Frequency of disease-associated and other nuclear autoantibodies in patients of the German Network for Systemic Scleroderma: correlation with characteristic clinical features

Laboratory at Rheumaklinik Aachen, Hauptstrasse 21, Aachen, D-52066, Germany.
Arthritis research & therapy (Impact Factor: 3.75). 10/2011; 13(5):R172. DOI: 10.1186/ar3495
Source: PubMed
ABSTRACT
In the present study, we analysed in detail nuclear autoantibodies and their associations in systemic sclerosis (SSc) patients included in the German Network for Systemic Scleroderma Registry.
Sera of 863 patients were analysed according to a standardised protocol including immunofluorescence, immunoprecipitation, line immunoassay and immunodiffusion.
Antinuclear antibodies (ANA) were detected in 94.2% of patients. In 81.6%, at least one of the autoantibodies highly associated with SSc or with overlap syndromes with scleroderma features was detected, that is, anti-centromere (35.9%) or anti-topoisomerase I (30.1%), followed in markedly lower frequency by antibodies to PM-Scl (4.9%), U1-ribonucleoprotein (U1-RNP) (4.8%), RNA polymerases (RNAPs) (3.8%), fibrillarin (1.4%), Ku (1.2%), aminoacyl-transfer RNA synthetases (0.5%), To (0.2%) and U11-RNP (0.1%). We found that the simultaneous presence of SSc-associated autoantibodies was rare (1.6%). Furthermore, additional autoantibodies were detected in 55.4% of the patients with SSc, of which anti-Ro/anti-La, anti-mitochondrial and anti-p25/p23 antibodies were most frequent. The coexistence of SSc-associated and other autoantibodies was common (43% of patients). SSc-associated autoantibodies disclosed characteristic associations with clinical features of patients, some of which were previously not acknowledged.
This study shows that five autoantigens (that is, centromere, topoisomerase I, PM-Scl, U1-RNP and RNAP) detected more than 95% of the known SSc-associated antibody responses in ANA-positive SSc patients and characterise around 79% of all SSc patients in a central European cohort. These data confirm and extend previous data underlining the central role of the determination of ANAs in defining the diagnosis, subset allocation and prognosis of SSc patients.

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RESEARC H ARTIC LE Open Access
Frequency of disease-associated and other
nuclear autoantibodies in patients of the German
network for systemic scleroderma: correlation
with characteristic clinical features
Rudolf Mierau
1*
, Pia Moinzadeh
2
, Gabriela Riemekasten
3
, Inga Melchers
4
, Michael Meurer
5
, Frank Reichenberger
6
,
Michael Buslau
7,8
, Margitta Worm
9
, Norbert Blank
10
, Rüdiger Hein
11
, Ulf Müller-Ladner
12
, Annegret Kuhn
13,14
,
Cord Sunderkötter
14
, Aaron Juche
15
, Christiane Pfeiffer
16
, Christoph Fiehn
17
, Michael Sticherling
18
,
Percy Lehmann
19
, Rudolf Stadler
20
, Eckhard Schulze-Lohoff
21
, Cornelia Seitz
22
, Ivan Foeldvari
23
, Thomas Krieg
2
,
Ekkehard Genth
24
and Nicolas Hunzelmann
2
Abstract
Introduction: In the present study, we analysed in detail nuclear autoantibodies and their associations in systemi c
sclerosis (SSc) patients included in the German Network for Syst emic Scleroderma Registry.
Methods: Sera of 863 patients were analysed according to a standardised protocol including immunofluorescence,
immunoprecipitation, line immunoassay and immunodiffusion.
Results: Antinuclear antibodies (ANA) were detected in 94.2% of patients. In 81.6%, at least one of the
autoantibodies highly associated with SSc or with overlap syndromes with scleroderma features was detected, that
is, anti-centromere (35.9%) or anti-topoisomerase I (30.1%), followed in markedly lower frequency by antibodies to
PM-Scl (4.9%), U1-ribonucleoprotein (U1-RNP) (4.8%), RNA polymerases (RNAPs) (3.8%), fibrillarin (1.4%), Ku (1.2%),
aminoacyl-transfer RNA synthetases (0.5%), To (0.2%) and U11-RNP (0.1%). We found that the simultaneous
presence of SSc-associated autoantibodies was rare (1.6%). Furthermore, additional autoantibodies were detected in
55.4% of the patients with SSc, of which anti-Ro/anti-La, anti-mitochondrial and anti-p25/p23 antibodies were most
frequent. The coexistence of SSc-associated and other autoantib odies was common (43% of patients). SSc-
associated autoantibodies disclosed characteristic associations with clinical features of patients, some of which
were previously not acknowledged.
Conclusions: This study shows that five autoantigens (that is, centromere, topoisomerase I, PM-Scl, U1-RNP and
RNAP) detected more than 95% of the known SSc-associated antibody responses in ANA-positive SSc patients and
characterise around 79% of all SSc patients in a central European cohort. These data confirm and extend previous
data underlining the central role of the determination of ANAs in defining the diagnosis, subset allocation and
prognosis of SSc patients.
Keywords: systemic sclerosis, scleroderma, autoantibodies, antinuclear antibodies
* Correspondence: mierau.rmn@t-online.de
1
Laboratory at Rheumaklinik Aachen, Hauptstrasse 21, Aachen, D-52066,
Germany
Full list of author information is available at the end of the article
Mierau et al. Arthritis Research & Therapy 2011, 13:R172
http://arthritis-research.com/content/13/5/R172
© 2011 Mierau et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (htt p://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Page 1
Introduction
Autoantibodies targeting characteristic nuclear antigens
are one of the hallmarks of systemic sclerosis (SSc)
[1-3]. The occurrence of different antinuclear an tibodies
(ANAs) is associated with distinct disease subtypes and
with differences in disease severity, including extent of
skin involvement, internal organ manifestation and
prognosis. Although the current SSc criteria of the
American College of Rheumatology [4] do not include
thepresenceofANAs,Thedetectionofscleroderma-
associated ant ibodies may be a valuable tool in the diag-
nosis of patients with very ear ly SSc or onl y subtle
symptoms [5,6]. For instance, in a recent study of
patients with Raynaud s phenomenon, the presence of
ANAs (adjusted HR = 5.67) a nd SSc-associated antibo-
dies (HR = 4.7) was the strongest independent predictor
of definite SSc [6]. Some of the autoantibodies in SSc
are regarded as disease-specific and can be correlated
with genetic, demographic, diagnostic, clinical and prog-
nostic aspects of the disease [1,3]. Therefore, autoanti-
bodies are pivotal tools in the diagnosis of SSc by
helping clinicians make deci sions whether to perform
further, more detailed and efficient diagnostic proce-
dures, as well as decisions addressing disease
management.
For frequently occurring antibodies such as anti-cen-
tromere (ACA) and anti-topoisomerase I (ATA), reliable
detection systems based on ELISA or other binding tests
have been devel oped. Other antibodies (that is, t o fibril-
larin, RNA polymerases (RNAPs) and so on) are not
identified by common test procedures, but rather by
laboratories able to perform sophisticated proced ures to
confirm the results on the basis of more than one inde-
pendent method. Even for the most common autoanti-
bodies, the choice of the detection method used is
critical to the sensitivity and specificity of the results
and hence their diagnostic value [7].
Researchers in numerous studies have examined the
presence of antibodies to single predefined antigens in
SSc and their cli nical as sociations, whereas many of the
investigators who have comprehensively examined large
SSc pati ent c ohorts have often restricted their autoanti-
body analyses to the most common SSc antibodies,
ACA and ATA [8-12], or analys ed only a few additional
antibodies [13-17]. The aim o f this study was therefore
to characterise all known non-organ-specific, SSc-asso-
ciated autoantibodies, as well as other, potentially new
nuclear autoantibo die s by using a standardised protocol
inthelargeSScpatientcohortincludedintheGerman
Network for Systemic Scleroderma Regi stry, and to cor-
relate these findings with the clinical characteristics of
these patients.
