in a computer; (2) Regarding the genomic DNA purified in (1) above,
Determining the genotype of at least one polymorphism selected from the group consisting of: (a) c. In the human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. 695 genotype; and (4) based on the genotype determined in (2) above
The genotype of the polymorphism described in 1. is searched for the score table stored in the storage means for storing the score table in which a predetermined score is associated, and the corresponding score data is extracted and extracted. Outputting all of the score data and the total score of the scores; (5) Predicting the onset risk of systemic lupus erythematosus in a subject from the total score obtained in (4); .

7. A method of predicting the risk of developing systemic lupus erythematosus in a subject, the method comprising:

(1) Purifying genomic DNA; (2) Regarding the genomic DNA purified in (1) above,
Determining the genotype of at least one polymorphism selected from the group consisting of: (a) c. In the human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) the number of repeats; and (d) c. In human FCGR2B. Genotype of 695; (4) based on the genotype determined in (2) above (2)
The score table in which a predetermined score is made to correspond to the polymorphism of the polymorphism described in is extracted, and the corresponding score data is extracted, all of the extracted score data,
And outputting the total score of the above scores; and predicting the risk of developing systemic lupus erythematosus in the subject from the total score obtained in (5) and (4).

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides CD19, a molecule that regulates B cell activation in B cells.
Based on previous findings that and the FcγR2B receptor may be associated with SLE pathogenesis, CD19 and Fc
Detecting mutations in the γR2B receptor gene and analyzing the relationship between the detected mutations and the onset of SLE, thereby
It was achieved by first identifying the LE susceptibility gene.

1. Glossary of terms As used herein, “susceptibility gene”
ne) refers to an onset-related gene that influences the risk of developing SLE. As used herein, “SLE risk of onset” refers to the relative degree of risk of SLE.

The term "polymorphic gene" as used herein refers to a group of alleles occupying one locus, or an individual allele belonging to such a group of alleles. In addition, among the polymorphic sites, those that differ by only one base are particularly referred to as “single nucleotide polymorphism” (Single Nucleotide Polymo
rphism, hereinafter referred to as SNP). Further, the term "genotype" used herein indicates the presence state of alleles at the gene locus of interest.

The term "polynucleotide" as used herein is used to mean a general term for polynucleotides and oligonucleotides, etc. {Here, "oligonucleotide" refers to several to several tens of nucleosides. It means a substance obtained by polymerizing a phosphate ester (that is, a nucleotide) by a phosphodiester bond, and includes, but is not limited to, oligoribonucleotides and oligodeoxyribonucleotides. The term "polynucleotide" used herein means a substance in which two or more nucleosides are linked by a phosphate bond. Nucleosides include, but are not limited to, deoxyribonucleosides and ribonucleosides. Furthermore, in the present invention, "polynucleotide" means peptide nucleic acid,
Artificially synthesized nucleic acids such as morpholino nucleic acid, methylphosphonate nucleic acid and S-oligo nucleic acid are also referred to.

2. Relationship between polymorphism and SLE onset (1) CD19 gene The polymorphism in the CD19 gene is screened for single-stranded DNA conformation polymorphism PCR (PCR-single).
STRAND CONFORMATION POLYM
orphism; hereinafter abbreviated as PCR-SSCP method) and direct sequencing method. Furthermore, the association of the detected polymorphisms with the disease was analyzed by the case-control association analysis method in the Japanese population.
The analysis was carried out by Sociation analysis).

As a result, the present inventors have identified six novel polymorphisms in the CD19 gene, and further revealed that three of the identified polymorphisms are associated with the development of SLE. I chose Hereinafter, three polymorphisms newly identified by the inventor and revealed to be associated with the onset of SLE, that is, c. 705G> T (P235P), IVS14-3
0C> T, and c. ^* 132 (GT) _12-18 will be described.

As used herein, the term "c.705G>T" means a genotype that may exist in the SNP existing at position 705 of the translation region of the CD19 gene, that is, thymine (T.
And a guanine (abbreviated as G) of genotype T. In addition, of these genotypes, the base at the relevant position in the conventionally generally known sequence is G. c. If the genotype of 705 is T, whether it is homozygous or heterozygous, S
Low risk of developing LE (ie, low risk genotype). In this polymorphism, whether in G or T, the amino acid at position 235 of the corresponding codon is proline (abbreviated as P), which is a synonymous substitution. As used herein, "c.705T" refers to the allele c. It shows that 705 is genotype T.

As used herein, "IVS14-30C>
The term "T" refers to the genotypes that can exist in the SNP present at position 30 of the 14th intron region counted from the transcription start site of the CD19 gene, namely cytosine (abbreviated as C) and thymine (abbreviated as T). The gene of genotype T is shown. In addition, of these genotypes, the base at the corresponding position in the conventionally generally known sequence is C. When the genotype of IVS14-30 is T, the risk of developing SLE is low, whether it is a homozygote or a heterozygote. As used here, "IVS14
"-30T" indicates that the allele IVS14-30 of the gene is genotype T.

The allele c. 705 and IVS
14-30 have a haplotype relationship in which they are linked in linkage disequilibrium. Therefore, if either one exists, the other one is very likely to exist. Therefore, it is possible to predict the risk rate of SLE onset in a patient by detecting either.

As used herein, "c. ^* 132 (GT)"
The term " _12-18 " refers to the untranslated region of CD19 (ie,
The polymorphism of 12 to 18 GT repeats in the genomic DNA corresponding to position 132 of the 3'UTR) is shown. In the case of individuals with this number of repetitions extended to 15 or more, SLE
The risk of onset is high. No such association was observed in RA and Crohn's disease. "C. ^* 132
(GT) _{15 −} ”denotes a gene in which the number of repeats of the microsatellite polymorphism at the locus is 15 or more.

These polymorphisms were registered in GenBank / EMBL / DDBJ on Dec. 19, 2000 with registration number AB0.
5799, AB052814, AB052815, AB
052816, AB052817 and AB05281
Registered as 8 by the inventors.

(2) Fcγ receptor IIB (FCGR2B
Gene) Human low-affinity Fcγ receptor genes are FcγRII
It constitutes a gene family consisting of A, IIB, IIC, IIIA and IIIB and is located on the chromosome 1q23 region. They differ from each other in ligand affinity, cell distribution and effector function (see refs. 37, 38). In particular, FcγRIIB is an immunoreceptor tyrosine group inhibitory motif (ITI) in its cytoplasmic domain.
It is unique in its ability to transduce inhibitory signals via M).

The present inventors have now found that a novel SNP in the human FCGR2B gene, namely c. 695T> C was identified and the relationship between the genotype of the polymorphism and SLE was clarified. c. 695T> C is a polymorphism located in exon 5.

As used herein, the term "c.695T>C" refers to a genotype which may be present in the SNP present at position 695 of the translation region of the human FCGR2B gene, ie, T.
And the gene of genotype C of C is shown. In addition, among these genotypes, it is shown that the base at the relevant position in the conventionally generally known sequence is T. As used herein, "c.695C" refers to the allele c. It shows that 695 is genotype C.

This polymorphism also confers two alleles at codon 232 in the transmembrane domain of Fcγ receptor IIB. That is, when the genotype is C, the codon 232 is encoded by threonine (abbreviated as Thr or T), that is, 232T. On the other hand, when the genotype is T, the codon 232 is encoded by isoleucine (abbreviated as Ile or I), that is, 232I.

The number of individuals having the 232T / T genotype in SLE patients is remarkably high as compared with healthy individuals. That is,
The risk of developing SLE is high when the polymorphisms of the two alleles are both homozygous for C.

In addition, strong linkage disequilibrium is due to FcγRIIB
-232I / T and the FcγRIIIB-NA1 / 2 polymorphism were detected, but comparison of the strength of the association revealed that in the context of SLE, the above-mentioned FCGR2B polymorphism was the primary susceptibility gene. Was thought to be. Furthermore, it seems that a synergistic effect exists between the FCGR2B 232T / T genotype and HLA-DRB1 ^* 1501. Taken together with the results of recent animal studies demonstrating the role of FcγRIIB in the regulation of autoimmunity, this study strongly suggests a role for FcγRIIB in gene susceptibility to human SLE.

The polymorphisms in the FCGR2B gene determined by the present inventors are identified by accession numbers AB050934 and AB.
Registered in GenBank at 051387 on November 8, 2000 and November 20, 2000 by the inventors. The known FCGR2B gene sequences used here are AH005422 and NM004001, FC
GR2A is AH003095, FCGR2C is AH00
2832.

3. Polymorphism According to one aspect of the present invention, a novel polymorphic gene selected from the following groups: (1) c. 705G>T; (2) IVS14-30C in human CD19 gene
>T; (3) c. In human CD19 gene ^* 132 (G
T) _12-18 ; and (4) c. In human FCGR2B. 695T>C; is provided.

As mentioned above, these are polymorphisms that are susceptible or have a low risk of SLE, especially SLE in Japanese.
It is a polymorphism that is sensitive or resistant to. Therefore, determining at least one of these genotypes can predict the relative risk of developing SLE in a subject.

4. Prediction Method According to one aspect of the present invention, a method of predicting the risk of developing SLE is provided. That is, the SLE that comprises:
(1) Preparing a genomic DNA sample from a subject; (2) Determining at least one genotype of the obtained genomic DNA sample; (a) Human CD19 gene C. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) the number of repeats; and (d) c. In human FCGR2B. 695 genotypes; and (3) predicting the risk of developing systemic lupus erythematosus in a subject by determining the risk of developing SLE based on the determined genotype.

The individuals to which the present method is preferably applied are humans, and Japanese are particularly preferable.

As a means for preparing a genomic DNA sample from a subject, a phenol chloroform method, a salting out method or a commercially available kit etc. is used from blood cells such as leukocytes, monocytes, lymphocytes and granulocytes in peripheral blood obtained from the subject. Any generally used means may be used, such as a method of extraction using the same.

As the means for determining the genotype, any commonly used means such as direct sequencing method, SSCP method, oligonucleotide hybridization method and specific primer method may be used.

The genotype and the risk of developing SLE are judged as follows. When the genotype of the polymorphism of (1) above is detected in the first subject in the prediction target, the risk of developing SLE is higher than that in the second individual in which the genotype of the polymorphism is G. The rate is low. When the genotype of the polymorphism in (2) above is T, the risk of developing SLE is low. Also,
As described above, the polymorphisms of (1) and (2) form haplotypes with each other. Therefore, either one may be detected, or both genotypes may be determined for determination.

When the number of repetitions of GT in the polymorphism of (3) above is 15 or more in the prediction target, the risk of SLE onset is higher than that in the case of less than that number.

