ARTHRITIS & RHEUMATOLOGY
Vol. 66, No. 2, February 2014, pp 407–417
DOI 10.1002/art.38219
© 2014, American College of Rheumatology
Prevalence of Anti–RNA Polymerase III Antibodies
in Systemic Sclerosis
New Data From a French Cohort and a Systematic Review and Meta-Analysis
Vincent Sobanski,1 Luc Dauchet,2 Guillaume Lefèvre,1 Marc Lambert,3
Sandrine Morell-Dubois,3 Thierno Sy,3 Eric Hachulla,1 Pierre-Yves Hatron,3
David Launay,1 and Sylvain Dubucquoi4
Objective. Studies assessing the prevalence of
anti–RNA polymerase III (anti–RNAP III) antibodies in
systemic sclerosis (SSc) have yielded a wide range of
results. The aim of the present study was to describe a
new SSc cohort tested for presence of anti–RNAP III
and perform a systematic review and meta-analysis to
assess the prevalence of anti–RNAP III in patients
worldwide and the potential factors of variability.
Methods. Seropositivity for anti–RNAP III was
evaluated in a French cohort of SSc patients. A system-
atic review of the literature was carried out in PubMed
and EMBase. Meta-analysis was performed using avail-
able data on prevalence, clinical characteristics of SSc
1
Vincent Sobanski, MD, Guillaume Lefèvre, MD,
Eric Hachulla, MD, PhD, David Launay, MD, PhD: Université Lille
Nord de France, EA 2686, IMPRT IFR 114, Centre National de
Référence de la Sclérodermie Systémique, and Hôpital Claude
Huriez, Centre Hospitalier Régional Universitaire de Lille, Lille,
France; 2Luc Dauchet, MD, PhD: Université Lille Nord de France,
Hôpital Calmette, Centre Hospitalier Régional Universitaire de Lille,
INSERM 744, and Institut Pasteur de Lille, Lille, France; 3Marc
Lambert, MD, PhD, Sandrine Morell-Dubois, MD, Thierno Sy, MD,
Pierre-Yves Hatron, MD: Université Lille Nord de France, Centre
National de Référence de la Sclérodermie Systémique, and Hôpital
Claude Huriez, Centre Hospitalier Régional Universitaire de Lille,
Lille, France; 4Sylvain Dubucquoi, MD, PhD: Université Lille Nord de
France, EA 2686, IMPRT IFR 114, and Centre Hospitalier Régional
Universitaire de Lille, Lille, France.
Drs. Launay and Dubucquoi contributed equally to this work.
Dr. Launay has received consulting fees, speaking fees, and/or
honoraria from GlaxoSmithKline, Pfizer, and Actelion (less than
$10,000 each).
Address correspondence to David Launay, MD, PhD, Uni-
versité Lille Nord de France, Service de Médecine Interne, Centre
National de Référence de la Sclérodermie Systémique, Hôpital
Claude Huriez, CHRU Lille, 59037 Lille Cedex, France. E-mail:
david.launay@chru-lille.fr.
Submitted for publication April 2, 2013; accepted in revised
form September 26, 2013.
407
patients, and the types of assays used for anti–RNAP III
testing.
Results. One hundred thirty-three French SSc
patients were tested for anti–RNAP III, and a preva-
lence of 6–9% was found in these patients. Thirty
studies representing a total population of 8,437 SSc
patients were included in the meta-analysis. Prevalence
of anti–RNAP III in this population was highly variable
(range 0–41%). The overall pooled prevalence of anti–
RNAP III was 11% (95% confidence interval 8–14),
but heterogeneity was high among studies (I2 ⴝ 93%,
P < 0.0001). Geographic factors such as continent or
country of study origin partially explained this hetero-
geneity and correlated with the prevalence. No other
baseline SSc characteristics were significantly corre-
lated with the prevalence of anti–RNAP III.
Conclusion. Data on our new cohort and our
meta-analysis of the literature confirmed that anti–
RNAP III prevalence in SSc varies among centers.
Geographic factors were significantly associated with
prevalence, which underscores the probable implication
that genetic background and environmental factors play
a role. Heterogeneity among studies remained largely
unexplained.
Systemic sclerosis (SSc) is a chronic connective
tissue disorder characterized by vascular involvement,
fibrosis, and autoimmunity. Autoantibodies against
multiple cellular components are used in daily prac-
tice as disease biomarkers for both positive diagnosis
and prognosis. The most common antinuclear auto-
antibodies found in SSc are anticentromere (ACA),
anti–topoisomerase I (anti–topo I), and anti–RNA poly-
merase III (anti–RNAP III) antibodies (1). Although
408
anti–RNAP III was identified in 1993 (2,3), data about
the clinical impact of these antibodies were limited until
enzyme-linked immunosorbent assay (ELISA) kits be-
came available (4,5). Since then, many reports on the
prevalence and clinical association of anti–RNAP III in
SSc have been published (2–4,6–17).
Although there is global consensus on the clinical
association of anti–RNAP III with the diffuse cutaneous
form of SSc and renal crisis (1,18), important and
unexplained discrepancies in the prevalence of anti–
RNAP III are still present. For example, anti–RNAP III
has been found in 8% of patients in a US cohort and
16% of patients in a Canadian cohort (19,20). In Japan,
prevalence was estimated at 5% in one study, and 11%
in a later study (2,15). In Europe, important variations
exist between countries: a prevalence of 3–9% was seen
in France and Italy, while a prevalence of 20–22% was
seen in Denmark and the UK (12,14,16,21,22). The aims
of the present study were to assess the prevalence of
anti–RNAP III in a new French cohort and to perform a
systematic review and meta-analysis of published reports
to assess the prevalence of anti–RNAP III in patients
worldwide and the potential factors explaining the ob-
served heterogeneity.
PATIENTS AND METHODS
New French cohort of SSc patients. One hundred
thirty-three consecutive and unselected patients with SSc were
evaluated in our department between May and November
2009 and retrospectively studied. Patients fulfilled the follow-
ing criteria for inclusion: age ⬎18 years and a diagnosis of SSc
according to the American College of Rheumatology criteria
and/or the criteria established by LeRoy and Medsger (23,24).
