A new classification of HLA-DRB1 alleles differentiates predisposing and protective alleles for autoantibody production in rheumatoid arthritis
Arthritis Research & Therapy
VRol9No2 esearch article Open Access A new classification of HLA-DRB1 alleles differentiates predisposing and protective alleles for autoantibody production in rheumatoid arthritis
Pierre-Antoine Gourraud 2
Philippe Dieud 1
Jean-Frdric Boyer 0 6
Leonor Nogueira 5
Anne Cambon-Thomsen 3
Bernard Mazires 0 6
Franois Cornlis 4
Guy Serre 5
Alain Cantagrel 0 6
Arnaud Constantin 0 2 6
0 GRCB40, UFR Sciences Medicales Rangueil , 1 avenue du Professeur Jean Poulhes, Toulouse Cedex 9, 31059 , France
1 Service de Rhumatologie , CHU Bichat Claude-Bernard, 46 rue Henri Huchard, Paris, 75018 , France
2 Service d'Epidemiologie CHU Toulouse, INSERM, U558, Universite Paul Sabatier Toulouse III, Faculte de Medecine , 37 allees Jules Guesde, Toulouse Cedex 7, 31073 , France
3 INSERM, U558, Faculte de Medecine , 37 allees Jules Guesde, Toulouse Cedex 7, 31073 , France
4 GenHotel , Genopole, 2 rue Gaston Cremieux, Evry Cedex, 91057 , France
5 Laboratoire de Biologie Cellulaire et Cytologie , CHU Toulouse Purpan, Place du Docteur Baylac, Toulouse cedex 9, 31059 , France
6 Service de Rhumatologie , CHU Toulouse Rangueil, 1 avenue du Professeur Jean Poulhes, Toulouse Cedex 9, 31059 , France
The HLA-DRB1 gene was reported to be associated with anticitrullinated protein/peptide autoantibody (ACPA) production in rheumatoid arthritis (RA) patients. A new classification of HLA-DRB1 alleles, reshaping the shared epitope (SE) hypothesis, was recently found relevant in terms of RA susceptibility and structural severity. We investigated the relevance of this new classification of HLADRB1 SE+ alleles in terms of rheumatoid factor (RF) and ACPA production in a sample of French RA patients. We studied 160 early RA patients included in a prospective longitudinal cohort of French Caucasian patients with recentonset arthritis. RF, anticyclic citrullinated peptide 2 (anti-CCP2) and antideiminated human fibrinogen autoantibodies (AhFibA) were assessed in all patients at inclusion. The HLA-DRB1 gene was typed by PCR-sequence specific oligonucleotides probes
(PCR-SSOP), and SE+ alleles were classified into four groups
(S1, S2, S3P, S3D) according to the new classification.
The new classification of HLA-DRB1 SE+ alleles distinguishes
predisposing and protective alleles for RF, anti-CCP2 or AhFibA
production. The presence of S2 or S3P alleles is associated
with both RF, anti-CCP2 or AhFibA positivity, whereas the
presence of S3D or S1 alleles appears to be protective for RF,
anti-CCP2 or AhFibA positivity.
The new classification of HLA-DRB1 SE+ alleles is relevant in
terms of autoantibody production in early RA patients by
differentiating predisposing and protective alleles for RF or
Since early rheumatoid arthritis (RA) is often indistinguishable
from other inflammatory joint diseases, recent-onset
inflammatory synovitis poses a diagnostic and prognostic challenge to
rheumatologists . The identification and validation of
immunologic and genetic markers with strong diagnostic and
prognostic value in early RA may help rheumatologists to meet this
ACPA = anticitrullinated protein/peptide autoantibody; AhFibA = antideiminated human fibrinogen autoantibodies; anti-CCP = anticyclic citrullinated
peptide; ELISA = enzyme-linked immunosorbent assay; HLA = human leukocyte antigen; MHC = minor histocompatibility complex, PCR =
polymerase chain reaction; RA = rheumatoid arthritis; RF = rheumatoid factor; SE = shared epitope.