Further, in the prediction target, when the two genotypes of the polymorphism of (4) in the two alleles are both C, that is, the two alleles of the target are 232 T / T homozygous. The risk of developing SLE is higher in the case of the body than in the case of the case where the allele is 232I / T heterozygote or 232I / I homozygote. Furthermore, when the allele to be predicted is a 232T / T homozygote and the HLA-DRB1 ^* 1501 locus is also present, compared to the case where the 232T / T genotype is present alone. The risk of developing SLE is even higher.

Here, "HLA-DRB1 ^* 1501"
Is a class I of human leukocyte antigen (abbreviated as HLA) gene.
The allele of the DRB1 locus contained in I is shown.

For predicting the risk of developing SLE, whether all the genotypes of the polymorphisms (1) to (4) above are determined or only one of them is performed, a combination of these is performed. May grow. For example, by determining the genotype of the polymorphism of any one of the above (1) and (2) which is a haplotype and the three polymorphisms of the above polymorphisms (3) and (4) Good. Alternatively, any one of the above (1) to (4) may be combined and determined. Furthermore, the polymorphism of HLA may be determined according to the genotype of (4), and the prediction may be performed by further considering this.

5. Apparatus for Predicting SLE Onset Risk According to one aspect of the present invention, an apparatus for predicting SLE onset risk is provided. That is, an apparatus for predicting the risk of developing SLE using a computer, comprising: (1) an apparatus for purifying genomic DNA; (2) the genomic DNA obtained in (1) above. A device for determining the genotype of a polymorphism selected from the group consisting of: (1) c. 705 genotype; (2) IVS14-30 genotype in human CD19 gene; (3) c. ^* 132 (G
T) repeat count; (4) c. In human FCGR2B. 695 genotypes; and (5) presence or absence of human leukocyte HLA-DRB1 ^* 1501; (3) storing a score table in which a predetermined score is associated with the polymorphic genotype described in (2) above. A storage means for storing the score data based on the genotype determined in (4) and (2), and extracting the corresponding score data, and summing all the score data and the score. Means for outputting the score;

FIG. 1 is a block diagram of one embodiment of the SLE onset risk rate prediction apparatus of the present invention. All components are bus lines 1
1 is connected by a bus connection. As shown in FIG. 1, means 1 for purifying a genomic DNA sample from a sample from a subject, means 2 for determining a genotype in a desired polymorphism from the nucleic acid obtained by means 1, a display device 3, And input means 4 such as a keyboard and a mouse
Are connected to the bus line 11.

The CPU 5 executes the purification program 12 to control the genomic DNA sample purification device 1 to purify the genomic DNA from the sample. Purified genome D
NA is transported to the genotyping device 2 connected to the purifying device 1, where genotyping is performed. In this genotyping, the CPU 5 executes the genotyping program 1
3 and control the genotyping device 2.

The information on the genotype of the genotyping device 2 is converted into an information form that can be handled by a computer. For example, such information is typically
Since it can be obtained from the fluorescence intensity, the radioactivity level, the absorbance, etc., information such as the optical signal may be converted into an electric signal by a general means. In addition, this step may be performed by the operator viewing the result of the electrophoresis or the result of the hybridization performed manually and judging the positive or negative from the result.

Based on the obtained information on the genotype, C
The PU 5 executes a prediction program to instruct image data to be displayed, search for a score based on a genotype and a combination thereof, and the like. The RAM 6 temporarily stores image data for display, and the image processing unit 7 generates necessary image data according to an instruction from the CPU 5 and displays the image on the display device 3.

The memory in the present SLE onset risk rate prediction apparatus stores roughly three files.

The first file 8 stores a refining program 12, a genotyping program 13, and image data related to refining and genotyping. In the second file 9, a prediction program 15 and image data related to prediction are stored. Furthermore, a score table is stored in the third file 10.

FIG. 2A shows an example of the score table. FIG. 2B is a diagram showing a part of an example of the display screen. This display is on the right side of the score table in FIG.
The data obtained by the genotyping apparatus 2, the scores retrieved by searching the score table, and the total score “3” obtained by totalizing the scores are displayed.

According to the flow chart of FIG. 3, this SLE is executed.
The operation of the onset risk rate prediction device will be described. (S1) When the instruction to start the prediction is issued, the refining is executed according to the refining program. (S2) When the purification is completed, the No. 1 to No. Genotyping is performed for up to 4 polymorphisms. (S3) N
o. If the polymorphism of 4 is a gene that gives the codon 232T / T, the process proceeds to (S4), and if not, the process proceeds to (S5). (S4) No. Five genotypes are detected. (S5) The score table is searched, the score corresponding to the determined genotype is read, and the total score is totaled. (S6) The determined genotype, the acquired score, and the total score are displayed on the display device 3.

The sample from the subject may be a liquid sample such as blood containing blood cells and lymph, or any tissue sample, or genomic DNA from these samples to a desired degree in advance. It may be purified.

An apparatus for purifying a genomic DNA sample from the sample is a DNA automatic extraction apparatus (for example, AB
I or available from Toyobo Co., Ltd.). Of course, this step may be manually performed by an operator by a general means known per se using an extraction kit or the like.

The apparatus for genotyping is a real-time P using an automatic sequencer and the FRET method.
Any commonly used device such as a CR device (for example, ABI7700, LghyCyclex, etc.) may be used. In addition, as a matter of course, this step may also be manually performed by an operator by a general means known per se using an extraction kit or the like.

Further, here, an example of an apparatus for automatically performing purification and genotyping of a genomic DNA sample by the genomic DNA sample purifying apparatus 1 and the genotyping apparatus 2, respectively, is shown. 1 and the genotyping apparatus 2 are also included in the scope of the present invention even if both or only one of them is not provided. In the following, an example in which neither of the devices is provided is shown.

According to one aspect of the present invention, an apparatus for predicting the risk of developing SLE from a genotype of a novel polymorphism that is susceptible to SLE is provided. That is, a device that uses a computer to predict the risk of developing SLE, the device comprising: (1) A score in which a predetermined score is associated with a combination of genotypes of SLE susceptibility polymorphisms. Storage means for storing a table; and (2) searching the score table based on the genotype determined for the selected polymorphism, extracting corresponding score data, and extracting all the score data, And means for outputting a total score of said scores. The configuration of this aspect is shown in FIG. S
Manually determining the genotype in the LE susceptible polymorphism,
Input the data from the input means, based on the data,
Predict SLE risk.

The apparatus described in detail is a novel polymorphism of the present invention, namely (1) c. 705G>
T, (2) IVS14-3 in the human CD19 gene
0C> T, (3) c. In human CD19 gene. ^* 1
32 (GT) _12-18 , (4) in human FCGR2B c. In addition to 695T> C, other analyzes such as other disease-related genes and / or polymorphisms, and polymorphisms related to blood groups may be programmed to be performed simultaneously.

With such a device, it is possible to easily predict the risk of developing SLE.

According to one aspect of the present invention, the following polynucleotides are provided. That is, (a) a polynucleotide represented by the base sequence of 1 selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 8; (b) the polynucleotide described in (a) above, wherein A modified polynucleotide in which one or several nucleotides are deleted, substituted or added at a site other than where the polymorphic gene is present; (c) a sequence containing one nucleotide at the 2553th position or the 8518th position of SEQ ID NO: 1 A fragment consisting of a polynucleotide having a continuous nucleotide sequence of 10 to 30 bases contained in No. 1; (d) A continuous 15 contained in SEQ ID No. 5 containing 15 nucleotides present at positions 194 to 223 of SEQ ID No. 5; (E) SEQ ID NO: 6 is from 194 to 2 A fragment consisting of a polynucleotide having a contiguous base sequence of 18 to 30 bases contained in SEQ ID NO: 6 containing 18 nucleotides existing up to position 29; (f) SEQ ID NO: containing 1 nucleotide located at position 304 of SEQ ID NO: 7 A fragment consisting of a polynucleotide having a continuous base sequence of 15 to 30 bases contained in 7; (g) a continuous base of 15 to 30 bases contained in SEQ ID NO: 8 containing one nucleotide at position 386 of SEQ ID NO: 8; A fragment consisting of a polynucleotide depending on the sequence; and (h) a polynucleotide represented by the base sequence of SEQ ID NO: 9.

The polynucleotide set forth in SEQ ID NO: 1 is c. Human CD including 705 and IVS 14-30
It is a part of 19 genes. Of this base sequence, SLE
It is the SNP sites of the sequence, that is, positions 2553 and 8518, which are involved in the onset of the disease. Therefore, when the polynucleotide of the present invention is a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO: 1 containing a SNP site containing at least one nucleotide present at the site is more preferred.

The polynucleotides shown in SEQ ID NOs: 2 to 6 are c. ^* Human CD gene containing 132 (GT) repeats. In particular, SEQ ID NOS: 5 and 6 have a repetition number of 15 or more, and are highly useful in determining the risk rate of SLE onset.

SEQ ID NO: 7 is c. Human FC containing 695
GR2B gene. Of this base sequence, the SNP site involved in the development of SLE is the SNP site of the sequence, that is,
221 and 304. Therefore, when the polynucleotide of the present invention is a fragment of SEQ ID NO: 7, S containing at least one nucleotide present at the site is
It is preferably a fragment containing the NP site.

Similar to SEQ ID NO: 7, SEQ ID NO: 8 is c. 6
Human FCGR2B gene containing 95. In this base sequence, it is the SNP site of the sequence, that is, position 386, which is involved in the development of SLE. Therefore, when the polynucleotide of the present invention is a fragment of SEQ ID NO: 8,
It is preferably a fragment containing an SNP site containing one nucleotide present at the site.

These polynucleotides may be used, for example, as primers for various PCR amplifications for determining the genotype of interest, or as probes for use in DNA chips. In this case, the present polynucleotide preferably has 10 or more nucleotides and 30 or less nucleotides. If the length of the polynucleotide fragment is too long, it will be difficult to distinguish single nucleotide differences. Further, basically, when the length of the polynucleotide is excessively short, it becomes difficult to determine the base sequence of the polynucleotide contained in the sample.

In particular, the polynucleotide shown in SEQ ID NO: 9 is a sequence useful as a primer specific to FCGR2B. In particular, this polynucleotide is useful for use as a primer for downstream PCR.

[0077]

EXAMPLES Example 1. Association of systemic lupus erythematosus with CD19 polymorphism in Japan In Example 1, the polymorphism sequence of CD19 was screened to detect the polymorphism, and the association between the detected polymorphism and SLE was examined.

