AU785071B2 - Method of diagnosing rheumatoid arthritis - Google Patents
Method of diagnosing rheumatoid arthritis Download PDFInfo
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- AU785071B2 AU785071B2 AU10530/01A AU1053001A AU785071B2 AU 785071 B2 AU785071 B2 AU 785071B2 AU 10530/01 A AU10530/01 A AU 10530/01A AU 1053001 A AU1053001 A AU 1053001A AU 785071 B2 AU785071 B2 AU 785071B2
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Description
FROM 2002- 4 268()18:05/017:57/X -4503499396 P 3
DKSCRIWTON
Method for Dkaonmuag Rheumatoid Arthrits Techuical Field The invention of the present application relates to a method for diagnosing disease gene in human rheumatoid arthritis.
Backpound Art The condition of arthritis and joint destruction that are the cause of rheumatoid arthritis, particularly their pathological process, has gradually been elucidated through diverse studies. Many autoimmune diseases to which the rheumatoid arthritis belongs are first developed into a disease and become worse through overlapping many causal factors. In this situation, in order to make correct investigation of the diseases and an appropriate therapy therefor, the true form of multi-factorial interaction per ae has to be elucidated.
Though the incidence rate of rheumatoid arthritis (RA) is 1% or less worldwide Engl. J. Med. 322: 1277-1289, 1990), the ratio of the risk for sibling of pati-nts is approximately 8% or more (Cell. 85: 311-318. 1996), and accordingly some genetic factors have been assumed as causative factors. The molecular genetic techniques or genetic engineering techniques usually used in identification of the genetic factors of the diseases, however, do not function effectively for autoimmune diseases. The reason is that the incident mechanism of autoimmune diseases is quite different from a biologically simple mechanism in cancers in which abnormal proliferation is caused by a single gene mutation. On F RO0M NRW 20024 4M2B(118:05/0417:57/MX 4503499396 P 4 the other~ haind, the so far classical genetic techniques investigating the hereditary base of diseases have elucidated that the autoinmiune diseases are caused by a multiple factorial inheritance, but it has not be possible to follow after the inner part or the true form. Thus, in fact, with respect to the genes predisposing to rheumatoid arthritis, not only their entities but also their gene loci on the chrbmosome has not yet been elucidated.
In this situation, the inventors of the present application conducted a linkage analysis using a niicrosatelite marker on a patient with rheumatoid arthritis and his blood relatives and identified the 3 genetic loci at which the diseaae genes of the rheumatoid arthritis are located. (International Immunology 10(12): 1891-1895, 1998; Journal of Clinical Rheumatology 156-158, 1998).
The following disease genes have been filed as a Patent Application PCT/JP98/0 1665.
is Disease gene of rheumatoid arthritis in the human chromosome I whichis located within *1 centi morgan (cM) from the DNA sequence to which a rmcrosatelite marker D1IS214 and/or D1S253 hybridize.
Disease gene of rheumatoid arthritis in the human chromosome 8 which is located within :1 1 cM from the DNA sequence to wvhich a microeatelite markcer D8S556 hybridize.
Disease gene of rheumatoid arthritis in the human chromosome X which is located within il cM from the DNA sequence to which zmcrosatelite markers DXSlOO1, DXS1O47, DXS12OS, DXS1227 and/or DXS1232 hybridize.
The present inventors continued to investigate the respective genes of the diseases in the above-described earlier patent application. As results, they found that the physical positions of the markers DIS214 and DIS253 on the 084 Radiation hybrid map relating to the above disease genes are mapped on 21. 14cR3000 and 23.56cR3000, respectively, and that the death receptor gene 04/08 '06 FRI 14:06 FAX 61299255911 GRIFFITI HACK IAj008 DKl3 is located at dub flAighboT 2-2. 1 4cR3000 (Nature 384; 372-375, 1996; .Proc.
Natl; Acad. Sci. USA 94: 4615-4619. 1997; Gccovics 49: 38&-393. 1998).