Among immunologic markers, anticitrullinated protein/peptide
antibodies (ACPAs) constitute relevant tools in the diagnosis
and prognosis of early RA. Citrulline is a nonstandard amino
acid, generated by post-translational modifications of several
proteins by deimination of arginine residues by
peptidylarginine deiminases [3,4]. The citrulline moiety is the true
determinant in proteins recognized by antiperinuclear factor
, antikeratin antibodies , antifilaggrin antibodies ,
anticyclic citrullinated peptide (anti-CCP) antibodies  and
antideiminated human fibrinogen autoantibodies (AhFibA) [9-11].
ACPAs may be detected in healthy individuals, years before
the onset of symptoms of RA [12,13], and may predict
progression to persistent erosive arthritis or to RA in patients with
undifferentiated arthritis [14-16]. ACPAs are as sensitive as,
and more specific than, rheumatoid factor (RF) for early RA
diagnosis [17-19]. Furthermore, ACPAs represent a
prognostic factor for erosive disease in early RA [20-24].
Among genetic markers, the HLA-DRB1 gene has been
clearly involved in the pathogenesis of RA [25,26]. The
association between HLA-Dw4 and RA was first reported in 1976
. The development of HLA-DRB1 genotyping led to the
demonstration that different HLA-DR4 alleles were not equally
associated with RA and that several non-DR4 HLADRB1
alleles were also associated with the disease. The shared
epitope (SE) hypothesis, first proposed in 1987, represents an
approach to understand the molecular genetics of
susceptibility to RA. The SE hypothesis assumes that HLA-DRB1 alleles
encoding a highly conserved amino acid sequence, known as
the SE which is characterized by the RAA pattern at
positions 7274 of the third hypervariable region of different
HLADR1 chains are associated with susceptibility to RA .
HLA-DRB1 alleles encoding the SE were then associated with
structural severity of RA  and have been more recently
associated with production of ACPAs [9,12,24,30-32].
As was done in previous attempts [33,34], du Montcel and
colleagues recently introduced a new classification of
HLADRB1 alleles that reconsiders the SE hypothesis . In
terms of susceptibility to RA, this new classification suggests
that the risk of developing RA depends on whether the RAA
sequence occupies positions 7274 but the risk is modulated
by the amino acids at position 71 (K confers the higher risk, R
an intermediate risk, A and E a lower risk) and at position 70
(Q or R confers a higher risk than D) [35-37] complexifying the
classical SE epitope classification based on the presence of
RAA in positions 7274. In terms of structural severity of RA,
this new classification allowed the differentiation of
predisposing or protective alleles (two effects) respectively
characterized by the DRRAA or by the DERAA amino acid pattern at
positions 7074  which was not possible using the
classical SE epitope classification based on the only presence of
RAA in positions 7274.
In the present study, we investigated the relevance of this new
classification of HLA-DRB1 alleles in terms of RF and ACPA
production in a cohort of French Caucasian patients with early
RA. Interestingly, the new classification of HLA-DRB1 alleles
allows the differentiation between predisposing and protective
alleles for autoantibody production.
Materials and methods
One hundred and sixty Caucasian outpatients were selected
from the Rangueil Midi-Pyrnes cohort, which involved
patients with early arthritis who attended the Rangueil Hospital
Department of Rheumatology between November 1992 and
December 1997, according to the following criteria: the
American College of Rheumatology 1987 criteria for RA ,
disease duration <1 year from the first clinical manifestation of
RA, and age over 16 years.
Each individual included in the Rangueil Midi-Pyrnes cohort
signed an informed consent form. The protocol was initially
approved by the Committee for the Protection of Persons
Participating in Biomedical Research (French law 881138
December 20, 1988).
Detection of RF and ACPAs
Blood samples were collected at baseline, immediately
centrifuged and stored at -80C until assayed. RF was quantified by
nephelometry according to the manufacturer's
recommendations (RF Reagent, IMMAGE immunochemistry system;
Beckman Coulter, Inc., Fullerton, CA, USA). Anti-CCP2 antibodies
were detected by ELISA according to the instructions of the
manufacturer (IMMUNOSCAN RA; Euro-Diagnostica,
Arnhem, The Netherlands). AhFibA were detected with a recently
developed inhouse ELISA, using in vitro deiminated human
fibrinogen as immunosorbent [9,10]. The cut-off points of the
two ELISAs were previously set so they reached the same
diagnostic specificity of 98.5%.