1. Materials and Methods (1) Patients and Healthy Subjects First, 127 RA patients and SLE patients (first S
LE group: hereafter referred to as SLE1 group) 87 cases, 156 patients with Crohn's disease, and 247 healthy subjects, case-control association study (case-control association study)
y) went. Furthermore, in order to re-examine the association detected in the SLE1 group, an independent second SLE group consisting of 99 SLE patients (hereinafter referred to as SLE2 group) was constructed. Therefore, the total number of SLE patients is 186.

The healthy subjects in the control group are 145 males and 102 females (average age: 36.6 years) consisting of researchers, laboratory staff and students from the University of Tokyo and the Japanese Red Cross Central Blood Center. . RA patients, 18 males and 168 females (mean age: 57.8 years) were classified according to the American College of Rheumatology classification criteria (see Reference 46). SLE1
And SLE2 consisted of 12 males and 75 females, respectively.
(Average age: 40.3 years), 6 males and 93 females (average age: 40.0 years). SLE patients were classified according to the American College of Rheumatology classification criteria (see Ref. 47). Cloned patients, 124 males and 32 females (average 3
1.1 years old) was clinically diagnosed by radiography, endoscopy and histological examination of biopsy specimens. CVI
There were 3 males and 1 female D patient. The percentage of CD19-positive B cells in peripheral blood mononuclear cells from CVID patients was 10%, 3.3%, 3.0% and 0%, respectively.

All the healthy subjects and patients used in the case control study are Japanese people living in the Kanto region who are not related to each other. The central part of Japan has already been shown to be relatively homogeneous in terms of genetic background (Reference 48
(See), the case control study used in this study will be possible.

The clinical characteristics of the patient were obtained from the clinical record of each patient. If it did not provide sufficient information, the patient was excluded from further analysis. Nephritis here means persistent proteinuria of 0.5 g / day or 3+ or more, or there are findings of cell casts. Central nervous system disease refers to convulsions or psychosis, but these are ACR diagnostic criteria. (Ref. 47).

This study was approved by the Research Ethics Committee of the Human Subject Protection Committee of the University of Tokyo, Juntendo University and UCLA.

(2) Genomic DNA Genomic DNA was extracted from peripheral blood leukocytes of patients and healthy subjects by using the QIAamp Blood kit.
t, Qiagen, Hilden, Germany).

(3) PCR Single-Stranded Polymorphism (PCR-single
strand conformation polymorphism; hereinafter referred to as PCR-SSCP) Table 1 shows the primers used for PCR and the annealing temperature.
Shown in.

[0085]

[Table 1]

These were designed according to the genomic DNA sequence of human CD19 (GenBank accession number M84371). Each exon was amplified by setting primers specific for the flanking intron sequences. Moreover, from the previous studies, the optimal fragment size for this method is 200-400 bp.
Exons 2 and 4 were divided into two fragments, and exons 11 and 12 and 13 and 14 were amplified as one fragment, respectively. Since the amplification efficiency was low in one-step PCR, the promoter region was analyzed using the two-step PCR method. The first primer set was designed to amplify -1351 to 136 using long PCR. After the first denaturation (96 ° C., 10 minutes), denaturation (96 ° C., 1 minute), annealing (58 ° C., 30 seconds) and extension reaction (72 ° C., 3 minutes) were performed in 35 cycles under the conditions of thermal cycler. (Thermal cycler MP; Takara, Kyoto, J
apan) was used to perform PCR amplification. PCR-SSCP
For the three long fragments (3
00-450 bp). PCR amplification consisted of an initial denaturation (96 ° C, 10 min) followed by denaturation (96 ° C, 30 min).
Second), annealing (60 ° C., 30 seconds) and extension reaction (72 ° C., 30 seconds) in 35 cycles.
Thermal cycler MP; Takara or
GeneAmp PCR system 9600; Perkin-Elmer Applied Biosy
stems; Foster City, CA).
The promoter region up to −1050 bp was analyzed by such a method.

This amplified DNA was subjected to PCR-SSCP
Method was used to analyze. 1 μL of the solution containing the PCR product was added to 7 μL of denaturing solution (95% formamide, 20 mM E
DTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF). The mixture was denatured at 96 ° C for 5 minutes and immediately cooled on ice. 1 μL of the mixture was analyzed for exon 15 using 7.5% polyacrylamide gel (acrylamide: bisacrylamide = 49: 1) and 10% polyacrylamide gel (acrylamide: bisacrylamide = 49: 1). And other parts were analyzed. For better separation, 10% glycerol was added to the gel for analyzing exons 2B and 4A. Electrophoresis is 0.5 x TBE (4
5 mM Trisborate [pH 8.0], 1 mM EDTA)
Mini gel electrophoresis device (90 x 80 x 1 mm, AE-6410 and A with a constant temperature control system under a constant current of 20 mA / gel)
E-6370; ATTO, Tokyo). The optimum temperature and electrophoresis time were determined by preliminary experiments. The single-stranded DNA fragment in the gel was visualized by silver staining (Daiichi Pure Chemicals, Tokyo).

(4) PCR restriction fragment length polymorphism (hereinafter referred to as PCR-R
The IVS14-30C> T polymorphism in intron 14 was determined by the PCR-RFLP method. Specific primer set (CD19-3'UTRF: 5'-AGAGGGAA
CAGGGTTCCTAG-3 ', CD19ex15R: 5'-AGGAATACAAAGGGGACTGG
2 from the CD19 gene containing the polymorphic site using
A 56 base pair fragment was amplified. Moreover, this amplification used the same conditions as the above-mentioned PCR-SSCP. After the PCR reaction, the amplified product was digested with BamHI for 2 hours, and further SYBR Gold (trade name Cyber Gold, Molec
ular probes, Inc., Engene, OR) staining was performed on a 10% polyacrylamide gel.

(5) Direct Sequencing Method The PCR product was amplified, and the same primers as those used in the PCR-SSCP described above were used to directly sequence both the sense strand and the antisense strand. ABI310
Or ABI377 sequencer (ABI PRISM, PE Biosys
(ABI PRISMTM dRhodamine Terminato) using the die terminator method according to the manufacturer's instructions.
r Cycle Sequencing-Ready Reaction Kit) Automated cycle sequencing was performed based on the fluorescence of the PCR product.

(6) Genotyping of dinucleotide repeat polymorphisms The genotypes of dinucleotide repeat polymorphisms contained in the 3'UTR were determined by PCR amplification, sequencer and GENSC.
Performed using AN ^™ software. A 256 base pair fragment containing the polymorphic site was labeled with Fam or Hex CD19-
Amplification was performed using the 3'UTRF primer and the CD19ex15R primer. In addition, the amplification is performed by the above-mentioned PC.
It performed on the same conditions as R-SSCP. The amplified product is 4
Electrophoresed on a% polyacrylamide gel (acrylamide: bisacrylamide = 19: 1) using an ABI377 sequencer (ABI-PRISM, PE Biosystems). P
1.5 μL of the solution containing the CR product was added to 2.5 μL of 99.5% formamide, 0.5 μL of loading buffer (50 mg / mL blue dextran, 25 mM EDTA) and 1.0 μL of Gene. Scan (Gene Scan; registered trademark)-
500 [ROX] size standard (ABI-PRISM, PE Biosyste
ms) 1.0 μL. The mixture was denatured at 96 ° C. for 2 minutes and immediately cooled on ice. 2 μL of the mixture
Was loaded on a 4% polyacrylamide gel. Data were collected with ABI 377 collection software and size analysis was performed using Genescan-500 ROX.
500 ROX) was used as a size standard (ABI-PRISM).

(7) Statistical Analysis Japanese patients and healthy subjects were compared by case-control-related analysis. Positive rate was defined as the percentage of individuals with alleles (homozygous and heterozygous) in the total number of individuals. Chi-square test and Fisher's exact calculation were used to analyze the association between CD19 polymorphisms and susceptibility to each disease, or between CD19 polymorphisms and clinical features in the Japanese population . The P value for comparing the presence or absence of individual alleles was corrected by the number of alleles by multiplying by 13 (corrected P
The value is noted as P _corr ). That is, 10 types of repetitive sequence polymorphisms having 9 types of SNPs and 5 alleles were analyzed (ie, 9 + 5-1).

Caucasoid SLE kindreds were analyzed as usual using a transmission imbalance test (hereinafter abbreviated as TDT) (see Reference 51). In families with 2 or 3 affected children, only the first child was selected for analysis. TDT
Of significance was tested using the NcNemar's test. The χ ² value was calculated by the following formula: χ ² = (T−NT) ² / (T + NT) where T is the number of alleles inherited and NT is the number of non-inherited alleles. is there. Haplotype frequency and linkage disequilibrium parameters were evaluated from typing results using the EH program (see reference 52). The relative linkage disequilibrium (hereinafter abbreviated as RLD) value is the maximum possible value of linkage disequilibrium (hereinafter abbreviated as LD) (LD> 0).
Value) or the smallest possible value (when LD <0)
It is defined as the ratio of LD to the absolute value of. As a result, RLD varies from -1 to 1.

2. Results (1) Identification of CD19 polymorphism Full length of DC19 translation region, promoter region (ie, ~
-1050 bp) and 3'untranslated region (ie, 3'-
UTR) systematic displacement screening was performed using 32 Japanese healthy subjects and 36 patients (ie 16 SLE patients, RA
Genomic DN from 16 patients and 4 CVID patients)
For sample A, PCR-SSCP was used. The nucleotide sequences of the detected mutations were determined using the direct sequencing method. Through the screening, 3 SNPs in the translation region and 2 SNPs in the intron sandwiched between exon-intron junctions
And 4 SNPs in the promoter region and 3 '
A GT repeat polymorphism in the UTR was identified (Figure 5). FIG. 5 shows the genomic arrangement of the human CD19 gene and the mutations detected in Japanese. In FIG. 5, boxes indicate exons. The name of the mutation was based on the literature (see literatures 53 and 54). Mutations other than common mutations are CVID
It was not detected in the patient.

(2) Relationship between SLE and CD19 mutation in Japanese. Next, we found that which of the CD19 polymorphisms detected in (1) was RA, SLE and Crohn's disease in the Japanese population. A case-control analysis was performed to analyze whether it was related to the susceptibility of the. RA patient 127
The genotypes of these polymorphisms were determined in Examples, 87 patients with SLE, 156 patients with Crohn's disease and 247 healthy Japanese people.

Table 2 shows the positive rate of CD19 SNPs detected in the promoter and translation region.

[0096]

[Table 2]

C. In exon 4 705T (P23
5P) (P = 0.0096, Pcorr = 0.12, odds value [OR] = 0.49, 95% confidence interval [CI]:
0.29-0.84) and IV in intron 14
S14-30T (P = 0.028, Pcorr = 0.36,
OR = 0.56, 95% CI: 0.33-0.94)
Showed a negative association with SLE. No other significant association was observed between any of the other SNPs and SLE, nor between any of the other SNPs and RA and Crohn's disease.

