Abstract:
A composition and method for the diagnosis of autoimmune hepatitis. The composition, which contains SLA antigens detects soluble liver antigen (SLA) auto-antibodies, which occur in sera of patients suffering from chronic hepatitis.

Description:
This is a division of application Ser. No. 09/249,020 filed Feb. 12, 1999. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to a composition and method for diagnosing auto-immune hepatitis, which use an immune reaction to detect soluble liver antigen (“SLA”) auto-antibodies. 
     Auto-immune hepatitis (AIH) is a chronic inflammation of the liver that when left untreated leads to cirrhosis, but when treated has a very good prognosis. For this reason, a timely diagnosis is important. It is estimated that 5% of all patients in Western countries who have chronic hepatitis have AIH. At the present time, there is no specific diagnostic test for AIH. Rather the diagnosis for AIH is undertaken by a plurality of diagnosis, such as excluding viral hepatitis, recognizing [hyper] immunoglobulinaemia, recognizing the tissue type (HLA type), and detecting auto-antibodies. Auto-antibodies are found in about 90% of patients with chronic hepatitis, and most of the detectable auto-antibodies are also present, at least in low titers, in other inflammations of the liver as well. In particular, these auto-antibodies are antibodies to nuclear antigens (ANA) and unstriated muscles (SMA), as well as the very rare antibodies to cytochrome p450 (LKM). SLA auto-antibodies were described for the first time in 1987 (Manns M. et al., Lancet 1987;1:292-4). Tests have shown that SLA auto-antibodies occur in about 25 to 30% of patients having AIH, but hardly ever occur in patients having other diseases, including other auto-immune diseases (Lohse A. W. et al., Z. Gastroenterol 1995;33:527-33)Detecting SLA auto-antibodies therefore provides a significant diagnostic procedure for recognizing AIH. 
     The present invention is directed to use and detection of SLA antigens, which are a prerequisite for developing a specific immuunoassay for detecting SLA auto-antibodies in patients&#39; sera. Previous methods known in the art could not detect such SLA auto-antibodies in patients&#39; sera. Liver cytokeratins 8 and 18 were described in 1990 as target antigens of the SLA auto-antibodies (Journal of Repatology, 1990; 11: 232 to 239), however, these findings were never confirmed, and were even refuted (Wies I. et al., Z. Gastroenterol. 1998;36:93). 
     SUMMARY OF THE INVENTION 
     The invention is directed to a composition for detecting the presence of SLA auto-antibodies in blood serum, said composition comprising an antigen that is recognized by SLA auto-antibodies. In particular the invention is directed to antigens having amino acid sequence SEQ ID NO:2, SEQ ID NO:4, or antigenic derivatives thereof, or encoded by DNA having DNA sequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivatives thereof. Another embodiment of the invention is a purified protein or polypeptide recognized by SLA auto-antibodies. In a preferred embodiment of the invention the purified protein has the amino acid sequence SEQ ID NO:2, SEQ ID NO:4, or antigenic derivatives thereof, or encoded by DNA having DNA sequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivatives thereof, or is a fusion protein containing either SEQ ID NO:2, SEQ ID NO:4, or antigenic derivatives thereof, or encoded by DNA having DNA sequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivatives thereof. In another aspect of the invention, there is disclosed a cDNA having a nucleotide sequence that codes for an antigen recognized by SLA auto-antibodies in blood serum. In a preferred embodiment of this aspect of the invention the cDNA codes for an antigen having the amino acid sequence SEQ ID NO:2, SEQ ID NO:4, or antigenic derivatives thereof, or has DNA sequence SEQ ID NO:1, SEQ ID NO;3, or antigenic derivatives thereof. 
     Another aspect of the invention is directed to a method of detecting the presence of SLA auto-antibodies in a blood sample by binding the composition of the invention to a matrix, detecting the binding of SLA auto-antibodies bound to the antigens in the composition, and correlating such binding to the presence of SLA auto-antibodies in the sample, Examples of suitable methods in which the present compositions can be used to detect SLA auto-antibodies in blood include immunoassays such as, but not limited to, radioimmunoassay, chemiluminescence, immunoassay, immunoblot assay, enzyme assay and inhibition immunoassay. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to a composition and a method for diagnosing auto-immune hepatitis, by enabling one to detect in the blood serum of a patient, antibodies to two proteins or polypeptides (SLA antigens), which antigens wholly or partially contain the amino acid sequence corresponding to SEQ ID NO:2, SEQ ID NO:4, or antigenic derivatives thereof, or encoded by DNA having DNA sequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivatives thereof of the SLA antigens, which are recognized by SLA auto-antibodies. These polypeptides are referred to as SLA-1 and as SLA-2. One embodiment of the invention is directed to synthetic or natural variants of SLA-1 or SLA-2, or synthetic or natural variants of partial or incomplete polypeptides of SLA-1 or SLA-2, which correspond wholly or partially to the amino acid sequences or antigenic derivatives thereof and are likewise recognized by SLA auto-antibodies. 