Patients were further classified as having diffuse cutaneous SSc
(dcSSc), limited cutaneous SSc (lcSSc), or limited SSc accord-
ing to the LeRoy and Medsger criteria for early SSc (24).
Age at onset of disease was defined as age at the first
non–Raynaud’s phenomenon symptom. Interstitial lung dis-
ease (ILD) was defined as the presence of at least 1 usual sign
of SSc-associated ILD (i.e., subpleural pure ground-glass opac-
ities and/or interstitial reticular pattern with or without trac-
tion bronchiectasis and/or honeycomb cysts) on high-
resolution computed tomography. Pulmonary hypertension
was diagnosed on right-sided heart catheterization (mean
pulmonary arterial pressure ⱖ25 mm Hg) and considered to be
SSc-associated precapillary pulmonary arterial hypertension if
the pulmonary capillary wedge pressure was ⱕ15 mm Hg.
Heart involvement was considered if abnormal findings on
electrocardiography, echocardiography (systolic or diastolic
dysfunction), or cardiac magnetic resonance imaging were
found in association with symptoms, such as malaise or syn-
cope, dyspnea, chest pain, or palpitations. Renal crisis was
defined as acute renal insufficiency or accelerated severe
systemic hypertension with proteinuria (25). Myositis was
diagnosed if there was muscle weakness associated with an
SOBANSKI ET AL
increased creatine phosphokinase level, or abnormal findings
on electromyography, muscle magnetic resonance imaging, or
muscle biopsy.
Laboratory methods. Identification of antinuclear
antibody specificities using both specific immunofluorescence
patterns on HEp-2 cells and the Luminex approach (Bio-Plex
2200; Bio-Rad) for anti–topo I, ACA, anti–U1 RNP, anti-SSA/
Ro, and anti-SSB/La antibodies was performed as part of
routine clinical care. Anti–PM-Scl antibodies were identified
by immunodot (Euroline Systemic Sclerosis [Nucleoli] Profile
[IgG]; Euroimmun). Antimitochondrial antibodies were iden-
tified based on typical immunofluorescence patterns on rat
liver, kidney, and stomach tissues (Bio-Rad) and the immuno-
dot method (Euroimmun). Two different ELISAs were used
to test every serum sample for anti–RNAP III, each according
to the manufacturer’s instructions: Quanta Lite RNA Pol III
(cutoff 20 arbitrary units [AU]/ml; Inova Diagnostics) and
EliA RNA Pol III Well (cutoff 10 AU/ml; Phadia Laboratory
Systems, Thermo Fisher Scientific) were kindly provided by the
respective manufacturers. Calibration and control sample test-
ing were performed in duplicate. A positive result was con-
firmed in each case by an additional assay.
In accordance with French legislation, written informa-
tion was provided and consent was obtained from each patient.
The study was conducted in accordance with the Declaration
of Helsinki and complied with the requirements of the French
Commission Nationale Informatique et Libertés (no. DC-
2008-642).
Systematic review and meta-analysis. The statement
on Preferred Reporting Items for Systematic Reviews and
Meta-Analyses (26) was used as a guide to conduct the review
and analysis.
Data sources and searches. Two of the authors (VS and
DL) performed independent searches of the electronic data-
bases, PubMed and EMBase. All records (articles and confer-
ence abstracts) published before April 2012 were included in
the search (Figure 1A). We used combinations of the terms
“systemic sclerosis,” “scleroderma,” “anti–RNA polymerase
III,” “DNA-directed RNA polymerases,” and “autoanti-
bodies.” We adapted the search strategy to meet the specific-
ities of each database. The reference lists of the retrieved
reports were searched to identify additional relevant publica-
tions.
Study selection. Studies were included if they met the
following criteria: original data were reported in English or
French, patients were ⱖ18 years old, patients were diagnosed
as having SSc, and ⬎30 patients with SSc were tested for
anti–RNAP III. Reports that failed to provide sufficient infor-
mation for the data analysis were excluded. Two of the authors
(VS and DL) independently screened the titles and abstracts of
the articles that were retrieved and applied the selection
criteria to identify relevant material to be read in full. The
reviewers’ selections were compared and, in cases of disagree-
ment, decisions were made by consensus. The reviewers inde-
pendently read the complete articles and applied the selection
criteria to determine whether the studies would be included in
the meta-analysis. The selections were again compared, and in
cases of disagreement, decisions were made by consensus.
Since the studies that were initially selected included some
overlapping cohorts for a given center assessed during the
same period, we chose to include 1 study per center (whichever
PREVALENCE OF ANTI–RNAP III IN SSc
Figure 1. A, Flow chart showing the search strategies used to identify publications for inclusion in the study. B, Results of the methodologic
assessment using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool showing the proportion of studies with low, high, or
unclear risk of bias. C, Modified QUADAS-2 items used in the study. ARA ⫽ anti–RNA polymerase III.
study included the highest number of patients). Studies of 2 or
more cohorts were included if extraction of data for each
cohort was feasible. In this case, each cohort was analyzed as
an independent cohort. Multicenter studies were excluded if
participating centers had published single-cohort reports that
were already included. Therefore, for each center, only 1
source of information was analyzed in order to avoid duplicate
data.
Assessment of study quality and risk of bias. The authors
(VS and DL) independently assessed the quality of the studies
(risk of bias) using the Quality Assessment of Diagnostic
Accuracy Studies (QUADAS-2) tool (27). In accordance with
the QUADAS-2 user guidelines (28), items were modified for
this study (Figure 1B) (see also Supplementary Table 1,
available on the Arthritis & Rheumatology web site at http://
onlinelibrary.wiley.com/doi/10.1002/art.38219/abstract). In do-
main 1 (Patient selection), the item “Was a case–control
design avoided?” was omitted. In domain 2 (Index test), the
items “Were the index test results interpreted without knowl-
edge of the results of the reference standard?” and “If a
threshold was used, was it pre-specified?” were substituted
with the item “Was the method of antibody determination
described?” This substitution was made because we included
articles regardless of the technique used to test for anti–RNAP
409
III. In domain 3 (Reference standard), the items “Is the
reference standard likely to correctly classify the target condi-
tion?” and “Were the reference standard results interpreted
without knowledge of the results of the index test?” were
omitted. In domain 4 (Flow and timing), the item “Was there
an appropriate interval between index test and reference
standard?” was omitted, and the item “Did all patients receive
the same reference standard?” was substituted with the item
“Were all patients tested for anti–RNAP III?” In accordance
with the QUADAS-2 guidelines, articles were assessed for
each item according to the following rating scale: high risk of
bias, low risk of bias, or unclear (see Supplementary Table 1
and Figure 1C).