HLA-DRB1 genotyping and allele classification
Genomic DNA was extracted from ethylenediamine
tetraacetic acid anticoagulated peripheral blood, using a standard
proteinase K digestion and phenol/chloroform extraction method,
in all patients at the time of inclusion. HLA-DRB1 typing and
subtyping were performed by a PCR-based method, using a
panel of sequence-specific oligonucleotide probes .
HLA-DRB1 alleles were pooled according to the new
classification proposed by du Montcel and colleagues [35,36].
Briefly, the HLA-DRB1 alleles were first divided into two
groups according to the presence or absence of the RAA
sequence at positions 7274 and were denoted S and X
alleles, respectively. The S alleles were subsequently divided into
four groups according to the amino acid at position 71: an
alanine (A), a glutamic acid (E), a lysine (K), or an arginine (R).
Different groups were thus defined in the new classification:
S1 for ARAA and ERAA, S2 for KRAA, S3 for RRAA, and X for
all non-RAA patterns. Since an aspartic acid (D) at position 70
was reported to be protective against RA susceptibility in
comparison with a glutamine (Q) or an arginine (R) at the same
position , two additional groups were defined: S3D for
DRRAA, and S3P for QRRAA or RRRAA [35,36] (Table 1).
Agreements with HardyWeinberg equilibrium were tested
using Pearson's chi-square test and Fischer's exact test when
relevant. The association between the HLA-DRB1 gene
polymorphism and RF or ACPAs was tested by comparing (by the
chi-square test or the exact Fisher test when relevant) the
distribution of positive or negative patients for RF or anti-CCP2
antibodies or AhFibA among carriers and noncarriers for each
of the four groups of HLA-DRB1 alleles encoding the SE,
defined according to the new classification of HLA-DRB1
alleles (S1D, S2D, S3P, S3D). Odds and odds ratios (95%
confidence intervals) were also calculated. The dose effect was
investigated for alleles positively or negatively associated with
immunological markers using tests for the trend of the log
Statistical analyses were performed using Stata Statistical
Software (release 9.1 SE; Stata Corporation, College Station,
TX, USA). All P values were two-sided, and P < 0.05 was
considered statistically significant after correcting when relevant
for multiple testing according to the BenjaminiYekutieli 2001
Demographic and immunologic characteristics of RA
The main baseline demographic and immunologic
characteristics of the 160 patients with early RA included in the present
study were the following: 120 women (75%) and 40 men
(25%); mean ( standard deviation) age, 50.31 ( 14.03)
years; mean ( standard deviation) disease duration, 0.55 (
0.02) years; number (%) RF-positive, 110/160 (68.75%);
number (%) anti-CCP2 antibody-positive, 110/160 (68.75%);
and number (%) AhFibA-positive, 108/160 (67.25%).
Allele frequencies for HLA-DRB1 polymorphisms
The frequencies of HLA-DRB1 alleles, classified into five
groups according to the new classification, were as follows:
S1, 59/320 (18.4%); S2, 65/320 (20.3%); S3D, 42/320
(13.3%); S3P, 89/320 (27.81%); and X, 65/320 (20.31). No
departures from HardyWeinberg equilibrium were found for
HLA-DRB1 alleles classified into the five groups (P = 0.7171;
10 degrees of freedom).
Relationship between HLA-DRB1 allele carrier status
and RF status
Table 2 presents the status for RF among patients carrying the
different HLA-DRB1 alleles encoding the SE classified into
four groups according to the new classification. On the one
hand, S2 carriers had a higher frequency of RF in comparison
with noncarriers (odds ratio > 1 and P < 0.05). On the other
hand, S3D and S1 carriers had a lower frequency of RF in
comparison with noncarriers (odds ratio < 1 and P < 0.05).
These results support the hypothesis of an association
between HLA-DRB1 gene polymorphisms and RF, and the
results point out the interest of the new classification of
HLADRB1 alleles in order to differentiate predisposing and
protective alleles for RF production in early RA.