     These polypeptides are referred to as SLA-1 and as SLA-2. One embodiment of the invention is directed to synthetic or natural variants of SLA-1 or SLA-2, or sythetic or natural variants of partial or incomplete polypeptides of SLA-1 or SLA-2, which correspond wholly or partially to the amino acid sequences or antigenic derivatives thereof and are likewise recognized by SLA auto-antibodies. 
     In a preferred embodiment, the peptides of the invention are fusion proteins. Such fusion proteins can be made by known methods in the art (Maniatis, T. et al.,  Molecular Cloning , 1982: 412-430). 
     Another embodiment of the invention is directed to CDNA, which encodes a natural or synthetic variant of one of the SLA antigens SLA-1or SLA-2, having a nucleotide sequence corresponding to SEQ ID NO:1 or to SEQ ID NO:3 or to antigenic derivatives thereof. The cDNAs are present as two spliced variants, the longer of these (SEQ ID NO:3) having an insertion of 156 nucleotides., SLA-1 and SLA-2 have similar nucleotide sequences, except that SLA-2 contains a 156 nucleotide insert. 
     SLA-positive sera, i.e. SLA auto-antibody containing sera, produces a double band on a Western blot when probed with the SLA antigens. The double band measures 50 kDa, which corresponds to the molecular weight of the SLA antigens, SLA-1 and SLA-2. When SLA-positive serum is incubated with the fusion protein according to the present invention, e.g. SLA-1 or SLA-2 fused to another protein or polypeptide, the double band corresponding to the SLA-antigen is not detected. 
     Incubation of SLA-positive sera with a control fusion protein, which does not contain either SLA-1 or SLA-2 does not eliminate the appearance of a double band on Western blots. 
     The cDNA according to the present invention allows for the production of large quantities of the SLA antigens in a recombinant fashion. Further, the concentration of SLA antigens can be measured using known SLA auto-antibody methods. 
     In another aspect of the invention there is disclosed a method for determining the presence of SLA auto-antibodies in a blood sample using the composition of the invention in known assay methods such as, but not limited to, radioimmunoassay (RIA), chemiluminescence immunoassay (CIA), immunoblot assay or enzyme immunoassay (EIA). In these assays, one of the SLA antigens (SLA-1or SLA-2) is bound to a matrix, such as a microtiter plate or a blot membrane. Then the patient&#39;s serum to be tested is applied to the matrix with the bound SLA antigen. Following incubation, the test serum is washed off. The specific binding of SLA auto-antibodies (in the test serum) to the matrix-bound SLA-antigen is detected and verified by using a secondary antibody (anti-human immunoglobulin or an immunoglobulin subclass, for example) that has been labeled with a tracer. Tracers commonly used include enzyme compounds such as peroxidase or alkaline phosphatase, radioactive compounds, or a chemiluminescing substances. The more SLA auto-antibodies present in the test serum, the more tracer is bound to the matrix. Normal serum from healthy blood donors can be used as a negative control. Additional controls include the use of SLA auto-antibody positive serum that has SLA auto-antibodies present at known various concentrations. 
     Alternatively, an inhibition immunoassay can also be performed using the SLA antigens of the present invention. In such an assay the SLA antigens bind to a selected SLA auto-antibody, which has been labeled with a tracer. The amount of binding of the labeled SLA auto-antibody to the SLA antigens is measured as the control. A patient&#39;s sera is then tested against the control. The patient&#39;s serum is added along with the labeled SLA auto-antibodies. SLA auto-antibodies present in the patient&#39;s serum compete with the labeled SLA auto-antibodies in binding to the SLA antigens. Thus, if the patient&#39;s serum contains SLA auto-antibodies, the amount of labeled SLA auto-antibodies present will be less than the control. Therefore the detection of fewer bound labeled SLA auto-antibodies reveals the presence of competing SLA auto-antibodies in the test serum. 
    