Table 3 shows the frequency of 3'UTR dinucleotide repeat polymorphisms.

[0099]

[Table 3]

C. ^* 132 (GT) ₁₈ (ie, cDN
132 counted from the 3'base of the stop codon in the A sequence
The positive rate of 18 alleles encoding the GT repeat starting from the th base) was significantly higher in SLE (15/87, 17.2%) than in healthy subjects (17/247, 6.9%). It was high (P =
0.005, Pcorr = 0.065, OR = 2.8
2, 95% CI: 1.37-5.79). Also, SLE
1 and the control group were significantly different, c. ^* 132 (GT)
₁₈ allele frequencies (χ ² = 7.6, df = 1, P =
0.006, P _corr = 0.078), genotype distribution (χ ² = 19.0, df = 10, P = 0.04, P
_corr = 0.36) and allele frequency (χ ² =
9.8, df = 4, P = 0.04, P _corr = 0.3
Obtained in 6). To test whether this association could be replicated, an independent S for GT repeat polymorphisms was tested.
The LE patient group (ie SLE2) was analyzed. SLE2
C. ^* 132 (GT) ₁₈ positive rate is SLE1
Lower than, but c. ^* 132 (GT) ₁₅
The positive rate of was significantly increased (χ ² = 4.8, df
= 1, p = 0.03, P _corr = 0.39).

From these results, the inventors have found that GT
G in 3'UTR when the number of iterations is 15 or more
We thought that T repeat was associated with SLE. Therefore, c. ^* 1
32 (GT) _15-18 positive rate and frequency, SLE
1, SLE2 and control were compared (Table 4).
c. ^* 132 (GT) _{15-18 has} a positive rate of SLE1
(P = 0.016) and SLE2 (P = 0.049)
Significantly increased in both groups. When the two groups were combined, a more significant difference was observed with the control group (P = 0.0096). Also, c. ^* 132 (GT)
The frequency of the _15-18 allele was significantly higher in SLE (P = 0.0012).

[0102]

[Table 4]

For RA and Crohn's disease, the GT
No significant association with repeat polymorphisms was observed.

(3) Linkage disequilibrium between polymorphic sites Polymorphic sites showing significant association with SLE c. 705, I
VS 14-30 and c. In order to investigate the relationship between * 132, we analyzed based on the results of genotyping of healthy Japanese individuals. Significant linkage disequilibrium is found in CD19c. 70
Observed between 5T and IVS 14-30T (relative linkage disequilibrium value [RLD] = 0.83, χ ² = 93.8, P
< ^10-10 ). In addition, the strong association is c. 705G
And c. ^{* Between} 132 (GT) ₁₈ (RLD = 1.0, χ
² = 8.2, P = 0.004), and c. 705G and c. ^{* Between} 132 (GT) ₁₅ (RLD = 0.84, χ
² = 1.12, P = NS), but the latter did not result in a statistically significant difference due to the small number of individuals carrying this allele.

(4) Relationship with clinical features Next, the present inventors analyzed whether the GT repeat polymorphism was associated with the clinical features of SLE. Interestingly, c.
^* Compared to patients without the 132 (GT) _15-18 allele, c. ^* 132 (GT) ₁₅ or c. ^* 1
32 _(GT) may have towards the patient an anti-Sm antibodies with _{1 8} was low (P = 0.02). No association was observed for parameters such as age of onset, presence or absence of nephropathy, neurological abnormalities (stroke or psychosis), serositis, hypocomplementemia or anti-dsDNA antibody (Table 5).

[0106]

[Table 5]

3. Discussion In this study, we identified a number of nucleotide sequence variations in human CD19, three of which were SLE.
It was shown to be significantly related to. Two showed a negative association with SLE and the rest showed a significant positive association.
The former two were in negative linkage disequilibrium with the latter. Furthermore, since SNPs encoding synonymous substitutions and single base substitutions in introns are unlikely to be associated with any functional alteration, the 3′UTR GT repeat polymorphism is primarily functionally important. Is likely to be.

No previous studies have reported that CD19 gene polymorphisms are associated with SLE. For example,
The polymorphism analyzed in an Italian case-control study may be the same as the polymorphism reported by the present inventors, but the study did not find an association with SLE. This suggests that the association of CD19 3'UTR with SLE may be a characteristic feature of the Japanese population and neighboring groups. Therefore, it would be interesting to analyze the presence or absence of this association in non-Japanese Asian populations.

At this time, it is only speculative how extended GT repeat alleles are associated with SLE. If such an allele is present in the peripheral CD
If associated with a higher expression level of 19, a lower threshold of B cell activation may occur which leads to the production of low affinity autoantibodies to self antigens. CD19
Phenotype of B cells from mice overexpressing
-1 protein tyrosine phosphatase (see References 55 and 56)
And Lyn (see reference 57) are similar to B cells with a defect.
Since these two molecules are negative regulators of the B cell response, lowering their signal thresholds may cause the induction of autoimmunity. Or, for example,
c. ^* 132 (GT) _15-18 allele is premature B
Involvement in low surface CD19 expression in cells may cause defects in tolerance induction to self-antigens in bone marrow (see ref. 19). The expression level of CD19 in peripheral B cells was reported to be slightly decreased in a small number of SLE patients (see reference 29). In fact, the majority of CD19-deficient mice show antinuclear antibodies (Sat
o S., personal communication). Therefore, one possible mechanism is that defective CD19 leads to a lack of tolerance induction to self-antigens, probably due to reduced BCR signaling in immature B cells (Reference 58).
reference). For example, SL in combination with genetic polymorphisms of negative regulators that result in inadequate negative signals, or when other genetic and / or environmental conditions are provided.
It is also possible that polymorphisms leading to low levels of CD19 expression in E patients may lead to the persistence and activation of autoreactive B cells.

In this study, c. ^* 132 (GT)
It was observed that SLE patients with the _15-18 allele were significantly less likely to have anti-Sm antibodies. Of particular interest is that the expression level of CD19 is
In mice expressing a heavy chain transgene, B-1
It has recently been reported that it regulates differentiation of B-2 cell group and autoantibody production (see Reference 59). Thus, CD19 expression levels may be associated with regulation of nuclear autoantigen-specific B cell differentiation and activation.

Since many polymorphic sites were detected and statistical analysis was performed for each of the alleles detected, the P-values were statistically significant after correction for multiple comparisons. I didn't reach sex. However, since a significant association of GT repeat polymorphisms was observed both in RA and Crohn's disease, but in two independent groups of SLE, this association is unlikely to be false positive.

Example 2. Relationship between polymorphism in FCGR2B gene and human systemic lupus erythematosus In order to analyze the relationship between polymorphism in FCGR2B gene and human systemic lupus erythematosus, mutation screening of FCGR2B was performed by PCR single-chain polymorphism (PCR-singl
e strand conformation polymorphism (SSCP).

As shown in FIG. 6, the FcγRIIB protein consists of a signal peptide (SP), two Ig-like extracellular domains (in FIG. 6, labeled EC1 and EC2, respectively), a transmembrane region (in FIG. 6, TM). And cytoplasmic end (indicated as C1, C2, and C3 in FIG. 6, respectively).
(Fig. 6). Through initial mutation screening of the entire translation region using an exon-specific primer set and genomic DNA template, four possible mutation sites were detected in exon 4 and exon 5 (data not shown). However, through exons 1-5, Fc
The amino acid and nucleotide sequences of γRIIB are Fc
It has high homology with γRIIA and IIC (Reference 6
1,62), and therefore some of the mutations are actually FCGR
It could also be derived from the 2A or 2C polymorphism.

In consideration of the above situation, in order to amplify only the FCGR2B gene, two-step PCR was performed using cDNA prepared by using RNA extracted from peripheral blood mononuclear cells as a template. As previously reported, two fragments of different sizes were observed in the first PCR, which is due to the spliced variant FcγRIIB.
1 and FcγRIIB2 (Reference 63)
reference).

1. Method (1) Patients and Healthy Subjects 75 SLE patients and 94 healthy subjects who were unrelated Japanese were analyzed. SLE patients are 12 males and 63 females aged 16 to 78 years (mean age 40.0 ± 13.7 years). Also, these SLE patients
A patient who is visiting the University of Tokyo Hospital and Juntendo University Hospital. The healthy subjects are 49 males and 45 females aged 22 to 50 years (mean age 31.3 ± 8.3 years), researchers and laboratories of the University of Tokyo and the Japanese Red Cross Central Blood Center. It consists of staff and students.
Residents in the Kanto region of Japan have been shown to be relatively homogenous with respect to their genetic background (see Ref. 48), which enables the case-control study employed in this study.
This study was approved by the Research Ethics Committee of the University of Tokyo and Juntendo University.

(2) RNA and genomic DAN RNA are available from RNeasy Mini Kit (Qiagen, Hilde
n, Germany) and were purified from peripheral blood mononuclear cells of SLE patients and healthy subjects and reverse transcribed into cDNA. In addition, genomic DNA was obtained from peripheral blood leukocytes of patients and
IAamp Blood kit, Qia
(Gen, Hilden, Germany).

(3) Determination of FCFR2B genotype FCGR2B exon 4, exon 5 and exon 6
Genotyping was performed using a two-step PCR and SSCP method. 5'primer for the first PCR
UTR (2B5'UTR-F: 5'-GAGAAGGCTGTGACTGC
TGT-3 ') and the ITIM region of exon 8 (2BITI
M-R: 5'-CGGGTGCATGAGAAGTGAAT-3 '), FC
A 944 bp cDNA fragment of GR2B was amplified (FIG. 6). PCR conditions were as follows: 0.2 μL cDNA, 0.2 μM each primer, 0.4 mM d.
NTPs, 2 mM MgCl ₂ , 2.5 U LA Ta
50 μL containing qDNA polymerase (Takara)
Reaction liquid. After the first denaturation (3 min at 96 ° C), denaturation (96
35 cycles of PCR reaction of 30 seconds at ℃), annealing (30 seconds at 60 ℃), extension (90 seconds at 72 ℃),
Furthermore, the final extension reaction (5 minutes at 72 ° C.) was performed. First P
The CR product was confirmed to be FCGR2B by the direct sequencing method.

