WO2007136205A1

WO2007136205A1 - Markers for diagnosis of autoimmune diseases and method for diagnosis using the same

Info

Publication number: WO2007136205A1
Application number: PCT/KR2007/002444
Authority: WO
Inventors: Young-Wook Song; Sung-Hoon Kim; Jung-Min Han
Original assignee: Seoul National University Industry Foundation; Imagene Co Ltd
Current assignee: Seoul National University Industry Foundation; Imagene Co Ltd
Priority date: 2006-05-19
Filing date: 2007-05-18
Publication date: 2007-11-29
Anticipated expiration: 2008-11-19

Abstract

The present invention relates to a marker for diagnosis of autoimmune diseases and a method for diagnosis of autoimmune diseases using the same, and more particularly to a marker for diagnosis of autoimmune diseases that is based on polymorphism of gp96, and a kit, a microarray and a method for diagnosis of autoimmune diseases using the same. The marker for diagnosis of autoimmune diseases according to the present invention and the kit, the microarray and the method for diagnosis of autoimmune diseases using the same may be useful to promptly and accurately diagnose the autoimmune diseases.

Description

MARKERS FOR DIAGNOSIS OF AUTOIMMUNE DISEASES AND METHOD FOR DIAGNOSIS USING THE SAME

Technical Field

The present invention relates to a marker for diagnosis of autoimmune diseases and a method for diagnosis using the same, and more particularly to a marker for diagnosis of autoimmune diseases on the basis of polymorphism of gp96, and a kit, a microarray and a method for diagnosis of autoimmune diseases using the same .

Background Art

Generally, it has been known that gp96, which belongs to a family of heat shock proteins (HSP) , activates dendritic cells to induce adaptive immune response . The gp96 is a protein belonging to a family of

HSP90 (heat shock protein 90) that is present in endoplasmic reticulum. Dendritic cells are matured and activated if the gp96 is translocated and secreted into a cell surface with the attacks of necrosis or infections. If the gp96 is expressed in tumor cells, the tumors are degenerated by the immune response by T lymphocyte, indicating that the translocation of the gp96 plays an important role in mediating immune response.

Meanwhile, autoimmune response in human body takes part in inducing various diseases or conditions, for example systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, Hashimoto's thyroiditis, psoriasis, systemic sclerosis inflammatory bowel diseases and myasthenia gravis. The diseases caused by this autoimmune response are referred to as autoimmune diseases. However, reliable methods which can diagnose the autoimmune diseases remain to be proposed.

A plurality of treatises and patents are cited through this entire application and its contexts are presented in parentheses. Disclosures of the cited treatises and patents are incorporated herein by reference in their entirety, and therefore the scopes and spirits in the art to which the present invention belongs and the contexts of the present invention will be described in more detail .

Disclosure of the Invention

Technical Problem

The present inventors have conducted ardent attempts to develop a method capable of easily diagnosing autoimmune diseases (particularly, systemic lupus erythematosus) , and found a single nucleotide polymorphism (SNP) and a deletion in a gp 96 gene which can discern the autoimmune diseases at a genetic level, and that anti-gp96 antibody is present at a large amount in sera of patients with the autoimmune diseases. Therefore, the present invention was completed on the basis of the above-mentioned facts.

Technical solution

Accordingly, the present invention has been made to solve the above-mentioned problems in the prior art, and an object of the present invention is to provide polynucleotides or a marker for diagnosis of autoimmune diseases which are useful to diagnose autoimmune diseases

Another object of the present invention is to provide a method for diagnosis of autoimmune diseases.

Still another object of the present invention is to provide a kit for diagnosis of autoimmune diseases, or a microarray for diagnosis of autoimmune diseases.

Yet another object of the present invention is to provide a composition for diagnosis of autoimmune diseases .

The other objects and advantages of the present invention will be described in more detail with reference to the detailed description, the appended claims and the accompanying drawings of this application.

In accordance with one aspect of the present invention, there is provided a marker for diagnosis of autoimmune diseases, comprising at least one isolated polynucleotide selected from the group consisting of: an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base C at a position 1471 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 3991 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base A at a position 13679 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including 2476^th-2478^th nucleotide deletion from a polynucleotide set forth in SEQ ID NO: 2; and their complementary polynucleotides. In accordance with another aspect of the present invention, there is provided a kit for diagnosis of autoimmune diseases, the kit comprising a primer set that is able to amplify one isolated polynucleotide selected from the group consisting of the isolated polynucleotide including the 194^th nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 1471^st nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 3991^st nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 13679^th nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 17115^th nucleotide of SEQ ID NO: 1; and the isolated polynucleotide including the region in which 2476^th-2478^th nucleotides are deleted from a polynucleotide set forth in SEQ ID NO: 2.

In accordance with still another aspect of the present invention, there is provided a microarray for diagnosis of autoimmune diseases, comprising at least one isolated polynucleotide selected from the group consisting of: an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base C at a position 1471 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 3991 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base A at a position 13679 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including 2476^th-2478^th nucleotides deletion from a polynucleotide set forth in SEQ ID NO: 2; and their complementary polynucleotides.

In accordance with still another aspect of the present invention, there is provided a method for diagnosis of autoimmune diseases, the method comprising: a) obtaining a nucleic acid sample from a specimen; b) amplifying a polynucleotide sequence including at least one polymorphic site in the nucleic acid sample obtained in the step a) , the polynucleotide sequence being selected from the group consisting of a polynucleotide sequence including a 194^th nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 1471^st nucleotide of SEQ ID NO : 1, a polynucleotide sequence including a 3991^st nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 13679^th nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 17115^th nucleotide of SEQ ID NO: 1, and a polynucleotide sequence including a region in which 2476^th-2478^th bases are deleted from a polynucleotide sequence set forth in SEQ ID NO: 2; and c) determining a base of the amplified polymorphic site of the step b) .