    
     EXAMPLE 1 
     Detecting SLA auto-antibodies in the ELISA test using the recombinant SLA antigen (SEQ ID No:2) 
     The recombinant SLA antigen was purified using the his-tag. 50 μg of the SLA antigen in 50 μl phosphate buffered saline (“PBS”)(pH 7.0) was placed in a microtiter plate and left over night. After pipetting off and washing the microtiter plate, a post-coating was carried out for one hour using a 1% Bovine Serum Albumin (“BSA”)/PBS solution at room temperature. 50 μl each of normal sera, patients&#39; sera, and control sera were diluted 1:100 with PBS. 50μl of each diluted serum was incubated for 30 minutes at room temperature on its respective microtiter plate. The plates were then washed 1-5 times and a peroxidase-labeled secondary antibody, an antihuman immunoglobulin, diluted 1:8000 was then added and allowed to incubate. After washing out the excess secondary antibody, ABTS solution [55 mg of 2, 2′-azidodi-3-ethyl-benzthiazine-6-sulphonic acid (Serva, Heidelberg) in 5 ml 0.01 M K 2 HPO 4  (pH 6.0), containing 50 μl H 2 O 2 , diluted in a 1:300 proportion in PBS] was added. The optical density of the microtiter plates was measured with a Titertec Multiscan MC (Flow Laboratories, Meckenheim). Table 1 reveals that all of the patients&#39; sera containing SLA auto-bodies exhibited distinctly elevated values of light absorption, as compared to normal serum, and serum of patients who suffered from other liver diseases. 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Serum 
                 OD 
               
               
                   
                   
               
             
             
               
                   
                 normal serum 1 
                 0.476 
               
               
                   
                 normal serum 1 
                 0.471 
               
               
                   
                 SLA-serum 1 
                 1.863 
               
               
                   
                 SLA-serum 2 
                 1.995 
               
               
                   
                 SLA-serum 3 
                 1.791 
               
               
                   
                 SLA-serum 4 
                 1.867 
               
               
                   
                 AMA-pos. serum (PBC) 
                 0.615 
               
               
                   
                 LKM-pos. serum (hep. C) 
                 0.769 
               
               
                   
                   
               
               
                   
                 Explanations: OD = optical density (extinction); AMA = anti-mitochondrial antibody; PBC = primary biliary cirrhosis; LKM = liver-kidney-microsomal; BSA = bovine serum albumin; PBS = phosphate-buffered saline  
               
             
          
         
       
     
     
       
         
               
             
           
               
                   
               
               
                                                SEQUENCE LISTING 
               
               
                   
               
               
                   
               
               
                 &lt;160&gt; NUMBER OF SEQ ID NOS: 4 
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 1 
               
               
                 &lt;211&gt; LENGTH: 1335 
               
               
                 &lt;212&gt; TYPE: DNA 
               
               
                 &lt;213&gt; ORGANISM: Homo sapiens 
               
               
                 &lt;220&gt; FEATURE: 
               
               
                 &lt;221&gt; NAME/KEY: CDS 
               
               
                 &lt;222&gt; LOCATION: (1)...(1335) 
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 1 
               
               
                   
               
               
                 tcg cgg cgg gag agc ggc tgg tgt cgc cgg ctt acg tgc ggc agg gct       48 
               
               
                 Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
               
               
                  1               5                   10                  15 
               
               
                   
               
               
                 gtg agg ccc gcc gct cgc atg agc acc tca tac ggc tgc ttc tgg aga       96 
               
               
                 Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
               
               
                              20                  25                  30 
               
               
                   
               
               
                 agg ttc att cat ggc att gga cga tcc ggt gat att tct gct gtg caa      144 
               
               
                 Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile Ser Ala Val Gln 
               
               
                          35                  40                  45 
               
               
                   
               
               
                 cca aaa gct gca ggc tct agc ctt ttg aac aaa att acc aat tct ttg      192 
               
               
                 Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile Thr Asn Ser Leu 
               
               
                      50                  55                  60 
               
               
                   
               
               
                 gtc ctg gac att ata aag ctg gct ggt gtc cat aca gta gcc aac tgc      240 
               
               
                 Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr Val Ala Asn Cys 
               
               
                  65                  70                  75                  80 
               
               
                   
