Marker sequences for diagnosing and stratifying systemic sclerosis patients

Abstract

The present invention relates to methods for identifying markers for systemic sclerosis (also scleroderma; SSc) and to the markers identified with the aid of this method, which can differentiate between SSc and other autoimmune diseases on the one hand and between different SSc subgroups on the other hand. The invention also relates to panels, diagnostic devices and test kits which comprise these markers, and to the use and application thereof, for example for the diagnosis, prognosis and therapy control of SSc. The invention also relates to methods for screening and for validating active substances for use in SSc.

Claims

1. A fusion protein for detecting systemic sclerosis (SSc), comprising: a peptide sequence selected independently of one another from any one of SEQ ID NOs: 1-9, 13, and 16-18, and at least one protein tag bound to the peptide sequence.

2. The fusion protein of claim 1, wherein the peptide sequence has a length of no more than 35 amino acids.

3. The fusion protein of claim 1, wherein the peptide sequence is selected independently of one another from any one of SEQ ID NOs: 2-8, 13, and 16-18.

4. The fusion protein of claim 1, wherein the tag is selected from c-myc, His tag, Arg tag, FLAG, alkaline phosphatase, V5 tag, T7 tag or Strep tag, HAT tag, NusA, S tag, SBP tag, thioredoxin, and DsbA.

5. The fusion protein of claim 1, wherein the fusion protein has one or more additional domains selected from a cellulose-binding domain, green fluorescent protein, maltose-binding protein, calmodulin-binding protein, glutathione S-transferase and lacZ.

6. The fusion protein of claim 1, wherein the peptide sequence is a binding region.

7. The fusion protein of claim 1, wherein the peptide sequence is an epitope.

8. The fusion protein of claim 1, wherein the fusion protein binds to autoantibodies that are present during the course of development, establishment, or therapy of SSc.

9. The fusion protein of claim 8, wherein the fusion protein binds to autoantibodies that are up-regulated or down-regulated during the course of development, establishment, or therapy of SSc.

10. A pharmaceutical composition for the treatment of systemic sclerosis (SSc), comprising a peptide sequence selected independently of one another from any one of SEQ ID NOs: 1-9, 13, 15-18, at least one protein tag bound to the peptide sequence, and at least one additive or auxiliary agent in the pharmaceutical composition.

11. A method for screening active substances for diagnosis of SSc, comprising: a) contacting a substance to be tested with at least one fusion protein of claim 1; and b) detecting an interaction of the substance to be tested with the one or more fusion proteins.

Description

(1) The following examples explain the invention, but do not limit the invention to the examples. In the following figures, systemic sclerosis is denoted by PPS (progressive systemic sclerosis).

(2) FIG. 1 shows the intensity plots of the CENPA peptides that were achieved with the patient samples PG520-P01-2012-076, PG520-P01-2012-090, PG520-P01-2012-100 and PG520-PG1-2012-138 at a dilution of 1:5000.

(3) FIG. 2 shows the intensity plots of the CENPA peptides that were achieved with the patient sample PG520-P01-2012-076.

(4) FIG. 3 shows the intensity plots of the CENPA peptides that were achieved with the patient sample PG520-P01-2012-090.

(5) FIG. 4 shows the intensity plots of the CENPA peptides that were achieved with the patient sample PG520-P01-2012-100.

(6) FIG. 5 shows the intensity plots of the CENPA peptides that were achieved with the patient sample PG520-P01-2012-138.

(7) FIG. 6 shows the intensity plots of the KDM6B peptides that were achieved with the patient samples PG520-P01-2012-076, PG520-P01-2012-090, PG520-PG1-2012-100 and PG520-P01-2012-138 at a dilution of 1:5000.

(8) FIG. 7 shows the intensity plots of the KDM6B peptides that were achieved with the patient sample PG520-P01-2012-138.

EXAMPLES

Example 1

Description and Objective of the Study

(9) The objective of the study was to determine the relevant epitope/the relevant, epitopes of the novel autoantigen KDM6B. Within the scope of this study, peptide microarrays from PEPperPRINT were used.

(10) The CENPA antigen was analysed as the target antigen and used for the evaluation of the technology. In addition, the novel KDM6B antigen was used as an antigen for the diagnosis of SSc. While this antigen originally has a very large scope, it was only expressed as a short N-terminal fragment.