Subsequently, the amplified fragment was used as a template for the second PCR reaction, using two primer sets, one of which was (2B exon 3-F: 5'-GCATC).
TGACTGTGCTTTCTG-3 ', 2B Exon 4-R: 5'-CTTGGACAGTG
Exon 4 full length 275b using ATGGTCACA-3 ')
The p fragment was amplified, and the other was (2B exon 4.5-F: 5 '
-TCCAAGCTCCCAGCTCTTCA-3 ', 2B exon 6-R: 5'-TGGTTTC
TCAGGGAGGGTCT-3 ') was used to amplify a 176 bp fragment encompassing exon 5 and exon 6 (Figure 6). PC
R conditions are as follows: 0.5 μL of initial PCR
Product, 0.4 μM each primer, 0.2 mM dNT
Ps, 1.5 mM MgCl ₂ , 1 U AmpliTa
q Gold DNA Polymerase (Perkin-Elmer Applied Biosystems, Norwalk,
25 μL of reaction solution containing CT, USA). After denaturation at 96 ° C for 10 minutes, denaturation (96 ° C, 30 seconds), annealing [60 ° C (for amplification of 275bp fragment) or 62 ° C (for amplification of 175bp fragment), 30 seconds],
The PCR reaction of the extension reaction (72 ° C, 30 seconds) was performed for 25 cycles, and the final extension reaction (72 ° C, 1 minute) was performed.

Amplified 275 bp and 176 bp DN
The A fragment was analyzed using the SSCP method as described in our previous report (see References 40 and 69). 10% polyacrylamide gel (acrylamide: bis = 49: 1)
For 90 minutes at 21 ° C. and 10
Each was carried out at 75 ° C. for 75 minutes. The separated fragments were visualized by silver staining.

(4) Determination of genotypes of FCGR2A, 3A and 3B FCGR2A-131H / R, FCGR3A-176F
/ V and FCGR3B-NA1 / 2 with SL of 75 cases
Determined using genomic DNA from E patients and 94 healthy controls. FCGR2A-131H / R gene determination was performed using PCR-RFLP with mismatched primers (see reference 40), and FCGR3A-176 / V
PCR-SSCP was used to determine the gene for the gene (see reference 40), and the gene for FCGR3B-NA1 / 2 was determined using the PCR-PHFA method (see reference 64) and the PCR-SSP method.
(See Reference 65).

(5) Direct Sequence Method The direct sequence of FCGR2B is converted to ABI PRISM.
^TM 310 Gene Analyzer (Perkin-Elmer
Applied Biosystems) and the dye-terminator method.

(6) Statistical analysis Calculation for the related analysis is performed by Stat for Macintosh.
View-J4.11 (Abacus Concepts Inc., Berkele
y, CA, USA). Association between polymorphisms in the four FcγR gene groups and SLE susceptibility was analyzed using the χ ² test. When the number of samples was small, Fisher's exact method was used. The EH program was used to estimate haplotype frequencies and linkage disequilibrium parameters.

2. Results Second PCR amplified with two primer sets
The product is (FIG. 6) two mutation sites, c. 612G> A (synonymous substitution) and c. 772T> G (Y258D), which are
It has been reported as a mutation site detected in a preliminary experiment using the human B lymphoblast cell line Raji (see document 66) (the name of the mutation site is based on document 53). A biallelic polymorphism (SNP) was detected in exon 5 by the PCR-SSCP method (FIG. 7), and further by the direct sequencing method, codon 232 in the transmembrane region of FcγRIIB.
A novel SNP that causes a non-conservative amino acid substitution (I232T) in S. It was revealed that 695T> C (exon 5).

Table 6 lists 75 SLE patients and 94 SLE patients.
Frequency of FCGR2B-232I / T genotype in healthy subjects
Indicates. The frequency of the genotype is between SLE and healthy subjects.
Significantly different (χ^Two= 6.4, P = 0.04). SLE
Odds value of T / T and I / T genotype (O
R) is 3.9 (χ) for the I / I genotype, respectively. ^Two
= 6.3, P = 0.02, 95% confidence interval [CI]: 1.
4-11.2) and 1.1 (χ^Two= 0.1, P = 0.
73, 95% CI: 0.6-2.2), 232T
A significant association between / T genotype and SLE was shown. Opposition
The positive rate for the 232I allele was
Significantly decreased (χ^Two= 6.3, P = 0.02, OR:
0.3, 95% CI: 0.1-0.7). Furthermore, 232
The I and 232T allele frequencies were also between SLE and healthy individuals.
Was significantly different (χ^Two= 5.1, P = 0.02). healthy
Genotype frequencies of individuals are suitable for Hardy-Weinberg equilibrium.
It matched.

[0125]

[Table 6]

The inventors next turned on FcγRIIB-23.
The relationship between the 2I / T polymorphism and the age of onset in SLE patients, the presence or absence of lupus nephritis, disease phenotypes such as anti-dsDNA and anti-Sm was analyzed. No significant differences in genotype frequencies were observed between patients with positive or negative clinical and immunological features (data not shown).

To confirm that the FcγR family of genes are primarily associated with SLE, FCG
The genotypes of R2A-131H / R, FCGR3A-176F / V and FCGR3B-NA1 / 2 were determined to be FCGR.
Comparisons were made in the same set of patients and normals analyzed for 2B. CDNA for FCGR2B genotyping
The number of individuals analyzed was lower than that of ours, since samples were needed and cDNA samples were available in the majority of patients, but in about half of the healthy subjects. (Reference 40).
As shown in Table 7, with the exception of FCGR2B, no significant association was detected in these sets of samples, with FcγRIIB being the most SLE among the four FcγR gene clusters.
It was shown to be strongly related to.

[0128]

[Table 7]

Linkage disequilibrium between the polymorphisms of these four FcγR gene groups was analyzed using data from healthy subjects. As a result, FCGR2B and FCGR3B (χ ² = 7) were analyzed.
FCGR2A and FC during 9.7, P <10 ⁻⁶ )
Strong linkage disequilibrium was detected between GR3A (χ ² = 6.4, P = 0.01), but not in the other combinations (Table 8).

[0130]

[Table 8]

These results are in agreement with reported physical maps showing that FCGR2B is located adjacent to 3B and FCGR2A is located adjacent to 3A. In addition, in the SLE patient, a strong association was FCGR2B.
Observed between the −232T allele and the 3B-NA2 allele (χ ² = 58.8, P <10 ⁻⁶ ), which was previously reported by the inventors using a larger number of samples. Consistent with the association of SLE with the FCGR3B-NA2 allele.

Although it was explained that the previously detected association with the FCGR3B-NA2 allele resulted from linkage disequilibrium with the FCGR2B-232T allele, FCGR3B-NA2 was associated with low clearance of immune complexes. It may also have independent contributions. To evaluate such a possibility, FCGR2B and 3B
2 loci were analyzed. A significant increase in odds value (OR) was detected only in individuals with 2B-232T / T and 3B-NA2 / 2 genotypes (OR: 3.4).
1, 95% CI: 1.1-10.9, P = 0.05),
It was not detected in other combinations including 2B-232I / T, 3B-NA2 / 2 group (FIG. 8). This result indicates that FCGR2B polymorphism is unique and FCGR3B
It was supported that the relationship of was secondary.

In order to rule out the presence of other mutations not detected by SSCP, the sequence of the full-length translational region of FCGR2B was set in 10 individuals with FCGR2B-232I / I genotype, 10 with 232I / T. Individual,
And cDN from 20 individuals with 232 T / T
Determined using As. Two synonymous substitutions, namely within exon 3 c. 216G> T (R72R) and c. 612G> A (L204L) is 232-I
Only one individual of the / I and 232-I / T genotypes was detected. On the other hand, previously reported c. 772T> G (Y258D) is a mutation of human FCGR2B whose function may be altered (Reference 67).
), Was not detected in our subject, and
It has also been shown to be rare in Caucasian and African American populations. In addition, sequences registered in the past (GenBank accession number AH005422, respectively)
And NM_004001) c. 614T and A (F2
05Y), but all samples analyzed by the inventors were c. Since it was a homozygote of 614A (205Y), it became clear that 205Y was high in frequency, at least in Japanese.

Furthermore, the inventors have found that HLA-DRB1 ^* 1
501 (Ref. 68) and TNFR2-196R (Ref. 6)
(See 9) and the possibility of interaction between FCGR2B polymorphism was analyzed. These are genes that have previously been shown to be associated with Japanese SLE. As shown in Table 9, FCG
R2B-232T / T genotype and at least one HL
The odds value (ie, risk of onset) for individuals with both the A-DRB1 ^* 1501 allele was very high compared to that predicted from the independent effects of each locus (OR: 20). 9.9, 95% CI: 2.9-15
2.7, P = 0.004). This is a very interesting result that suggests a synergistic effect between these genes. No such effect was observed between FCGR2B and TNFR2 (data not shown).

[0135]

[Table 9]

From the above, the present inventors' findings are F
It strongly suggests that FCGR2B in the cγR family is the major susceptibility gene for SLE in the Japanese population. Fcγ as a negative regulator of B cells
Taking into account in vitro and animal studies that clearly demonstrate the role of RIIB, the FcγRIIB-232T polymorphism is associated with a decline in FcγRIIB function, thereby reducing S
The mechanism by which autoantibodies specific to LE are produced is presumed. FCGR2B-232I / T in the transmembrane region
The functional differences associated with substitution are still speculative, but recently Fcγ in the initiation of B cell apoptosis
The role of the transmembrane domain of RIIB has been demonstrated. Therefore, it is considered that one possibility is that the FcγRIIB-232I / T polymorphism influences apoptotic signal transduction, allowing survival of B cells that produce autoantibodies and causing the autoimmune disease. It is an interesting subject to verify this hypothesis experimentally in the future.

[0137]

The present inventors have for the first time determined a new SLE susceptibility polymorphism gene. This determination allowed a more detailed prediction of the risk of developing SLE in the subject.

[0138] The polymorphic genotype can be easily determined by using a polynucleotide containing such a polymorphic gene. Further, by using the SLE onset risk prediction method and the apparatus for performing this method,
It is possible to easily predict the risk rate of developing SLE.

