Anti-citrullinated peptide antibody-negative RA is a genetically distinct subset: a definitive study using only bone-erosive ACPA-negative rheumatoid arthritis.
ABSTRACT ACPA is a highly specific marker for RA. It was recently reported that ACPA can be used to classify RA into two disease subsets, ACPA-positive and ACPA-negative RA. ACPA-positive RA was found to be associated with the HLA-DR shared epitope (SE), but ACPA negative was not. However, the suspicion remained that this result was caused by the ACPA-negative RA subset containing patients with non-RA diseases. We examined whether this is the case even when possible non-RA ACPA-negative RA patients were excluded by selecting only patients with bone erosion.
We genotyped HLA-DRB1 alleles for 574 ACPA-positive RA, 185 ACPA-negative RA (including 97 erosive RA) and 1508 healthy donors. We also tested whether HLA-DR SE is associated with RF-negative or ANA-negative RA.
ACPA-negative RA with apparent bone erosion was not associated with SE, supporting the idea that ACPA-negative RA is genetically distinct from ACPA-positive RA. We also tested whether these subsets are based on autoantibody-producing activity. In accordance with the ACPA-negative RA subset, the RF-negative RA subset showed a clearly distinct pattern of association with SE from the RF-positive RA. In contrast, ANA-negative as well as ANA-positive RA was similarly associated with SE, suggesting that the subsets distinguished by ACPA are not based simply on differences in autoantibody production.
ACPA-negative erosive RA is genetically distinct from ACPA-positive RA.
Anti-citrullinated peptide antibody-negative RA is a
genetically distinct subset: a definitive study using
only bone-erosive ACPA-negative rheumatoid
Koichiro Ohmura1, Chikashi Terao1,2, Etsuko Maruya3, Masaki Katayama1,
Kenichiro Matoba4, Kota Shimada5, Akira Murasawa6, Shigeru Honjo7,
Kiyoshi Takasugi4, Shigeto Tohma5, Keitaro Matsuo8, Kazuo Tajima8,
Naoichiro Yukawa1, Daisuke Kawabata1, Takaki Nojima1, Takao Fujii1,
Ryo Yamada2, Hiroo Saji3, Fumihiko Matsuda2and Tsuneyo Mimori1
Objectives. ACPA is a highly specific marker for RA. It was recently reported that ACPA can be used to
classify RA into two disease subsets, ACPA-positive and ACPA-negative RA. ACPA-positive RA was found
to be associated with the HLA-DR shared epitope (SE), but ACPA negative was not. However, the sus-
picion remained that this result was caused by the ACPA-negative RA subset containing patients with
non-RA diseases. We examined whether this is the case even when possible non-RA ACPA-negative RA
patients were excluded by selecting only patients with bone erosion.
Methods. We genotyped HLA-DRB1 alleles for 574 ACPA-positive RA, 185 ACPA-negative RA (including
97 erosive RA) and 1508 healthy donors. We also tested whether HLA-DR SE is associated with
RF-negative or ANA-negative RA.
Results. ACPA-negative RA with apparent bone erosion was not associated with SE, supporting the idea
that ACPA-negative RA is genetically distinct from ACPA-positive RA. We also tested whether these
subsets are based on autoantibody-producing activity. In accordance with the ACPA-negative RA
subset, the RF-negative RA subset showed a clearly distinct pattern of association with SE from the
RF-positive RA. In contrast, ANA-negative as well as ANA-positive RA was similarly associated with
SE, suggesting that the subsets distinguished by ACPA are not based simply on differences in autoanti-
Conclusions. ACPA-negative erosive RA is genetically distinct from ACPA-positive RA.