In accordance with still another aspect of the present invention, there is provided an isolated polynucleotide selected from the group consisting of: an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a region in which 247β^th-2478^th nucleotides are deleted from a polynucleotide set forth in SEQ ID NO: 2; and their complementary polynucleotides.

In accordance with still another aspect of the present invention, there is provided a composition for diagnosis of autoimmune diseases, comprising an antigen protein specific to autoantibody against gp96.

In accordance with yet another aspect of the present invention, there is provided a method for diagnosis of autoimmune diseases, the method comprising:

(a) contacting an antigen protein specific to autoantibody against gp96 with a detection sample;

(b) forming an autoantibody-antigen complex; and (c) comparing an amount of the formed autoantibody- antigen complex with that of the control .

Hereinafter, the present invention will be described in detail .

First, the present inventors have found a single nucleotide polymorphism (SNP) and a deletion which are able to discern autoimmune diseases at a genetic level in patients with the autoimmune diseases, particularly systemic lupus erythematosus (SLE) (see Example 1) . Meanwhile, the present inventors confirmed that an AMIPl protein binds to gp96 in screening a protein binding to the AIMPl protein (results not shown) . The AIMPl (ARS- interacting multi-functional protein 1) protein was known to be p43 protein in the prior art, and renamed by the present inventors (Sang Gyu Park, et al . , Trends in Biochemical Sciences, 30:569-574, 2005). The binding of the AIMPl to gp96 was re-confirmed using coimmunoprecipitation (see FIG. 4) . The AIMPl protein consists of 312 amino acids and binds to a mult-tRNA synthetase complex (Deutscher, M. P., Method Enzymol , 29, 577-583, 1974; Dang C. V. et al . , Int. J. Biochem. 14, 539-543, 1982; Mirande, M. et al . , EMBO J. 1, 733-736, 1982; Yang D. C. et al . , Curr. Top Cell. Regul . 26, 325- 335, 1985) to enhance a catalytic activity of the mult- tRNA synthetase (Park S. G. et al . , J^". Biol. Chem. 274, 16673-16676, 1999) .

Because a deleted gp96 which is deleted at a position 791 amino acid was found in SLE patients, the present inventors examined a binding affinity of the AIMPl to the deleted gp96. As a result, they confirmed that the affinity of the AIMPl to the deleted gp96 is significantly reduced, compared to wild-type gp96 (see FIG. 5) . From the above results, it was considered that mutation of the gp96 reduces the affinity to the AMPl to break the binding to the AIMPl, and then the expression level of the gp96 in the cell surface increases, which leads to the induction of autoimmune response. In order to confirm the above results more specifically, an expression level of the gp96 in the cell surface was analyzed on peripheral blood mononuclear cells (PBMCs) of SLE patients. As a result, it was revealed that the gp96 was expressed in the cell surface at a more increased level than the normal control (see FIG. 6) . Also, the present inventors confirmed that the autoantibody against gp96 is present at a large amount in sera of the SLE patients (see FIG. 7) . Therefore, the autoantibody against gp96 may be used as an effective marker to diagnose autoimmune diseases.

The SNP or deletion of the present invention may be applied to the diagnosis of all the autoimmune diseases, preferably systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, Hashimoto's thyroiditis, psoriasis, systemic sclerosis, inflammatory bowel diseases and myasthenia gravis, more preferably systemic lupus erythematosus and rheumatoid arthritis, and the most preferably systemic lupus erythematosus.

According to one preferred embodiment of the present invention, the following polynucleotides are more effectively used in the above-mentioned SNP and deletion as the marker for diagnosis of autoimmune diseases, wherein the polynucleotides are selected from the group consisting of an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a which is substituted with a base C at a position 1471 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a base sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 3991 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base A at a position 13679 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a site in which 2476^th-2478^th nucleotides are deleted from a polynucleotide set forth in SEQ ID NO: 2 ; and their complementary polynucleotides.

According to one preferred embodiment of the present invention, a nucleotide sequence encoding gp 96 deletion is a polynucleotide sequence including the region in which 2476^th-2478^th nucleotidesare deleted from a polynucleotide set forth in SEQ ID NO: 2. As used herein, the term "nucleotide" is referred to as deoxyribonucleotide or ribonucleotide that is present in a single-strand or double-strand form, and includes natural nucleotide analogues, unless stated otherwise (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543- 584 (1990) ) .

As used herein, the term "isolated" means a material that is separated from its original environment (i.e., a natural environment if it occurs naturally) . For example, naturally occurring nucleic acids or polypeptides, or cells present in living animals are not something isolated, but the same polynucleotides, polypeptides or cells separated from some or all of coexisting materials are something isolated if they are reintroduced into the natural system later.

Meanwhile, the present invention relates to alleles including a nucleotide selected from the group consisting of a nucleotide which is changed into a base G at a position 194; a nucleotide which is changed into a nucleotide C at a position 1471; a nucleotide which is changed into a base T at a position 3991; a nucleotide which is changed into a base A at a position 13679; and a nucleotide which is changed into a base T at a position 17115 in the polynucleotide sequence of a gp 96 gene set forth in SEQ ID NO: 1, as described above, but it is interpreted that the alleles include polynucleotide sequences complementary to the above-mentioned polynucleotide sequences if these allelic polynucleotides are found in double-strand gDNA (genomic DNA) .

In this aspect, it is understood that all the sequences of this specification are based on the sense strands within the gDNA, unless particularly stated otherwise .