References

[Table 10]

[0140]

[Table 11]

[0141]

[Table 12]

[0142]

[Table 13]

[0143]

[Table 14]

[0144]

[Table 15]

[0145]

[Table 16]

[0146]

[Sequence list]                                SEQUENCE LISTING <110> OLYMPUS OPTICAL CO., LTD. <120> Susceptive gene to systemic lupus erythematosus and use thereof <130> A000101594 <140> <141> <160> 9 <170> PatentIn Ver. 2.0 <210> 1 <211> 8743 <212> DNA <213> Homo sapiens gene for CD19 <220> <221> source <222> (1) .. (8743) <223> / organism = "Homo sapiens"       / db_xref = "taxon: 9606" <220> <221> variation <222> (541) <223> / replace = "g" <220> <221> variation <222> (908) <223> / replace = "t" <220> <221> variation <222> (1104) <223> / replace = "t" <220> <221> variation <222> (1391) <223> / replace = "a" <220> <221> variation <222> (2367) <223> / gene = "CD19"       / replace = "t" <220> <221> variation <222> (2481) <223> / gene = "CD19"       / replace = "c" <220> <221> variation <222> (2553) <223> / gene = "CD19"       / replace = "t" <220> <221> variation <222> (2785) <223> / gene = "CD19"       / replace = "g" <220> <221> variation <222> (7248) <223> / gene = "CD19"       / replace = "g" <220> <221> variation <222> (8518) <223> / replace = "t" <300> <303> GenBank / EMBL / DDBJ <308> AB052799 <400> 1 ggatcctctc gcctcggcct cctaaagtat tgggattaca ggcatgagcc tctgtgcctg 60 gctgtaactg acatgtttta agcaggggaa tgacatgctc tagtgaaagc cagtctgggc 120 agctgggtag ctaatgaggg gattagagag attttgttga atgaaaggca gattgagtcc 180 tgctactcgc ccccttcatt ccccttcatt catgcctcat tcttccgcct cccagccgcc 240 tcaactggcc aaagggaagt ggaggccctg ccacctgtag ggagggtccc ctggggcttg 300 cccacagcaa acaggaagtc acagcctggt gagatgggcc tgggaatcag ccactgagaa 360 agtgggtctc ttgggtccct gaattctttt tctgagtccc tgcagcagtg aaaaagacac 420 agaggcacat agagagtgac agagaaagag agagacagag aggagaggca tggggcagaa 480 taagaacaga tttaggagtt agaactcctg ggttctttta aaacaatttt tcttttagag 540 acagggtctt gttgtgttgc ccggactgga gcacagtggc tattcccagg cataatcatg 600 gtgcactgca gccttgaact cctgggctca agcgatcctt ctacctcagc ctcccaagga 660 cctgggacca taggcgtgta ccactgtgcc tggcttttgc ctggttttaa actgaggcag 720 tatgacttga gctcttaggc attaattgaa gctgtatctc attaactgag ggcttatgat 780 gtgctggaca ctgggctaat agtgctgaac atattgtcatcat ttttaatctt cacaaacaat 840 atttgtatag gactgttttc ttttcttttt tttttttgaa acagagtctc actctggtgc 900 ccaggctgga gtgcagtggt gtgatctcgg ctcactgcaa cctccgcctc ctggtttcca 960 gtgattctcc tgcctcagcc tcctaagtag ctgggattac aggtgtgcgc caccatgccc 1020 ggctaatttt tttttttttt tttgagaagg agtctatgtg cccagcattg ttctagagca 1080 cttgcaatta gtggtgaaca acacggtctc tactccaagg ggctcacatt cttgtgcaga 1140 aaacagaaat gaacaaataa acacacaaga tcatttcccg tggtagtgag agctgggatg 1200 aaaataaaac agcgtggcag ggaggaggca agtgttgtga gtctggaggg ttcctggaga 1260 atggggcctg aggcgtgacc accgccttcc tctctggggg gactgcctgc cgcccccgca 1320 gacacccatg gttgagtgcc ctccaggccc ctgcctgccc cagcatcccc tgcgcgaagc 1380 tgggtgcccc ggagagtctg accaccatgc cacctcctcg cctcctcttc ttcctcctct 1440 tcctcacccc catggaagtc aggcccgagg aacctctagt ggtgaaggtg gaaggtatgt 1500 ccaaagggca gaaagggaag ggattgaggc tggaaacttg agttgtggct gggtgtcctt 1560 ggctgagtaa cttaccctct ctgagcctcc attttcttat ttgtaaaatt caggaaaggg 1620 ttggaaggac tctgccggct cctccactcc cagcttttgg agtcctctgc tctataacct 1680 ggtgtgagga gtcggggggc ttggaggtcc cccccaccca tgcccacacc tctctccctc 1740 tctctccaca gagggagata acgctgtgct gcagtgcctc aaggggacct cagatggccc 1800 cactcagcag ctgacctggt ctcgggagtc cccgcttaaa cccttcttaa aactcagcct 1860 ggggctgcca ggcctgggaa tccacatgag gcccctggca tcctggcttt tcatcttcaa 1920 cgtctctcaa cagatggggg gcttctacct gtgccagccg gggcccccct ctgagaaggc 1980 ctggcagcct ggctggacag tcaatgtgga gggcagcggt gagggccggg ctggggcagg 2040 ggcaggagga gagaagggag gccaccatgg acagaagagg tccgcggcca caatggagct 2100 ggagagaggg gctggaggga ttgagggcga aactcggagc taggtgggca gactcctggg 2160 gcttcgtggc ttcagtatga gctgcttcct gtccctctac ctctcactgt cttctctctc 2220 tctgcgggtc tttgtctcta tttatctctg tctttgagtc tctatctctc tccctctcct 2280 gggtgtctct gcatttggtt ctgggtctct tcccagggga gctgttccgg tggaatgttt 2340 cggacctagg tggcctgggc tgtggcctga agaacaggtc ctcagagggc cccagctccc 2400 cttccgggaa gctcatgagc cccaagctgt atgtgtgggc caaagaccgc cctgagatct 2460 gggagggaga gcctccgtgt gtcccaccga gggacagcct gaaccagagc ctcagccagg 2520 gtatggtgat gactggggag atgccgggaa gctggggtcc agagacagag gggaggggaa 2580 actgaagagg tgaaaccctg aggatcaggc tttccttgtc ttatctctcc ctgtcccaga 2640 cctcaccatg gcccctggct ccacactctg gctgtcctgt ggggtacccc ctgactctgt 2700 gtccaggggc cccctctcct ggacccatgt gcaccccaag gggcctaagt cattgctgag 2760 cctagagctg aaggacgatc gcccggccag agatatgtgg gtaatggaga cgggtctgtt 2820 gttgccccgg gccacagctc aagacgctgg aaagtattat tgtcaccgtg gcaacctgac 2880 catgtcattc cacctggaga tcactgctcg gccaggtaga gtttctctca actgggaggc 2940 atctgtgtgg gggtactggg aagaagtgga agccagtcaa tcttagattc ccccaacccg 3000 agggctactc ccagcctcac cccaaacccc aacttccaca cagaacactg actccaagtc 3060 tttctttttt ttgacagagt ctcgctctgt tgcctaggct ggagtgcagt ggtgccatct 3120 tgtcttggct cactgcaacc tccgcctccc aggttcaagt gattcccctg cctcagcctc 3180 ctgagtagct gggattacag gtgcccacca ccacgcctgg ctaatttttt tttttttttt 3240 gagacggagt cttgcactgt cacccaggct ggagtgcagt ggcacgatct cagctcactg 3300 caacctccac cttccaggtt caagtgattc tcctgcctca gcctcccgag tagctgggat 3360 taaagcctgg ctaatttttt ttgtattttt agtagagatg gggtttcatt atgttggcca 3420 ggctggtctc aaactcctga cctcgtgatc cacccgcctc ggcctcccaa agtgctggga 3480 ttacagacat gagccacagg gccgggccaa gcctaatttt gtatttttag tagagatggg 3540 gtttctccct gttggaccag gctggtcttg aactcctgac ttcaggtgat ctgcctgcct 3600 tggcctccca aagtactggg attacaggca taagccaccg cacctggcct agacttcaag 3660 tctttcttcc ctcgcttcca agacactact tttctgggtc ttcacctacc attgcttgcg 3720 cctgcccacc agcttgggtg gagtcttcct tcctccccaa ctcctcactc ttggagccct 3780 gggccctctt cttatccctg tctgcacact ttcctatttg aacttgactc tcaatggctt 3840 cttgggtcac catgccttgg tgactctatt ccaggctcca tactcagcca tctcctgtgc 3900 catttgatat cccatggaca cctcaggctc aacagataca aaatcaaact caatgtcttc 3960 cccaagtata gtcttcttgg tggcccagtg taagcagagg gcaccaccac ctgctccctc 4020 gcccaggcta agaacctggg catccttctt tttcctcacc ccgtccaaca aactggtcac 4080 agtgttctgc caattctctc tccatgcaat cctatcatgc tatcctaact gcaattcaca 4140 aacccaaccc caactttcac tccaaacttg atccaagcaa tgtgctggat cccaactgta 4200 accttgcaaa ctcaactctg cccttcactt tgaccgtgac tatccttaat tgcagcagga 4260 aactgatcat tatgctcccc tcaatccaca cattgcctct gagtacagcc atggtttgtc 4320 cacgatttgc tcaaagacac tgcccatgtc ctgtgccagg gtctgtgaca atccctgacc 4380 tcctgggaca tggctcctta gagagaggag agcctttctc acagcttggg actttgagtc 4440 tgtgtctttt tttttttctt gagacggagt tttgctgtgg ttgcccaggc tggagtgcag 4500 tgatctcggc tcactgaaac ctccgcctcc cgggttcaaa cgattctcct gcctcagcct 4560 cccaagtagc tgggattaca ggcacccacc accatgccca gctaattttt ttgtattttt 4620 agtagagatg gggtttcacc atgttggcca ggctggtctc gaactcctga cctcaggtga 4680 tccacccgcc tttgcctccc aaagtgctgg gattacaggc gtcaaccacc gcgcccggcc 4740 gagtctgtgt cttgcctctg tgcctcagac ttgcggttcc ttgagatctc aggattggga 4800 cgtaagatgc cagcctgggg tcctcgtctc atagcccctt ccccctagta ctatggcact 4860 ggctgctgag gactggtggc tggaaggtct cagctgtgac tttggcttat ctgatcttct 4920 gcctgtgttc ccttgtgggc attcttcatc ttcaaagagg tgagtcatgt ccccagtggg 4980 tctgtccaaa ccctactcca tcttccccag gataagccgg ctctggccag tctgacaacc 5040 atctttcttt cctcccatcc ctcccttcaa gaccccagaa tcctgttctc cccagtcttc 5100 ctctagcctc cctcaaactt cccaagcctc ttgcaatttt tttttttttt ttgagacagg 5160 gtctcattct gtcaccccag ctggagtgca gtggcacaat ctgagctcac tgtaacctct 5220 gcctcccagg cttaagtgat tcttgtgctt cagcctcccg agtacctggg actacaagtg 5280 tatgccacca cacccggcca attttttata tttttagtag agacgaggtt tcaccatgtt 5340 ggccagactg gtctcgaact cttgacctca aatgatccgc ccacctcggc ctcccaaagt 5400 gctgggatta caggcacgag ccaccgcgcc cgtccgcctc gcaatttgaa ctcctgtctc 5460 ctttgttgaa ccaagtgacc tccccagcac ctggccccac aaatcctcac cctgccaagc 5520 agcccctcct ctgatcacgc cctttaactc ccaccagccc tggtcctgag gaggaaaaga 5580 aagcgaatga ctgaccccac caggaggtaa tgcaaccagt gcaccccgcg gtaacaccct 5640 ccaccttcac tttatgcctt gcacttactg tttcctctgc ccaggggttc tttgctccgt 5700 ctctactgtt tcaaatactg cccaacctca aagcccagct ccaaagctac ctcctctgtg 5760 aagaactcct tggaaatgat catctcagac tcctctattg gctgtcccag cacaagtgat 5820 cacgtttaac ttctgaaggc ctggacagaa tcttgagtgg gtccgccatt ccattccaag 5880 tcggccctca ccgtgcactt cctcttctcc cgccagattc ttcaaagtga cgcctccccc 5940 aggaagcggg ccccagaacc agtacgggaa cgtgctgtct ctccccacac ccacctcagg 6000 cctcggtaag aggcaccgcc cctccagcct atagctccgc cccagatccg gggctccacc 6060 cccactctcc tcatccctcc aatccgctgt gcgccaagcc ttctggagct cggaactccg 6120 cccccggggc ggggagtccc gcccagctat gagccccgcc tctagaacca gaccccgcct 6180 ccagggctca gagccacgcc cccaggaccc agagcctgaa gtcgtaatca agagcagaac 6240 ttcgccccag aactgaaggc ctcggcccta gatttagatt ccgccccagg gttcaaggcc 6300 gggttcctag acccagagtc cattcgcaga gcccaaaaca tcctcttccc gtgccccgcc 6360 gcgcggaccc