Key words: Rheumatoid arthritis, Anti-citrullinated peptide antibody, HLA, Shared epitope, Subset, Genetics,
RA is an inflammatory arthritic disorder that is character-
ized by inflammatory cell infiltration, synovial cell prolifer-
ation and destruction of cartilage and subcartilageous
bones, which can lead to joint deformity. However, the
clinical course of RA varies from patient to patient, as
do autoantibody profiles such as RF and ACPA. Such het-
erogeneity may be derived from genetic and environmen-
tal factors. In the early stage of arthritis, the diagnosis of
RA is often difficult and such patients can be classified as
1Department of Rheumatology and Clinical Immunology,2Center for
Genomic Medicine, Graduate School of Medicine, Kyoto University,
3HLA Laboratory, Kawabata-Marutamachi, Sakyo-ku, Kyoto,
4Department of Rheumatology, Dohgo Spa Hospital, Dohgo
Himezuka, Matsuyama,5Department of Rheumatology, Sagamihara
National Hospital, National Hospital Organization, Sakuradai,
Sagamihara,6Department of Rheumatology, Niigata Rheumatic
Center, Shibata, Niigata,7Department of Rheumatology, Saiseikai
Takaoka Hospital, Takaoka, Toyama and8Division of Epidemiology
and Prevention, Aichi Cancer Center Hospital and Research Institute,
Chikusa-ku, Nagoya, Japan.
Correspondence to: Koichiro Ohmura, Department of Rheumatology
and Clinical Immunology, Graduate School of Medicine, Kyoto
University, 54 Shogoin-Kawahara-cho Sakyo-ku, Kyoto 606-8507,
Japan. E-mail: firstname.lastname@example.org
Submitted 30 March 2010; revised version accepted 19 July 2010.
! The Author(s) 2010. Published by Oxford University Press on behalf of The British Society for Rheumatology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Advance Access publication 9 September 2010
undifferentiated arthritis (UA). According to Thabet et al.
, about half of the UA patients remit spontaneously,
while ?30% develop RA. At baseline, 28.6% of UA pa-
tients have bone erosions and it is a good prognostic
value for the development of RA.
ACPA is an autoantibody that recognizes peptides or
proteins whose arginine residues are changed to citrulline
by post-translational modification. The target protein is
not a single protein, but filaggrin , vimentin , fibrin
, a-enolase  and so on. ACPA is a useful diagnostic
marker for RA because of its very high specificity (>95%)
and reasonably high sensitivity (65–88%) [6–8]. It has also
been proposed that ACPA is a useful marker for predicting
destructive RA [9, 10].
Genetic predisposition to RA has been investigated in-
tensively. HLA is a major determinant of RA susceptibility
and HLA-DRB1 *0101, *0102, *0401, *0404, *0405, *0408,
*0410, *1001, *1303 and *1402 are reported to be asso-
ciated with RA development. There is a common amino
acid sequence among such HLA-DR molecules at the
70th–74th residues of the HLA-DRb1 chain, which is
called a shared epitope (SE) . The association of car-
rying this SE and developing RA has been repeatedly
reported for different ethnic groups. However, recently,
a Dutch group reported that the association of SE was
only exhibited with ACPA-positive RA and no association
was seen with the ACPA-negative RA patients . They
also showed that the influence of SE on joint damage was
abrogated when stratified by ACPA. In addition to HLA-
DRB1 (SE), other RA susceptibility genes such as
PTPN22, CTLA4, TRAF1/C5 and STAT4 were also inves-
tigated for association by stratifying RA with ACPA [13–
16]. In almost all cases, such susceptibility genes were
found to be associated with ACPA-positive RA but not
with ACPA-negative RA. Although genetic differences
are clear between ACPA-positive and ACPA-negative
RA, there still remains the possibility that such differences
might be caused by the contamination of non-RA dis-
eases such as seronegative SpA and PMR in the
ACPA-negative RA subset. In this article, we re-evaluated
the association analysis by selecting only patients with
bone-eroding arthritis for the ACPA-negative population.
Materials and methods
Patients and healthy control subjects
A total of 1411 patients who were diagnosed with RA in
five hospitals (Kyoto University Hospital, Dohgo Spa
Hospital, Sagamihara National
Rheumatic Center and Saiseikai Takaoka Hospital) were
enrolled in this study. All patients were Japanese and
fulfilled the ACR (formerly ARA) 1987 revised criteria for
the classification of RA. RA patients overlapped with other
collagen vascular diseases were excluded. SS was not
excluded because the prevalence of SS in our cohort
was quite low (<2%) compared with the reported preva-
lence of 10–24%, probably due to incomplete clinical
information. The ethics committee of each hospital
approved the study and genomic DNA was extracted
from peripheral blood of patients and healthy individuals
after written informed consent was obtained. Out of
1411 RA patients, 1182 (83.8%) were ACPA positive and
229 (16.2%) were ACPA negative. Five hundred and
seventy-four ACPA-positive and 185 ACPA-negative RA
patients were selected and genotyped for HLA-DRB1.