(11) For epitope mapping, 15-mer peptides were generated, with an overlap of 14 amino acids from peptide to peptide.

(12) All peptides that cover the sequence of both antigens were printed on a flat microarray by way of PEPperPRINT. In total, four microarrays were generated. For analysis, three sera from SSc patients which exhibited reactivity to both antigens were selected and incubated on the arrays. One serum from an SSc patient which exhibited no reactivity to these two antigens was used as the negative control.

(13) The primary objective of the present study was to identify the relevant immunogenic regions/epitopes of CENPA, labelled by SSc sera, by way of peptide microarrays, and to compare the results to the previously published state of the art.

(14) The secondary objective of the present study was to identify putative immunogenic epitopes of a novel antigen (KDM6B).

(15) The third objective was to analyse whether the three disease sera behave identically or whether they exhibit biological diversity based on polyclonal immune response.

Example 2

Selection of the SSc Patient and Control Samples

(16) In total, four different samples from SSc patients were selected on the basis of the reactivity thereof to the respective antigens CENPA and KDM6B (Table 1).

(17) Three of the SSc patient samples (PG520-P01-2012-076, PG520-P01-2012-090, PG520-P01-2012-100), which tested positive for anti-CENPA and anti-KDM6B protein fragments, were selected as positive samples.

(18) An SSc control sample (PG520-P01-2012-138), which showed negative test results for anti-CENPA and anti-KDM6B, was included. However, this does not preclude the possibility that this sample may react negatively to an epitope located at a distance from the region that was tested in the previous screen.

(19) TABLE-US-00002 TABLE 1 Overview of positive and negative control series/plasma samples that were incubated on peptide arrays. Age Sample Tube Sample Gen- SCL Ana of external Donor Barcode Identifier w der Indication Diagnose Antiz + 70+ pos donor identifier identifier 116654445 PG520- PSS F Progressive diffuse F. x — x   52   21 PG520-P01- P01- 2012-090D 2012- 090 116654423 PG520- PSS F Progressive limitierte x — x   553   7 PG520-P01- P01- F. 2012- 076 2012-076D 116654420 PG520- PSS F Progressive limitierte x — x   557   31 PG520-P01- P01- F. 2012- 100 2012-100D 116654439 PG520- PSS F Progressive limiterte — — x   553   69 PG520-P01- P01- F. 2012- 076 2012-138D Tube Date of BBA16.344_ BBA17.083_ BBA25.203_ BBA16.344_ BBA17.083_ BBA25.203_ Barcode Birth 0105510253 1047890025 1066859716 0105510253 1047890025 1066859716 GeneID 23135 23135 1058 23135 23135 1058 Gene KDM6B KDM6B CENPA KDM6B KDM6B CENPA Symbol Gene lysine (K)- lysine (K)- centromere lysine (K)- lysine (K)- centromere Name specific specific protein A specific specific protein A demethylase demethylase demethylase demethylase 6B 6B 6B 6B HV  776  2108  1760  458  1191  925 Mean HVSD  991  2563  3822  914  3064  1829 SLE SLE SLE HV HV HV Cutoff  2757.7321  7234.02335  9404.01883  2286  7319  4583 mean + 2 SD Cutoff  2758  7234  9404  2286  7319  4583 mean + 2 SD Cutoff  3749  9797 13226  3200 10383  6411 mean + 3 SD 116654445 NA 18576.4937 49161.9785 17368.5453 18576 49162 17369 116654423 NA 29769.6421 49161.9785 27676.8844 29770 49162 27677 116654420 NA 10340.0046 17180.4974 27676.8844 10340 17180 27677 116654439 NA  368  878  206  368  878  206

Example 3

Epitope Mapping by Way of Peptide Microarrays

(20) 3.1 Description of Peptide Microarrays

(21) The antigens CENPA and KDM6B were converted into 15-mer peptides with an overlap between peptides of 14 amino acids, which resulted in 1,831 different peptides, printed in duplicates (a total of 3,662 peptide spots). The corresponding peptide microarrays were additionally formed by FLAG and HA control peptides (124 spots each).

(22) 3.2 Experimental Conditions and Procedure

(23) The experimental conditions were listed as indicated by PEPperPRINT:

(24) Incubation buffer: PBS, pH 7.4 with 0.05% Tween 20 and 10% Rockland's blocking buffer

(25) Washing buffer: PBS, pH 7.4 with 0.05% Tween 20 (2×1 min after each assay)

(26) Blocking buffer: Rockland's blocking buffer MB-070 (60 min prior to first assay)

(27) Conditions for the assay: serum dilutions of 1:5000 and 1:1000 in the incubation buffer; incubation for 16 hrs at 4° C. and shaking at 500 rpm.