If the genetic marker of the present invention is used to diagnose autoimmune diseases, nucleic acid molecules are isolated from a specimen in advance. As used herein, the term "diagnosis" includes all kinds of diagnoses used to predict and determine outbreaks of diseases or to deduce the outbreaks of diseases.

As used herein, the term "polymorphism" means occurrences of at least two substituted sequences or substituted alleles in the genetically determined populations. A polymorphic marker preferably has two alleles having a frequency of at least 1 %, more preferably at least 10 % or 20 % in the selected populations.

As used herein, the term "nucleic acid molecule" means a molecule that comprehensively includes DNA (gDNA and cDNA) molecules and RNA molecules.

In the present invention, the nucleic acid molecules may be obtained from various specimens, for example tissues, cells, blood, lymph, bone marrow aspirate, saliva, urine, feces, tears, semen, brain extracts, spinal fluid, joint fluid, thymic fluid, ascitic fluid, amniotic fluid and cellular tissue fluid.

If the genetic marker of the present invention is used to diagnose autoimmune diseases, the isolation of gDNA is carried out according to the conventional methods known in the art if the gDNA is used as a starting material (see Rogers & Bendich (1994)).

If mRNA is used as a starting material, the total RNA is isolated according to the conventional methods known in the art (see Sambrook, J. et al . , Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al . , Plant MoI. Biol. Rep., 9:242 (1991); Ausubel, F.M. et al . , Current Protocols in Molecular Biology, John Willey & Sons (1987) ; and Chomczynski, P. et al . , Anal .Biochem. 162:156 (1987)). The isolated total RNA is synthesized into cDNA using reverse transcriptase. Because the total RNA is isolated from plant cells, mRNA has a poly-A tail at its terminus, and cDNA may be easily synthesized with oligo-dT primers and reverse transcriptase, wherein the oligo-dT primers are designed and synthesized on the basis of these sequence characteristics (see PNAS USA, 85:8998 (1988); Libert F, et al . , Science, 244:569 (1989); and Sambrook, J. et al . , Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) .

The examination on the presence of a target gene using the genetic marker of the present invention may be carried out according to the various modified methods known in the art. For example, techniques that may be used in the present invention includes, but is not particularly limited to, fluorescence in situ hybridization (FISH) , direct DNA sequencing, PFGE analysis, Southern blotting analysis, single-strand conformation analysis (SSCA, Orita et al . , PNAS, USA 86:2776 (1989)), RNase protection assay (Finkelstein et al . , Genomics, 7:167 (1990)), dot-blot assay, denaturing gradient gel electrophoresis (DGGE, Wartell et al . ,

Nucl .Acids Res., 18:2699 (1990)), a method using proteins

(i.e., mutS protein from E. coli) which recognize nucleotide mismatches (Modrich, Ann. Rev. Genet., 25:229-

253 (1991)), and allele-specific PCR.

The changes in sequences lead to the difference in the binding of single-stranded intracellular bases, resulting in appearance of bands with different mobility. At this time, the bands are detected using the SSCA. The sequences having mobility different from that of a wild- type sequence are also detected using the DGGE analysis or TDGS (Two-Dimensional Gene Scanning) analysis.

Other techniques are generally carried out using probes or primers which are complementary to the nucleotide sequence including the SNP nucleotide of the present invention.

For example, a riboprobe that is complementary to the sequence including the SNP nucleotide of the present invention is used in the case of the RNase protection assay. The isolated DNA or mRNA is hybridized with the riboprobe, and then digested with an RNase A enzyme that can detect nucleotide mismatches. Smaller bands are observed if the nucleotide mismatches are recognized by the RNase A.

A probe complementary to the sequence including the SNP nucleotide of the present invention is used in the case of the analysis using a hybridization signal. Hybridization signals of the probe and a target sequence are detected to directly determine the presence of autoimmune diseases in this technique. As used herein, the term "probe" means a natural or modified monomer, or a linear oligomer having a bond(s), wherein the natural or modified monomer includes deoxyribonucleotides and ribonucleotides that can be hybridized with a specific nucleotide sequence. Preferably, the probe is present in a single-strand form for the purpose of the maximum efficiency in hybridization. The probe is preferably deoxyribonucleotide . A nucleotide sequence that is perfectly complementary to the sequence including the SNP nucleotide may be used as the probe used in the present invention, but nucleotide sequences that are substantially complementary to the sequence including the SNP nucleotide may be used without obstructing the specific hybridization. Preferably, the probe used in present invention includes sequences that may be hybridized with the sequences including the above- mentioned genetic marker. More preferably, a 3'- or 5'- terminus of the probe has a nucleotide that is complementary to the SNP base. Generally, stability of a duplex formed through the hybridization tends to be determined by the consensus of the terminal sequences, and therefore the duplex may be broken down under stringent conditions if the terminal region of the probe having a nucleotide complementary to the SNP nucleotide is not hybridized with the 3'- or 5' -terminus of the probe . The condition that is suitable for the hybridization may be determined with reference to the context disclosed in Joseph Sambrook, et al . , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al . , Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D. C. (1985) . The stringent condition used in the hybridization may be determined by adjusting the temperature, the ionic strength (concentration of buffer) and the presence of compounds such as organic solvent . This stringent condition may be differently determined, depending on the sequences to be hybridized.