Materials and methods
Patients
Serum samples from 863 consec utive patien ts included
in the German Network for Systemic Scleroderma Reg-
istry between 20 04 and 2007 from 23 different clinical
centres were analysed. Patient data are gathered and
registered using a consensual registration form and
reference documents with item definitions and recom-
mendations for organ-specific diagnostic procedures as
previously described [18,19]. O f the patients included,
82.9% we re female, their mean age ± SD was 58.0 ±
13.4 years (median = 60 years, range 12 to 93). The
patients age at disease onset ranged from 3 to 87 years
(median = 49 years, mean = 47.7 ± 14.2).
The registry defines five subsets, that is, limited cuta-
neous and diffuse cutaneous SSc [20], overlap syndrome
[21,22], systemic sclerosis sine scleroderma [23,24] and
undifferentiated connective tissue disease with features
of scleroderma [25,26], as recently described [18]. The
latter subset corresponds largely to the subgroup early
SSc as described by LeRoy and Medsger [5] but may
also include patients who will never develop definite
SSc. The study, including the patients informed consent
regarding data storage, was approved b y the lead Ethics
committee of the Cologne University Hospital and by
the respective ethics committees of the contributing
centres.
Autoantibody analysis
To detect SSc-asso ciated autoantibodies in a compre-
hensive way, we used the search strategy commonly per-
formed in diagnostic procedures for connective tissue
diseases based on a HEp-2 cell immunofluorescence
assay followed by tests using cellular extracts and/or
recombinant antigens . This strategy is focused on circu-
lating antibodies against non-organ-specific cellular
autoantigens. C ell- or tissu e-specific autoantibodies,
which have also been described in scleroderma patients
[3], were not included in the analytical protocol. At least
one serum draw from each patient (N = 863) was ana-
lysed for circulating autoantibodies by a predefined pro-
tocol (Figure 1) with at least four assay systems
performed in a single laboratory by a single group of
technologists.
Indirect immunofluorescence using fluorescein iso-
thiocyanate-con jugated goat anti-h uman immunoglobu-
lin G was performed as a screening method for the
detection of ANAs on HEp-2 cells (HEp-20-10; Euroim-
mun, Lübeck, Germany) seeded onto a microscope slide
[27]. Titres of at least 1:80 dilution were regarded as
positive. Different nuclear and cytoplasmic fluorescence
patterns were documented.
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Page 2
Alineimmunoassay(EUROLINEANAProfile3;
Euroimmun) was p erformed according to the manufac-
turers instructions. This assay is able to detect, by bind-
ing to recombinant or purified antigens, the following
autoantibodies: U1-ribonucleoprotein (U1-RNP), Sm,
Ro60, Ro52, La (SS-B), Scl -70, PM-Scl, ce ntromere pro-
tein B (CENP-B), proliferating cell nuclear antigen
(PCNA), double-stranded DNA (dsDNA), nucleosomes,
histones, ribosomal P proteins and the mitochondrial
M2 antigen.
Immunoprecipitation (IP) of radiolabelled HeLa cell
extract was performed as described [28,29] with slight
modifications. In brief, HeLa S3 cells in suspension cul-
ture in methionine -deficient RPMI medium wit h 10%
dialyzed foetal bovine serum were incubated with
35
S
methionine (to a final activity of 0.3 MBq/ml) overnight.
They were (1) washed in Tris-buffered saline and ( 2)
lysed by resuspension in IP buffer (10 mM Tris·HCl, pH
8.0, with 500 mM NaCl, 0.1% Igepal (Sigma, Munich,
Germany) and 2 mM phenylmethylsulfonyl fluoride) and
sonication on ice. IP was performed by incubation of
patient sera (10 μl) with protein A Sepharose beads (2
mg in 500 μl; Sigma) for two hours, three short washing
steps, end-over-end rotation with the radiolabelled cell
extract overnight, five washi ng steps, separation of the
precipitates on 5 % to 20% gradient SDS-PAGE gels and
subsequent autoradiography for six to ten days. Bands
typical of autoantibodies were identified according to
their apparent molecular weight and comigration with
bands produced by reference sera with known autoanti-
body specificity. After we completed this procedure, the
autoantibodies to the following antigens were routinely
detectable: topoisomerase I (Scl-70), RNAPs (I and/or
III), Ku, fibrillarin, To, NOR-90, Pl-7, Pl-12, EJ, OJ, KS,
Mi-2, signal recognition particle, ribon ucleoprotein
(usually U1-RNP), SL, PCNA, ribosomal P proteins a nd
p25/p23, also known as anti-chromo [30,31]. In our
hands, the detection of antibodies to Ro, La, PM-Scl, Jo-
1, centromere antigens and the mitochondrial M2 anti-
genwasunreliablebythismethod.Bandsofunknown
nature were registered and entered into the database of
autoantibody results.
Immunodiffusion (ID) was performed in A garose gels
with rabbit and/or calf thymus extract (Pel-Freez Biolo-
gicals, Rogers, AR, USA) as described previously [32].
Autoantibodies were identified by the identity of precipi-
tation lines with patient sera compared with monospeci-
fic prototype sera with known autoantib ody specificity.
The use of prototype serum was guided by results of the
immunofluorescence pattern on HEp-2 cells. The fol-
lowing auto antibodies were detectable: topoisomerase 1
(Scl-70), PM-Scl, Ku, SL, Jo-1, Pl-7, U1-RNP, Sm and La
(SS-B). Precipitation bands that did not merge with any
of t he prototype sera were registered as unknown a uto-
antibodies and entered into the database of autoantibody
results.
In selected sera, confirmatory assays using recombi-
nant or synthetic antigens (see Table 1 Antibody det ec-
tion criteria) were performed. More than one serum
sample was available from 213 patient s (from 2 to 25
samples). At least one serum sample per pa tient was
tested with the whole protocol described in Figure 1,
whereas in most cases th e follow-up se ra drawn were at
least partially charac terised with, for example, the HEp-
Figure 1 Protocol for serological analysis of systemic sclerosis patient sera.
Mierau et al. Arthritis Research & Therapy 2011, 13:R172
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Page 3
Table 1 Methodological criteria for assignment of autoantibodies
Autoantibody
against
Findings classifying patients as antibody-positive Usual additional findings
Centromere Centromeric immunofluorescence pattern on HEp-2 cells (308 of
310 were positive) or a CENP-B band in line assay (309 of 310
positive)
Topoisomerase
I
At least two of three findings: Scl-70-positive signal in line assay
(258 of 260 positive), a band comigrating with a prototype band
in IP (all positive), a line of identity in ID with the Scl-70
prototype serum (244 of 260 positive)
Typical HEp-2 cell immunofluorescence pattern: fine granular
karyoplasmic, weakly nucleolar, metaphase chromosome-positive
RNA
polymerases
Characteristic IP pattern comigrating with the pattern of a
prototype serum mainly consisting of four bands: Ia, Ib, IIIa and
IIIb [58]
Confirmation by ELISA in 32 of 32 cases with the
immunodominant epitope of RNA polymerase III subunit
RPC155 according to Kuwana et al. [71], provided by Matritec,
Freiburg, Germany. ANA immunofluorescence on HEp-2 cells
was predominantly fine granular only sometimes (five cases) in
addition nucleolar [72].