               
               
                 ttt gta gtt cct atg gca act ggt atg agt cta act ctg tgt ttc tta      288 
               
               
                 Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr Leu Cys Phe Leu 
               
               
                                  85                  90                  95 
               
               
                   
               
               
                 aca tta cga cac aaa aga cca aag gca aag tat att ata tgg cca cga      336 
               
               
                 Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile Ile Trp Pro Arg 
               
               
                             100                 105                 110 
               
               
                   
               
               
                 ata gac cag aag tcc tgc ttt aaa tcc atg atc act gca ggt ttt gag      384 
               
               
                 Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr Ala Gly Phe Glu 
               
               
                         115                 120                 125 
               
               
                   
               
               
                 cct gtg gtg ata gaa aat gtt ttg gaa ggt gac gag ctg cgt aca gac      432 
               
               
                 Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu Leu Arg Thr Asp 
               
               
                     130                 135                 140 
               
               
                   
               
               
                 ctg aaa gca gtg gag gct aaa gtc cag gaa ctt ggg cct gat tgc att      480 
               
               
                 Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly Pro Asp Cys Ile 
               
               
                 145                 150                 155                 160 
               
               
                   
               
               
                 ctg tgt att cat tct act aca tcc tgt ttt gct cca agg gtg cct gat      528 
               
               
                 Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro Arg Val Pro Asp 
               
               
                                 165                 170                 175 
               
               
                   
               
               
                 aga tta gaa gaa ctg gct gtg att tgt gct aat tat gac att cca cat      576 
               
               
                 Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr Asp Ile Pro His 
               
               
                             180                 185                 190 
               
               
                   
               
               
                 ata gtt aat aat gct tat gga gtg cag tct tca aag tgt atg cat ctc      624 
               
               
                 Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys Cys Met His Leu 
               
               
                         195                 200                 205 
               
               
                   
               
               
                 att cag cag ggg gct cga gtt ggt aga ata gat gct ttt gtt cag agc      672 
               
               
                 Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala Phe Val Gln Ser 
               
               
                     210                 215                 220 
               
               
                   
               
               
                 ttg gac aaa aat ttt atg gtt cca gta ggt ggt gct ata att gct ggc      720 
               
               
                 Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala Ile Ile Ala Gly 
               
               
                 225                 230                 235                 240 
               
               
                   
               
               
                 ttt aat gat tca ttc att cag gaa atc agc aag atg tat cca gga aga      768 
               
               
                 Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met Tyr Pro Gly Arg 
               
               
                                 245                 250                 255 
               
               
                   
               
               
                 gct tca gct tca cct tct tta gat gtc ctt att act tta ttg tca ctt      816 
               
               
                 Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr Leu Leu Ser Leu 
               
               
                             260                 265                 270 
               
               
                   
               
               
                 gga tca aat ggc tat aag aag cta cta aaa gaa aga aag gaa atg ttt      864 
               
               
                 Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg Lys Glu Met Phe 
               
               
                         275                 280                 285 
               
               
                   
               
               
                 tca tat ttg tcc aac caa ata aag aag ttg tca gaa gcc tac aat gaa      912 
               
               
                 Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu Ala Tyr Asn Glu 
               
               
                     290                 295                 300 
               
               
                   
               
               
                 aga ctg ttg cat aca cct cac aat ccc ata tct tta gct atg aca ctt      960 
               
               
                 Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu Ala Met Thr Leu 
               
               
                 305                 310                 315                 320 
               
               
                   
               
               
                 aaa aca cta gat gaa cac cgt gac aaa gct gtc act cag ctt ggc tcg     1008 
               
               
                 Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr Gln Leu Gly Ser 
               
               
                                 325                 330                 335 
               
               
                   
               
               
                 atg ctt ttt acc aaa cag gtt tct gga gcc agg gtt gtg cct ctt ggg     1056 
               
               
                 Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val Val Pro Leu Gly 
               
               
                             340                 345                 350 
               
               
                   
               
               
                 tcc atg caa act gtg agt ggc tat act ttc aga ggc ttt atg tca cat     1104 
               
               
                 Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly Phe Met Ser His 
               
               
                         355                 360                 365 
               
               
                   
               
               
                 aca aat aat tac cct tgt gct tac ctc aat gct gca tca gcc atc gga     1152 
               
               
                 Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala Ser Ala Ile Gly 
               
               
                     370                 375                 380 
               
               
                   