Out of the 185 ACPA-negative RA patients, radiographic
data were available in 160 patients, of whom 97 patients
had typical bone erosions. Such patients are denoted as
ACPA-negative erosive RA patients in this article. DNA
samples from 1508 healthy control subjects were col-
lected at Aichi Cancer Center Hospital and from the
DNA banks for healthy Japanese volunteers of the
Pharma SNP Consortium  after written informed
consent was obtained.
Genotyping and autoantibody detection
HLA-DRB1 genotyping was carried out with a high-
sequence-specific oligonucleotide probe protocols with
system to quantify fluorescently labelled oligonucleotides
attached to colour-coded microbeads. The following
DRB1*0101, *0102, *0401, *0404, *0405, *0408, *0413,
*0416, *1001, *1303 and *1402.
ACPA in sera or plasma was detected using a
second-generation anti-CCP antibody (Ab) ELISA kit
(MESACUP CCP; Medical & Biological Laboratories Co.
Ltd, Nagoya, Japan) in accordance with the manufactur-
er’s instructions. A cut-off value of 4.5U/ml was used for
anti-CCP Ab positivity. RF was quantified by latex immu-
noturbidimetry and the cut-off values of each detection kit
in each hospital were employed. ANA was semi-quantified
by IIF for most samples, but some were measured by
Laboratories Co. Ltd). The cut-off values of each hospital
Chi-squared test, Student’s t-test, Jonckheere–Terpstra
trend test and the 95% CI of odds ratio (OR) were used
to assess the statistical significance and magnitude of
association for categorical outcomes.
ACPA-positive RA is distinct from ACPA-negative RA
on the basis of SE association
One hundred and eighty-five ACPA-negative patients and
574 ACPA-positive patients, as well as 1508 healthy indi-
viduals, were genotyped for HLA DRB1. SE was deter-
mined as described in the ‘Materials and methods’
section. ACPA was not tested for healthy individuals be-
cause its positivity among healthy people was reported to
be only ?1% [6, 18]. As shown in Tables 1 and 2, SE was
the clear risk factor for ACPA-positive RA development
ACPA-negative erosive RA as a distinct subset
ACPA-negative RA development (P=0.43 and 0.16 for
double- and single-SE carriers, respectively). There was
also a dose effect of SE number for ACPA-positive RA
(ORs were 6.6 and 3.2 for double- and single-SE carriers,
respectively), but not for ACPA-negative RA (ORs were
0.71 and 1.3 for double- and single-SE carriers, respect-
ively). When combining the double- and single-SE carriers,
P-values for ACPA-positive RA vs control, ACPA-negative
RA vs control and ACPA-positive RA vs ACPA-negative
RA were 1.8?10?37, 0.28 and 3.3?10?11, respectively.
These results are similar to those obtained for Caucasian
 and Japanese subjects .
SE was not associated with ACPA-negative RA even
when selecting only bone-destructive RA patients
As reported previously , no association was observed
between SE and ACPA-negative RA. However, some of
the patients who were diagnosed with ACPA-negative RA
might be non-RA patients, such as those with seronega-
tive SpA, PMR, palindromic rheumatism, OA and other
collagen vascular diseases. Indeed, during a survey of
the medical records, we found three patients in the
ACPA-negative RA subset who had been diagnosed
with MCTD, SLE or PMR and were subsequently recorded
as presenting with RA. Although we cannot tell which
diagnosis is correct, such cases led us to the idea that it
is important to exclude possible non-RA patients in
ACPA-negative RA subset in order to reveal whether SE
is really not associated with ACPA-negative RA. We first
excluded the patients who had suffered from RA for
<3 years in order to exclude patients with potentially
false-negative results for ACPA, on the basis of the fact
that the sensitivity of ACPA is lower in the early stage of
RA than in the established stage of RA (disease duration
53 years) . Then, we excluded possible non-RA pa-
tients who do not have bone erosions by X-ray. Ninety-
seven ACPA-negative RA patients showed typical bone
erosions and were denoted as ACPA-negative erosive
RA patients. As shown in Table 3, the baseline character-
istics of ACPA-negative erosive RA patients are similar to
those of ACPA-positive RA patients. However, an associ-
ation of SE with ACPA-negative erosive RA was not
observed (Tables 1 and 2). The P-value for ACPA-negative
erosive RA against the control was 0.26; in contrast, that
for ACPA-positive RA against the control was 1.8?10?37.