Fibrillarin An IP band (approximately 34 kDa) comigrating with a
prototype serum band, plus a nucleolar immunofluorescence
pattern on HEp-2 cells
Confirmation by investigational ELISA kindly provided by
Euroimmun, Lübeck, Germany, positive in 11 and borderline in 1
of the 12 cases
To An IP band of approximately 40 kDa plus a nucleolar
immunofluorescence pattern on HEp-2 cells; confirmation by
immunoblot analysis with recombinant To antigen kindly
supplied by Dr M Blüthner, Labor Seelig, Karlsruhe, Germany
PM-Scl A line of identity in ID with a PM-Scl prototype serum (41 of 42
cases) and/or positive result of ELISA with the synthetic peptide
PM-1a [73] (Dr Fooke Laboratorien GmbH, Neuss, Germany) (12
of 13 cases)
Positive reaction in 37 of 41 cases for PM-Scl by line assay. ANA
immunofluorescence on HEp-2 cells usually was nucleolar plus
fine granular karyoplasmic.
Ku Two prominent IP bands at about 70 and 80 kDa comigrating
with prototype bands
In 3 of 10 cases, a line identical to a Ku prototype band in ID.
ANA immunofluorescence was finely granular, usually at a high
titre.
U1-RNP A positive signal for RNP/Sm in line assay, with or without a
positive signal for Sm, plus a typical IP pattern consisting of at
least antigen A (about 33 kDa), antigen B/B (about 28/29 kDa)
and antigen C (about 22 kDa)
In 37 of 41 cases, a line of identity with a U1-RNP prototype in
ID. ANA pattern on HEp-2 cells usually was coarsely speckled.
Sm A positive signal for RNP/Sm as well as for Sm in line assay In two of four cases, a band identical to a Sm prototype in ID
with ribonuclease-digested calf thymus extract. IP and
immunofluorescence patterns were similar to those found for
anti-U1-RNP.
Jo-1 A positive signal for Jo-1 in line assay plus a band identical to a
Jo-1 prototype band in ID
Immunofluorescence on HEp-2 cells was inconsistent.
Pl-7 IP band of about 80 kDa comigrating with prototype band plus
a band identical to a Pl-7 prototype band in ID
Cytoplasmic immunofluorescence on HEp-2 cells
OJ Typical triplet band in IP comigrating with prototype bands Cytoplasmic immunofluorescence on HEp-2 cells
U11-RNP
[74,75]
An RNP-like IP pattern and coarsely speckled ANA
immunofluorescence, without any U1-RNP signals in line assay
and ID; U11-RNP specificity detected by C Will and R Lührmann,
Marburg, Germany
p25/p23 [76,77] Doublet IP bands of about 25 and 23 kDa, with the 25 kDa
band comigrating with the precipitate of rabbit anti-p25 kindly
provided by E Chan, Gainesville, FL, USA
HEp-2 cell immunofluorescence pattern was always centromeric
because anti-p25/p23 was exclusively found together with
anticentromere.
SL A band identical to the SL prototype band in ID plus an IP band
at about 31 kDa comigrating with the precipitate of the SL
reference serum
HEp-2 cell immunofluorescence pattern was fine granular, but in
this study often was masked because of other coexisting
antibodies
NOR-90 Doublet IP bands at about 90 kDa comigrating with the
precipitate of a NOR-90 reference serum [78]
The nucleolar immunofluorescence pattern expected on HEp-2
cells was hard to detect in the sera examined in this study,
because NOR-90 antibodies in all cases coincided with other
autoantibodies visible on HEp-2 cells.
Mitochondrial
M2
AMA M2-positive signal by line assay (40 of 41 positive) and/or
AMA typical cytoplasmic immunofluorescence on HEp-2 cells
and/or rat kidney sections (27 of 41 positive)
In IP, a band of around 70 kDa was present in 36 of 41 cases.
Sp100 Multiple nuclear dot pattern on HEp-2 cells [79] plus Sp100
signal in the line assay HUMAN IMTEC-Liver Line Immunoassay
(HUMAN Diagnostics GmbH, Wiesbaden, Germany)
Ro52 Ro52-positive signal by line assay
Ro60 Ro60-positive signal by line assay
La La-positive signal by line assay
AMA = antimitochondrial antibodies; ANA = antinuclear antibodies; CENP-B = centromere protein B; ID = immunodiffusion; IP = immunoprecipitation; PM-Scl =
polymyositis and scleroderma; RNAP = RNA polymerase; RNP = ribonucleoprotein;.
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Page 4
2 cell test. The criteria for classifying sera as positive for
autoantibodies are listed in Table 1 together with addi-
tional serological results commonly found and helpful to
identify the antibodies.
In addition to the autoantibodies defined in Tabl e 1
other circulating au toantibodies detected by at least one
of the above-mentioned procedures, either known (for
example, anti-histone, anti-dsDNA) or unknown (for
example, either unidentified bands in IP or ID or anti-
nuclear or anticytoplasmic antibodi es on HEp-2 cells
without subsequent identification), were registered. Sera
which were negative for ANAs in immunofluorescence
on HEp-2 cells but exhibited cytoplasmic fluorescence
in that assay and/or a positive signal in any of the other
assays were grouped together as ANA-negative. Sera
without any positive signal, neither defined nor unde-
fined, in all four assay systems described above were
listed as autoantibody-negative.
Statistics
The data were analysed using Microsoft Excel (Microsoft
Corp, Redmond, WA, USA) and SPSS version 14.0 soft-
ware (SPSS, Inc, Chicago, IL, U SA) for tabular and gra-
phic representation. Statistical evaluation was performed
using contingency table tests with the help of GraphPad
Prism version 3.02 software (GraphPad, La Jolla, CA,
USA). We calculated OR and 95% CI data. P-values were
calculated using Fisher s exact test. When multiple tests
were performed, P-values below 0.005 were recorded
without performing strict Bonferroni correction. For
most vari ables, l ess than 5% of data were missing. Quan-
titative data (erythrocyte sedimentation rate (ESR), age at
disease onset and Rodnan skin score), depending on the
presence or absence of different autoantibodies, were
analysed using the Mann-Whitney rank-sum test.
Results and discussion
Of the 863 SSc patients studied, 513 (59.4%) were classi-
fied as having limited disease and 173 (20.1% ) we classi-
fied as having diffuse cutaneous disease. Another 108
patients (12.5%) had a scleroderma overlap syndrome,
64 (7.4%) had undifferentiated connective tissue disease
with scleroderm a featu res and 5 (0.6 %) had sys temic
sclerosis sine scleroderma.
The frequency of autoantibodies de tected in these
patients is shown in Table 2. Overall, ANAs were
detected in 94.2% of patients. This frequency was similar
to data previously publish ed [12,14,15,33,34] in which
ANA frequencies reported w ere between 85% and 99%.
Among our patients with ANAs, 86.6% (704 of 813) had
autoantibodies known to be highly associated with SSc,
and am ong these latter patients, 96.4% (679 of 704) had
antibodies that detected five antigens: centromere,
topoisomerase I, PM-Scl, U1-RNP and RNAPs.
A coincidence of SSc-associated au toantibodies (Tabl e
3) was rare, being detected in only 1.6% patients (11 of
704). The presence of a SSc-associated ant ibody without
any other autoantibo dy detectable by the methods used
was found at varying frequencies, being highest for anti-
PM-Scl (7 3.8%) and lower for, for example, anti -centro-
mere (33.9%) and anti-fibrillarin (33.3%) (see Table 3).