Relationship between HLA-DRB1 allele carrier status
and anticitrullinated protein/peptide autoantibody
Table 3 presents the status for anti-CCP2 antibodies or
AhFibA among patients carrying the different HLA-DRB1
alleles encoding the SE classified into four groups according to
the new classification. On the one hand, S2 and S3P carriers
had a higher frequency of anti-CCP2 antibodies or AhFibA in
comparison with noncarriers (odds ratio > 1 and P < 0.05).
On the other hand, S3D and S1 carriers had a lower frequency
of anti-CCP2 antibodies or AhFibA in comparison with
noncarriers (odds ratio < 1 and P < 0.05).
The interest of the new classification is that both predisposing
and protective alleles for the production of ACPA are found.
The effects remain significant after correction for multiple
testing using the BenjaminiYekutieli 2001 procedure
implemented in STATA 9.0 (State Corporation), which corrects for
an overall false discovery rate (5% here) (see Table 3). In the
present analysis based on carrier status, a potential bias may
be introduced by the presence of an adverse effect allele in the
control group. In the analysis of the S2 effect, for example, the
association may be overestimated by the presence of S3D
carriers in the control group (noncarrier of S2). The effect of
S2 may similarly be underestimated by the presence of S3P
carriers. After controlling for the adverse effect of S3D and S1
in the analysis of S2, the association with the positivity of
Ahfiba remains significant (P < 0.05). After controlling for the
adverse effect of S2 and S3P in the analysis of S3D, the
association with negativity of anti-CCP2 remained significant.
These results support the hypothesis of an association
between HLA-DRB1 gene polymorphisms and ACPAs, and
point out the interest of the new classification of HLA-DRB1
alleles in order to differentiate predisposing and protective
alleles for ACPA production in early RA.
The results of the present study confirm previous evidence of
an association between HLA-DRB1 gene polymorphisms and
RF or ACPAs in RA. Furthermore, the results point out the
interest of the new classification of HLA-DRB1 alleles in order
Relationship between HLA-DRB1 allele carrier status and rheumatoid factor status in French patients with early rheumatoid
Odds ratio (95% confidence interval)
to differentiate predisposing and protective alleles for
autoantibody production in early RA.
The results of the present study confirm previous evidence of
an association between HLA-DRB1 gene polymorphisms and
autoantibody production in RA. We found a positive
association between carriers of HLA-DRB1*SE+ alleles
(HLADRB1*0401, HLA-DRB1*0404, HLA-DRB1*0405,
HLADRB1*0408, HLA-DRB1*1001) and RF or ACPA production,
while we did not find any negative association between
carriers of HLADRB1*SE- alleles and RF or ACPA production (data
not shown). An association between HLA-DRB1*04 or
HLADRB1*SE+ alleles and RF has been reported in some studies
[12,30,40] but rejected in others [12,32,41]. An association
between HLA-DRB1*01, HLA-DRB1*04 or HLA-DRB1*SE+
alleles and ACPAs was more constantly reported in European
or North American RA patients [9,12,24,30,32,40,42-45].
Since the presence of RF was strongly correlated with that of
ACPAs in most of these studies, several groups investigated
whether these associations between HLA-DRB1 gene
polymorphisms and RF or ACPAs were independent. These
studies showed that the association between HLA-DRB1*SE+
alleles and ACPAs is constantly stronger than the association
between HLA-DRB1*SE+ alleles and RF. Furthermore, they
suggested that the association between HLA-DRB1*SE+
alleles and ACPAs is independent of the RF status, leading to the
conclusion that HLA-DRB1*SE+ alleles are primarily
associated with the presence of ACPAs, but not with the presence
of RF [24,32,41].
Data presented as n (%). Status for rheumatoid factor among 160 patients with early rheumatoid arthritis, carrying the different HLA-DRB1 alleles
encoding the shared epitope classified into four groups according to the new classification. Odds ratios, 95% alpha-risk confidence interval and
P value for exact Fisher test. The dose effect was investigated for alleles positively or negatively associated with immunological markers using
tests for trend of the log odds. *Significant after correcting for multiple testing according to the BenjaminiYekutieli 2001 method at an overall
critical P value of 5%.