(28) Secondary antibody: F(ab′)2 goat anti-human IgG(H+L) conj. DyLight680; 30 min staining at RT and a dilution of 1:5000

(29) Control antibody: monoclonal anti-HA (12CA5)-DyLight680, monoclonal anti-FLAG (M2)—DyLight800; staining in the incubation buffer for 1 h at RT and a dilution of 1:1000

(30) Scanner: LI-COR Odyssey Imaging System; scanning offset 1 mm, resolution 21 μm, scanning intensity green/red 7/7

(31) Microarray data: MicroarryData_PG520-P01-2012-076.xlsx, MicroarrayData_PG520-P01-2012-090.xlsx, MicroarrayData_PG520-PO1-2012-100.xlsx, MicroarrayData_PG520-P01-2012-138.xlsx, MicroarrayData_Summary.xlsx

(32) Microarray Identification: 000616_02, 000616_03, 000646_05, 000646_06 (two array copies per microarray)

(33) The pre-staining of one of the peptide arrays was carried out with the F(ab′)2 goat anti-human IgG(H+L) conj. DyLight680 antibody at a dilation of 1:5000 to analyse the background interactions with the peptides that included no antigens, which could impair the primary assays. The subsequent incubation of the peptide microarrays with the human sera PG520-P01-2012-076, PG520-P01-2G12-090, PG520-P01-2012-100 and PG520-P01-2012-138 at dilutions of 1:5000 and 1:1000 in the incubation buffer was followed by staining with the secondary antibody and the readout at a scanning intensity of 7 (red). HA and FLAG control peptides, which frame the peptide arrays, were finally stained for internal quality control to verify the quality of the assay and the integrity of the peptide microarray (scanning intensities red/green 7/7).

Example 4

Data Analysis

(34) The data analysis was carried out by way of PEPperPRINT as described hereafter. The quantification of the spot intensities and the peptide detection were carried out with the aid of the PepSlide® analyser. A software algorithm breaks down the fluorescence intensities of every spot into raw, foreground and background signals and calculates the standard deviation from mean foreground intensities. Intensity maps were generated based on averaged mean foreground intensities, and the binders were labeled in the peptide maps by a red intensity color code for high spot intensities and in white for low spot intensities, PEPperPRINT additionally recorded averaged spot intensities of all assays against the linked antigen sequences from the N-terminus of CENPA to the C-terminus of KDM6B so as to visually represent all the spot intensities and the relationship between signal and sound.

(35) The intensity plots were correlated with peptide and intensity maps and with a visual inspection of the microarray scans so as to identify peptides and consensus patterns that interacted with the plasma samples.

(36) Where it was unclear whether a certain amino acid contributed to the binding of antibodies, the corresponding letters were written in gray color.

Example 5

Peptide Mapping of CENPA

(37) As shown in FIG. 1, the intensity plots (dilution 1:5000) of the CENPA peptides underscored the very strong and almost identical reaction of the sera PG520-P01-2012-076, PG520-P01-2012-090 and PG520-P01-2012-100 with the two primary epitopes RSPSPTPTPGPSR (SEQ ID No. 13) and GPSRRGPSLGAS (SEQ ID No. 11) and a third, but superimposed (overlapping) epitope TPTPGPSRRGPS (SEQ ID No. 12).

(38) All these three individual peptides are represented by the amino acids (aa) 16-36 (FIG. 1). The serum PG520-P01-2012-076 showed a minor difference in the intensity ratio of the two primary epitopes and caused an additional N-terminus epitope RSPSPTPTPGPSR (SEQ ID No. 15) or PRRRSRKPEAPR (SEQ ID No. 14) (represented by aa 3-14). This epitope also has a weak reaction in the sample PG520-P01-2012-090 at a dilution of 1:1000 (FIG. 3). Moreover, the sample PG520-P01-2012-100 exhibits an additional reaction to this N-terminus region at a dilution of 1:1000 (FIG. 4).