That SSc-associated autoantibodies are largely mutually
exclusive is well-known [14,15,35]. Coincidences in indi-
vidual patients do occur but are rare [33,36,37]. Our
study shows that this statement holds true even if all
known non-organ-specific, SSc-associated autoantibodies
are sought using a rigorous protocol in all patients. On
the other hand, additional (mainly not SSc-specific)
autoantibodies are common and were detected in about
53% in our patient cohort with SSc-associated antibodies
(and in 55.4% of all of our patients). In 14.0% of patients
(n = 121), none of the above-mentioned SSc-associated
but other (defined or undefined) autoantibodies were
found, whereas in 4.4% no autoantibodies at a ll were
Table 2 Prevalence of autoantibodies in 863 scleroderma
patients
Autoantibodies Patients, n
(%)
Positive for antinuclear antibodies 813 (94.2)
Antibodies highly associated with SSc or scleroderma
overlap syndromes
704 (81.6)
Anti-centromere 310 (35.9)
Anti-topoisomerase I 260 (30.1)
Anti-PM-Scl 42 (4.9)
Anti-U1-RNP 41 (4.8)
Anti-RNA polymerase 33 (3.8)
Anti-fibrillarin 12 (1.4)
Anti-To 2 (0.2)
Anti-Ku 10 (1.2)
Anti-Jo-1/-Pl-7/-OJ 4 (0.5)
Anti-U11-RNP 1 (0.1)
Other autoantibodies
Anti-Ro and/or anti-La 206 (23.9)
Anti-Ro52 187 (21.7)
Anti-Ro60 59 (6.8)
Anti-La 16 (1.9)
Anti-mitochondrial M2 41 (4.8)
Anti-p25/p23 28 (3.2)
Anti-NOR-90 6 (0.7)
Anti-SL 9 (1.0)
Anti-Sm 4 (0.5)
Anti-Sp100 4 (0.5)
Other (known or unknown) 363 (42.1)
Negative for all highly SSc-associated antibodies 159 (18.4)
Negative for antinuclear antibodies 50 (5.8)
Autoantibody-negative by all criteria used 38 (4.4)
ANA = antinuclear antibodies; SSc = systemic sclerosis.
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Table 3 Coincidence* of autoantibodies in 863 individual systemic sclerosis patients
Antibodies ACA ATA Anti-
PM-Scl
Anti-U1-
RNP
Anti-
RNAP
Anti-
fibrilla-rin
Anti-
To
Anti-
Ku
Anti-Jo-1/
Pl-7/OJ
Anti-U11-
RNP
Anti-
Ro52
Anti-
Ro60
Anti-
La
AMA Anti-
p25/p23
Anti-
NOR-90
Anti-
SL
Anti-
Sm
Anti-
Sp100
Other
ACA 1 0 1 0 0 0 0 0 0 92 11 1 31 28 4 2 0 3 144
ATA 1 2 0 0 0 4 0 0 36 26 6 4 0 1 4 1 0 77
Anti-PM-Scl 0 0 0 0 1 0 0 6 2 0 0 0 0 0 0 0 4
Anti-U1-
RNP
1 0 0 0 0 0 11 6 1 2 0 1 0 4 0 11
Anti-RNAP 0 0 0 0 0 5 1 1 1 0 0 1 0 0 5
Anti-
fibrillarin
00 0 0 0 0 01 0 0 00 0 8
Anti-To 0 0 0 1 1 0 0 0 0 0 0 0 2
Anti-Ku 002200001002
Anti-Jo-1/
Pl-7/OJ
030000 00001
Anti-U11-
RNP
00000 00000
Anti-Ro52 41 15 14 11 2 5 2 2 97
Anti-Ro60 14 6 0 0 0 1 1 33
Anti-La 10 00108
AMA 2011229
Anti-p25/
p23
110118
Anti-NOR-
90
01 0 4
Anti-SL 003
Anti-Sm 00
Anti-Sp100 2
Other
Isolated
a
104 153 31 19 23 4 0 4 1 1 2 1 0 0 0 0 1 0 0 79
Total
b
310 260 42 41 33 12 2 10 4 1 187 59 16 41 28 6 9 4 4 363
*Number of patients with co-occurrences of autoantibodies.
a
Isolated: presence without coincidence of any other autoantibody by all detection methods used.
b
Total number of individuals with the respective
autoantibody; these numbers mostly are smaller than the sum of all co-occurrences plus the isolated individuals listed because of some triple, quadruple and higher-order coincidences. ACA = anti-centromere
antibodies; AMA = antimitochondrial antibodies; ATA = anti-topoisomerase I antibodies;; RNAP = RNA polymerase; RNP = ribonucleoprotein.
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Page 6
detected by the methods used. Defined autoantibodies
not regarded as SSc-specific, such as anti-Ro/La, anti-
NOR-90 or AMAs rarely occurred without the evidence
of SSc-associated autoantibodies (Table 3). Antibodies
to p25/p23 were detected exclusively in conjunction
with ACA.
From 213 patients, more than one serum sample was
available (from 2 to 25 samples). In the majority (86.4%)
of cases, the results of follow-up testing remained essen-
tially the same and differed in ANA titre by up to only
two titre steps. In 11.3% (24 patients), ANA titre
changes exceeded two steps (up to eight steps), in two
cases ANAs turne d negative, in one case the ANA pat-
tern changed from finely granular to nucleolar and in
only two cases new, additional typical SSc autoantibo-
dies emerged.
The detection of antibodies in different disease subsets
is shown in Table 4. It is obvious that ACA and ATA
are not exclusive to either the limited or the diffuse sub-
set. In patients with overlap syndrome, anti-U1-RNP,
anti-PM-Scl and anti-synthetase antibodies are
characteristic.
The correlation of demographic features or signs and
symptoms of SSc w ith the presence or absence of
defined autoantibodies was investigated by contingency
table analysis. The frequencies of these features and
their positive or negative correlations with specific auto-
antibodies are listed in Table 5. For the purpose of
clarity, only those comparisons that led to P-values
below 0.05 derived by Fishers exact test are shown. Sig-
nificance was calculated without correction of P-values
for multiple comparisons, because not all variables used
were independent; however, we are aware of the fact
that some of the w eak associations listed in Table 5
might have arisen by chance due to the high number of
comparisons made. Therefore, we focused on those dif-
ferences calcul ated that w ere highly significant (P <
0.005; OR printed in bold in Table 5).