In order to diagnose autoimmune diseases, a primer set, which is designed to amplify the above-mentioned genetic marker, is basically used to amplify a genetic marker. The amplification techniques that may be used in the present invention include, but is not particularly limited to, PCR amplification (see Miller, H. I. (WO89/06700) and Davey, C. et al . (EP 329,822)), ligase chain reaction (LCR, Wu, D. Y. et al . , Genomics 4:560

(1989) ) , polymerase/ligase chain reaction (Barany, PCR

Methods and Applic, 1:5-16 (1991)), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), 3SR (Kwoh et al., PNAS, USA, 86:1173 (1989)) and NASBA (U.S. Pat.No. 5,130,238). The gene amplification is the most preferably carried out according to the PCR amplification steps.

The design of primers is important if the amplification technique is applied to the method according to the present invention. Preferably, a forward or reverse primer in the primer set includes a nucleotide sequence including a nucleotide selected from the group consisting of a nucleotide which is changed into a base G at a position 194; a nucleotide which is changed into a base C at a position 1471; a nucleotide which is changed into a base T at a position 3991; a nucleotide which is changed into a base A at a position 13679; and a nucleotide which is changed into a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1, the nucleotide sequence being able to be annealed to a nucleotide sequence adjacent to an autoimmune diseases-related single nucleotide polymorphism (SNP) base, and the 3' -terminus of the forward primer has a nucleotide complementary to the SNP base. More preferably, the 3 '-terminus of the forward primer has a nucleotide complementary to the SNP nucleotide and the immediately adjacent nucleotide sequence has a non-complementary nucleotide that is not matched to the SNP base . The conditions of the PCR method and the reagents and enzymes used in the PCR method widely known in the art may be used herein. Meanwhile, a product amplified by the PCR method may be confirmed using the conventional electrophoretic methods such as agarose-gel electrophoresis. That is to say, in order to determine the presence of autoimmune diseases, the PCR-amplified product is loaded onto an agarose gel to observe the presence of bands . The present invention provides a kit for diagnosis of autoimmune diseases. The kit of the present invention includes the above-mentioned primers as essential components, and therefore the detailed description of the primers is applicable in the same manner as in the primers used for the kit of the present invention. Accordingly, the detailed description of the primers is omitted in order to avoid excessive complexity of this specification caused by the repeated description of the same context. If the kit of the present invention is used in the PCR amplification procedure, the kit of the present invention may selectively" include reagents required for PCR amplification, for example buffer, DNA polymerase (for example, thermostable DNA polymerase obtained from Thermus aquaticus (Taq) , Thermus thermophilus (Tth) , Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu) ) , DNA co-polymerase and dNTPs . The kit of the present invention may be made of a plurality of packagings or compartments including the above reagent components.

The present invention provides a microarray for diagnosis of autoimmune diseases, the microarray including at least one isolated polynucleotide selected from the group consisting of an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base C at a position 1471 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 3991 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base A at a position 13679 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a site in which 2476^th-2478^th nucleotides are deleted from a polynucleotide set forth in SEQ ID NO: 2; and their complementary polynucleotides.

The microarray may include DNA or RNA polynucleotides. The microarray includes the same components with the conventional microarray, except using the polynucleotide of the present invention as the probe.

A method for preparing a microarray by immobilizing the probe polynucleotides onto a substrate has been widely known in the art. A process of immobilizing the autoimmune disease-related probe polynucleotides of the present invention onto a substrate may also be easily carried out using these conventional techniques. Also, the hybridization of nucleic acids onto the microarray and the detection of the hybridization product have been widely known in the art. The detection of the hybridization product may be carried out, for example by labeling a nucleic acid sample with a marker that can generate a detectable signal including fluorescent materials, for example materials such as Cy3 and Cy5, followed by hybridizing the nucleic acid sample onto a microarray and detecting a signal generated from the marker .

The present invention provides a method for diagnosis of autoimmune diseases, the method including: a) obtaining a nucleic acid sample from a specimen; b) amplifying a polynucleotide sequence including at least one polymorphic site in the nucleic acid sample obtained in the step a) , the polynucleotide sequence being selected from the group consisting of a polynucleotide sequence including a 194^th nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 1471^st nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 3991^st nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 13679^th nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 17115^th nucleotide of SEQ ID NO: 1, and a polynucleotide sequence including a region in which 2476^th-2478^th nucleotides are deleted from the polynucleotide sequence set forth in SEQ ID NO: 2; and c) determining a base of the amplified polymorphic site of the step b) .

As used herein, the term "polymorphic site" refers to a site in which a single nucleotide polymorphism or a deletion occurs in the polymorphic sequence.

The step of obtaining nucleic acids from a specimen (Step a) may be carried out according to the conventional DNA isolation method.

The step of amplifying a polymorphic site (Step b) may be carried out according to the conventional amplification method. For example, a target nucleic acid may amplified using a PCR method and obtained through a purification process. In addition, ligase chain reaction

(LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren, et al . , Science 241, 1077 (1988)), transcription amplification (Kwoh, et al . , Proc.Natl .Acad. Sci . USA 86,

1173 (1989) ) , self-sustained sequence replication

(Guatelli, et al . , Proc. Natl. Acad. Sci. USA 87, 1874 (1990)) and nucleic acid-based sequence amplification

(NASBA) may be used herein. The determination of the nucleotide of the polymorphic site in the step c) may be carried out using at least one method selected from the group consisting of sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization (DASH) , PCR extension assay, PCR-RFLP assay and TaqMan technique .