Data extraction. Relevant data were extracted from
the selected studies using a standard form that included
information about the following items: continent, country,
center, ethnic origin of the patients, study design, recruit-
ment period, mid–cohort year, length of followup, disease
duration, sex ratio, cutaneous form of SSc (percentage of
diffuse form), age of patients, percentage of patients with ILD,
percentage of patients with scleroderma renal crisis, method
of antibody determination, kit used for ELISA, substrate used
for immunoprecipitation, number of SSc patients tested for
410 SOBANSKI ET AL

Table 1. Characteristics of the patients included in the study*

SSc patients (n ⫽ 133)

dcSSc (n ⫽ 36) lcSSc (n ⫽ 97) P

Sex, no. (%) female 22 (61) 90 (93) ⬍0.001
Age, mean ⫾ SD years 56.6 ⫾ 13.3 64.5 ⫾ 13.5 0.003
Ethnicity, no.† 0.196
Caucasian 32 92
African 3 5
Asian 0 –
Age at onset of disease, mean ⫾ SD years‡ 46.4 ⫾ 14.3 50.3 ⫾ 14 0.172
Disease duration, mean ⫾ SD years‡ 10.0 ⫾ 5.2 13.9 ⫾ 8.3 0.013
Pulmonary arterial hypertension, no. (%) 4 (11) 21 (22) 0.215
Interstitial lung disease, no. (%) 26 (72) 31 (32) ⬍0.001
Digital ulceration, no. (%) 22 (61) 33 (34) 0.006
Joint involvement, no. (%) 15 (42) 29 (30) 0.218
Myositis, no. (%) 12 (33) 6 (6) ⬍0.001
Esophageal involvement, no. (%) 30 (83) 82 (85) ⬎0.999
Renal crisis, no. (%) 4 (11) 1 (1) 0.019
Heart involvement, no. (%) 3 (8) 6 (6) 0.703
Overlap syndromes, no.
Sjögren’s syndrome 5 23
Primary biliary cirrhosis – 10
SLE – 2
ANA specificity, no.†
Anti–topo I 17§ 14¶
Anti–PM-Scl 3 1
Anti-Ro/La 3 –
ACA – 67#
Anti–U1 RNP – 4
AMA – 1
Unidentified 12 10

* Three patients with limited cutaneous systemic sclerosis (lcSSc) had limited SSc. SLE ⫽ systemic lupus
erythematosus; ANA ⫽ antinuclear antibody; anti–topo I ⫽ anti–topoisomerase I antibody; ACA ⫽
anticentromere antibody.
† Data were missing for 1 patient with diffuse cutaneous SSc (dcSSc).
‡ For dcSSc, 35 patients are represented; for lcSSc, 84 patients are represented. Age at onset of disease
and disease duration are as of the first appearance of a non–Raynaud’s phenomenon symptom.
§ Isolated in 15 patients; present along with anti–PM-Scl in 1 patient and along with anti-Ro/La in 1
patient.
¶ Isolated in 13 patients; present along with antimitochondrial antibodies (AMA) in 1 patient.
# Isolated in 59 patients; present along with AMA in 6 patients and along with anti-Ro/La in 2 patients.

anti–RNAP III, number of SSc patients positive for anti–
RNAP III.
Statistical analysis. For the analysis of the new French
cohort, results were expressed as the mean ⫾ SD for contin-
uous variables and as numbers with percentages for categorical
variables. XLSTAT software (Addinsoft) was used to perform
statistical analyses. Comparisons between groups were con-
ducted using Student’s t-test for normally distributed continu-
ous variables, Mann-Whitney U test for non-normally distrib-
uted variables, and either the chi-square test or Fisher’s exact
test for discrete variables. The R coefficient of correlation was
estimated by Spearman’s method. P values less than 0.05 were
considered significant.
For the meta-analysis, we calculated weighted pooled
summary estimates of anti–RNAP III prevalence. Analyses
were performed if at least 2 studies evaluating the same
outcome could be combined. For each meta-analysis, the
DerSimonian-Laird method was used. Accordingly, studies
were considered to be a random sample from a popula-
tion of studies. Heterogeneity was quantified using a chi-
square heterogeneity statistic and by means of an I2 statistic for
each analysis. A random-effects model was used to combine
data. The overall effect was estimated using a weighted
average of the individual effects, with weights inversely pro-
portional to variance in observed effects. Freeman-Tukey
transformation was used. Meta-regression was performed
to assess the impact of continent and country of origin, types
of test (ELISA or immunoprecipitation), manufacturer
of ELISA, substrate of immunoprecipitation, year of pub-
lication, mean age of patients, percentage of patients who
were men, percentage of patients who were Caucasian, per-
centage of patients who were Asian, percentage of patients
who were African, disease subtype (dcSSc versus lcSSc),
mean disease duration, percentage of patients with ILD, and
PREVALENCE OF ANTI–RNAP III IN SSc
percentage of patients with renal crisis. All analysis was
performed using R software and the metafor package (29).
RESULTS
Data from the cohort study. Clinical and biologic
features. Clinical and biologic characteristics of the 133
SSc patients are summarized in Table 1. Thirty-six
patients (27%) had dcSSc, and 97 patients (73%) had
lcSSc (3 of the lcSSc patients had limited SSc). Anti–
topo I was found in 17 of 36 dcSSc patients (47%) and in
14 of 97 lcSSc patients (14%). ACA was found in 67 of
97 lcSSc patients (69%).