Patients with ACA represented 35.9% of all SSc
patients and 38.1% of ANA-positive SSc patients. In
accordance with previous reports [8,9,12,15,33, 36,38,39],
these patients were less often male, were older at disease
onset and had a more limited extension of cutaneous
involvement, as documented by a much lower OR for a
Rodnan skin score (RSS) above 10 (Table 5) and by a
very significantly lower mean RSS (Table 6). They had
less involvement of internal organs (pulmonary fibrosis,
cardiac, muscu loskeletal and oral involvement), with the
excepti on of pu lmonary hypertension. An association of
ACA with pulmonary hypertension has been observed
in several previous reports [2,12,33,40], but not a ll of
them [13,36,38,39]. Digital ulcers in our patients with
Table 4 Autoantibodies in different disease subsets in 863 individual systemic sclerosis patients
Limited (N = 513) Diffuse (N = 173) Overlap (N = 108) Undifferentiated (N = 64)
Antibodies n (%) OR
a
(P-value) n (%) OR (P-value) n (%) OR (P-value) n (%)
ACA 253 (49.3) 5.00 (P < 0.0001) 12 (6.9) 16 (14.8) 28 (43.8)
ATA 141 (27.5) 98 (56.6) 4.26 (P < 0.0001) 11 (10.2) 9 (14.1)
Anti-RNAP 14 (2.7) 14 (8.1) 3.11 (P = 0.0029) 2 (1.9) 2 (3.1)
Anti-U1-RNP 7 (1.4) 0 (0.0) 31 (28.7) 30.00 (P < 0.0001) 2 (3.1)
Anti-PM-Scl 16 (3.1) 2 (1.2) 22 (20.4) 9.40 (P < 0.0001) 2 (3.1)
Anti-fibrillarin 3 (0.6) 8 (4.6) 8.32 (P = 0.0005) 1 (0.9) 0 (0.0)
Anti-To 1 (0.2) 0 (0) 1 (0.9) 0 (0.0)
Anti-Jo-1/Pl-7/OJ 0 (0.0) 0 (0) 4 (3.7) 65.07 (P = 0.0002) 0 (0.0)
Anti-U11-RNP 0 (0.0) 1 (0.6) 0 (0.0) 0 (0.0)
Anti-Ku 5 (1.0) 1 (0.6) 3 (2.8) 1 (1.6)
Anti-SL 5 (1.0) 3 (1.7) 1 (0.9) 0 (0.0)
Anti-Sm 0 (0.0) 0 (0.0) 3 (2.8) 21.54 (P = 0.0069) 0 (0.0)
Anti-NOR-90 5 (1.0) 0 (0.0) 1 (0.9) 0 (0.0)
AMA 28 (5.5) 4 (2.3) 4 (3.7) 5 (7.8)
Anti-Sp100 3 (0.6) 0 (0.0) 0 (0.0) 1 (1.6)
Anti-Ro52 125 (24.4) 1.50 (P = 0.023) 20 (11.6) 27 (25.0) 15 (23.4)
Anti-Ro60 28 (5.4) 14 (8.1) 13 (12.0) 2.11 (P = 0.0382) 4 (6.3)
Anti-La 9 (1.8) 3 (1.7) 1 (0.9) 3 (4.7)
Anti-p25/p23 24 (4.5) 4.25 (P = 0.0031) 0 (0.0) 2 (1.9) 2 (3.1)
Other 209 (40.7) 74 (42.8) 44 (40.7) 34 (53.1)
ANA-negative 32 (6.2) 6 (3.5) 5 (4.6) 7 (10.9)
ACA, anti-centromere antibodies; AMA, antimitochondrial antibodies; ANA, antinuclear antibodies; ATA, anti-topoisomerase I antibodies; RNAP, RNA polymerase;.
a
OR for antibody in that subset compared with all other subsets. Only significant positive associations are documented by OR, and those with P -values < 0.005
are printed in bold.
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Table 5 Correlations of clinical features with SSc associated autoantibodies
ACA
(310)
ATA
(260)
Anti-PM-
Scl
(42)
Anti-U1-
RNP
(41)
Anti-RNA
-P
(33)
Anti-
Fibrillarin
(12)
Anti-Ku
(10)
Anti-Ro52
(187)
Anti-Ro60
(59)
Anti-La
(16)
AMA
(41)
Anti-p25/
23
(28)
ANA-
negative
(50)
no SSc
associated
abs
(161)
male sex
148 (17.1%)
17
0.19 (0.11
to 0.32)
P < 0.0001
64
2.02 (1.4
to 2.91)
p = 0.0002
6984223
0.62 (0.38
to 0.997
p = 0.049
12 2 1 1 10 41
1.90 (1.261
to 2.863)
p = 0,0035
age at disease
onset < 50 y
399/781 (51.1%)
115
0.57 (0.43
to 0.77)
p = 0.0003
135
1.39 (1.02
to 1.88)
p = 0.0431
22 29
2.92 (1.40
to 6.07)
p = 0.0029
13 8 4 77 31 10 20 8 18 73
Rodnan skin
score > 10
294/750 (39.2%)
58
0.27 (0.19
to 0.38)
P < 0.0001
132
3.10 (2.24
to 4.27)
P < 0.0001
14 9 18
3.24 (1.44
to 7.31)
p = 0.0042
7 1 58 23 6 10 11 14 56
Raynauds
phenomenon
819 (94.9%)
301
2.26 (1.07
to 4.77)
p = 0.0349
252 37 41 32 12 10 184
3.96 (1.2 to
12.94
p = 0.0133
58 16 41 28 43
0.29 (0.12
to 0.70)
p=
0.0104
140
0.23 (0.12 to
0.42)
P < 0.0001
Digital ulcers
216/840 (25.7%)
55
0.50 (0.36
to 0.71)
P < 0.0001
106
3.18 (2.30
to 4.41)
P < 0.0001
9 11 7 4 1 48 19 6 13 6 4
0.26 (0.09
to 0.76)
p=
0.0076
28
0.60 (0.39 to
0.94)
p = 0.024
Pulmonary
hypertension
126 (14.6%)
57
1.58 (1.08
to 2.32)
p = 0.0208
36 2 9 4 1 0 31 14 2 2 7 2
0.23 (0.06
to 0.97)
p=
0.0232
17
Pulmonary
fibrosis
287 (33.3%)
39
0.18 (0.12
to 0.26)
P < 0.0001
151
4.76 (3.48
to 6.50)
P < 0.0001
16 11 7 2 6 69 30
2.20 (1.29
to 3.75
p = 0.0040
96
0.33 (0.14
to 0.79)
p = 0.01
4
0.33 (0.11
to 0.95)
p = 0.0393
11 55
Lung restrictive
defect
218/833 (26.2%)
41
0.31 (0.21
to 0.45)
P < 0.0001
104
2.96 (2.14
to 4.09)
P < 0.0001
9 7 8 3 4 45 19 4 8 4 10 34
Esophageal
involvement
535 (62.0%)
198 175
1.39 (1.02
to 1.89)
p = 0.039
14
0.29 (0.152
to 0.56)
p = 0.0001
29 20 7 6 120 39 13 24 21 27 87
0.67 (0.47 to
0.94)
p = 0.0243
Proteinuria
90/830 (10.8%)
23
0.56 (0.34
to 0.92)
p = 0.0207
34 4 4 4 1 1 15 9 2 3 1 5 19
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Table 5 Correlations of clinical features with SSc associated autoantibodies (Continued)
Cardiac
involvement
114 (13.2%)
27
0.51 (0.32
to 0.81)
p = 0.0033
45
1.62 (1.08
to 2.44)
p = 0.0216
57512209141522
Musculoskeletal
involvement
421/852 (49.4%)
131
0.64 (0.48
to 0.85)
p = 0.0022
130 20 28
2.49 (1.25
to 4.96)
p = 0.009
17 10
5.22 (1.14
to 23.97)
p = 0.0202
680
0.71 (0.51
to 0.99)
p = 0.0468
32 12
3.13 (1.002
to 9.79)
p = 0.0447
17 9 25 79
Synovitis
157/837 (18.8%)
35
0.43 (0.29
to 0.65)
P < 0.0001
62
1.71 (1.19
to 2.45)
p = 0.0049
813
2.27 (1.14
to 4.53)
p = 0.0326
822321552
0.21 (0.05
to 0.89)
p=
0.0216
210 29
Joint
contractures
253/840 (30.1%)
54
0.36 (0.25
to 0.50)
P < 0.0001
107
2.26 (1.65
to 3.08)
P < 0.0001
9 9 10 6 2 49 23 6 9 9 20
1.93 (1.05
to 3.54)
p=
0.0436
56
Tendon friction
rubs
88/840 (10.5%)
14
0.30 (0.16
to 0.53)
P < 0.0001
32 2 5 6 3 1 21 5 2 2 0 6 25
1.95 (1.18 to
3.22)
p = 0.0122
CK elevation
74/835 (8.9%)
14
0.37 (0.21
to 0.68)
p = 0.0009
20 19
3.56 (1.67
to 7.57)
p = 0.0023
56
2.60 (1.03
to 6.55)
p = 0.0485
03
5.32 (1.30
to 21.72)
p = 0.038
21 8 2 1 0 6 16
Sicca syndrome
366/858 (42.7%)
150
1.44 (1.19
to 6.61)
p = 0.0119
98 14 18 12 5 4 96
1.57 (1.13
to 2.17)
p = 0.0075
33
1.85 (1.08
to 3.17)
p = 0.0275
12
4.14 (1.32
to 12.93)
p = 0.0102
19 20
3.50 (1.52
to 8.03)
p = 0.0029
23 66
Mouth
involvement
223/829 (26.9%)
64
0.61 (0.44
to 0.85)
p = 0.0034
93
2.1 (1.53
to 2.93)
P < 0.0001
10 7 10 3 4 49 18 6 11 6 12 34
ESR > 25 mm/h
199/741 (26.9%)
58
0.70 (0.49
to 0.99)
p = 0.046
75
1.55 (1.10
to 2.19)
p = 0.015
4
0.33 (0.11
to 0.94)
p = 0.0325
14 5 0 0 60
1.76 (1.21
to 2.54)
p = 0.0039
28
3.39 (1.92
to 5.97)
P < 0.0001
9
3.62 (1.33
to 9.86)
p = 0.0447
10 5 15 42
ACA, anti-centromere antibodies; AMA, antimitochondrial antibodies; ANA, antinuclear antibodies; ATA, anti-topoisomerase I antibodies; CK, creatine kinase; ESR, erythrocyte sedimentation rate; RNAP = RNA
polymerase; RNP = ribonucleoprotein; SSc, systemic sclerosis. Dichotomous variables are expressed as raw numbers, OR (95% CI) and P values.