               
               
                 atg aar atg cag gat gtg gac ctg ttc ata aac ara ctt gac agg tgt     1200 
               
               
                 Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa Leu Asp Arg Cys 
               
               
                 385                 390                 395                 400 
               
               
                   
               
               
                 tta aag gca gta aga aaa gaa cga agt aaa gag agt gat gac aat tat     1248 
               
               
                 Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser Asp Asp Asn Tyr 
               
               
                                 405                 410                 415 
               
               
                   
               
               
                 gac aaa act gaa rat gtg gat att gaa gaa atg gct tta aaa cta gat     1296 
               
               
                 Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala Leu Lys Leu Asp 
               
               
                             420                 425                 430 
               
               
                   
               
               
                 aat gta ctt ctt gac aca tac cag gat gct tct tca tga                 1335 
               
               
                 Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser  * 
               
               
                         435                 440 
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 2 
               
               
                 &lt;211&gt; LENGTH: 444 
               
               
                 &lt;212&gt; TYPE: PRT 
               
               
                 &lt;213&gt; ORGANISM: Homo sapiens 
               
               
                 &lt;220&gt; FEATURE: 
               
               
                 &lt;221&gt; NAME/KEY: VARIANT 
               
               
                 &lt;222&gt; LOCATION: (1)...(444) 
               
               
                 &lt;223&gt; OTHER INFORMATION: Xaa = Any Amino Acid 
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 2 
               
               
                   
               
               
                 Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
               
               
                  1               5                  10                  15 
               
               
                   
               
               
                 Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
               
               
                             20                  25                  30 
               
               
                   
               
               
                 Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile Ser Ala Val Gln 
               
               
                         35                  40                  45 
               
               
                   
               
               
                 Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile Thr Asn Ser Leu 
               
               
                     50                  55                  60 
               
               
                   
               
               
                 Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr Val Ala Asn Cys 
               
               
                 65                  70                  75                  80 
               
               
                   
               
               
                 Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr Leu Cys Phe Leu 
               
               
                                 85                  90                  95 
               
               
                   
               
               
                 Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile Ile Trp Pro Arg 
               
               
                             100                 105                 110 
               
               
                   
               
               
                 Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr Ala Gly Phe Glu 
               
               
                         115                 120                 125 
               
               
                   
               
               
                 Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu Leu Arg Thr Asp 
               
               
                     130                 135                 140 
               
               
                   
               
               
                 Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly Pro Asp Cys Ile 
               
               
                 145                 150                 155                 160 
               
               
                   
               
               
                 Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro Arg Val Pro Asp 
               
               
                                 165                 170                 175 
               
               
                   
               
               
                 Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr Asp Ile Pro His 
               
               
                             180                 185                 190 
               
               
                   
               
               
                 Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys Cys Met His Leu 
               
               
                         195                 200                 205 
               
               
                   
               
               
                 Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala Phe Val Gln Ser 
               
               
                     210                 215                 220 
               
               
                   
               
               
                 Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala Ile Ile Ala Gly 
               
               
                 225                 230                 235                 240 
               
               
                   
               
               
                 Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met Tyr Pro Gly Arg 
               
               
                                 245                 250                 255 
               
               
                   
               
               
                 Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr Leu Leu Ser Leu 
               
               
                             260                 265                 270 
               
               
                   
               
               
                 Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg Lys Glu Met Phe 
               
               
                         275                 280                 285 
               
               
                   
               
               
                 Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu Ala Tyr Asn Glu 
               
               
                     290                 295                 300 
               
               
                   
               
               
                 Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu Ala Met Thr Leu 
               
               
                 305                 310                 315                 320 
               
               
                   
               
               
                 Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr Gln Leu Gly Ser 
               
               
                                 325                 330                 335 
               
               
                   
               
               
                 Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val Val Pro Leu Gly 
               
               
                             340                 345                 350 
               
               
                   
               
               
                 Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly Phe Met Ser His 
               
               
                         355                 360                 365 
               
               
                   
               
               
                 Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala Ser Ala Ile Gly 
               
               
                     370                 375                 380 
               
               
                   
               
               
                 Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa Leu Asp Arg Cys 
               
               
                 385                 390                 395                 400 
               
               
                   
               
               
                 Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser Asp Asp Asn Tyr 
               
               
                                 405                 410                 415 
               
               
                   
               
               
                 Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala Leu Lys Leu Asp 
               
               
                             420                 425                 430 
               
               
                   
               
               