The present application. is intended to provide a method for hiby predsely diagnosing a sign of rheumatoid arthritis aimning at the DR3 gene based on the above fndings made by the present inventors.
DbWclosizz of Inventiou As the invention for solving the above-described problems, there is provided in a first aspect a method for diagnosing rheumatoid arthritis which comprises ~digesting humWU genouzc DNA with a rmtficlion enzyme &zRI and dtctibng an about 7 kb DNA fragment which hybridi'ws 'with a probe having the base sequece~ oftepsto 5-2 nSQI
O
in a second aspect, the invention provides a method for diagnosing rheumatoid arthritis which comprises. digesting human. genomic DNA with a restiibon enZYMC, Dr and detcctiAng an about 1.5 kb DNA fragmertt wbich bybr2idi~ ualb, aL probe hAving th~e base uequcflCB of the positian IS9 -424 to SM ID NO: 1.
in a third aspect, the invention provides a method for diagnosing rhe~natoid arthritis which comprises stimulating the peripheral blood Mononuclear call in a subject with phorbol 12-myristate 13-acetate (PMA) and/or phytohemagglutihin (M1A), and comparing the amount of =RNA in the peripheral blood mononuclear ci-Ils 'with the armount of the same mRNA in the peripheral blood mononuclear cels in a healthy person stimulated by PMA arnd/or
PHA,
wherein the m.RNA is. expressed from the base sequence of at the least 689-760 3D posiion i SEQID NO: I 3 COMS ID No: SBMI-04365356 Received by IP Australia: Time 13:13 Date 2006-08-04 04/D8 '06 FRI 14:07 FAX 61299255911 GRIFFITH HACK 1dJ009 a In a fourth aspect, the invention provides a method for diagnosing rhuumataid ardwitis which comprises stimulating the Peripheral blood montonuclear cells in a subjec t with Phorbol 12-myristate 13-acetate (PM) MndOM S phytohemagglutii (PMA, and comparing thc anmut of a PolypePtide exRevssed in the pniheral blood mononudlear eefli vith- the amouxit of the sm polypepid exprissed in the peripheral Wlood wconnuclear aell i a'healthY peruon stzmudated by PMA and/or pHA. wherein the polpeptide has the -amino ar-id mequenee of at the least 201-224 poiiion in SEQ ID NO: 1.
7n the above diagnostic methods, the diagwass is made accoding to a criterion that the DINA or polypeptide expressed lin the RA patients is greater in quantity than that expre-ned in healthy persons Iconcretply, 2 times or more).
DiE! Demiption Of Dmwtflg mig, 1 shows the results of tbe Southern analysis for RobRl fragnzxits from the respective genonllic DNAa of RA pafients and healthy persons.
n0 Fig. 2 shows the reslts of the Soauen analysis for DrcI hmaprerts ftof the respective genomiC DNAS of RA patients and healthy persons.
Fig. 3 shows the results of quantitatively determined m'RNAB amount for 2S the =Ul transmembaie rcgom and death domain =Sion of the DR3 gene of RA patients and healthy persons.
Best Mods for Carrying Out the invenationi 4 COMS ID No: SBMI-04365356 Received by IP Australia: Time 13:13 Date (Y-Md) 2006-08-04 FROM RJRN 200 268 1:60175/ i40499 P 7 The following examples serve to explain the best mode for carrying out the invention specifically and in detail but are not intended to limit the invention.
bmaple 1 RFLP Anmlymis of ZcoRI hagment Genornic DNAs were prepared from the peripheral blood of healthy persons and RA patients according to the guanidine thiocyanate method (Nippon Yuketsu Gakkai Zasabi (Journal of the Japan Society of Blood Transfusion), 40(2): 413, 1994). Briefly, 20 ml of cell membrane lysis solution (solution 1: 0.32M sucrose, 1%v/v Triton X-100, 5mM MgCb2, 12mM This-HCI pH 7.6) was added to ml of peripheral blood containing EDTA. The mixture was aptated under upsetting and centrifuged at 3000 rpm for 10 minutes to recover the nuclei. Five ml of nuclear membrane lysis solution (solution 11: 4M guanidine thyocyanate, 12mM EDTA, 375mM NaCi, 0.5% N-lauroyl sarcosinate, 0. 1M P-mercaptoerthanol, 12mM Tris-HC1 pH 7.6) was added to the recovered nucli. The mixture was incubated at 55*C for 10 minutes and precipitated with ethanol to give the genomic: DNA.