The results of the present study indicate the interest of the
new classification of HLA-DRB1 alleles to differentiate
predisposing and protective alleles for autoantibody production in
early RA. This new classification, which is based on an initial
split of HLA-DRB1 alleles into two groups according to the
presence (S alleles) or absence (X alleles) of the RAA
sequence at positions 7274, subsequently divides S alleles
into four groups according to the amino acids at positions 71
and 70. Most of the previous studies, based on the common
classification, identified HLA-DRB1*101, HLA-DRB1*0401,
HLA-DRB1*404 and HLA-DRB1*1001 as predisposing
alleles for ACPA production in RA, with a significant dose effect
in patients carrying two of these predisposing alleles
[9,12,32,44]. Only a few association studies reported an
HLADRB1 allelic protective effect for ACPA production in RA. In
these studies, HLA-DRB1*03 was associated with
ACPAnegative RA and decreased titers of ACPAs, even in the
presence of an SE allele [32,45]. In the new classification of
HLADB1 allelles, HLA-DRB1*03 is not taken into account
separately since it is classified into the X group of alleles, which do
not encode the SE sequence. In the present study,
complementary analysis did not show any association between
HLADRB1*03 carrier status and RF or ACPA production (data not
shown). The use of the classification by du Montcel and
colleagues suggests a risk hierarchy in ACPA production in early
RA patients: the S2 (KRAA at positions 7174) and S3P
(QRRAA or RRRAA at positions 7074) alleles conferring
predisposition, while the S1 (ARAA or ERAA at positions 71
74) and S3D (DRRAA at positions 7074) alleles confer
proArthritis Research & Therapy Vol 9 No 2
Gourraud et al.
Relationship between HLA-DRB1 allele carrier status and anticitrullinated protein/peptide autoantibody status in French patients
with early rheumatoid arthritis
Odds ratio (95% confidence interval)
tection, in comparison with X (non-RAA patterns at positions
The use of the new classification of HLA-DRB1 alleles
proposed by du Montcel and colleagues seems to provide
different pictures of the relative contribution of the HLA-DRB1
locus to RA pathogenesis. This relative contribution is not
restricted to ACPA production, but also includes risk hierarchy
for RA susceptibility and structural severity [35-37].
tions between HLA-DRB1 molecules and citrullinated
peptides may impact RA pathogenesis in several ways. For
example, a previous study conducted in DR4-IE transgenic
mice demonstrated that the conversion of arginine to citrulline
at the peptide side-chain position interacting with the SE
significantly increases peptideMHC affinity and leads to the
activation of CD4+ T cells, suggesting that HLA-DRB1 alleles
encoding the SE could initiate an autoimmune response to
citrullinated self-antigens .
Trying to understand the findings of genetic
association/linkage studies in complex multifactorial diseases, such as RA, in
light of the amino acid alignment of a protein encoded by a
candidate gene remains a challenging task. In fact, the
Although no formal conclusions on causality can be drawn
from the present association study, our findings indicate the
interest of this new classification of HLA-DRB1 alleles in order
to differentiate predisposing and protective alleles for
autoantibody production in RA.
The authors declare that they have no competing interests.
P-AG and ACo took the leadership of the study in both clinical
immunological and statistical aspects. FC and PD contributed
specifically to the genotyping. GS and LN were specifically in
charge of the autoantibody study. AC-T contributed to the
statistical analysis. BM, ACa and J-FB contributed through the
assessment of the RMP cohort.
The authors acknowledge the contribution of Delphine Nigon as a
clinical research data manager as well as the help of the Computational
platform for Clinical research and Analysis in Epidemiology & Public Health
1. El-Gabalawy HS , Duray P , Goldbach-Mansky R : Evaluating patients with arthritis of recent onset: studies in pathogenesis and prognosis . JAMA 2000 , 284 : 2368 - 2373 .
2. Scott DL : The diagnosis and prognosis of early arthritis: rationale for new prognostic criteria . Arthritis Rheum 2002 , 46 : 286 - 290 .
3. Girbal-Neuhauser E , Durieux JJ , Arnaud M , Dalbon P , Sebbag M , Vincent C , Simon M , Senshu T , Masson-Bessiere C , Jolivet-Reynaud C , et al.: The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues . J Immunol 1999 , 162 : 585 - 594 .