                 Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser 
               
               
                         435                 440 
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 3 
               
               
                 &lt;211&gt; LENGTH: 1491 
               
               
                 &lt;212&gt; TYPE: DNA 
               
               
                 &lt;213&gt; ORGANISM: Homo sapiens 
               
               
                 &lt;220&gt; FEATURE: 
               
               
                 &lt;221&gt; NAME/KEY: CDS 
               
               
                 &lt;222&gt; LOCATION: (1)...(1491) 
               
               
                 &lt;221&gt; NAME/KEY: misc_feature 
               
               
                 &lt;222&gt; LOCATION: (1)...(1491) 
               
               
                 &lt;223&gt; OTHER INFORMATION: n = A,T,C or G 
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 3 
               
               
                   
               
               
                 tcg cgg cgg gag agc ggc tgg tgt cgc cgg ctt acg tgc ggc agg gct       48 
               
               
                 Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
               
               
                  1               5                   10                  15 
               
               
                   
               
               
                 gtg agg ccc gcc gct cgc atg agc acc tca tac ggc tgc ttc tgg aga       96 
               
               
                 Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
               
               
                              20                  25                  30 
               
               
                   
               
               
                 agg gcw nnn tgt cca aag aat ggc tgg gat gaa agt aca ctt gaa ctc      144 
               
               
                 Arg Xaa Xaa Cys Pro Lys Asn Gly Trp Asp Glu Ser Thr Leu Glu Leu 
               
               
                          35                  40                  45 
               
               
                   
               
               
                 ttt tta cat gaa ctt gca atc atg gac agc aac aat ttc tta ggc aat      192 
               
               
                 Phe Leu His Glu Leu Ala Ile Met Asp Ser Asn Asn Phe Leu Gly Asn 
               
               
                      50                  55                  60 
               
               
                   
               
               
                 tgt ggt gtg gga gaa agg gaa ggg aga gtg gca tcc gca ctg gtt gct      240 
               
               
                 Cys Gly Val Gly Glu Arg Glu Gly Arg Val Ala Ser Ala Leu Val Ala 
               
               
                  65                  70                  75                  80 
               
               
                   
               
               
                 cgt cgt cat tac agg ttc att cat ggc att gga cga tcc ggt gat att      288 
               
               
                 Arg Arg His Tyr Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile 
               
               
                                  85                  90                  95 
               
               
                   
               
               
                 tct gct gtg caa cca aaa gct gca ggc tct agc ctt ttg aac aaa att      336 
               
               
                 Ser Ala Val Gln Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile 
               
               
                             100                 105                 110 
               
               
                   
               
               
                 acc aat tct ttg gtc ctg gac att ata aag ctg gct ggt gtc cat aca      384 
               
               
                 Thr Asn Ser Leu Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr 
               
               
                         115                 120                 125 
               
               
                   
               
               
                 gta gcc aac tgc ttt gta gtt cct atg gca act ggt atg agt cta act      432 
               
               
                 Val Ala Asn Cys Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr 
               
               
                     130                 135                 140 
               
               
                   
               
               
                 ctg tgt ttc tta aca tta cga cac aaa aga cca aag gca aag tat att      480 
               
               
                 Leu Cys Phe Leu Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile 
               
               
                 145                 150                 155                 160 
               
               
                   
               
               
                 ata tgg cca cga ata gac cag aag tcc tgc ttt aaa tcc atg atc act      528 
               
               
                 Ile Trp Pro Arg Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr 
               
               
                                 165                 170                 175 
               
               
                   
               
               
                 gca ggt ttt gag cct gtg gtg ata gaa aat gtt ttg gaa ggt gac gag      576 
               
               
                 Ala Gly Phe Glu Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu 
               
               
                             180                 185                 190 
               
               
                   
               
               
                 ctg cgt aca gac ctg aaa gca gtg gag gct aaa gtc cag gaa ctt ggg      624 
               
               
                 Leu Arg Thr Asp Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly 
               
               
                         195                 200                 205 
               
               
                   
               
               
                 cct gat tgc att ctg tgt att cat tct act aca tcc tgt ttt gct cca      672 
               
               
                 Pro Asp Cys Ile Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro 
               
               
                     210                 215                 220 
               
               
                   
               
               
                 agg gtg cct gat aga tta gaa gaa ctg gct gtg att tgt gct aat tat      720 
               
               
                 Arg Val Pro Asp Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr 
               
               
                 225                 230                 235                 240 
               
               
                   