Seventy-five urut of EcoRl (Takara Shuzo) and 20 pl of l~xil buffer tTakara, Shuzo) was added to 10 pg of the genomic DNA and the whole was made up to 200 pl with sterile water. The resulting solution was incubated at 37*C for 18 hours to digest the genoinic DNA to give EcoRi fragments. Then, 100 p1 of phenol and 100 p1 of chloroform were added to the reaction mixture, and centrifuged at 12000 rpm for 10 minutes to recover the supernatant, from which was recovered fragmented DNAs by means of a conventional ethanol precipitation.
The resulting DNA fragments were electrophoresed on 0.7% Agarose L (Nippon Gene) along with a molecular weight maker )./HindllI (Nippon Gene) in a TAE buffer at 50V for about 2 hours, The gel was immersed in a denaturation 'ROM NO 97 200X~ 4M26(BI8:6/17:57/M. 4503499396 P 8 solution (0.5M NaOH, 1.5M NaCl) for 25 minutes and then in a neutralization solution (0.5M Tris-HCl pH 7T5, 1.5M NaCi) for 30 minutes. The DNA in the gel was transferred to a nylon membrane (Hybond-N4-, Amersham FharmacWa overtnight by means of a conventional capillary technique and immobilized on the mdinbrane with a UV croas-linker (Japan Genetic). As a capillary buffer. I1OxSSC wag used.
Hybridization and detection of the DNA fragments were conducted using a Gene ImagesN labeling detection system (Amersham Pharrnaca) as follows. The membrane fixed DNA was immersed in a hybridization buffer (5xSSC, 0.l0/w/v SDS, S%w/v dextran sulfate (mol.wt. 500,000; Sigma), 5%/v/v blacing reagent (attached to the kcit)] and shaken at 60*C for 30 minutes for pre-hybridization.
DR3 cDNA was amplified by Rlr-PCR from the total RNA obtained from the peripheral blood mononuclear cells of healthy persons. An oligonucleotide having the base sequence of the 159-424 position of SEQ ID NO: 1 was prepared from this cDNA, and this was labeled with fluorescein to give a probe. This probe was heat denatured under heating at 95 0 C for 5 minutes, then added to a pre-hybridization solution at a concentration of 10 ng/rnl and the membrane was shaken at 60*C overnight for hybridization. This was washed with a buffer (lxSSC, 0.1%/w/v SDS) pre-heated at 60*C for 15 minutes, and further washed withi another buffer lxSSC, 0. 1%/w/v SDS) for 1S minutes. The membrane was rnsed roughly with Buffer A (100mM Ths-HC pH 9.5, 300mM NaCi) and blocked with a blocing agent (attached to the kit) diluted 10 times with Buffer A at room temperature for 1 hour. The membrane was rin sed roughly with Buffer A, then immersed in an antibody-diluted solution [alkaline phosphatase-labeled anti-fluorescein antibody (attached to the kIt) diluted 5,000 times with Buffer A containing 0,5%w/v BSA] and incubated at room temperature for 1 hour, The membrane was washed 3 times with Buffer A containing O.3%/v/v Thween 20 at room temperature with shaldng for 10 minutes. The membrane was rinsed FROM NI W 2OO2* 4 6()80/.A1:7 440499 P 9 roughly with Buffer A. then immersed in a detection reagent (attached to the kit) and exposed to an X-ray film (Fuji Film).