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ACA were less common compared to American patients
[2] and more similar to European [9,12,38] and Japanese
patients [34]. Nevertheless, the presence of ACA does
notbyanymeansprecludedigitalulcers.Todateno
marker constellation allows the identification of patients
prone to this co mplication [41-43]. ACAs are frequ ently
associated with other antibodies, such as anti-Ro
[44- 46], a nti-mitochondrial (M2) [1 5,44,47,48] and anti-
p25/p23 [31,49,50] antibodies. These associations were
confirmed by this study. The reasons for this frequent
co-occurrence are unknown. There is no known anti-
genic relationship between the individual targets of the
antibodies. Probably t he (unknown) aetiopathogenetic
pathways marked b y these antibodies have common
components, including common genetic predispositions.
The prevalence of ATA in our cohor t (30.1%) is i n
line with the numbers published by others, w hich hav e
varied between 13% and 36%
[2,8,9,12,14,15,17,33,34,36,51]. The patients with ATA in
our study were more likely to be male and had higher
RSSs (Tab les 5 and 6). More common in this patient
group were digital ulcers, pulmonary fibro sis, dyspnoea,
lung restrictive defect and joint involvement (synovitis,
contractur es) a s well as mout h involv ement. However,
renal involvement, as measured by proteinuria or renal
insufficiency, was not more prevalent in this subgroup, a
finding reported by most other researchers
[8,9,14,15,33,36,51,52] but not all of them [2,34].
The frequency of RNAP antibodies in our cohort was
3.8%, which is at the lower end of the frequency range
reported by others, namely, 10% to 25% in North Amer-
ica [33,52-55], 4% to 31.5% in Europe
[14,16,33,35,56-59 ] and 5 % to 11% in Japan [34,3 6,60].
This may have several reasons: (1) Our cohort is com-
posed of a broad s pectrum of SSc pat ients, including
patients with milder forms and with overlap or undiffer-
ent iated subtypes of the disease , (2) regional differences
due to genetic background and/or environment al influ-
ences and (3) different techniques used to ascertain the
presence of RNAP antibodies. A high mean RSS, reflect-
ing diffuse skin involvement, was evident for patients
with anti-RNAP antibodies (Tables 5 and 6) as pre-
viously observed [2,14,33,34,36,40,54-60]. In addition, we
found creatine kinase (CK) elevation to be more fre-
quently associated with the presence of anti-RNAP anti-
bodies. This has not been noted before; association with
muscular involveme nt has been found to be nonsignifi-
cant [14,34,40,53,54] or even inverse [2,36,55] in pre-
vious publications. Our result in this study therefore
might b e a chance finding due to multiple compar isons.
We did not find any significant positive association of
RNAP antibod ies, or any autoantibody evaluated in this
study, with renal involvement. In the German Network
for Systemic Scleroderma Registry, renal involvement is
defined as renal insufficiency in the form of decreased
creatinine clearance and/or proteinuria, as well as a con-
sequence of acute renal crisis. The registry did not
include renal crisis as a separate item at that time,
which may underestimate the possible correlation of
antibodies with renal crisis [ 18]. Within our network,
the frequency of re nal crisis currently does not exceed
2% to 3% per year (Hunzelmann N, unpublished obser-
vation). The prevale nce of renal crisis among patients
with RNAP antibodies reported in the literature (for
review, see Meyer et al. [57]) varies considerably,
between 0% and 43%.
Antibodies to fibrillarin (U3-RNP) were most promi-
nent in patients with the diffuse subtype (table 4), which
is in accordance with the findin gs published in previous
reports [2,13,34,40,61]. In fact, patients with anti-fibril-
larin antibodies, on average, had the highest RSS of all
patients in our cohort (Tables 5 and 6). The significance
of this finding, however, is less pronounced because of
the lower number of patients. The detected anti-fibril-
larin antibody frequency of 1.4% was conside rably lower
than that reported in previous cohorts (2.5% to 19%)
[2,13 ,34,36,40,61-63], which may reflect our broad spec-
trum of SSc patients that i ncluded patients with overlap
syndrome and undifferentiated forms, as well as
Table 6 Correlations of clinical features with systemic
sclerosis-associated autoantibodies
Quantitative traits Clinical data
Age at disease onset n Mean ± SD (years) P*
Total 781 47.7 (14.2)
Anti-centromere 273 51.3 (12.5) < 0.0001
Anti-topoisomerase I 238 46.0 (14.0) 0.0076
Anti-fibrillarin 12 38.8 (16.0) 0.0404
Anti-U1-RNP 39 38.2 (15.0) < 0.0001
Anti-La 15 37.9 (18.1) 0.0431
Autoantibody-negative 36 52.9 (14.7) 0.0205
Rodnan skin score n Mean score ± SD P
Total 750 10.2 (9.4)
Anti-centromere 275 6.4 (6.0) < 0.0001
Anti-topoisomerase I 227 14.1 (9.7) < 0.0001
Anti-RNA polymerase 27 15.7 (11.7) 0.0091
Anti-fibrillarin 10 21.2 (15.0) 0.0108
Anti-U1-RNP 35 6.9 (9.2) 0.0053
Erythrocyte sedimentation rate n Mean ± SD (mm/hour) P
Total 741 19.46 (16.7)
Anti-topoisomerase I 227 22.95 (19.1) 0.0002
Anti-PM-Scl 36 12.19 (9.4) 0.0014
Anti-Ro52 167 22.05 (18.8) 0.0374
Anti-Ro60 53 28.47 (19.6) < 0.0001
Anti-La 16 28.56 (20.1) 0.0447
PM-Scl = polymyositi s and scleroderma; RNP = ribonucleoprote in; SSc,
systemic sclerosis. *P-value calculated by Mann-Whitney rank-sum test for
comparison of antib ody-positive vs antibody-negative patients.
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methodological differences and/or the central European
background of the patients. Ethnic heterogeneity with a
higher frequency of anti-fibrillarin antibodies in black
patients has been described previ ously [ 2,13,40,6 1-63].
Our findings of a lower age at disease onset and a
higher prevalence of musculoskeletal involvement
(Tables 5 and 6) are in line w ith most previous results
[2,36,61,63].