ELISAs for anti–RNAP III. Results of the Phadia
ELISA were positive in 8 (6%) of 133 SSc patients (4
with dcSSc and 4 with lcSSc). Results of the Inova
ELISA were positive in 12 (9%) of 133 SSc patients (7
with dcSSc and 5 with lcSSc). Mean ⫾ SD anti–RNAP
III titers measured by Phadia ELISA were 20 ⫾ 8 AU/ml
versus 45 ⫾ 37 AU/ml (P ⫽ 0.143) in patients with lcSSc
and dcSSc, respectively. Titers measured by Inova
ELISA were 72 ⫾ 54 AU/ml versus 56 ⫾ 48 AU/ml (P ⫽
0.876) in patients with lcSSc and dcSSc, respectively.
Results were positive for anti–RNAP III on both the
Phadia and Inova ELISAs in 7 patients, 1 of whom was
also anti–topo I positive. Tests showed discordance
between results of the 2 ELISAs in 6 patients: 1 patient
(who had lcSSc and was ACA positive) had weakly
positive results on the Phadia ELISA only (14.5 AU/ml);
5 patients (3 with dcSSc and 2 with lcSSc) had positive
results on the Inova ELISA only (1 with anti–U1 RNP
antibodies and 4 with unidentified ANA). Titers were
low for 4 of these 5 patients (range 23–34 AU/ml), but 1
patient (with lcSSc) had a high titer of 150 AU/ml. Tests
were compared for agreement with regard to findings of
positivity (Cohen’s ␬ ⫽ 0.68) and titer measurements
(R ⫽ 0.729, P ⬍ 0.0001).
Studies included in the systematic review. Data-
base search results. Using defined keywords, 1,961 refer-
ences were retrieved as a result of an electronic search of
the PubMed and EMBase databases. Review of the
references did not return any additional material. After
reading the titles and abstracts, 99 articles were selected
for complete reading. Of these articles, 69 were assessed
for eligibility, and a further 39 articles were excluded
(essentially because of duplicate data) (Figure 1A).
Ultimately, 30 studies (5–14,16,17,20–22,30–43), includ-
ing the present one, representing a total population of
8,437 adult patients with SSc, were included in the
411
Table 2. Results of meta-regression analysis of the association be-
tween characteristics of cohorts and anti–RNA polymerase III
prevalence*
Variable P
Continent 0.0077
Country ⬍0.0001
Type of test (ELISA, IP, not specified) 0.1438
ELISA kit manufacturer 0.0624
Year of publication 0.7716
Mid–cohort year 0.6170
Age of patients 0.9769
Sex ratio 0.3641
Ethnicity
Caucasian 0.3314
Asian 0.1207
African 0.2246
Disease subtype 0.8322
Disease duration 0.4992
Interstitial lung disease 0.2516
Scleroderma renal crisis 0.8010
* Residual heterogeneity after inclusion of each factor in the analysis
was significant at P ⬍ 0.0001. ELISA ⫽ enzyme-linked immunosor-
bent assay; IP ⫽ immunoprecipitation.
meta-analysis (see Supplementary Table 1, available on
the Arthritis & Rheumatology web site at http://
onlinelibrary.wiley.com/doi/10.1002/art.38219/abstract
and Figure 1C).
Overall prevalence of anti–RNAP III and its
determinants. The overall pooled prevalence of anti–
RNAP III was 11% (95% confidence interval [95% CI]
8–14), with a high degree of heterogeneity (I2 ⫽ 93%,
P ⬍ 0.0001). We assessed whether geographic factors,
characteristics of anti–RNAP III testing methods, and
clinical characteristics of patients could explain the
observed heterogeneity between studies. Table 2 sum-
marizes the results of all meta-regression analyses.
Geographic influence. Meta-regression revealed a
significant association between the continent and anti–
RNAP III prevalence (P ⫽ 0.0077) (Table 2), suggesting
that the continent could explain a part of the heterogen-
eity. When continent was entered as a variable, residual
heterogeneity between studies was still significant (P ⬍
0.0001). Meta-analysis stratified by continent showed a
prevalence of 7% (95% CI 5–9) in Asia, 9% (95% CI
6–13) in Europe, and 14% (95% CI 8–21) in North
America. Prevalence was 15% (95% CI 12–19) in Oce-
ania and 41% (95% CI 31–52) in South America, but
both results were based on single studies (Figure 2).
Meta-regression also revealed a significant correlation
between country and prevalence (P ⬍ 0.0001) (Table 2).
Residual heterogeneity was still highly significant (P ⬍
0.0001) after inclusion of country as a variable. Meta-
analysis stratified by country showed that heterogeneity
412 SOBANSKI ET AL

Figure 2. Forest plots showing the prevalence of anti–RNA polymerase III in systemic sclerosis patients, represented by the pooled prevalence in
the whole population and separated by continent. Each square represents an individual prevalence, with the size of the square being proportional
to the weight given to the study. Lines represent the 95% confidence interval for the point estimate in each study. Diamonds represent the combined
prevalence. Phet ⫽ P value for heterogeneity.

was still strong within some countries such as the US and
France, while anti–RNAP III prevalence was more homo-
geneous in countries such as Japan or Italy (Figure 3).
Characteristics of anti–RNAP III tests. Meta-
regression did not reveal a significant association be-
tween the type of test (ELISA or immunoprecipitation)
and anti–RNAP III prevalence (P ⫽ 0.14). Meta-
analysis of ELISA results showed a prevalence of 13%
(95% CI 9–19), but heterogeneity was present. Within
studies using ELISAs, meta-regression showed a trend
toward a correlation between the manufacturer of the
ELISA (MBL International, Inova, Phadia, and in-
house) and prevalence of anti–RNAP III (P ⫽ 0.06).
However, there was no association between manufac-
turer and prevalence after inclusion of the geographic
variable in the model (P ⫽ 0.14). Results of immuno-
precipitation showed a prevalence of 7% (95% CI 5–9).
Characteristics of the patients included in studies.