The result is as shown in Fig. 1. In the healthy persons, only a band indicating the presence of about 9 kb DNA fragment was detected. On the other hand, in the RA patients, a band indicating the presence of about 7 kb DNA fragment in addition to the about 9 kb DNA fragment was detected (polytypic bands).
From the above results, it was confirmed that the RA patient can be dutgnosed by fragmenting the genomic DNA with EcoRI and confirming the presence of the about 7 kb DNA fragmnt hybridizing with said probe.
Rumple 2 RFL? Analysis of Dm1d fragments The analysis was conducted in the same manner as in Example 1 except that the restriction enzyme DraI (Takara Shuzo) and lOxM buffer flTakara. Shuzo) were used.
The result is as shown in Fig. 2. In the healthy persons, bands of about kb or more DNA fragments were detected. On the other hand, in the RA patients, a polytypic band indicating the presence of about 1.5 kb DNA fragment in i&ddition to the about 10 kb or more DNA fragments was detected.
From the above results, it was confirmed that the RA patient can be diagnosed by fragmenting the gcnomic DNA with Dral and conirning the presence of the about 1.5 kcb DNA fragment hybridizing with said probe.
FROM NIWJ Vj FR ~2O2* 4 26B(i)l8:07/-qI7:57/Xt.#453499396 ftnuple 3 Quantificatlon of DR3 =RNA S Heparinized peripheral blood (10 ml each) collected from 6 healthy persons anid 7 RA patients was mixed with an equal volume of a phosphate buffer (PBS) and layered on 20 ml of Lymphoprer (Daiichi Pure Chemicals). The mixture was centrifuaged at 1,500 rpm for 30 minutes to collect the peripheral blood monionuclear cells, which were then washed with PBS and suspended in a medium (RPM 1640/ 10% FCS). The cell suspension (5 x 105 cells/mI; 10 m4) was distributed on a 10 am dish, and incubated under stimulation with 20 ng/ml of PMA (Sigma) and I pig/rnl of PH-A (Difco Laboratories) in a Condition of 5% C02, 37*C, for 48 hours. The cells were recovered, washed with PBS, and treated with Isogen (Nippon Gene) to prepare the total RNAs.
The total RNAs were subjected to a quantitative RT-PCR using a kIt taqMan Cold RZT-PCR Reagents)(Perldn Elmer Using reagents attached to the kit. 100 rig of the total RNA was reverse-transcribed with a program of 10 minutes, 48*C/30 minutes, and 9s*C/S minutes. A variety of primers, probes and reagents attached to the it were added in a defuLnite amount to 1 /100 of this solution as a template, and the mixture was subjected to PCR. The amount mRNA was determined by an ABI PRISM 7700 PCR was conducted in a condition- 1 cycle, 50*C/2 minutes; 1 cycle, 95*C/ 10 minutes; and then cyd ea, 9 5C/ 15 seconds for thermal denaturation and 60*C/ 1 minute for annealing and extension reaction. Quantification of DR3 was conducted aiming at mRNA expressed from the DR3 gene cDNA containing the cell tranemembrane region (the base sequence of the 689-760 position of SEQ ID NO: 1) and rnRNA expressed from the DR3 gene cDNA containing the death domain region (the base sequence of the 1091-1330 position of SEQ ID NO:1). The probes and the PCR primers were as follows.
FROM ER"TMP 2OO2* 4M I80/A757 40499 P 11 Cell trartmembrane region Probe: Oligonucleotide comprising the base sequence of the 691-7 position in SEQ ID NO:1 (6-carboxy-fluorescein attached at 5-end and 6-carboxy-tetramethyl-rhodamine attached at 3'-end).
Sense primer: Oligonucleotide corresponding to the base sequence of the 46"-85 position in SEQ ID NO: 1.
Antisense primer- Oligonucleotide corresponding to the base sequence of the 743-762 position in SEQ ID NO: 1.