This result clearly shows that ACPA-negative erosive RA is
a distinct subset from ACPA-positive RA (P=1.1?10?6),
and HLA-DRs containing SE are not causative alleles for
developing ACPA-negative RA.
RF, but not ANA, positivity classified RA in terms
of SE association
Since it was previously reported that SE was associated
only with RF-positive RA [20, 21], we also tested this with
our cohort. RF data were available for 843 RA patients
and 85.6% were positive for RF. As shown in Table 4,
SE was significantly associated with RF-positive RA
(P=1.0?10?44, OR 3.7), while the association was much
weaker with RF-negative RA (P=2.2?10?4, OR 2.0),
showing similar results to Caucasian subjects.
We hypothesized that SE may be related to autoanti-
body production in general, because not only ACPA and
RF but also anti-calpastatin antibodies are reported to be
TABLE 2 P-values for association of SE between each group
Groups for comparison
SE (+/+) vs SE (?/?)
SE (+/?) vs SE (?/?)
SE (+) vs SE (?)
Control vs ACPA-positive RA
Control vs ACPA-negative RA
Control vs ACPA-negative erosive RA
ACPA-positive RA vs ACPA-negative RA
ACPA-positive RA vs ACPA-negative erosive RA
ACPA-negative RA vs ACPA-negative erosive RA
P-values were calculated by chi-squared test.
TABLE 1 Association of SE with ACPA-positive or ACPA-negative RA
ACPA-positive RA (n=574) ACPA-negative RA (n=185) ACPA-negative erosive RA (n=97)
n (%)OR (95% CI)n (%) OR (95% CI)n (%) OR (95% CI)
6.6 (4.7, 9.3)
3.2 (2.6, 4.0)
0.7 (0.3, 1.7)
1.3 (0.9, 1.7)
0.7 (0.2, 2.3)
1.4 (0.9, 2.1)
SE (+/+): double-SE carrier; SE (+/?): single-SE carrier; SE (?/?): no SE carrier.
Koichiro Ohmura et al.
associated with SE . Therefore, we further examined
the association between SE and ANA positivity in RA. ANA
data were available for 491 RA patients: 385 (78.4%) pa-
tients were ANA positive (Table 5). In contrast with ACPA
and RF results, SE was equally associated with both
ANA-positive and ANA-negative RA (P=3.1?10?29, OR
3.8 and P=6.4?10?9, OR 3.2, respectively), indicating
that ANA does not classify RA in terms of SE. Even
when the cut-off value of ANA was set higher, the result
was similar (data not shown).
SE (especially DRB1*0405) is associated with ACPA
titre but not RF nor ANA titre
We also investigated whether SE is related to autoanti-
body titres. ACPA, RF and ANA titres were measured
only for the sera from the Kyoto University cohort. The
sera with an ACPA titre >100IU/ml were further diluted
to obtain a correct titre. Among those for whom HLA data
were available, 252, 248 and 173 RA patients were posi-
tive for ACPA, RF and ANA, respectively. Only samples
positive for each autoantibody were selected and the as-
sociation of each autoantibody titre with SE number was
tested by Jonckheere–Terpstra trend test. As shown in
Fig. 1A–C, the number of SEs is associated with ACPA
titre, but not with RF or ANA titre. When we focused on the
DRB1*0405 allele (the most popular SE allele in Japanese
subjects), the association of ACPA titre and DRB1*0405
allele number was statistically significant (P=0.000127) as
shown in Fig. 2.