Meta-regression revealed that no other factors were
significantly associated with prevalence (Table 2). Char-
acteristics that were nonsignificant were as follows: year
of publication (P ⫽ 0.77), mid–cohort year (P ⫽ 0.62),
age of patients (P ⫽ 0.98), sex (P ⫽ 0.36), ethnicity
(percentage of Caucasians [P ⫽ 0.33], percentage of
Asians [P ⫽ 0.12], and percentage of Africans [P ⫽
0.22]), disease subtype (P ⫽ 0.83), disease duration (P ⫽
0.50), and prevalence of ILD (P ⫽ 0.25) or of renal crisis
(P ⫽ 0.80).
PREVALENCE OF ANTI–RNAP III IN SSc 413

Figure 3. Forest plots showing the prevalence of anti–RNA polymerase III in systemic sclerosis patients, represented by the pooled prevalence in
the whole population and separated by country. Each square represents an individual prevalence, the size of the square being proportional to the
weight given to the study. Lines represent the 95% confidence interval for the point estimate in each study. Diamonds represent the combined
prevalence. Phet ⫽ P value for heterogeneity.

Influence of study quality on anti–RNAP III prev-
alence and its determinants. Since quality bias is an
important concern in meta-analyses, the aforemen-
tioned analyses were performed in duplicate for the
studies that received the highest quality rating according
to the QUADAS-2. By selecting the 13 studies (5–
7,10,12–14,22,37,41,42, as well as the present study)
rated “yes” for items 1, 2, and 4 of QUADAS-2, we
found an overall pooled prevalence of 9% (95% CI
7–12) with lower heterogeneity (I2 ⫽ 73%). Meta-
regression still showed that the continent of study origin
was a factor that was associated with anti–RNAP III
prevalence (P ⫽ 0.0013). When continent was entered as
variable in the model, residual heterogeneity was not
significant (P ⫽ 0.071).
DISCUSSION
The main results of our study are as follows:
1) the prevalence of anti–RNAP III in our new cohort of
133 French patients was 6–9%, 2) the prevalence of
anti–RNAP III in the existing literature (which included
⬎8,000 patients from various countries worldwide) is
highly variable (range 0–41%), and 3) the overall pooled
prevalence of anti–RNAP III was 11% (95% CI 8–14).
The marked heterogeneity among studies was only
414
Figure 4. Estimation of the worldwide prevalence of anti–RNA polymerase III in systemic sclerosis patients, according to the meta-analysis.
partially explained by the geographic origin and was not
explained by the baseline characteristics of patients.
Our study provides data to estimate the fre-
quency of anti–RNAP III in SSc patients. Estimation of
the worldwide prevalence of anti–RNAP III in SSc
(Figure 4) is challenging because published studies
include different sample sizes and use different method-
ologies. The estimated pooled prevalence of anti–RNAP
III was 11% (95% CI 8–14%), in accordance with the
estimation made by Koenig et al in their study of 4,672
patients from different countries (44). In comparison,
prevalence of ACA was estimated at 26% in that same
study, and at 33% in the 2003 American College of
Rheumatology guidelines for antibody testing in SSc
(45). Prevalence of anti–topo I was estimated at between
20 and 29% (44,45). Antifibrillarin (U3 RNP) antibodies
are found in 4–10% of SSc patients, anti–U1 RNP
antibodies in 6%, anti–PM-Scl in 4–11%, and anti-
Th/To antibodies in 2–5% (18). Anti–RNAP III is often
mutually exclusive of anti–topo I and ACA. Anti–RNAP
III is therefore one of the most frequent antinuclear
antibodies in SSc after ACA and anti–topo I. Moreover,
anti–RNAP III is known to be associated with some
clinical characteristics associated with poor prognosis in
SSc, such as diffuse cutaneous involvement and renal
crisis (1,18,19). Taken together, these data and our
results indicating the high prevalence of anti–RNAP III
provide evidence of the benefit of including systematic
SOBANSKI ET AL
anti–RNAP III testing in SSc patients who tested nega-
tive for anti–topo I and ACA.
The considerable variation in the prevalence of
anti–RNAP III that was reported in the literature was,
not surprisingly, associated with a high heterogeneity
among studies included in our meta-analysis. Several
hypotheses were worth testing to seek an explanation for
this heterogeneity: geographic origin of patients, type of
assays used, and baseline characteristics of SSc.
First, the most obvious variable related to the
prevalence of anti–RNAP III appears to be the geo-
graphic origin. Results of our study confirm this, since
location of the studies partially explained heterogeneity,
and we found a significant correlation between continent
of origin and anti–RNAP III prevalence by meta-
regression analysis as well (e.g., we found a lower
prevalence in Europe than in North America [9% versus
14%]). Moreover, within continents, prevalence was also
significantly associated with specific countries. Reasons
for these variations between continents and even be-
tween countries have not been well established. Inter-
estingly, observational studies have suggested that SSc
manifestations can vary between patients from different
countries. Analysis of the European League Against
Rheumatism Scleroderma Trial and Research group
database showed a large variability of disease presenta-
tion in Europe (46). These differences could be ascribed
to genetic or environmental factors, which could influ-
PREVALENCE OF ANTI–RNAP III IN SSc
ence anti–RNAP III prevalence (47–49). As an example,
Liu and coworkers have recently generated a composite
interferon-inducible chemokine score that correlated
with the interferon gene signature (50). This score
correlated with SSc activity and organ involvement
(lung, skin, and muscle). Interestingly, it was negatively
associated with the presence of anti–RNAP III, suggest-
ing that antibody patterns could be associated with
specific genetic backgrounds and different cytokine pat-
terns. Whether other genetic backgrounds or cytokine
production patterns could explain the variation of anti–
RNAP III prevalence among the different continents
warrants further study.
We could not explain variability in prevalence
of anti–RNAP III by ethnicity in our overall analysis.
However, 2 recent studies have shown that anti–RNAP
III was less prevalent in patients of African origin than in
white patients (51,52). In a sensitivity analysis, we reran
the model including these 2 studies; heterogeneity be-
tween centers still remained highly significant (data not
shown). These observations highlight several points.