Death domain region Probe: Oligonucleotide comprising the base sequence of the 115441173 position in SEQ ID NO: 1 (6-carboxy-fluorescein attached at 5'-end and 6-carboxcy-tctrazzethyl-rhodaxnine attached at 3'-end).
is Sense prnmer: Oligonucleotide correnponding to the base sequence of the 9 12-932 position in SEQ ID NO: 1.- Antisense primer: Oligonucleotide corresponding to the base sequence of the 1209-1232 position in SEQ ID NO; 1.
in th-,is operation, rRNA was quantiied as an internal standard at the same tixe and used for correction. The probe used in the quantification of rRNA is an oligonucleotide comprising the base sequence of SEQ ID NO:2 (2 ,7-dimethow -,S-dichloro-6-carboxy-fluorescein attached at 5'-end and 6-crboxy-tetra-methyl-rhodamine attached at 3'-end). The PCR primers used in the quantification of rRNA are oligonucleotides comprising the base sequences of SEQ ID) NO:3 (sense primer) and SEQ ID NO:4 (antisense primer), respectively.
The calibration curves used for determination of DR3 and rRNA were prepared using plasmids as templates obtained by cloning cDNAs of DR3 and rRNA, respectively. In each case, the same sample was measured 3 times. The FROM 2002* 426B()18:08/1l7:57/A 14503499396 P 12 amount of DR3 mRNA was indicated by the correction value derived by dividing by the value of rRNA measured simultaneously.
The results are as shown in Fig. 3. In the death domain region, the amount of DR3 mRNA was 0.90 0.26 (mean SE) for the RA patients and 0.92 t 0.43 for the healthy persons, and no significant difference was observed betweenm their amounts. To the contrary, in the cell transmembrane region, the amount was 1.84 0.27 for the RA patients and 0.82 1 0.25 for the healthy persons, and there was observed a significant increase of DR3 mRNA expression in the RA patients.
From the above results, it was confirmed that the RA patients can be diagnosed by stimulating the peripheral blood mononuclear cells with PMA and PHA, determining the amount of mRNA from the base sequence of the 689-760 position of SEQ ID NO:1 I or the amount of a polypeptide (having the amnino acid sequence of the 201-224 position of SEQ ID NO: 1) transcribed from said mRNA, and judging whether the expressed amount is at least 2 or more of that of healthy persons.
Industrial Applicability As described above in detail, according to the invention of the present application, it is possible to diagnose rheumatoid arthritis conveniently and exctly. Such diagnoses are useful in development of a novel therapy or therapeutics for the rheumatoid arthritis.
04/08 '06 FRI 14:07 FAX 61299255911 GRIFFITH HACK 1010 In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.
00 o COMS ID No: SBMI-04365356 Received by IP Australia: Time 13:13 Date 2006-08-04 EDITORIAL NOTE APPLICATION NUMBER 10530/01 The following Sequence Listing pages 1/4 to 4/4 are part of the description. The claims pages follow on page "11".