The frequency of PM-Scl antibodies (4.9%) detected in
our cohort is in accord with previously published studies
in which frequencies between 2% and 6% were noted for
SSc patients [1-3,17,33]. These antibodie s are most well-
known as being typical in patients with dermatomyosi-
tis-scleroderma overlap syndrome [1,3]. Accordingly, CK
elevation was highly associated with anti-P M-Scl i n our
cohort. On the other hand, these patients were markedly
less likely to have oesophageal involvement (Table 5)
and had a low mean ESR ( Tables 5 and 6). We found
elevated ESR leve ls in an earlier series of SSc patients
with anti-PM-Scl antibodies [32], but others, to the best
of our knowledge, did not. (Patients with anti-PM-Scl
have rarely been analysed in the large SSc series
reported previously.) Theref ore, this fin ding has to be
reproduced by independent work in the future. That
these patients have a relatively benign progno sis has
been mentioned several times before [64-66]. Accord-
ingly, 31% of our patients with anti-PM-Scl antibodies
were devoid of any internal organ involvement, com-
pared with 13% of the patients without anti-PM-Scl (P =
0.0023, data not shown).
Antibodies to Ku, when found in SSc patie nts, are
often associated with scleroderma overlap syndrome and
with muscular involvement [17,36]. Accordingly, we
registered a high OR, but with low significance because
of the relatively low patient number, for CK elevation
associated with anti-Ku antibody (Table 5). The com-
plete absence of ACA and the occasional presence of
ATA described in a large previous study that focused on
anti-Ku in patients with SSc [17] were nicely reproduced
in our cohort (Table 3). Most of our anti-Ku sera (7 of
10) wer e positive only by IP and negat ive in a classical
precipitation test w ith native ant igen. In a previou s
study, on the contrary, a similar test (counterimmunoe-
lectrophoresis) was even more sensitive than a line assay
to anti-Ku. A poss ible explanatio n for th is discrepancy
might be the source of the antigen , which was of rabbit
origin, in our ID assay. Ku autoantibodies are known to
tend to be nonreactive with nonhuman antigens [67].
Antibodies to p25/p23 ( anti-chromo ) charact erise a
patient s ubset within the group of ACA-positive SSc
patients. The clinical findings among these patients were
heterogeneous in previous report s. Soriano et al. [49]
found an elevated prevalence of erosive arthritis and
Furuta et al. [50] reported more interstitial lung d isease
and liver involvement, whereas Japanese groups
[30,31,68] disc overed cytopenias, Sjögren ssyndrome,
overlap with systemic lupus erythematosus and higher
ESR l evels. We confirmed the relatively strong associa-
tion with Sjögrens syndrome on the basis of our finding
that 20 (71.4%) of 28 patients with p25/p23 antibodies
had s icca symptoms, compared to only 41.7% of SSc
patients w ho were negative for these autoantibodies
(Table 5). In fact, the weak association of A CA with
sicca symptoms (OR = 1.44) (Table 5) lost significance
when those patients with co-occurring anti-p25/p23
were eliminated. Like wis e, the low mean RSS calculated
for ACA-positive p atients (6.4) (see Table 6) turned
even lower (6.2) after exclusion of patients with anti-
p25/p23.
Antibodies to Ro and/or La, as expected and as pre-
viously reported [44,45,69,70], were associated with
sicca s yndrome. This association was only margin ally
significant; in fact, the antibodies with the most promi-
nent association with t he sicca complex were, as men-
tioned above, anti-p25/p23 antibodies. Anti-Ro and/or
anti-La antibodies showed a particularly high correla-
tion with elevated ESR (Tables 5 and 6). An unexpect-
edly strong association of anti-Ro60 with pulmonary
fibrosis (Table 5) was mainly secondary to the rela-
tively high c o-occurrence of this antibody with ATA
(see Table 3).
No highly significant differences for any of the autoan-
tibody-defined subgroups could be found for gastric,
intestinal or renal i nvolvement, including hypertension
and reduced renal function. Furthermore, no significant
differences dependent on an tibodies against aminoacyl-
transfer RNA synthetases, To, Sm, SL or NOR-90 were
detected, probably because of the low numbers of
patients positive for these antibodies. Patients without
highly SSc-associated antibodies were more often male
and less frequently had Raynauds phenomenon.
Conclusions
The occurrence of SSc-related autoantibodies has never
been analysed in such detail in a cohort as large as this
one. We have shown that five antigens appear to be suf-
ficient to dete ct more than 95% of the known SSc-asso-
ciated autoantibody responses in ANA-positive SSc
patients. In more than half of patients with a SSc-asso-
ciated antibody, other nuclear autoantibodies were
detected and a considerable patient g roup (around 40%)
still displayed uncharacterised ANAs of as yet unknown
significance.
To the best of our knowledge, this is the largest com-
prehensive analysis of the presence of SSc-associated as
well as other non-organ-specific autoantibodies in SSc
patients that was performed in a single central labora-
tory and demonstrates the complexity and heterogeneity
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of the autoimmune response underlying the pathogen-
esis of this still enigmatic disease.
Abbreviations
ACA: anti-centromere antibody; AMA: antimitochondrial antibody; ANA:
antinuclear antibody; ATA: anti-topoisomerase I antibody; CK: creatine kinase;
ELISA: enzyme-linked immunosorbent assay; ESR: erythrocyte sedimentation
rate; HR: hazard ratio; ID: immunodiffusion; IP: immunoprec ipitation; OR:
odds ratio; RNAP: RNA polymerase; RSS: Rodnan skin score; SSc: systemic
sclerosis.
Acknowledgements
The expert technical assistance of Marie-Claire Vondegracht and Rita
Bernstein is gratefully acknowledged. Furthermore, we are indebted to Dr M
Blüthner, Laboratory Prof Seelig, Karlsruhe, Germany, for confirming the anti-
To antibodies; to Dr C Will and Professor R Lührmann, Marburg, Germany,
for identification of the anti-U11 RNP antibodies; and to Dr EK Chan,
Gainesville, FL, USA; Prof B Liedvogel, Diarect, Freiburg, Germany; and Dr W
Schlumberger, Euroimmun, Lübeck, Germany, for supplying rabbit anti-sera
to p25/p23, recombinant PM-Scl antigens, and investigational anti-fibrillarin
ELISA kits, respectively. We also thank A Fehr and B Damm for data
acquisition and management and Hildegard Christ and PD Dr Martin
Hellmich for statistical advice. This study was supported by the German
Federal Ministry of Education and Research (BMBF) (grants 01GM0310 and
01GM0630).
Author details
1
Laboratory at Rheumaklinik Aachen, Hauptstrasse 21, Aachen, D-52066,
Germany.
2
Department of Dermatology and Venerology, University of
Cologne, Kerpener Strasse 62, Cologne, D-50937, Germany.
3
Department of
Rheumatology and Clinical Immunology, Charité Universi tätsmedizin,
Humboldt University, Charitéplatz 1, Berlin, D-10117, Germany.
4
Clinical
Research Unit for Rheumatology, University Medical Center Freiburg,
Breisacher Strasse 66, Freiburg, D-79106, Germany.
5
Department of
Dermatology, Dresden University Hospital, Fetschers trasse 74, Dresden, D-
01307, Germany.
6
Department of Internal Medicine II, University of Giessen,
Klinikstrasse 33, Giessen, D-35392, Germany.
7
Clinic for Rheumatology,
Schneckenhalde 13, Bad Säckingen, D-79713, Germany.
8
Reha-Rheinfelden,
Salinenstrasse 98, Rheinfelden, CH-4310, Switzerland.
9
Department of
Dermatology, Venerology and Allergology, Charité Universitätsmedizin,
Humboldt University, Charitéplatz 1, Berlin, D-10117, Germany.
10
Department
of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 410,
Heidelberg, D-69120, Germany.
11
Department of Dermatology and
Allergology, Technical University of Munich, Biedersteiner Strasse 29, Munich,
D-80802, Germany.
12
Department of Rheumatology and Clinical
Immunology, Kerckhoff Clinic, Justus-Liebig University, Benekestrasse 2, Bad
Nauheim, D-61231, Germany.