ttagccttga ccgcccccat ctcttctgac cccgtcttac aatgcccctc 6420 tcaccaggac gcgcccagcg ttgggccgca ggcctggggg gcactgcccc gtcttatgga 6480 aacccgagca gcgacgtcca ggcggatgga gccttggggt cccggagccc gccgggagtg 6540 ggtgaatgac tgggagaggg aagggtcgtt ccccacatgg agggggttgg agcggtctgt 6600 ggcccgaata gtggactggg ccctggagga gagggggcat gactcggttc cccatcccca 6660 tccccaaacc cccaggccca gaagaagagg aaggggaggg ctatgaggaa cctgacagtg 6720 aggaggactc cgagttctat gagaacgact ccaaccttgg gcaggaccag ctctcccagg 6780 gtaaggctgc cctcccccgt ggccccccac ctctgcggtg gcctgtggac tcccatggac 6840 acccctcctt ctacaccaga tggcagcggc tacgagaacc ctgaggatga gcccctgggt 6900 cctgaggatg aagactcctt ctccaacggt aacttggggc ctttgtggga cctcagagac 6960 ttaggtgtaa ttgcagcgct gtgacactcc tagaagggga tccctggagt tctctctctt 7020 ctgccacagc tgagtcttat gagaacgagg atgaagagct gacccagccg gtcgccagga 7080 caatgggtgt gtgtgaggat ggcaacagtc caggggggag gcggaggaca cctggaggcc 7140 aggaggaata gtaacctccc tcttcccttt ccagacttcc tgagccctca tgggtcagcc 7200 tgggacccca gccgggaagc aacctccctg ggtgagagat gctttcaatc agactgcctt 7260 gcccagcttg ggtgacctgg cctcagctct gacaccagat ccaactttga cctgaccctg 7320 accccaaacc cgaacccaat cctgtgactc ctctcacctc aacactgagc cccatccccc 7380 atcctgagcc ccatccccca tcctgacccc caatatttac cccctcccta actgtgaata 7440 tcaacaccga tcccaatgca gtatcagcct ggacttgatc tccacctcac ctcagcccca 7500 gtgcagacct caacttggac cccagcttac tctgcagctt cttcatgact ctgactccga 7560 ctccctccag tttcttcttt ttctttttct tttttttgag acggagtctc cctctgttgc 7620 ccaggctgga gtgcagttgc cacctctgcc tcctaggttc aagcgattct catgcctcag 7680 cctcctgagt agctgggatt atagacgttt gccaccacac ctggctaatt tttgtatttt 7740 cagtagagac agggtttcgc catgttggcc agactggtct ccaactcctg gcctctagtg 7800 atctgcccgc ctttggcttc ccaaagtgct gggattacag gcatgagcca ccacgcccag 7860 cccagttctg ttcttgaccc cttccttagc cataatctaa cccatatcta accctgaccc 7920 tacagctaac tggggcccca aactcaatgc taaccaaatc accccttccc agcacagcat 7980 gggtaatgct cctcaccttc ctctgcccct cagtcttcct ccttaccgta ggctgtactt 8040 cccatgccct agcctccaat tctccatccc ccgcccaagc agggtcccag tcctatgagg 8100 atatgagagg aatcctgtat gcagcccccc agctccgctc cattcggggc cagcctggac 8160 ccaatcatga ggaaggtggg tgcttctgcc gctgtcccct gctgtcccct gggctgactt 8220 tgccttccag cctacttcca gtgccaccca tgttctcctc ctccctggtc ctatccagat 8280 gcagactctt atgagaacat ggataatccc gatgggccag acccagcctg gggaggaggg 8340 ggccgcatgg gcacctggag caccaggtga tcctcaggtg gccaggtgag ctgggactgc 8400 ccctagggaa agcggggagg gagggagata ggcacggatg gcagtggctg ctggctttca 8460 gggagggaga gggaacaggg ttcctagggc ctggtgggca gggggaggac tgctggatcc 8520 ctccccatca ccgtttcttc tgcatagcct ggatctcctc aagtccccaa gattcacacc 8580 tgactctgaa atctgaagac ctcgagcaga tgatgccaac ctctggagca atgttgctta 8640 ggatgtgtgc atgtgtgtaa gtgtgtgtgt gtgtgtgtgt gtgtatacat gccagtgaca 8700 cttccagtcc cctttgtatt ccttaaataa actcaatgag ctc 8743 <210> 2 <211> 252 <212> DNA <213> Homo sapiens gene for CD19 <220> <221> source <222> (1) .. (252) <223> / organism = "Homo sapiens"       / db_xref = "taxon: 9606"       / chromosome = "16"       /map="16p11.2 " <220> <221> satellite <222> (194) .. (213) <223> / note = "microsatellite GT repeat variation 5"       / rpt_type = tandem       / rpt_unit = gt <300> <303> GenBank / EMBL / DDBJ <308> AB052818 <400> 2 agagggaaca gggttcctag ggcctggtgg gcagggggag gactgctgga cccctcccca 60 tcaccgtttc ttctgcatag cctggatctc ctcaagtccc caagattcac acctgactct 120 gaaatctgaa gacctcgagc agatgatgcc aacctctgga gcaatgttgc ttaggatgtg 180 tgcatgtgtg taagtgtgtg tgtgtgtgtg tgtatacatg ccagtgacac ttccagtccc 240 ctttgtattc ct 252 <210> 3 <211> 258 <212> DNA <213> Homo sapiens gene for CD19 <220> <221> source <222> (1) .. (258) <223> / organism = "Homo sapiens"       / db_xref = "taxon: 9606"       / chromosome = "16"       /map="16p11.2 " <220> <221> gene <222> (194) .. (219) <223> gene = "CD19" <220> <221> satellite <222> (194) .. (219) <223> / gene = "CD19"       / note = "microsatellite GT repeat variation 1"       / rpt_type = tandem       / rpt_unit = gt <300> <303> GenBank / EMBL / DDBJ <308> AB052814 <400> 3 agagggaaca gggttcctag ggcctggtgg gcagggggag gactgctgga cccctcccca 60 tcaccgtttc ttctgcatag cctggatctc ctcaagtccc caagattcac acctgactct 120 gaaatctgaa gacctcgagc agatgatgcc aacctctgga gcaatgttgc ttaggatgtg 180 tgcatgtgtg taagtgtgtg tgtgtgtgtg tgtgtgtgta tacatgccag tgacacttcc 240 agtccccttt gtattcct 258 <210> 4 <211> 260 <212> DNA <213> Homo sapiens gene for CD19 <220> <221> source <222> (1) .. (260) <223> / organism = "Homo sapiens"       / db_xref = "taxon: 9606"       / chromosome = "16"       /map="16p11.2 " <220> <221> satellite <222> (194) .. (221) <223> / note = "microsatellite GT repeat variation 2"       / rpt_type = tandem       / rpt_unit = gt <300> <303> GenBank / EMBL / DDBJ <308> AB052815 <400> 4 agagggaaca gggttcctag ggcctggtgg gcagggggag gactgctgga cccctcccca 60 tcaccgtttc ttctgcatag cctggatctc ctcaagtccc caagattcac acctgactct 120 gaaatctgaa gacctcgagc agatgatgcc aacctctgga gcaatgttgc ttaggatgtg 180 tgcatgtgtg taagtgtgtg tgtgtgtgtg tgtgtgtgtg tatacatgcc agtgacactt 240 ccagtcccct ttgtattcct 260 <210> 5 <211> 262 <212> DNA <213> Homo sapiens gene for CD19 <220> <221> source <222> (1) .. (262) <223> / organism = "Homo sapiens"       / db_xref = "taxon: 9606"       / chromosome = "16"       /map="16p11.2 " <220> <221> gene <222> (194) .. (223) <223> / gene = "CD19" <220> <221> satellite <222> (194) .. (223) <223> / gene = "CD19"       / note = "microsatellite GT repeat variation 3"       / rpt_type = tandem       / rpt_unit = gt <300> <303> GenBank / EMBL / DDBJ <308> AB052816 <400> 5 agagggaaca gggttcctag ggcctggtgg gcagggggag gactgctgga cccctcccca 60 tcaccgtttc ttctgcatag cctggatctc ctcaagtccc caagattcac acctgactct 120 gaaatctgaa gacctcgagc agatgatgcc aacctctgga gcaatgttgc ttaggatgtg 180 tgcatgtgtg taagtgtgtg tgtgtgtgtg tgtgtgtgtgtg tgtatacatg ccagtgacac 240 ttccagtccc ctttgtattc ct 262 <210> 6 <211> 268 <212> DNA <213> Homo sapiens gene for CD19 <220> <221> source <222> (1) .. (268) <223> / organism = "Homo sapiens"       / db_xref = "taxon: 9606"       / chromosome = "16"       /map="16p11.2 " <220> <221> gene <222> (194) .. (229) <223> / gene = "CD19" <220> <221> satellite <222> (194) .. (229) <223> / gene = "CD19"       / note = "microsatellite GT repeat variation 4"       / rpt_type = tandem       / rpt_unit = gt <300> <303> GenBank / EMBL / DDBJ <308> AB052817 <400> 6 agagggaaca gggttcctag ggcctggtgg gcagggggag gactgctgga cccctcccca 60 tcaccgtttc ttctgcatag cctggatctc ctcaagtccc caagattcac acctgactct 120 gaaatctgaa gacctcgagc agatgatgcc aacctctgga gcaatgttgc ttaggatgtg 180 tgcatgtgtg taagtgtgtg tgtgtgtgtg tgtgtgtgtgtg tgtgtgtgtta tacatgccag 240 tgacacttcc agtccccttt gtattcct 268 <210> 7 <211> 369 <212> DNA <213> Homo sapiens gene for FCGR2B exon4,5 <220> <221> variation <222> (221) <223> / replace = "a" <220> <221> variation <222> (304) <223> / replace = "c" <300> <303> GenBank / EMBL / DDBJ <308> AB050934 <400> 7 agtggctggt gctccagacc cctcacctgg agttccagga gggagaaacc atcgtgctga 60 ggtgccacag ctggaaggac aagcctctgg tcaaggtcac attcttccag aatggaaaat 120 ccaagaaatt ttcccgttcg gatcccaact tctccatccc acaagcaaac cacagtcaca 180 gtggtgatta ccactgcaca ggaaacatag gctacacgct atactcatcc aagcctgtga 240 ccatcactgt ccaagctccc agctcttcac cgatggggat cattgtggct gtggtcactg 300 ggactgctgt agcggccatt gttgctgctg tagtggcctt gatctactgc aggaaaaagc 360 ggatttcag 369 <210> 8 <211> 567 <212> DNA <213> Homo sapiens gene for FCGR2B exon5 & flanking intron <220> <221> variation <222> (95) <223> / replace = "t" <220> <221> variation <222> (100) <223> / replace = "c" <220> <221> variation <222> (134) <223> / replace = "t" <220> <221> variation <222> (148) <223> / replace = "t" <220> <221> variation <222> (153) <223> / replace = "g" <220> <221> variation <222> (251) <223> / replace = "t" <220> <221> variation <222> (328) <223> / replace = "c" <220> <221> variation <222> (386) <223> / replace = "c" <220> <221> variation <222> (468) <223> / replace = "a" <220> <221> variation <222> (477) <223> / replace = "g" <300> <303> GenBank / EMBL / DDBJ <308> AB062416 <400> 8 aaggctgtgc tccatagagt aatgatgcct ccagctatgc gaggctttgg gcccaccctt 60 cccactgccc ctgagggcta aggggagccc ttccttctgc tcctgcctgc tcaccagtgt 120 gcctttatta gtttggtgga gaaaccttgg taggcaggag gcataagtcc agccacagaa 180 accctgtgca gatgaggctg gggatgtagt gagtgctgca gaagtgagtg actcagacac 240 agaagagctt tgggtgacaa gcactaggac atagcattgg atggggggga ggtgggacaa 300 ggagagtact gcctgtcctg atgtctgcct tccctagctc ccagctcttc accgatgggg 360 atcattgtgg ctgtggtcac tgggactgct gtagcggcca ttgttgctgc tgtagtggcc 420 ttgatctact gcaggaaaaa gcggatttca ggtttgtagc tcctcccagt ccctttggtt 480 atcagtttcc acttggccca ggccctaacc ccagacattg ccagaatccc tctctttggg 540 ctagatacac attcagatct aggcccg 567 <210> 9 <211> 20 <212> DNA <213> Homo sapiens gene for FCGR2B exon7 <400> 9 cccaactttg tcagcctcat 20