Also, the present invention provides a composition for diagnosis of autoimmune diseases, the composition including an antigen protein that is specific to autoantibody against gp96. Preferably, the antigen protein is a gp96 protein, and more preferably a gp96 protein having an amino acid sequence set forth in SEQ ID

NO: 3. The composition for diagnosis of autoimmune diseases according to the present invention includes tools, reagents and the like which are used for the immunological analysis and widely known in the art, as well as an antigen protein capable of selectively recognizing the autoantibody against gp96. The tools/reagents include, but are not particularly limited to, a suitable carrier, a marker that can generate a detectable signal, a solubilizing agent, a detergent, a buffer agent, a stabilizing agent, etc. If the marker is an enzyme, the tool/reagent may include a substrate that can detect activity of the enzyme, and a reaction terminator. The suitable carrier may include, but is not particularly limited to, a soluble carrier such as a physiologically available buffer known in the art, for example PBS; and an insoluble carrier such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorocarbon resin, crosslinked dextran, polysaccharide, polymers such as magnetic microparticles in which Latex is coated with a metal, other papers, glass, metals, agarose, and combination thereof . The composition for diagnosis of autoimmune diseases according to the present invention may have shapes of, but is not particularly limited to, an ELISA plate, a dipstick device, an immunochromatographic test strip, a radial partition immunoassay device, and a flow- through device.

Also, the present invention provides a method for diagnosis of autoimmune comprising contacting an antigen protein specific to autoantibody against gp96 with a detection sample; forming an autoantibody-antigen complex; and comparing an amount of the formed autoantibody-antigen complex with that of the control.

As used herein, the term "detection sample" means a biological sample, such as tissues, cells, whole blood, serum, plasma, saliva, semen, cerebrospinal fluid or urine, that can detect the difference in amount of expressed marker proteins caused by the autoimmune diseases induction, and the sample is prepared through the treatment according to the methods widely known in the art .

As used herein, the term "autoantibody-antigen complex" means an antigen complex that specifically recognizes autoantibody against gp96 in a biological sample. An experimental method used to confirm the formation of the autoantibody-antigen complex includes, but is not particularly limited to, Immunohistological staining, Radioimmunoassay (RIA) , Enzyme-Linked Immunosorbent Assay (ELISA) , Western Blotting, Immunoprecipitation Assay, Immunodiffusion Assay, Complement Fixation Assay, FACS, protein chip, etc.

A labeling method that may qualitatively or quantitatively measure the autoantibody-antigen complex includes, but is not essentially limited to, an enzyme, a fluorescent material, a ligand, a phosphor, microparticle, a redox molecule, a radioactive isotope, etc. The enzyme that may be used as a detection label includes, but is not particularly limited to, β- glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxydase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, β-lactamase, etc. The fluorescent material includes, but is not particularly limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescent carmine, etc. The ligand includes, but is not particularly limited to, biotin derivatives, etc. The phosphor includes, but is not particularly limited to, acrydium ester, luciferin, luciferase, etc. The microparticle includes, but is not particularly limited to, colloid gold, colored latex, etc. The redox molecule includes, but is not particularly limited to, ferrocene, ruthenium complexes, biologen, quinone, Ti ion, Cs ion, diimide, 1, 4-benzoquinone, hydroquinone, K₄W(CN)₈/

[Os(bpy)₃]²⁺, [RU(bpy)₃]₂ ⁺, [MO(CN)₈]₄ ^', etc. The radioactive isotope includes, but is not particularly limited to, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I , ¹³¹I , ¹⁸⁶Re , etc .

The autoimmune diseases may be diagnosed by examining, as an absolute difference (i.e., μg/mβ) or a relative difference (i.e., relative strength of a signal) , a difference in the amount of the formed autoantibody-antigen complex between the control and the detection sample.

According to the present invention, the diagnosis of autoimmune diseases, which is required for collecting complex data, may be carried out promptly and accurately.

Hereinafter, the preferred embodiments of the present invention have been described in detail . However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, as defined in the appended claims.

Advantageous Effects The maker for diagnosis of autoimmune diseases according to the present invention is relatively closely associated with the autoimmune diseases. The diagnosis of autoimmune diseases is required for collecting complex data, however, the maker for diagnosis of autoimmune diseases according to the present invention aids to promptly and accurately diagnose the autoimmune diseases. Accordingly, the kit for diagnosis of autoimmune diseases and the microarray and method for diagnosis of autoimmune diseases using the marker according to the present invention may be useful to promptly and accurately diagnose the autoimmune diseases. Brief Description of the Drawings

The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

FIG. 1 is a diagram showing a two-dimensional gene scanning (TDGS) process that is carried out so as to examine a single nucleotide polymorphism (SNP) and a deletion according to the present invention.

FIGs 2 to 4 are a photographic diagram showing typical results obtained according to the TDGS process.

FIG. 5 is a diagram showing a gene scan result obtained to confirm an amino acid deletion specifically. FIG. 6 is a diagram showing that proteins isolated from HL-60 cell are co-immunoprecipitated with autoantibody against gp96.

FIG. 7 is a diagram showing an immunoblotting result examining whether or gp96 deletion mutant (E791Δ) binds to AIMPl.

FIG. 8 is a graph showing an expression level of gp96 on a cell surface of PBMCs of healthy controls and

SLE patients.

FIG. 9 is a graph showing a titer of autoantibody against gp96 in sera isolated from healthy controls and SLE patients. Best Mode for Carrying Out the Invention

Hereinafter, preferred aspects of the present invention will be described with reference to the accompanying drawings, so that the present invention may be easily put into practice by those skilled in the art.

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

However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, so various changes and modifications may be made without departing from the spirit and scope of the invention

Analysis for Detection of SNP in SLE Patient

In order to examine a single nucleotide polymorphism (SNP) in a systemic lupus erythematosus (SLE) patient, possible SNPs of all exons in the gp96 were analysed for 216 SLE patients and 215 normal controls using the two-dimensional gene scanning (TDGS) process. The TDGS process is an effective technique that can find all of the possible SNPs of the entire genes in one gel. This technique is based on an automated two- dimensional (2 -D) DNA electrophoresis system according to the base-pair sequence and its size, wherein the electrophoresis system detects DNA fragments having all possible mutations. The 2 -D electrophoresis system provides enormous megaplex PCRs since it may be used to diagnose up to 40 fragments having 250 base pairs, which leads to the cost effectiveness.