Here, we have demonstrated that HLA-DRB1 SE is asso-
ciated with ACPA-positive RA, but not with ACPA-
negative RA in Japanese subjects. No association of SE
with ACPA-negative RA was observed even when elimi-
nating possible non-RA patients from the ACPA-negative
RA group. We further demonstrated that ANA did not clas-
sify RA into two subsets in terms of SE association, in
contrast with RF and ACPA.
The fact that ACPA-positive and ACPA-negative RA
are genetically distinct subsets was first reported by a
Dutch group studying Caucasian subjects , followed
by a group studying Japanese subjects . However,
the numberof patients enrolledin theJapanese
TABLE 4 Association of SE with RF-positive or RF-negative RA
SE statusControl (n=1508)
RF-positive RA (n=722)RF-negative RA (n=121)
n (%) OR (95% CI)n (%) OR (95% CI)P-value*
6.5 (4.7, 9.0)
3.3 (2.7, 4.1)
2.5 (1.3, 5.1)
1.9 (1.3, 2.8)
*P-value for RF-positive vs RF-negative RA by chi-squared test.
TABLE 3 Baseline characteristics of ACPA-positive RA
and ACPA-negative erosive RA
erosive (n=97) P-value*
Age, mean (S.D.),
Sex: women, %
mean (S.D.) years
Stage, n (%)
Class, mean (S.D.)
63.0 (12.8) 62.1 (12.6)0.83
As not all of the X-ray films for ACPA-positive RA patients
were available, the total number of patients and the sum of
patients for stage classification do not match. *Student’s
t-test was used for statistical analysis. The P-values for
stage and class classification are not shown because
non-erosive patients were intentionally excluded from the
TABLE. 5 Association of SE with ANA-positive or ANA-negative RA
SE statusControl (n=1508)
ANA-positive RA (n=385)ANA-negative RA (n=106)
n (%) OR (95% CI)n (%) OR (95% CI)P-value*
5.7 (3.8, 8.5)
3.5 (2.7, 4.5)
7.1 (3.9, 12.8)
2.7 (1.7, 4.1)
*P-value for ANA-positive vs ANA-negative RA by chi-square test.
ACPA-negative erosive RA as a distinct subset
study was only 110 RA patients (82 ACPA-positive and
28 ACPA-negative) and the P-values were 0.017 and
0.033 for double-SE and single-SE carriers, respectively.
Furthermore, both the Dutch and the Japanese groups
enrolled only early RA patients. Therefore, as we discuss
later, their cohorts might have contained non-RA patients,
especially in the ACPA-negative group. Since our ACPA-
negative RA cohort consisted only of patients with estab-
lished RA (disease duration >3 years) and the P-value
reached 3.3?10?11, our study may be the first that has
clearly shown that ACPA-positive and ACPA-negative RA
subsets are distinct based on SE association using an
established RA cohort.
One of the major issues that we aimed to clarify was the
suspicion that the lack of an association of SE with
ACPA-negative RA was due to ACPA-negative RA
groups including non-RA patients. Since the specificity
of ACR (formerly ARA) 1987 revised criteria for the classi-
fication of RA has been reported to be 89%  and it is
probable that many non-RA patients will fall into the
ACPA-negative group, it is clear that the ACPA-negative
RA patient group contains some non-RA patients, which
affects the calculated association. From our survey of
medical records, 77 out of 174 ACPA-negative patients
for whom records were available did not show any bone
erosion by X-ray. These patients might not have RA, al-
though we believe that many of these patients do have RA
because the group should include RA patients in remis-
sion as well as some with slightly active RA without the
exhibition of clear changes detectable by radiography.