FROM 9M W,1970 FROM 4 2O 2 6811:0. l:7V.l404 9 P 18 114 SEQUENCE LISTING (110> Shiozawa, shunnich (120 A Method for Diagnosing Rheumatoid Arthritis (130) NP90453-YS <140) PCT/JPOO/07690 (141> 2000-11-01 (150 JPII-310805 <151) 1999-11-01 (160 4 <170 Patent in Ver. 2. 0 <21 0> (211> 2D <212> (213> (220> (221> (222> 1634
DNA
Homo sapiens CDs (1342) (400> 1 cgggccc tgc gggcgcgggg gaagcccctg ggcgcccgtc ctgaaggcgg aaccacgacg ggcagagagc acggagccgg ggagggct atg gag cag egg ccg cgg uc tgc 112 Met Giu Gin Arg Pro Arg Gly Cys gcg gcg gtg gcg gcg gcg ctc ctc ctg gtg ctg ctg Egg gcc egg gcc 160 Ala AlaVal Ala Ala Ala Lcu Leu Leu Val Leu Leu GiyAla Arg Ala 15 cag gge ggc'act cgt agc ccc agg tgt g2C tgt gcc agt gac ttc cac 208 Gin Gly Giy Thr Arg Ser Pro Arg Cys Aso Cys Ala Gly ASD Phe His 30 35 aag aag att ggt ctg ttt tgt tgc aga ggc tgc cca gcg ggg cac tac 256 Lys Lys Ilie Giy Leu Phe Cys Cys Arg Giy Cys Pro Aia Giy His Tyr 45 s0 FROM RIM EMM FR~ ~2O2* 4 268(t)18:1O/i.qi17:57/t*I~t453499396 P 19 ctg sg Leu Lys tgt ccc S Cys Pro tgt gcc Cys Ala gag aac Glu Asn 105 tgg ttt Tro Phe is scc cct Ala Pro tgc acz Cys Thr gag ccc tgc ggc aac Giu Pro Cys Gly Asn caa sac ace ttc ttg Gin Asp Thr Phe Leu cie tie cgs gcc tgt Arg Cys Gin Ala Cys tgt tca gca iti gcc Cys Ser Ala Vat Ala 110 ati gag tgc cag gtc Val Glu Cys Gin Val tgg gag aac Trp Glu Asn gag cag gcc Giu Gin Ala tce ace Ser Thr cac cat His His tcc eag Ser Gin tgc ctt gtg Cys Leu Val aat tet gaa Asn Ser Giu Btff aca ctg Val Ala Leu gac acc cie Asp Thr Arg age caa tit Ser Gin Cys 130 tac ggg gcc Cys Gly Ala tgt aag eca ge Cys Lys Pro Giy 120 agt tca ccc ttc Ser Ser Pro Phe 135 gtc age Val Ser tac tie caa cca Tyr Cys Gin Pro 140 eta gac Leu Asp etg caC Cgc cac sea cgi Leu His Arg His Thr Arg 150 304 352 400 448 495 544 592 640 688 736 784 832 eta etc 2D Leu Leu tte tat Phe Tyr tat Cys 155 t cc Ser cgc aga gut Arg Ang Asp tgt ggg ace tgc eta ect ge Cys Gly Thr Cys Leu Pro Giy 165 tce tgc ccc aeg agc ace eta Ser Cys Pro Thr Ser Thr Leu ggg GIy 185 t tc Ph.
gaa cat ge gat sic Glu His Gly Asp Gly 175 tgt eca gag eec tit Cys Pro Glu Arg Cys 190 gte cii gtg etc ctg Val Gin Val Leu Leu 205 gee Al a 180 get gte tgt gac Ala Val Cys Gly tgg aig cagf atg Tro Arg Gin Met 200 ccc etc eta ctt Pro Leu Leu Leu gtg ValI t gg Trp get gac ctt Ala Gly Leu 210 gtc ValI 215 ggg gee ace Giy Ala Thr etg gtt act Leu Val Thr e ti Leu 220 tie aca tac Tyr Thr Tyr cac tic tgg ect eac aig ec His Cys Trp Pro His Lys Pro 230 gag get ctg ace cca cc& cci Glu Ala Leu Thr Pro Pro Pro as aca gat gaa get ggg Ala Asp Ciu Ala Gly 240 etg tea ccc ttg gac Leu Ser Pro Leu Asp 255 ace ace Ala Thr 250 agc ace cac Ser Ala His acc ctt eta aca cet Thr Leu Leu Ala Pro 250 cct gac aac aat gag iii ate tac ace ate ca tta 9t9 gat aac agc FROM NOW-10 RM~200X2 60V81/q1:7A. 14.3499 P Pro Asp 265 tog acc Trp Thr Ser Ser Glu cct ggc tic Pro Gl Tyr 285 lie Cys Thr Val Gin 275 Lem Val Gly Asn gag ace cag gag gcg Glu Thr Gin Glu Ala 290 ttg ccc agc aga gct Leu Pro Ser Arg Ala ctc tgc ccg cag gtg Leu Cys Pro Gin Val 295 