13
Department of Dermatology, Heinrich-Heine-
University, Moorenstrasse 5, Düsseldorf, D-40225, Germany.
14
Department of
Dermatology, University of Münster, Von-Esmarch-Strasse 58, Münster, D-
48149, Germany.
15
Center of Rheumatology of Brandenburg, Johanniter
Hospital in Fläming, Johanniterstrasse 1, Treuenbrietzen, D-14929, Germany.
16
Department of Dermatology and Allergology, University of Ulm, Maienweg
12, Ulm, D-89081, Germany.
17
Center for Rheumatology, Acura Hospital,
Rotenbachtalstrasse 5, Baden-Baden, D-76530, Germany.
18
Department of
Dermatology, Venerology and Allergology, University of Leipzig, Philipp-
Rosenthal-Strasse 23, Leipzig, 04103, Germany; present address: Department
of Dermatology, University Hospital Erlangen, Ulmenweg 18, Erlangen, D-
91054, Germany.
19
Department of Dermatology and Allergology, Helios
Klinikum, Heusnerstrasse 40, Wuppertal, D-42283, Germany.
20
Department of
Dermatology, Johannes-Wesling-Klinik, Hans-Nolte-Strasse 1, Minden, D-
32429, Germany.
21
Medical Clinic I, Hospital Cologne-Merheim,
Ostmerheimer Strasse 200, Cologne, D-51109, Germany; present address:
Medical Clinic 6, Marien-Hospital, Wanheimer Strasse 167a, Duisburg, D-
47053, Germany.
22
Department of Dermatology, Venerology and Allergology,
University of Würzburg, Josef-Schneider-Strasse 2, Würzburg, D-97080,
Germany; present address: Department of Dermatology, Venerology and
Allergology, Georg-August-University, Von-Siebold-Strasse 3, Göttingen, D-
37075, Germany.
23
Hamburg Centre for Pediatric and Adolescence
Rheumatology, Dehnhaide 120, Hamburg Eilbek, D-22081, Germany.
24
Rheumaklinik Aachen, Burtscheider Markt 24, Aachen, D-52066, Germany.
Authors contributions
RM designed the study, performed the serological analyses, had full access
to all of the data in the study, analysed the data, takes responsibility for the
integrity of the data and the accuracy of the data analysis, interpreted the
data, and drafted the manuscript. EG designed the study, was responsible
for overall project management, enrolled patients for the network, and
contributed data from one participating centre. TK designed the study and
was responsible for overall project management. PM, GR, MM, FR, MB, MW,
NB, RH, AK, CS, AJ, CP, CF, MS, PL, RS, ESL, CS and IF enrolled patients for the
network and contributed the data from the participating centres. IM and
UML were responsible for overall project management, enrolled patients for
the network and contributed the data from the participating centres. NH
interpreted the data and drafted the manuscript, was responsible for overall
project management and designed the study. All authors critically revised
the manuscript and read and approved the final manuscript for public ation.
Competing interests
The authors declare that they have no competing interests.
Received: 21 July 2011 Revised: 23 August 2011
Accepted: 21 October 2011 Published: 21 October 2011
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doi:10.1186/ar3495
Cite this article as: Mierau et al.: Frequency of disease-associated and
other nuclear autoantibodies in patients of the German network for
systemic scleroderma: correlation with characteristic clinical features.
Arthritis Research & Therapy 2011 13:R172.
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Mierau et al. Arthritis Research & Therapy 2011, 13:R172
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    • "The remainder of patients who had a clinical association with diffuse SSc were captured in cluster topo I (18%). Topo I frequencies were consistent with the frequencies reported in other international registries of similar ethnicity but varying geographic locations, and it should be noted that the frequencies of this autoantibody vary with ethnicity (13,14,20,21,28,30,37,38,40–42,47–49). Significant clinical associations in this cohort include high MRSS, ILD, joint contractures, and digital ulcers. "
    [Show abstract] [Hide abstract] ABSTRACT: To determine the relationships between systemic sclerosis (SSc) related autoantibodies (AA), and their clinical associations, in a well characterised Australian patient cohort. Serum from 505 Australian SSc patients were analysed with a line immunoassay (EUROLINE, Euroimmun, Lubeck, Germany) for AA to centromere (CENP) A and B, RNA polymerase III (RNAP3) (11 and 155 epitopes), NOR-90, Fibrillarin, Th/To, PMScl-75 and PMScl-100, Ku, Topoisomerase-1 (Topo1), TRIM21/Ro52 and PDGF-Receptor (PDGFR). Patient subgroups were identified by hierarchical clustering of the first two dimensions of a Principal Components Analysis (PCA) of quantitative AA scores. Results were compared with detailed clinical data. 449/505 patients were positive for at least one immunoblot AA. Heatmap visualisation of AA scores, and PCA clustering, demonstrated strong, mutually exclusive relationships between CENP, RNAP3 and Topo1. Five patient clusters were identified (CENP, RNAP3 'strong', RNAP3 'weak', Topo1, "Other"). Clinical features associated with CENP, RNAP3 and Topo1 were consistent with previously published reports concerning limited cutaneous and diffuse cutaneous SSc. A novel finding was the statistical separation of RNAP3 into two clusters. RNAP3 'strong' patients had an increased risk of gastric antral vascular ectasia, but a lower risk of oesophageal dysmotility. "Other" patients were more likely to be males and have a history of smoking and malignancy, but less likely to have telangiectasia, Raynaud's phenomenon and joint contractures. Five major autoantibody clusters, with specific clinical and serological associations, were identified in Australian SSc patients. Sub-classification and disease stratification utilising AAs, may have clinical utility, particularly in early disease. This article is protected by copyright. All rights reserved. © 2015, American College of Rheumatology.
    No preview · Article · Aug 2015 · Arthritis and Rheumatology
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    • "In addition, the discrimination between SSc and controls as measured by the AUC (0.73) derived from ROC analysis using anti-Th/To (Rpp25) antibody results was similar to the AUC (0.67) generated by ACA [34]. Further studies using large cohorts of SSc patients such as those collected by EULAR Scleroderma Trials and Research (EUSTAR) [35], the Canadian Scleroderma Research Group (CSRG) [36] or the German Network for systemic scleroderma [13] and the Australian cohort [37] are needed to analyze the clinical utility of antibodies to Rpp25. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method. Methods The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied. Results Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809). Conclusion Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.
    Full-text · Article · Apr 2013 · Arthritis research & therapy
    • "The remainder of patients who had a clinical association with diffuse SSc were captured in cluster topo I (18%). Topo I frequencies were consistent with the frequencies reported in other international registries of similar ethnicity but varying geographic locations, and it should be noted that the frequencies of this autoantibody vary with ethnicity (13,14,20,21,28,30,37,38,40–42,47–49). Significant clinical associations in this cohort include high MRSS, ILD, joint contractures, and digital ulcers. "
    [Show abstract] [Hide abstract] ABSTRACT: Autoantibodies directed against a variety of nuclear, cytoplasmic and extracellular autoantigens are a serological hallmark of systemic sclerosis. This review provides an overview of the history and clinical association of many of the autoantibodies identified in SSc sera to date. Some of these autoantibodies predate the clinical diagnosis of SSc, some are pathogenic while others have no apparent role in pathogenesis. It was once thought that the autoantibody spectrum of individual SSc sera were less complex than other systemic autoimmune rheumatic diseases with respect to heterogeneous B cell responses reflected in circulating autoantibodies. However, with the advent of array technologies, there is now an unprecedented capability to detect multiple autoantibodies in an individual serum and this long held tenet of clinical diagnostic immunology is being reexamined.
    No preview · Article · Jun 2012 · Autoimmunity reviews
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