               
               
                 gac att cca cat ata gtt aat aat gct tat gga gtg cag tct tca aag      768 
               
               
                 Asp Ile Pro His Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys 
               
               
                                 245                 250                 255 
               
               
                   
               
               
                 tgt atg cat ctc att cag cag ggg gct cga gtt ggt aga ata gat gct      816 
               
               
                 Cys Met His Leu Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala 
               
               
                             260                 265                 270 
               
               
                   
               
               
                 ttt gtt cag agc ttg gac aaa aat ttt atg gtt cca gta ggt ggt gct      864 
               
               
                 Phe Val Gln Ser Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala 
               
               
                         275                 280                 285 
               
               
                   
               
               
                 ata att gct ggc ttt aat gat tca ttc att cag gaa atc agc aag atg      912 
               
               
                 Ile Ile Ala Gly Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met 
               
               
                     290                 295                 300 
               
               
                   
               
               
                 tat cca gga aga gct tca gct tca cct tct tta gat gtc ctt att act      960 
               
               
                 Tyr Pro Gly Arg Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr 
               
               
                 305                 310                 315                 320 
               
               
                   
               
               
                 tta ttg tca ctt gga tca aat ggc tat aag aag cta cta aaa gaa aga     1008 
               
               
                 Leu Leu Ser Leu Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg 
               
               
                                 325                 330                 335 
               
               
                   
               
               
                 aag gaa atg ttt tca tat ttg tcc aac caa ata aag aag ttg tca gaa     1056 
               
               
                 Lys Glu Met Phe Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu 
               
               
                             340                 345                 350 
               
               
                   
               
               
                 gcc tac aat gaa aga ctg ttg cat aca cct cac aat ccc ata tct tta     1104 
               
               
                 Ala Tyr Asn Glu Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu 
               
               
                         355                 360                 365 
               
               
                   
               
               
                 gct atg aca ctt aaa aca cta gat gaa cac cgt gac aaa gct gtc act     1152 
               
               
                 Ala Met Thr Leu Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr 
               
               
                     370                 375                 380 
               
               
                   
               
               
                 cag ctt ggc tcg atg ctt ttt acc aaa cag gtt tct gga gcc agg gtt     1200 
               
               
                 Gln Leu Gly Ser Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val 
               
               
                 385                 390                 395                 400 
               
               
                   
               
               
                 gtg cct ctt ggg tcc atg caa act gtg agt ggc tat act ttc aga ggc     1248 
               
               
                 Val Pro Leu Gly Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly 
               
               
                                 405                 410                 415 
               
               
                   
               
               
                 ttt atg tca cat aca aat aat tac cct tgt gct tac ctc aat gct gca     1296 
               
               
                 Phe Met Ser His Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala 
               
               
                             420                 425                 430 
               
               
                   
               
               
                 tca gcc atc gga atg aar atg cag gat gtg gac ctg ttc ata aac ara     1344 
               
               
                 Ser Ala Ile Gly Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa 
               
               
                         435                 440                 445 
               
               
                   
               
               
                 ctt gac agg tgt tta aag gca gta aga aaa gaa cga agt aaa gag agt     1392 
               
               
                 Leu Asp Arg Cys Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser 
               
               
                     450                 455                 460 
               
               
                   
               
               
                 gat gac aat tat gac aaa act gaa rat gtg gat att gaa gaa atg gct     1440 
               
               
                 Asp Asp Asn Tyr Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala 
               
               
                 465                 470                 475                 480 
               
               
                   
               
               
                 tta aaa cta gat aat gta ctt ctt gac aca tac cag gat gct tct tca     1488 
               
               
                 Leu Lys Leu Asp Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser 
               
               
                                 485                 490                 495 
               
               
                   
               
               
                 tga                                                                 1491 
               
               
                  * 
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 4 
               
               
                 &lt;211&gt; LENGTH: 496 
               
               
                 &lt;212&gt; TYPE: PRT 
               
               
                 &lt;213&gt; ORGANISM: Homo sapiens 
               
               
                 &lt;220&gt; FEATURE: 
               
               
                 &lt;221&gt; NAME/KEY: VARIANT 
               
               
                 &lt;222&gt; LOCATION: (1)...(496) 
               
               
                 &lt;223&gt; OTHER INFORMATION: Xaa = Any Amino Acid 
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 4 
               
               
                   
               
               