Ctt ggc ccc sct gct Lou Gly Pro Ala Ala 976 1024 aca tg tcc Thr Trp Sir gcg Ccc aca Ala Pro Thr 315 ctg rag ccg Lem Gin Pro gac cag ASD Gin 310 tcg cca gag tec Ser Pro Glu Ser 320 ccg cii ctc tac Pro Gin Leu Tyr sec giC Ala Gly tcg cci scc atg atg Ser Pro Ala Met Met 325 gac gcg gtc cci gcg Asp Ala Vat Pro Ala 1072 ggc GI y gac gtg atg Asp Val Met 335 cgg Arg 345 atc I Ie cgc tgg aag gag Arg Trp Lys Glu gai gcc gtg gag Glu Ala Val Glu ttc Phe 350 gtg Val CgQ acg Gtg Arg Thr Leu gag atg etc aug Giu Met Lem Lys 380 gtg gag atc ggc Vai Giu Ile Gly tgg cgc cag cag Trp Ang Gin Gin 385 gag nCC atg ggg Glu Arg Met Gly ggg ctg cgc gag sea gag Gly Leu Arm Glu Ala Glu 355 360 ttc cmi gac cam cag tic Phe Ang Asp Gin Gin Tyr 375 ccc gcg ggc etc gina scc Pro Ala Gly Lem Gly Ala 390 gac ggc tgc gtg mua gac Asp Gly Cys Val Giu Asp 1120 1168 1216 1264 1312 1362 1422 1482 1542 1602 1634 gtt tac sg Vai Tyr Ala 39S ttg Cgc agc Leu Arg Sir gec Ala ctin Lem ctg Leu dOO cgc ctg cam cmc gic ccg Arm Leu Gin Arg Gly Pro 405 tga cacggcgccc act tgccacc 3D taggcgctct ttutktcact ctmntcgcrc gaagacattt uumaaaaeaa gticcctt tat taagccg ctatcgctcc ctcaacttct aaaaaaaaaa gcagaagccc c tggcacggc agccaaggcg cggccmgagt aaaaaaaaaa taagtaeggt cctgcgtagc aagaagcacg t tmmctgaga aa tacttatgcg tgtagacatt agcaccagcc ggccccaccc aacgaatgtc gagigggggt tcgcggtatt aaatctgtga (210> (211>) (212> (213> 2 22
DNA
Artificial seauence F R 0 M ROW., W rA RM~2OO2* 02B 1:0TA757 40499 P 21 (220> (223> synthesized oligonucleotide (400> 2 tgctggcacc agACttgccc tc (210 3 (211> <212) DNA <213> Artificial seauence (220> (223> synthesized oliganuclectide <400 3 cggctaccac atceaaggaz (21 0> 4 (21 1> 2D (212> DNA (213> Artificial 3eaUenCe (220> 18 (223> synthesized oliaonucleotide 400 4 gctggaatta ccgcugct
Claims (2)
1. A indhod for dagaslflg rheumatoid arthritis which compfils digvetifl hurban geiomnic DNA with a reshietion enzyme SwR1 and detecting an about 7 kb S DNA fragment whic-h hybridizes ith a probe having the base sequence of the pouition
159-424 in SEQ ID NO: 1. 2. A method for diaVO6i= rheumatoid arthrTi Which compnis digsting hu~ia genoii DNA with a restriction enzyme Prgl and detecurg an about kb DNA fragment which. hybridlizes with a probe having the base s~euee of the position 159-424 in SEQ ID NO; 1. 3. A method for &Wcsioing rheumatoid arthrifis. 'wbic'h comPrise's stimulating the peripheral blood mononu~clear cells in a subject with pborbol is 12-inyristate 13-acetate (PMA1 and/or phytohemaggltiflin MMHA, and comparing the amount of mzRNA in the peripheral blood mononuclear cells with the amcmint of the same znRNA- in the peripheral l1ood montonucdear cells in a healthy person stimulated by PMA and/or P1-kA wherein the =RNA is czprcsaed from the base sequence of at the least 689-760 position in SEQ ID*NO:. 1. 