[Brief description of drawings]

FIG. 1 is a configuration diagram of an SLE onset risk rate prediction apparatus according to one embodiment of the present invention.

FIG. 2 is a diagram showing an example of a score table used in the present invention.

FIG. 3 is a diagram showing a processing flow used in one embodiment of the present invention.

FIG. 4 is a configuration diagram of an SLE onset risk rate prediction device according to one embodiment of the present invention.

FIG. 5 shows the genomic arrangement of the human CD19 gene and the mutations detected in Japanese.

FIG. 6 shows the cDNA structure of the FCFR2B gene and the nested PCR strategy.

FIG. 7: Representative S of FCGR2B-232I / T polymorphism
Electrophoresis photograph showing SCP pattern.

FIG. 8 shows the main role of FCGR2B obtained by 2-locus analysis.

[Explanation of symbols]

1. Genome DNA sample purification device 2. Genotyping means 3. Input means 4. Display device 5. CPU 6. RAM 7. Image processing unit 8. Phil 11. Bus line

Continued front page    F-term (reference) 4B024 AA11 AA19 CA01 CA04 CA06                       CA12 CA20 HA11                 4B029 AA07 AA23 BB20 FA15                 4B063 QA13 QA17 QA19 QQ02 QQ42                       QQ53 QR08 QR14 QR32 QR35                       QR40 QR42 QR62 QS16 QS25                       QS36 QS39 QX01 QX10

Claims (15)

[Claims] 1. At least one novel polymorphic gene selected from the following group: (1) c. 705G>T; (2) IVS14-30C in human CD19 gene
>T; (3) c. In human CD19 gene ^* 132 (G
T) _12-18 ; and (4) c. In human FCGR2B. 695T> C. 2. A method for predicting the risk of developing systemic lupus erythematosus in a subject comprising: (1) preparing a genomic DNA sample from the subject; (2) at least the following for the obtained genomic DNA sample: Genotyping the polymorphism of 1; (a) c. In the human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. Genotype of 695; and (3) when the polymorphism selected in (2) above is the polymorphism of (d) above and the genotype is a polymorphism resulting in codon 232T / T And (e) human leukocytes HL
Determining the presence or absence of A-DRB1 ^* 1501; (4) Risk of developing systemic lupus erythematosus in a subject by determining the risk rate of developing SLE based on the genotypes determined in (2) and (3) above. Predict rates. 3. An apparatus for predicting the risk of developing systemic lupus erythematosus in a subject using a computer, comprising: (1) an apparatus for purifying genomic DNA; (2) the above (1). ), The genomic DNA purified in
Device for determining the genotype of at least one polymorphism selected from the group consisting of (a) c. In human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. 695 genotypes; and (3) storage means for storing a score table in which a predetermined score is associated with the polymorphic genotype described in (2) above; and (4) determined in (2) Searching the score table stored in the storage means of (3) based on the determined genotype,
Means for extracting corresponding score data, and outputting all of the extracted score data and the total score of the scores. 4. An apparatus for predicting the risk of developing systemic lupus erythematosus in a subject using a computer, comprising: (1) a systemic lupus erythematosus susceptibility selected from the group consisting of: Polymorphic gene; (a) c. In human CD19 gene. 705G>T; (b) IVS14-30C in human CD19 gene
>T; (c) c. ^* 132 (G
T) _12-18 ; and (d) c. In human FCGR2B. Storage means for storing a score table in which a predetermined score is associated with a genotype of 695T>C; and (2) the storage of (1) based on the genotype determined for the selected polymorphism. Means for searching the score table stored in the means, extracting corresponding score data, and outputting all of the extracted score data and the total score of the scores. 5. A computer, (1) determining the genotype of at least one of the following polymorphisms in the obtained genomic DNA sample; (a) c. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. Genotype of 695; and (2) when the polymorphism selected in (1) above is the polymorphism of (d) above and the genotype is a polymorphism resulting in codon 232T / T And (e) human leukocytes HL
Determining the presence or absence of A-DRB1 ^* 1501; (3) Risk of developing systemic lupus erythematosus in a subject by determining the risk of developing SLE based on the genotypes determined in (1) and (2) above. Predicting rate; a program for performing. 6. (1) Purifying the genomic DNA in a computer; (2) Regarding the genomic DNA purified in (1) above,
Determining the genotype of at least one polymorphism selected from the group consisting of: (a) c. In the human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) number of repetitions; (d) c. In human FCGR2B. 695 genotype; and (4) based on the genotype determined in (2) above
The genotype of the polymorphism described in 1. is searched for the score table stored in the storage means for storing the score table in which a predetermined score is associated, and the corresponding score data is extracted and extracted. Outputting all of the score data and the total score of the scores; (5) Predicting the onset risk of systemic lupus erythematosus in a subject from the total score obtained in (4); . 7. A method of predicting the risk of developing systemic lupus erythematosus in a subject, the method comprising: (1) Purifying genomic DNA; (2) Regarding the genomic DNA purified in (1) above,
Determining the genotype of at least one polymorphism selected from the group consisting of: (a) c. In the human CD19 gene. 705 genotype; (b) IVS14-30 genotype in the human CD19 gene; (c) c. ^* 132 (G
T) the number of repeats; and (d) c. In human FCGR2B. Genotype of 695; (4) based on the genotype determined in (2) above (2)
The score table in which a predetermined score is made to correspond to the polymorphism of the polymorphism described in is extracted, and the corresponding score data is extracted, all of the extracted score data,
And outputting the total score of the scores; and predicting the risk of developing systemic lupus erythematosus in the subject from the total score obtained in (5) and (4). 8. A polynucleotide represented by the nucleotide sequence 1 selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 1 to 8. 9. The modified polynucleotide according to claim 8, wherein one or several nucleotides are deleted, substituted or added at a site in the sequence other than where the polymorphic gene is present. 10. Position 2553 or 851 of SEQ ID NO: 1
A fragment consisting of a polynucleotide having a continuous nucleotide sequence of 10 to 30 nucleotides contained in SEQ ID NO: 1 containing 1 nucleotide at position 8. 11. A fragment consisting of a polynucleotide having a contiguous base sequence of 15 to 30 bases contained in SEQ ID NO: 5, which contains 15 nucleotides present at positions 194 to 223 of SEQ ID NO: 5. 12. A fragment consisting of a polynucleotide comprising a contiguous base sequence of 18 to 30 bases contained in SEQ ID NO: 6 which includes 18 nucleotides present at positions 194 to 229. 13. A fragment comprising a polynucleotide having a contiguous base sequence of 15 to 30 bases contained in SEQ ID NO: 7, which contains 1 nucleotide at the 304th position of SEQ ID NO: 7. 14. A fragment consisting of a polynucleotide having a contiguous base sequence of 15 to 30 bases contained in SEQ ID NO: 8, which contains 1 nucleotide at the 386th position of SEQ ID NO: 8. 15. A polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 9.