Genomic DNA used in this experiment is a (disease- free) sample of a normal human whose age/sex is identical to those of the SLE patient. The genomic DNA was amplified by using 0.5 μJL of DNA polymerase Taq (Roche), and 5 βl of 1OX buffer, 1 βg of DNA, 1 μJt of 10 nmol sense primer, 1 μJL of antisense primer were added to the amplified genomic DNA, and the dNTP mixture was adjusted to 1 mM, and then the total volume of the resultant mixture was adjusted to 50 βi with DNase/RNase-free distilled water (DW) . Sequences of the used primers are listed in the following Table 1. The PCR condition was total 35 cycles at an annealing temperature of 60 ^°C for 1 minute 30 seconds.

Table 1

Primer Set used for SNP amplification L Ot gp96

Primers Sequences SEQ ID NOs. gp96-l sense 5' - CGC GAT TGG TGG GTT CAT GT -3 ' 4 gp96-l antisense 5' - CTG CTC CTC CAG AAT CAC TC -3' 5 gp96-2 sense 5' - TCC TTG GAG AAG TGT GAT GT -3' 6 gp96-2 antisense 5¹ - GTT TTC ATC ACT CCT AGG GA — ^"} * 7 gp96-5 sense 5'- AGT TTG GTG TCG GTT TCT ATT -3' 8 gp96-5 antisense 5¹ - TAT AAG GAA TTG GTC ATA CTC AC 9 -3' gp96-14 sense 5' - TGC TCA ATG ACC TTA CCT GT -3¹ 10

A PCR product was confirmed in 1.5% agarose gel, and the unnecessary dNTP and primers were removed from the PCR product using a LABO PASS PCR purification kit (Cosmogenetech) and the purified PCR product was sequenced according to the Macrogen sequencing method.

Multiple alignments of the normal sample and the SLE sample were carried out, respectively, to diagnose a polymorphical difference between the samples. In order to effectively screen a single nucleotide polymorphism in each of the samples, the TDGS process was carried out according to the method as shown in FIG. 1. FIG 2 is a photographic diagram showing typical results obtained according to the TDGS process. As shown in FIG. 1, circles represent heteromutations .

A representative example of the TDGS results is shown in FIG. 2. Through the TDGS process, the present inventors identified 5 single nucleotide polymorphisms (g.l94C>G, g.l471G>C (reference SNP: rsl7034931) , g.3991C> T (reference SNP: rs6615) , g.l3679G> A

(reference SNP: rsl882019) , g.l7115C> T) and one amino acid deletion (+2476_8delGAA) in the genomic DNA of the gp96 gene. Among them, the single nucleotide polymorphisms (g.l94C> G (p=0.008), g.3991C> T (p=0.023), g.l3679G> A (p=0.008)) and the amino acid deletion (+2476_8delGAA, p=0.005) were closely associated with the outbreak of systemic lupus erythematosus (Table 2 and Table 3) .

Table 2

Table 3

Table 4

In order to identify the amino acid deletion, the gene scan was carried out using a more effective method. A fluorescent dye 6-FAM was bound to a gp96-17-sense primer (SEQ ID NO: 12) as listed in the Table 1 to synthesize a primer β-FAM-TTATACATATAAATGAGCAAAGTGG. PCR amplification was carried out using a gp96-17-antisense primer (SEQ ID NO: 13) as a reverse primer and the primer β-FAM-TTATACATATAAATGAGCAAAGTGG as a forward primer, and the dimers and dNTPs remaining after the PCR amplification were removed by PCR purification, and then the gene scan was carried out .

The genomic DNA was amplified by using 0.5 βi of DNA polymerase Taq (Roche) , and 5 fd of 1OX buffer, 1 βg of DNA, 1 μi of 10 nmol sense primer, 1 μJt of 10 nmol antisense primer were added to the amplified genomic DNA, and the dNTP mixture was adjusted to 1 mM, and then the total volume of the resultant mixture was adjusted to 50 μl with DNase/RNase-free distilled water (DW) . The PCR reaction consisted of: pre-denaturation of the template DNA at 95 ^°C for 7 minutes, followed by 30 cycles of 1 min at 95 ^°C , 1 min 30 sec at 53 ^°C , 1 min at 72 "C, and then a final extension step for 7 min at 72 ^°C .

From the gene scan results, it was confirmed that peaks corresponding to a 294bp fragment and a 297bp fragment were present, and the peaks of the PCR-amplified products may be classified into three groups: a single peak appears at 297 bp in the case of the wild/wild types at the amino acid position 791, two peaks appear at 297 bp and 294 bp in the case of the wild/deleted types at the amino acid position 791, and one peak appears at 294 bp in the case of the deleted/deleted types at the amino acid position 791 (FIG. 5) . From the gene scan results of the samples from the SLE patients and the normal controls, it was confirmed again that the amino acid deletion is closely associated with the induction of SLE

(see Table 4) .