4. A methoad for diagnosing rheUMatoi3d azthrits which cOmPrimsS stimulating the Peripheral blood morionuclea cells in a su1tect with pbmrbol 2-myristate 13-acetate (PM and/or phytoheiagglutininf WMiA, and comparing the amount af a polypeptide in the peripheral blood mononuclear cells with the 2S amzount of the some poly peptide aqnrcscd in the pcriphcral blood mnotudckar cells in a healthypersofl stimulated by PMA and/or PHA, wherein the polypePtidc haA the azmo acid sequence of at the least 201-224 position in SEQ MD NO: 1. The method according to any one of claims I to 4, substant ially as hereinbefoie described with reference to any one of the Examples. COMS ID No: SBMI.04365356 Received by IP Australia: Time (I-tm) 13:13 Date 2006-08-04
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11/310805 | 1999-11-01 | ||
| JP31080599 | 1999-11-01 | ||
| PCT/JP2000/007690 WO2001032921A2 (en) | 1999-11-01 | 2000-11-01 | Method of diagnosing rheumatoid arthritis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU1053001A AU1053001A (en) | 2001-05-14 |
| AU785071B2 true AU785071B2 (en) | 2006-09-14 |
Family
ID=18009651
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU10530/01A Expired AU785071B2 (en) | 1999-11-01 | 2000-11-01 | Method of diagnosing rheumatoid arthritis |
Country Status (4)
| Country | Link |
|---|---|
| KR (1) | KR100762444B1 (en) |
| AU (1) | AU785071B2 (en) |
| CA (1) | CA2390387A1 (en) |
| WO (1) | WO2001032921A2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002210926B2 (en) | 2000-10-24 | 2005-12-15 | Shunichi Shiozawa | Genomes participating in rheumatoid arthritis, method of the diagnosis thereof, method of evaluating the onset possibility thereof, diagnostic kit for detecting them and therapeutic method and remedies for rheumatoid arthritis |
| JP3710809B1 (en) * | 2004-02-26 | 2005-10-26 | 俊一 塩澤 | Polynucleotides involved in the development of rheumatoid arthritis and uses thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5110920A (en) * | 1982-01-22 | 1992-05-05 | Cetus Corporation | HLA typing method and DNA probes used therein |
| US5665548A (en) * | 1986-03-13 | 1997-09-09 | Roche Molecular Systems, Inc. | Characterization and detection of sequences associated with autoimmune diseases |
-
2000
- 2000-11-01 WO PCT/JP2000/007690 patent/WO2001032921A2/en active IP Right Grant
- 2000-11-01 KR KR1020027005592A patent/KR100762444B1/en active IP Right Grant
- 2000-11-01 AU AU10530/01A patent/AU785071B2/en not_active Expired
- 2000-11-01 CA CA002390387A patent/CA2390387A1/en not_active Abandoned
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5110920A (en) * | 1982-01-22 | 1992-05-05 | Cetus Corporation | HLA typing method and DNA probes used therein |
| US5665548A (en) * | 1986-03-13 | 1997-09-09 | Roche Molecular Systems, Inc. | Characterization and detection of sequences associated with autoimmune diseases |
Non-Patent Citations (1)
| Title |
|---|
| INTERNATIONAL IMMUNOLOGY 10(12): 1891-95(SHIOZAWA ET AL) * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU1053001A (en) | 2001-05-14 |
| KR20020065498A (en) | 2002-08-13 |
| CA2390387A1 (en) | 2001-05-10 |
| KR100762444B1 (en) | 2007-10-04 |
| WO2001032921A2 (en) | 2001-05-10 |
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