First, definitions of ethnicity can differ between studies,
and ethnic groups are often heterogeneous. Second,
data on ethnicity were missing for several studies and
have therefore not been extensively included in the
meta-regression analysis. Indeed, only 5 studies included
patients of African origin, and in 3 of those, ⬍10% of
patients were of African origin.
In studies comparing ELISA and immunopre-
cipitation for the diagnosis of SSc versus other connec-
tive tissue diseases, ELISA was reported to be more
sensitive and immunoprecipitation to be more specific
(4,5,13,14). In our study, ELISAs resulted in a higher
prevalence (13% [95% CI 9–19]) versus immunopre-
cipitation (7% [95% CI 5–9]) in the overall population,
but meta-regression did not reveal a significant associa-
tion between the type of test (ELISA or immunoprecipi-
tation) and prevalence. When the type of assay was
included in the model as a variable, the heterogeneity
remained very significant. The impact of the type of
assay on heterogeneity among studies is therefore prob-
ably negligible.
Anti–RNAP III has been associated with the
diffuse cutaneous form of SSc and renal crisis (1,19,44).
ILD seems to be less prevalent in this group of patients
(19). Therefore one might expect that the variability in
anti–RNAP III prevalence could influence the clinical
findings in case series of SSc patients from different
countries. In our study, we analyzed whether a different
proportion of these clinical manifestations in each study
could account for heterogeneity in the reported preva-
415
lence rates. Disease subtype, disease duration, ILD, or
frequency of renal crisis were not found to explain
heterogeneity among studies. The variation of anti–
RNAP III prevalence between studies was not explained
by differences in the percentage of patients with diffuse
SSc, renal crisis, or ILD at baseline in the included
studies.
Our meta-analysis has several limitations. First,
as a systematic review, we found a broad range in qual-
ity of the methodologic design of the studies. We chose
to include as many studies providing data from various
centers as possible rather than to select only studies
with the highest-quality methodologic design. For each
center, only 1 source of information was included to
avoid duplicate inclusion of patients. As we noted,
when selecting the 13 studies with the highest quality,
we found similar results with a lower heterogeneity. The
fact that residual heterogeneity was not significant after
inclusion of continent as a variable in the model should
be interpreted with caution, since reducing the number
of studies could have reduced the power of the study.
Second, the number of studies and the size of the
samples studied varied greatly among centers. Our first
aim was to estimate the most accurate worldwide prev-
alence. Then, for the selection of studies, we took into
account each center, regardless of the sample size.
Weighted pooled summary estimates allowed us to
consider the effect of the sample size. Nevertheless,
there were some discrepancies between population size
of the country and the sample size. For example, esti-
mation of prevalence in South America is based on
results of one study of 85 patients from Brazil, while
prevalence estimates in China are based on a study of
92 patients.
Third, some articles were not designed for the
estimation of prevalence of anti–RNAP III but provided
sufficient information to estimate it. In a few studies, the
total number of patients tested for anti–RNAP III
(denominator) was uncertain. We have extrapolated
this number from the total number of patients ana-
lyzed for antinuclear antibodies. Therefore, prevalence
of anti–RNAP III may have been underestimated in
these studies (30–33,36,38,43). To address this issue,
Supplementary Table 1 (available on the Arthritis &
Rheumatology web site at http://onlinelibrary.wiley.com/
doi/10.1002/art.38219/abstract) shows the results of the
quality evaluation. Moreover, when we ran additional
analysis on the most robust studies, we found a close
prevalence of 9% (95% CI 7–12).
Finally, some studies used ELISAs from different
manufacturers, or the reports did not provide informa-
416
tion on the cutoff used. We were therefore not able to
assess the impact of the cutoff value on prevalence.
In conclusion, this study provided a new estima-
tion of anti–RNAP III prevalence in patients with SSc
who were assessed at a French center. The systematic
review and meta-analysis highlighted known discrepan-
cies between centers around the world and provided an
overall pooled prevalence of anti–RNAP III of 11%
(95% CI 8–14). Geographic factors (continents and
countries) partially explained this heterogeneity, sug-
gesting that genetic background and/or environmental
factors may play a role. No other baseline characteristic
was found to be significantly associated with the preva-
lence of anti–RNAP III.
ACKNOWLEDGMENTS
We thank our patients for participating in this study.
We are also grateful to Mrs. Dominique Gyselynckx and
Mrs. Elisabeth Leleu for technical assistance.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Launay had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Sobanski, Lefèvre, Lambert, Hachulla,
Hatron, Launay, Dubucquoi.
Acquisition of data. Sobanski, Lefèvre, Lambert, Morell-Dubois, Sy,
Hachulla, Launay, Dubucquoi.
Analysis and interpretation of data. Sobanski, Dauchet, Lefèvre,
Lambert, Hachulla, Hatron, Launay, Dubucquoi.
REFERENCES
1. Nihtyanova SI, Denton CP. Autoantibodies as predictive tools in
systemic sclerosis. Nat Rev Rheumatol 2010;6:112–6.
2. Kuwana M, Kaburaki J, Mimori T, Tojo T, Homma M. Auto-
antibody reactive with three classes of RNA polymerases in
sera from patients with systemic sclerosis. J Clin Invest 1993;91:
1399–404.
3. Okano Y, Steen VD, Medsger TA Jr. Autoantibody reactive with
RNA polymerase III in systemic sclerosis. Ann Intern Med
1993;119:1005–13.
4. Kuwana M, Kimura K, Kawakami Y. Identification of an immu-
nodominant epitope on RNA polymerase III recognized by sys-
temic sclerosis sera: application to enzyme-linked immunosorbent
assay. Arthritis Rheum 2002;46:2742–7.
5. Kuwana M, Okano Y, Pandey JP, Silver RM, Fertig N, Medsger
TA Jr. Enzyme-linked immunosorbent assay for detection of
anti–RNA polymerase III antibody: analytical accuracy and clini-
cal associations in systemic sclerosis. Arthritis Rheum 2005;52:
2425–32.
6. Bardoni A, Rossi P, Salvini R, Bobbio-Pallavicini F, Caporali R,
Montecucco C. Autoantibodies to RNA-polymerases in Italian
patients with systemic sclerosis. Clin Exp Rheumatol 2003;21:
301–6.