Since all of the patients in our ACPA-negative erosive
RA cohort have bone erosion as determined by X-ray,
the number of non-RA patients should be minimal. As
shown in Table 3, 73% of ACPA-negative erosive RA pa-
tients are classified in Steinbrocker’s Stage III or IV with
joint deformity. Often ACPA-negative RA is described as a
less severe arthritic subset, but our erosive cohort con-
sists of patients with RA of a severity similar to that of
ACPA-positive RA. Nonetheless, it is interesting that
ACPA-negative RA is genetically distinct from ACPA-
such subsetsmay be
autoantibody-producing ability. Since it has already
been reported thatSE
RF-negative RA [20, 21] or anti-calpastatin-negative RA
, it appears that SE is related to autoantibody
FIG. 1 Association of number of SE alleles and titre of
ACPA, RF or ANA. ACPA-positive (A), RF-positive (B) or
ANA-positive (C) RA patients were selected from the
Kyoto University cohort, and the serum ACPA titre (A), RF
titre (B) or ANA titre (C) was plotted stratified by the
number of SE alleles present. The P-values were
calculated by Jonckheere–Terpstra trend test.
FIG. 2 Association of HLA-DRB1*0405 allele number and
ACPA titre. Only ACPA-positive RA samples were se-
lected from the Kyoto University cohort, and ACPA titres
and the number of HLA-DRB1*0405 alleles (the most
popular SE allele in Japanese subjects) in each sample
are box plotted. The P-value by Jonckheere–Terpstra
trend test for this association is 0.000127.
Koichiro Ohmura et al.
production in general. However, ANA did not classify RA
into two subsets on the basis of the association with SE.
Therefore, SE is related to at least ACPA, RF and
anti-calpastatin production, but not ANA, suggesting
that HLA-DR molecules with SE consensus amino acid
sequence present rather specific autoantigens. The
dosage effect of the DRB1*0405 allele for ACPA titre
(Fig. 2), but not RF titre, (data not shown) supports this.
Genetic polymorphisms of PTPN22, CTLA4, TRAF1/C5
and STAT4 are also reported to be associated with only
ACPA-positive RA but not with ACPA-negative RA [13–
16]. There is a circumstantial evidence that smoking
may promote citrullination of protein/peptides  and
the affinityof citrullinated
*0401 and *0404, is higher than that of non-citrullinated
vimentin peptide . From these findings, one may
assume that SE and other genetic polymorphisms, to-
gether with smoking, promote the production of ACPA,
resulting in joint inflammation . Although there are no
direct evidences that ACPAs cause arthritis, aggravation
of experimental arthritis by transferring anti-citrullinated
fibrinogen mAbs was demonstrated , suggesting an
arthritis-promoting activity of ACPA. So, we assume that
antigen-presenting cells expressing HLA with SE may
preferentially present citrullinated peptides to Th2 cells,
which may support ACPA-producing B lymphocytes to
differentiate into plasma cells. In contrast, there are no
ACPA-negative RA. Unknown autoantibodies under a dif-
ferent genetic background might cause arthritis in
ACPA-negative RA, or antibody-independent mechanism
might be a major pathogenesis in ACPA-negative RA.
HLA-DRB1*03 and *0901 were reported to be weakly
Caucasian [28, 29] and Japanese  groups, respective-
ly, and only a few genetic determinants of ACPA-negative
RA among non-HLA genes have been reported [30, 31].
ACPA-negative RA has been reported, and genetic and
environmental factors of ACPA-negative RA development
is to be elucidated.
Rheumatology key messages
. ACPA-negative RA, even of bone-erosive type, is
distinct subset from ACPA-positive RA.
. HLA-DRB1 SE is not associated with ACPA-
. SE is associated with ACPA titre, but not with RF or
We would like to thank Ms Miki Kokubo for extraction and
preparation of DNA. We would also like to thank Mr Taishi
Shigeki for his excellent work in making clinical database
software in Dohgo Spa Hospital. We would also like to
thank all the doctors and co-medical people who
worked together to collect patients’ samples.
Funding: This work was supported by Grants-in-aid from
the Ministry of Health, Labor and Welfare of Japan and
from the Ministry of Education, Culture, Sports, Science
and Technology of Japan as well as by research grants
from the Japan Rheumatism Foundation, the Waksman
Foundation. Funding to pay the Open Access publication
charges for this article was provided by the Japan
Disclosure statement: The authors have declared no
conflicts of interest.
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