<Example 2> Analyses of Binding of AIMPl to gp96 and to gp96 deletion

<2-l> Co-immunoprecipitation of AIMPl with gp96

The present inventors found that AIMPl binds to gp96 on the way of screening a protein binding to the AIMPl (data not shown) . The co-immunoprecipitation was carried out so as to confirm the binding of the AIMPl to gp96. Particularly, HL-60 cells (American Type Culture

Collection, Manassas, VA) which was transfected with a plasmid encoding an AIMPl (Ko YG, et . al . , J^" Biol Chem. , 22;276 (25) : 23028-33 , 2001) was dissolved in a lysis buffer (25 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10% glycerol,

ImM EDTA, 0.5% Triton X-IOO, 2 mM DTT, 1 mM PMSF and aprotinin) . The cells were homogenated for 5 seconds using a sonicator, and centrifuged at a rotary speed of 14,000 rpm for 15 minutes using a centrifuge, and then the resultant supernatant was taken and used as a protein extract. The extracted protein was mixed with a rabbit anti-gp96 antibody (Santa Cruz. CA) previously bound to protein A agarose, and then the precipitated protein was western-blotted with a rabbit anti-gp96 antibody (Santa Cruz. CA) and an anti-AIMPl antibody (Park S. G., et al . , J. Biol. Chem. 274:16673-16676, 1999). The results are shown in FIG. 6.

As shown in FIG. 6, it was confirmed that the gp96 binds to the AIMPl.

<2-2> Examination on Binding Affinity of AIMPl to gp96 deletion

Because the gp96 deletion in which the 791^st amino acid GIu is deleted was found in the SLE patients in the <Example 1>, a wild-type gp96- (288-799) fragment and a deleted gp96- (288-799, E791Δ) fragment were prepared, respectively, to examine whether or not the deleted gp96 binds to the AIMPl.

The wild-type gp96- (288-799) fragment and the deleted gp96- (288-799, E791Δ) fragment were synthesized by the PCR by using cDNA of the gp96 as a template and using a primer set (Table 1) specific to each of the fragments. The PCR reaction condition was as follows; a template DNA was pre-denatured by heating the template DNA at 95 ^°C for 2 minutes, the total 30 cycles were repeated at 95 ^°C for 30 seconds; at 56 ^°C for 30 seconds; and at 72 ^°C for 2 minutes, and then a final reaction was carried out at 72 ^°C for 5 minutes. The PCR products were cut with restriction enzymes EcoRI and Sail, and ligated into a pET28c vector (Novagen) previously cut with the same enzymes. E.coli BL21 was transformed with the resultant vector, cultured to induce expression of peptides. The peptide expressed as His-tag fusion proteins was purified using a nickel column. In order to remove off lipopolysaccharides, the protein solution was dialyzed in a pyrogen-free buffer (10 mM potassium phosphate buffer, pH 6.0, 100 mM sodium chloride). After the dialysis, the protein solution was loaded onto a Polymyxin resin (Bio-Rad) equilibrilized with the same buffer, cultured for 20 minutes, and then eluted to prepare fragments of the gp96, respectively.

Table 5 Primer set used to prepare fragments of gp96

The purified gp96 proteins were reacted with GST or GST-AIMPl (condition: a binding reaction solution (25 tnM Tris-HCl buffer (pH 7.4) including 120 tnM NaCl, 10 mM KCl and 0.5% Triton X-100) and iramunoblotted with a rabbit anti-gp96 antibody (Santa Cruz. CA) or an anti-AIMPl antibody (Park S. G., et al . , J. Biol. Chem. 274:16673- 16676, 1999). The results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that the gp96 deletion in which a 791^st amino acid GIu is deleted has significantly reduced binding affinity to the AIMPl, compared to the wild-type gp96. Accordingly, it was considered that the low binding affinity to the AMPl leads to the cleavage in the binding of the gp96 to the AIMPl, and therefore the gp96 is increasingly expressed onto a cell surface, thereby inducing autoimmune response.

Examinations on Expression of gp96 onto Cell Surface in SLE Patient and Production of Autoantibody against gp96

<3-l> Examination on Expression of gp96 onto Cell Surface in PBMC of SLE Patient

In order to examine an expression of gp96 onto a cell surface in the SLE patient, each of peripheral blood mononuclear cells (PBMCs) was isolated from normal controls (n=6) or SLE patients (n=12) using a Ficoll density gradient centrifugation method. The cells were washed with IxPBS, and then suspended in a FACS buffer

(Ix PBS containing 2% FBS, 1% BSA, and 0.1% sodium azide) . And, the cells was pre-treated with goat antibody, washed, and then pre-treated with a conventional goat antibody. After the washing, the gp96 and HLA-DR present in the cell surface were measured using a rabbit anti-gp96 antibody (Santa Cruz. CA) and a

HLA-DR antibody (BD Biosciences) . The results are shown in FIG. 8.

As shown in FIG. 8, it was revealed that the expression level of the gp96 is increased in the cell surface of PBMC from the SLE patients, compared to the normal controls. From these results, it was considered that the expression of the gp96 onto the cell surface is increased since the binding of the gp96 to the AIMPl was cleaved in the case of the patients with autoimmune diseases unlike the normal control.

<3-2> Analysis of Autoantibody against gp96 in Sera of SLE Patients

The titer of autoantibody against gp96 in blood was analysed using ELISA. Particularly, the gp96 was dissolved in a PBS buffer (pH 7.4) and a 96-well plate (Maxisorp., F96; Nunc) was coated with the resultant gp96 solution. The 96-well plate was washed and reacted with a blocking buffer (a PBS buffer including 1% BSA (bovine serum albumin)) for one hour. Sera were isolated from the blood samples obtained in the above, respectively, and 20 ul of each of the sera was put into each of the wells, and then 1 x PBS containg 1 % BSA was added to adjust the final volume to 100 ul . After culture for two hours, the plate was washed and cultured for 30 minutes with an HRP-bound rabbit anti-human IgG. Then the plate was washed, and a substrate reaction solution was added to each of the wells, and their absorbances were measured at 450 nm. The results are shown in FIG. 9. As shown in FIG. 9, it was revealed that the autoantibody against gp9β was produced at a larger amount in the sera of the SLE patients than those in the normal controls. From these results, it was considered that the gp96 is expressed onto the cell surface at a large amount in the case of the patient with autoimmune diseases, and therefore the autoantibody is also produced at a large amount. Accordingly, the autoantibody against gp96 may be used as an effective marker to diagnose autoimmune diseases.