7. Ceribelli A, Krzyszczak ME, Li Y, Ross SJ, Chan JY, Chan EK,
SOBANSKI ET AL
et al. Atypical clinical presentation of a subset of patients with
anti-RNA polymerase III: non-scleroderma cases associated with
dominant RNA polymerase I reactivity and nucleolar staining.
Arthritis Res Ther 2011;13:R119.
8. Airo P, Ceribelli A, Cavazzana I, Taraborelli M, Zingarelli S,
Franceschini F. Malignancies in Italian patients with systemic
sclerosis positive for anti-RNA polymerase III antibodies. J Rheu-
matol 2011;38:1329–34.
9. Maes L, Blockmans D, Verschueren P, Westhovens R, De Beeck
KO, Vermeersch P, et al. Anti-PM/Scl-100 and anti-RNA-
polymerase III antibodies in scleroderma. Clin Chim Acta 2010;
411:965–71.
10. Nikpour M, Hissaria P, Byron J, Sahhar J, Micallef M, Paspaliaris
W, et al. Prevalence, correlates and clinical usefulness of anti-
bodies to RNA polymerase III in systemic sclerosis: a cross-
sectional analysis of data from an Australian cohort. Arthritis Res
Ther 2011;13:R211.
11. Kang EH, Lee EB, Kim DJ, Im CH, Lee HJ, Song YW. Anti-RNA
polymerase antibodies in Korean patients with systemic sclerosis
and their association with clinical features. Clin Exp Rheumatol
2005;23:731–2.
12. Emilie S, Goulvestre C, Berezne A, Pagnoux C, Guillevin L,
Mouthon L. Anti-RNA polymerase III antibodies are associated
with scleroderma renal crisis in a French cohort. Scand J Rheu-
matol 2011;40:404–6.
13. Chang M, Wang RJ, Yangco DT, Sharp GC, Komatireddy GR,
Hoffman RW. Analysis of autoantibodies against RNA poly-
merases using immunoaffinity-purified RNA polymerase I, II, and
III antigen in an enzyme-linked immunosorbent assay. Clin Im-
munol Immunopathol 1998;89:71–8.
14. Faucher B, Stein P, Granel B, Weiller PJ, Disdier P, Serratrice J,
et al. Low prevalence of anti-RNA polymerase III antibodies in a
French scleroderma population: anti-RNA polymerase III sclero-
derma. Eur J Intern Med 2010;21:114–7.
15. Satoh T, Ishikawa O, Ihn H, Endo H, Kawaguchi Y, Sasaki T, et al.
Clinical usefulness of anti-RNA polymerase III antibody mea-
surement by enzyme-linked immunosorbent assay. Rheumatology
(Oxford) 2009;48:1570–4.
16. Parker JC, Burlingame RW, Webb TT, Bunn CC. Anti-RNA
polymerase III antibodies in patients with systemic sclerosis de-
tected by indirect immunofluorescence and ELISA. Rheumatol-
ogy (Oxford) 2008;47:976–9.
17. Harvey GR, Rands AL, McHugh NJ. Anti-RNA polymerase
antibodies in systemic sclerosis (SSc): association with anti-
topoisomerase I antibodies and identification of autoreactive
subunits of RNA polymerase II. Clin Exp Immunol 1996;105:
468–74.
18. Mehra S, Walker J, Patterson K, Fritzler MJ. Autoantibodies in
systemic sclerosis. Autoimmun Rev 2013;12:340–54.
19. Steen VD. Autoantibodies in systemic sclerosis. Semin Arthritis
Rheum 2005;35:35–42.
20. Hudson M, Fritzler MJ, Baron M. Systemic sclerosis: establishing
diagnostic criteria. Medicine (Baltimore) 2010;89:159–65.
21. Jacobsen S, Ullman S, Shen GQ, Wiik A, Halberg P. Influence of
clinical features, serum antinuclear antibodies, and lung function
on survival of patients with systemic sclerosis. J Rheumatol
2001;28:2454–9.
22. Meyer OC, Fertig N, Lucas M, Somogyi N, Medsger TA Jr.
Disease subsets, antinuclear antibody profile, and clinical features
in 127 French and 247 US adult patients with systemic sclerosis.
J Rheumatol 2007;34:104–9.
23. Subcommittee for Scleroderma Criteria of the American Rheu-
matism Association Diagnostic and Therapeutic Criteria Commit-
tee. Preliminary criteria for the classification of systemic sclerosis
(scleroderma). Arthritis Rheum 1980;23:581–90.
24. LeRoy EC, Medsger TA Jr. Criteria for the classification of early
systemic sclerosis. J Rheumatol 2001;28:1573–6.
PREVALENCE OF ANTI–RNAP III IN SSc
25. Penn H, Howie AJ, Kingdon EJ, Bunn CC, Stratton RJ, Black
CM, et al. Scleroderma renal crisis: patient characteristics and
long-term outcomes. QJM 2007;100:485–94.
26. Moher D, Liberati A, Tetzlaff J, Altman DG, the PRISMA Group.
Preferred reporting items for systematic reviews and meta-
analyses: the PRISMA statement. PLoS Med 2009;6:e1000097.
27. Whiting PF, Rutjes AW, Westwood ME, Mallett S, Deeks JJ,
Reitsma JB, et al. QUADAS-2: a revised tool for the quality
assessment of diagnostic accuracy studies. Ann Intern Med 2011;
155:529–36.
28. QUADAS-2 manual. Bristol (UK): University of Bristol; 2011.
URL: http://www.bris.ac.uk/quadas/quadas-2/.
29. Viechtbauer W. Conducting meta-analyses in R with the metafor
package. J Stat Software 2010;36:1–48.
30. Agarwal SK, Gourh P, Shete S, Paz G, Divecha D, Reveille JD,
et al. Association of interleukin 23 receptor polymorphisms with
anti-topoisomerase-I positivity and pulmonary hypertension in
systemic sclerosis. J Rheumatol 2009;36:2715–23.
31. Hamaguchi Y, Hasegawa M, Fujimoto M, Matsushita T, Komura
K, Kaji K, et al. The clinical relevance of serum antinuclear
antibodies in Japanese patients with systemic sclerosis. Br J
Dermatol 2008;158:487–95.