(39) The patient sample PG520-P01-2012-138, which tested negative for anti-CENPA in the Luminex Perl assay, exhibits a weak reaction (FIG. 5). Only three individual peptide interactions based on the peptides APRRRSPSPTPTPGP (SEQ ID No. 16), RRRSPSPTPTPGPSR (SEQ ID No. 17) and SPSPTPTPGPSRRGP (SEQ ID No. 18) were observed at a dilution of 1:1000, not, however, at a dilution of 1:5000.

Example 6

Distribution of the Epitopes of CENPA

(40) The distribution of the epitopes that were identified by way of peptide mapping is shown in Table 2.

(41) TABLE-US-00003 TABLE 2  The amino acid sequence of CENPA (Gen ID: 1058) MGPRRRSRICPEAPRR SHQHSRRRQGWLKE IRKLQKSTHLLIRKLPFSRLAREICVKFTRGVDFNWQAQALLALQEAAEA FLVHLFEDAYLLTLHAGRVTLFPKDVQLARRIRGLEEGLG

(42) The larger epitope identified by peptide mapping is double-underlined (aa 16-36), while the second, weaker epitope is single-underlined (aa 3-14).

(43) Muro et al. expressed as series of cut peptides in E. coli and conducted an immunoblot analysis with 91 ACA-positive sera (Muro et. al., 2000). Eighty of the sera (88%) with ACA reacted to the N-terminus region with 52 amino acids, while none of the sera reacted to the C-terminus. Two synthetic peptides (amino acid sequences aa 3±17 (peptide A) and aa 25±38 (peptide B)) reacted in ELISA to 78 (86%) and 79 (87%) of the ACA-positive sera. Peptide A corresponds to the second epitope (aa 3-14), which was less reactive in our presently tested, patient samples than described by Muro et al. (2000).

(44) Peptide B is part of the larger epitope (aa 16-36) identified by peptide arrays.

(45) By way of the systematic approach of peptide mapping, it is possible to identify not only individual epitopes in the order of magnitude of the presently used peptides (14 aa), but also larger regions that are covered by multiple, overlapping peptides.

(46) TABLE-US-00004 TABLE 3  DNA sequence of Homo sapiens centromere protein A (CENPA), transcription variant 1, mRNA (ref|NM_001809.3|) gray: coding sequence (base pair 184-606); blue: sequence of the expression clone 00700_007_E24, BBA25_203; red: the larger epitope identified by peptide mapping; black: sequence portions not represented by the expression clone.    1 ccgtgaagtg ggcggagcga gcgatttgaa cgcgagcggc gcggacttct gccaagcacc   61 ggctcatgtg aggctcgcgg cacagcgttc tctgggctcc ccagaagcca gcctttcgct  121 cccggacccg gcagcccgag caggagccgt gggaccgggc gccagcaccc tctgcggcgt  181 gtcatgggcc cgcgccgccg gagccgaaag cccgaggccc cgaggaggcg cagcccgagc  241 ccgaccccga cccccggccc ctcccggcgg ggcccctcct taggcgcttc ctcccatcaa  301 cacagtcggc ggagacaagg ttggctaaag gagatccgaa agcttcagaa gagcacacac  361 ctcttgataa ggaagctgcc cttcagccgc ctggcaagag aaatatgtgt taaattcact  421 cgtggtgtgg acttcaattg gcaagcccag gccctattgg ccctacaaga ggcagcagaa  481 gcatttctag ttcatctctt tgaggacgcc tatctcctca ccttacatgc aggccgagtt  541 actctcttcc caaaggatgt gcaactggcc cggaggatcc ggggccttga ggagggactc  601 ggctgagctc ctgcacccag tgtttctgtc agtctttcct gctcagccag gggggatgat  661 accggggact ctccagagcc atgactagat ccaatggatt ctgcgatgct gtctggactt  721 tgctgtctct gaacagtatg tgtgtgttgc tttaaatatt tttctttttt ttgagaagga  781 gaagactgca tgactttcct ctgtaacaga ggtaatatat gagacaatca acaccgttcc  841 aaaggcctga aaataatttt cagataaaga gactccaagg ttgactttag tttgtgagtt  901 actcatgtga ctatttgagg attttgaaaa catcagattt gctgtggtat gggagaaaag  961 gctatgtact tattatttta gctctttctg taatatttac attttttacc atatgtacat 1021 ttgtactttt attttacaca taagggaaaa aataagacca ctttgagcag ttgcctggaa 1081 ggctgggcat ttccatcata tagacctctg cccttcagag tagcctcacc attagtggca 1141 gcatcatgta actgagtgga ctgtgcttgt caacggatgt gtagcttttc agaaacttaa 1201 ttggggatga atagaaaacc tgtaagcttt gatgttctgg ttacttctag taaattcctg 1261 tcaaaatcaa ttcagaaatt ctaacttgga gaatttaaca ttttactctt gtaaatcata 1321 gaagatgtat cataacagtt cagaatttta aagtacattt tcgatgcttt tatgggtatt 1381 tttgtagttt ctttgtagag agataataaa aatcaaaata tttaatgaaa a