                 Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 
               
               
                  1               5                  10                  15 
               
               
                   
               
               
                 Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 
               
               
                             20                  25                  30 
               
               
                   
               
               
                 Arg Xaa Xaa Cys Pro Lys Asn Gly Trp Asp Glu Ser Thr Leu Glu Leu 
               
               
                         35                  40                  45 
               
               
                   
               
               
                 Phe Leu His Glu Leu Ala Ile Met Asp Ser Asn Asn Phe Leu Gly Asn 
               
               
                     50                  55                  60 
               
               
                   
               
               
                 Cys Gly Val Gly Glu Arg Glu Gly Arg Val Ala Ser Ala Leu Val Ala 
               
               
                 65                  70                  75                  80 
               
               
                   
               
               
                 Arg Arg His Tyr Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile 
               
               
                                 85                  90                  95 
               
               
                   
               
               
                 Ser Ala Val Gln Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile 
               
               
                             100                 105                 110 
               
               
                   
               
               
                 Thr Asn Ser Leu Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr 
               
               
                         115                 120                 125 
               
               
                   
               
               
                 Val Ala Asn Cys Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr 
               
               
                     130                 135                 140 
               
               
                   
               
               
                 Leu Cys Phe Leu Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile 
               
               
                 145                 150                 155                 160 
               
               
                   
               
               
                 Ile Trp Pro Arg Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr 
               
               
                                 165                 170                 175 
               
               
                   
               
               
                 Ala Gly Phe Glu Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu 
               
               
                             180                 185                 190 
               
               
                   
               
               
                 Leu Arg Thr Asp Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu Gly 
               
               
                         195                 200                 205 
               
               
                   
               
               
                 Pro Asp Cys Ile Leu Cys Ile His Ser Thr Thr Ser Cys Phe Ala Pro 
               
               
                     210                 215                 220 
               
               
                   
               
               
                 Arg Val Pro Asp Arg Leu Glu Glu Leu Ala Val Ile Cys Ala Asn Tyr 
               
               
                 225                 230                 235                 240 
               
               
                   
               
               
                 Asp Ile Pro His Ile Val Asn Asn Ala Tyr Gly Val Gln Ser Ser Lys 
               
               
                                 245                 250                 255 
               
               
                   
               
               
                 Cys Met His Leu Ile Gln Gln Gly Ala Arg Val Gly Arg Ile Asp Ala 
               
               
                             260                 265                 270 
               
               
                   
               
               
                 Phe Val Gln Ser Leu Asp Lys Asn Phe Met Val Pro Val Gly Gly Ala 
               
               
                         275                 280                 285 
               
               
                   
               
               
                 Ile Ile Ala Gly Phe Asn Asp Ser Phe Ile Gln Glu Ile Ser Lys Met 
               
               
                     290                 295                 300 
               
               
                   
               
               
                 Tyr Pro Gly Arg Ala Ser Ala Ser Pro Ser Leu Asp Val Leu Ile Thr 
               
               
                 305                 310                 315                 320 
               
               
                   
               
               
                 Leu Leu Ser Leu Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg 
               
               
                                 325                 330                 335 
               
               
                   
               
               
                 Lys Glu Met Phe Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu 
               
               
                             340                 345                 350 
               
               
                   
               
               
                 Ala Tyr Asn Glu Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu 
               
               
                         355                 360                 365 
               
               
                   
               
               
                 Ala Met Thr Leu Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr 
               
               
                     370                 375                 380 
               
               
                   
               
               
                 Gln Leu Gly Ser Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val 
               
               
                 385                 390                 395                 400 
               
               
                   
               
               
                 Val Pro Leu Gly Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly 
               
               
                                 405                 410                 415 
               
               
                   
               
               
                 Phe Met Ser His Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala 
               
               
                             420                 425                 430 
               
               
                   
               
               
                 Ser Ala Ile Gly Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn Xaa 
               
               
                         435                 440                 445 
               
               
                   
               
               
                 Leu Asp Arg Cys Leu Lys Ala Val Arg Lys Glu Arg Ser Lys Glu Ser 
               
               
                     450                 455                 460 
               
               
                   
               
               
                 Asp Asp Asn Tyr Asp Lys Thr Glu Xaa Val Asp Ile Glu Glu Met Ala 
               
               
                 465                 470                 475                 480 
               
               
                   
               
               
                 Leu Lys Leu Asp Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala Ser Ser 
               
               
                                 485                 490                 495