32. Hesselstrand R, Scheja A, Shen GQ, Wiik A, Akesson A. The
association of antinuclear antibodies with organ involvement and
survival in systemic sclerosis. Rheumatology (Oxford) 2003;42:
534–40.
33. Johnson SR, Fransen J, Khanna D, Baron M, van den Hoogen F,
Medsger TA Jr, et al. Validation of potential classification criteria
for systemic sclerosis. Arthritis Care Res (Hoboken) 2012;64:
358–67.
34. Low AH, Wong S, Thumboo J, Ng SC, Fong KY. Comparison of
a new line immunoassay with conventional methods for the
detection of systemic sclerosis-specific autoantibodies in an Asian
systemic sclerosis cohort [abstract]. Int J Rheum Dis 2010;13 Suppl
1:184–5.
35. Majone F, Olivieri S, Cozzi F, Montaldi A, Tonello M, Visentin
MS, et al. Increased apoptosis in circulating lymphocyte cultures of
anti-RNA polymerase III positive patients with systemic sclerosis.
Rheumatol Int 2009;29:891–5.
36. Mierau R, Moinzadeh P, Riemekasten G, Melchers I, Meurer M,
Reichenberger F, et al. Frequency of disease-associated and other
nuclear autoantibodies in patients of the German Network for
Systemic Scleroderma: correlation with characteristic clinical fea-
tures. Arthritis Res Ther 2011;13:R172.
37. Morozzi G, Bellisai F, Fineschi I, Scaccia F, Pucci G, Simpatico A,
et al. Prevalence of anti-histone antibodies, their clinical signifi-
cance and correlation with other autoantibodies in a cohort of
Italian scleroderma patients. Autoimmun Highlights 2011;2:29–33.
38. Muller Cde S, Paiva Edos S, Azevedo VF, Radominski SC, Lima
Filho JH. Autoantibody profile and clinical correlation in a group
of patients with systemic sclerosis in southern Brazil. Rev Bras
Reumatol 2011;51:314–8, 323–4.
39. Ogawa F, Shimizu K, Hara T, Muroi E, Komura K, Takenaka M,
et al. Autoantibody against one of the antioxidant repair enzymes,
methionine sulfoxide reductase A, in systemic sclerosis: associa-
417
tion with pulmonary fibrosis and vascular damage. Arch Dermatol
Res 2010;302:27–35.
40. Rodriguez-Reyna TS, Hinojosa-Azaola A, Martinez-Reyes C,
Nunez-Alvarez CA, Torrico-Lavayen R, Garcia-Hernandez JL,
et al. Distinctive autoantibody profile in Mexican Mestizo systemic
sclerosis patients. Autoimmunity 2011;44:576–84.
41. Van Praet JT, Van Steendam K, Smith V, De Bruyne G, Mimori
T, Bonroy C, et al. Specific anti-nuclear antibodies in systemic
sclerosis patients with and without skin involvement: an extended
methodological approach. Rheumatology (Oxford) 2011;50:
1302–9.
42. Wang Q, Xu D, Li MT, Hou Y, Zhao JL, Zeng XF. The clinical
significance of scleroderma-related autoantibodies in Chinese
systemic sclerosis patients: reports from a prospective registry
database [abstract]. Int J Rheum Dis 2010;13 Suppl 1:186.
43. Ziswiler HR, Urech R, Balmer J, Ostensen M, Mierau R, Villiger
PM. Clinical diagnosis compared to classification criteria in a
cohort of 54 patients with systemic sclerosis and associated disor-
ders. Swiss Med Wkly 2007;137:586–90.
44. Koenig M, Dieude M, Senecal JL. Predictive value of antinuclear
autoantibodies: the lessons of the systemic sclerosis autoanti-
bodies. Autoimmun Rev 2008;7:588–93.
45. Reveille JD, Solomon DH, and the American College of Rheu-
matology Ad Hoc Committee on Immunologic Testing Guidelines.
Evidence-based guidelines for the use of immunologic tests:
anticentromere, Scl-70, and nucleolar antibodies. Arthritis Rheum
2003;49:399–412.
46. Walker UA, Tyndall A, Czirjak L, Denton CP, Farge-Bancel D,
Kowal-Bielecka O, et al, and the EUSTAR co-authors. Geograph-
ical variation of disease manifestations in systemic sclerosis: a
report from the EULAR Scleroderma Trials and Research
(EUSTAR) group database. Ann Rheum Dis 2009;68:856–62.
47. Kuwana M, Okano Y, Kaburaki J, Tojo T, Medsger TA Jr. Racial
differences in the distribution of systemic sclerosis–related serum
antinuclear antibodies. Arthritis Rheum 1994;37:902–6.
48. McNearney TA, Reveille JD, Fischbach M, Friedman AW, Lisse
JR, Goel N, et al. Pulmonary involvement in systemic sclerosis:
associations with genetic, serologic, sociodemographic, and behav-
ioral factors. Arthritis Rheum 2007;57:318–26.
49. Kuwana M, Pandey JP, Silver RM, Kawakami Y, Kaburaki J.
HLA class II alleles in systemic sclerosis patients with anti-RNA
polymerase I/III antibody: associations with subunit reactivities.
J Rheumatol 2003;30:2392–7.
50. Liu X, Mayes MD, Tan FK, Wu M, Reveille JD, Harper BE, et al.
Correlation of interferon-inducible chemokine plasma levels with
disease severity in systemic sclerosis. Arthritis Rheum 2013;65:
226–35.
51. Steen V, Domsic RT, Lucas M, Fertig N, Medsger TA Jr. A
clinical and serologic comparison of African American and Cau-
casian patients with systemic sclerosis. Arthritis Rheum 2012;64:
2986–94.
52. Gelber AC, Manno RL, Shah AA, Woods A, Le EN, Boin F, et al.
Race and association with disease manifestations and mortality in
scleroderma: a 20-year experience at the Johns Hopkins Sclero-
derma Center and review of the literature. Medicine (Baltimore)
2013;92:191–205.