Example 6

Peptide Mapping of KDM6B

(47) As shown in FIG. 6, the intensity plots (dilution 1:5000) underscored the moderate to strong and partially similar reaction of the sera PG520-P01-2012-076, PG520-P01-2012-090 and PG520-P01-2012-100 with the primary epitopes LPAPLPPSHGSS (SEQ ID No. 2) and SPQPSASSSSQF (SEQ ID No. 3). The LPAPLPPSHGSS epitope (SEQ ID No. 2) (aa 55-67) was common to all positive serum samples.

(48) In contrast, the SPQPSASSSSQF (SEQ ID No. 3) aa 861-873) epitope was only observed in the sera PG520-P01-2012-076 and PG520-P01-2012-090. The neighboring epitope SSQFSTSGGPWAR (SEQ ID No. 4) (aa 870-881), however, was likewise detected in sample PG520-P01-2012-076 and GGPWARERRAGEEPV (SEQ ID no. 6) (aa 877-890) and was detected slightly by PG520-P01-2012-100 (1:1000 dilution). The result is an immunogenic region from aa 861 to 890.

(49) Other epitopes, such as REKLNPPTPSIYL (SEQ ID No. 5), were identified only in one of the samples.

(50) The patient sample PG520-P01-2012-138, which tested negative for KDM6B in the Luminex Perl assay, exhibited a weak to moderate reaction to several individual peptides at a dilution of 1:1000, and to two different peptides at a dilution of 1:5000. The majority of interactions were either based on highly alkaline peptides such as KRNYGAKRGGPPVKR (SEQ ID No. 19) or hydrophobic peptides comprising a proline at the C-terminus (such as FPKTPEVGPGPPPGP (SEQ ID No. 20) or GHPSKPYYAPGAPTP (SEQ ID No. 21). Given the indistinct morphology of the spots, the weak spot intensities and the general inconsistency of the data, all interactions were rated to be non-specific (FIG. 7).

(51) The distribution of the epitopes that were identified by way of peptide mapping is shown in Table 4. In the Sero-Tag Discovery Screens, three clones express KDM6B antigen (or fragments of KDM6B). All three expression clones express a similar coding region (aa 42-435) and comprise the largest LPAPLPPSHGSS (SEQ ID No. 2) epitope.

(52) The epitope mapping of KDM6B results in two larger epitopes and only one epitope that was detected in one of three KDM6B-positive samples. All three available expression clones of KDM6B express the similar code region (aa 42-435) and comprise the one of the larger epitopes (LPAPLPPSHGSS, SEQ ID No. 2). The second immunogenic region, which is covered by >3 peptides (aa 861-890), is not represented by these expression clones.