Industrial Applicability

The marker for diagnosis of autoimmune diseases according to the present invention is relatively closely associated with the autoimmune diseases. The diagnosis of autoimmune diseases is required for collecting complex data, however, the marker for diagnosis of autoimmune diseases according to the present invention aids to promptly and accurately diagnose the autoimmune diseases. Accordingly, the kit for diagnosis of autoimmune diseases and the microarray and method for diagnosis of autoimmune diseases using the marker according to the present invention may be useful to promptly and accurately diagnose the autoimmune diseases.

This application claims the benefit of Korean Patent Application No. 10-2006-45379, filed on May 19, 2006, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference.

As described above, the preferred aspects of the present invention have been described in detail . However, it should be understood that the detailed description and specific examples, while indicating preferred aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, as defined in the appended claims .

Claims

Claims :

1. A marker for diagnosis of autoimmune diseases, comprising at least one isolated polynucleotide selected from the group consisting of: an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including a nucleotide which is substituted with a base C at a position 1471 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 3991 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including a nucleotide which is substituted with a base A at a position 13679 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including 2476^th-2478^th nucleotide deletion from a polynucleotide set forth in SEQ ID NO: 2; and their complementary polynucleotides.

2. The marker according to claim 1, wherein the autoimmune diseases are selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, Hashimoto's thyroiditis, psoriasis, systemic sclerosis, inflammatory bowel diseases and myasthenia gravis.

3. A kit for diagnosis of autoimmune diseases, the kit comprising a primer set that is able to amplify one isolated polynucleotide selected from the group consisting of the isolated polynucleotide including the 194^th nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 1471^st nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 3991^st nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 13679^th nucleotide of SEQ ID NO: 1; the isolated polynucleotide including the 17115^th nucleotide of SEQ ID NO: 1; and the isolated polynucleotide including the region in which 2476^th-2478^th nucleotidesare deleted from a polynucleotide set forth in SEQ ID NO: 2.

4. The kit according to claim 4, wherein a forward primer or a reverse primer in the primer set includes a nucleotide sequence being annealed a nucleotide sequence adjacent to an autoimmune diseases- related single nucleotide polymorphism (SNP) nucleotide selected from the group consisting of a nucleotide which is changed into a base G at a position 194; a nucleotide which is changed into a base C at a position 1471; a nucleotide which is changed into a base T at a position 3991; a nucleotide which is changed into a base A at a position 13679; and a nucleotide which is changed into a base T at a position 17115 in a polynucleotide set forth in SEQ ID NO: 1, and the 3 '-terminus of the forward or reverse primer has a nucleotide complementary to the SNP base.

5. A microarray for diagnosis of autoimmune diseases, comprising at least one isolated polynucleotide selected from the group consisting of: an isolated polynucleotide consisting of a nucleotide sequence of 10-

100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-

100 continuous nucleotides including a nucleotide which is substituted with a base C at a position 1471 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-

100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 3991 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including a nucleotide which is substituted with a base A at a position 13679 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10- 100 continuous nucleotides including 2476^th-2478^th nucleotides deletion from a polynucleotide set forth in SEQ ID NO: 2; and their complementary polynucleotides.

6. A method for diagnosis of autoimmune diseases, the method comprising: a) obtaining a nucleic acid sample from a specimen; b) amplifying a polynucleotide sequence including at least one polymorphic site in the nucleic acid sample obtained in the step a) , the polynucleotide sequence being selected from the group consisting of a polynucleotide sequence including a 194^th nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 1471^st nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 3991^st nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 13679^th nucleotide of SEQ ID NO: 1, a polynucleotide sequence including a 17115^th nucleotide of SEQ ID NO: 1, and a polynucleotide sequence including a region in which 2476^th-2478^th nucleotidesare deleted from a polynucleotide sequence set forth in SEQ ID NO: 2; and

c) determining a base of the amplified polymorphic site of the step b) .

7. The method according to claim 6, wherein the determination of the nucleotide in the step c) is performed using at least one method selected from the group consisting of sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization (DASH) , PCR extension assay, PCR-RFLP assay and TaqMan technique.

8. An isolated polynucleotide selected from the group consisting of: an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base G at a position 194 in a polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a nucleotide which is substituted with a base T at a position 17115 in the polynucleotide set forth in SEQ ID NO: 1; an isolated polynucleotide consisting of a nucleotide sequence of 10-100 continuous nucleotides including a region in which 2476^th-2478^th nucleotides are deleted from a polynucleotide set forth in SEQ ID NO: 2 ; and their complementary polynucleotides.

9. A composition for diagnosis of autoimmune diseases, comprising an antigen protein specific to autoantibody against gp96.

10. The composition according to claim 9, wherein the antigen protein is self gp96 protein.

11. The composition according to claim 10, wherein the gp96 protein has the amino acid sequence set forth in SEQ ID NO: 3.

12. A method for diagnosis of autoimmune diseases, the method comprising:

(a) contacting an antigen protein specific to autoantibody against gp96 with a detection sample; (b) forming an autoantibody-antigen complex; and

(c) comparing an amount of the formed autoantibody- antigen complex with that of the control.