(53) TABLE-US-00005 TABLE 4  The amino acid sequence of CENPA (Gen ID: 23135) The larger epitope identified by way of peptide mapping is shown in bold and underlined (aa 55-67), while the second region is composed of > peptides and double-underlined (aa 861-890). A third epitope, which was detected in only one sample, is single-underlined. Bold: expressed sequence using expression clones in the Sero tag. MHRAVDPPGARAAREAFALGGLSCAGAWSSCPPHPPPRSAWLPGGRCSASIGQPPLPAPLPP SHGSSSGHPSKPYYAPGAPTPRPLHGKLESLHGCVQALLREPAQPGLWEQLGQLYESEHDSE EATRCYHSALRYGGSFAELGPRIGRLQQAQLWNFHTGSCQHRAKVLPPLEQVWNLLHLEHKR NYGAKRGGPPVKRAAEPPVVQPVPPAALSGPSGEEGLSPGGKRRRGCNSEQTGLPPGLPLPP PPLPPPPPPPPPPPPPLPGLATSPPFQLTKPGLWSTLHGDAWGPERKGSAPPERQEQRHSLP HPYPYPAPAYTAHPPGHRLVPAAPPGPGPRPPGAESHGCLPATRPPGSDLRESRVQRSRMDS SVSPAATTACVPYAPSRPPGLPGTTTSSSSSSSSNTGLRGVEPNPGIPGADHYQTPALEVS HHGRLGPSAHSSRKPFLGAPAATPHLSLPPGPSSPPPPPCPRLLRPPPPPAWLKGPACRAAR EDGEILEELFFGTEGPPRPAPPPLPHREGFLGPPASRFSVGTQDSHTPPTPPTPTTSSSNSN SGSHSSSPAGPVSFPPPPYLARSIDPLPRPPSPAQNPQDPPLVPLTLALPPAPPSSCHQNTS GSFRRPESPRPRVSFPKTPEVGPGPPPGPLSKAPQPVPPGVGELPARGPRLFDFPPTPLEDQ FEEPAEFKILPDGLANIMKMLDESIRKEEEQQQHEAGVAPQPPLKEPFASLQSPFPTDTAPT TTAPAVAVTTTTTTTTTTTATQEEEKKPPPALPPPPPLAKFPPPSQPQPPPPPPPSPASLLK SLASVLEGQKYCYRGTGAAVSTRPGPLPTTQYSPGPPSGATALPPTSAAPSAQGSPQPSASS SSQPSTSGGPWARERRAGEEPVPGPMTPTQPPPPLSLPPARSESEVLEEISRACETLVERVG RSATDPADPVDTAEPADSGTERLLPPAQAKEEAGGVAAVSGSCKRRQKEHQKEHRRHRRACK DSVGRRPREGRAKAKAKVPKEKSRRVLGNLDLQSEEIQGREKSRPDLGGASKAKPPTAPAPP SAPAPSAQPTPPSASVPGKKAREEAPGPPGVSRADMLKLRSLSEGPPKELKIRLIKVESGDK ETFIASEVEERRLRMADLTISHCAADVVRASRNAKVKGKFRESYLSPAQSVKPKINTEEKLP REKLNPPTPSIYLESKRDAFSPVLLQFCTDPRNPITVIRGLAGSLRLNLGLFSTKTLVEASG EHTVEVRTQVQQPSDENWDLTGTRQIWPCESSRSHTTIAKYAQYQASSFQESLQEEKESEDE ESEEPDSTTGTPPSSAPDPKNHHIIKFGTNIDLSDAKRWKPQLQELLKLPAFMRVTSTGNML SHVGHTILGMNTVQLYMKVPGSRTPGHQENNNFCSVNINIGPGDCEWFAVHEHYWETISAFC DRHGVDYLTGSWWPILDDLYASNIPVYRFVQRPGDLVWINAGTVHWVQATGWCNNIAWNVGP LTAYQYQLALERYEWNEVKNVKSIVPMIHVSWNVARTVKISDPDLFKMIKFCLLQSMKHCQV QRESLVRAGKKIAYQGRVKDEPAYYCNECDVEVFNILFVTSENGSRNTYLVHCEGCARRRSA GLQGVVVLEQYRTEELAQAYDAFTLVRARRARGQRRRALGQAAGTGFGSPAAPFPEPPPAFS PQAPASTSR

LITERATURE

(54) Mehra S, Walker J, Patterson K, Fritzler M J (2013). Autoantibodies in systemic sclerosis. Autoimmun Rev. 12(3):340-54. Mierau R, Moinzadeh P, Riemekasten G, Melchers I, Meurer M, Reichenberger F, Buslau M, Worm M, Blank N, Hein R, Müller-Ladner U, Kuhn A, Sunderkötter C, Juche A, Pfeiffer C, Fiehn C, Sticherling M, Lehmann P, Stadler R, Schulze-Lohoff E, Seitz C, Foeldvari I, Krieg T, Genth E, Hunzelmann N (2011). Frequency of disease-associated and other nuclear autoantibodies in patients of the German Network for Systemic Scleroderma; correlation with characteristic clinical features. Arthritis Res Ther, 13(5):R172 LeRoy E C, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger T A Jr, Rowell N, Wollheim F (1988), Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol. 15(25:202-5. Watts R., (2006). Autoantibodies in the autoimmune rheumatic diseases, Medicine, 34 (11); 441-444