4. Schellekens GA , de Jong BA , van den Hoogen FH , van de Putte LB , van Venrooij WJ : Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritisspecific autoantibodies . J Clin Invest 1998 , 101 : 273 - 281 .
5. Nienhuis RL , Mandema E: A new serum factor in patients with rheumatoid arthritis; the antiperinuclear factor . Ann Rheum Dis 1964 , 23 : 302 - 305 .
6. Young A , Jaraquemada D , Awad J , Festenstein H , Corbett M , Hay FC , Roitt IM : Association of HLA-DR4/Dw4 and DR2/Dw2 with radiologic changes in a prospective study of patients with rheumatoid arthritis . Preferential relationship with HLA-Dw rather than HLA-DR specificities . Arthritis Rheum 1984 , 27 : 20 - 25 .
7. Simon M , Girbal E , Sebbag M , Gomes-Daudrix V , Vincent C , Salama G , Serre G : The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies,' autoantibodies specific for rheumatoid arthritis . J Clin Invest 1993 , 92 : 1387 - 1393 .
8. Schellekens GA , Visser H, de Jong BA , van den Hoogen FH , Hazes JM , Breedveld FC , van Venrooij WJ : The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide . Arthritis Rheum 2000 , 43 : 155 - 163 .
9. Auger I , Sebbag M , Vincent C , Balandraud N , Guis S , Nogueira L , Svensson B , Cantagrel A , Serre G , Roudier J : Influence of HLADR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen . Arthritis Rheum 2005 , 52 : 3424 - 3432 .
10. Chapuy-Regaud S , Nogueira L , Clavel C , Sebbag M , Vincent C , Serre G : IgG subclass distribution of the rheumatoid arthritisspecific autoantibodies to citrullinated fibrin . Clin Exp Immunol 2005 , 139 : 542 - 550 .
11. Masson-Bessiere C , Sebbag M , Girbal-Neuhauser E , Nogueira L , Vincent C , Senshu T , Serre G : The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin . J Immunol 2001 , 166 : 4177 - 4184 .
12. Berglin E , Padyukov L , Sundin U , Hallmans G , Stenlund H , Van Venrooij WJ , Klareskog L , Dahlqvist SR : A combination of autoantibodies to cyclic citrullinated peptide (CCP) and HLADRB1 locus antigens is strongly associated with future onset of rheumatoid arthritis . Arthritis Res Ther 2004 , 6 : R303 - R308 .
13. Nielen MM , van Schaardenburg D , Reesink HW , van de Stadt RJ , van der Horst-Bruinsma IE , de Koning MH , Habibuw MR , Vandenbroucke JP , Dijkmans BA : Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors . Arthritis Rheum 2004 , 50 : 380 - 386 .
14. van Gaalen FA , Linn-Rasker SP , van Venrooij WJ , de Jong BA , Breedveld FC , Verweij CL , Toes RE , Huizinga TW : Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis: a prospective cohort study . Arthritis Rheum 2004 , 50 : 709 - 715 .
15. Visser H , le Cessie S , Vos K , Breedveld FC , Hazes JM : How to diagnose rheumatoid arthritis early: a prediction model for persistent (erosive) arthritis . Arthritis Rheum 2002 , 46 : 357 - 365 .
16. von Essen R , Kurki P , Isomaki H , Okubo S , Kautiainen H , Aho K : Prospect for an additional laboratory criterion for rheumatoid arthritis . Scand J Rheumatol 1993 , 22 : 267 - 272 .
17. Jansen AL , van der Horst-Bruinsma I , van Schaardenburg D , van de Stadt RJ , de Koning MH , Dijkmans BA : Rheumatoid factor and antibodies to cyclic citrullinated peptide differentiate rheumatoid arthritis from undifferentiated polyarthritis in patients with early arthritis . J Rheumatol 2002 , 29 : 2074 - 2076 .
18. Kroot EJ , de Jong BA , van Leeuwen MA , Swinkels H , van den Hoogen FH , van't Hof M , van de Putte LB , van Rijswijk MH , van Venrooij WJ , van Riel PL : The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis . Arthritis Rheum 2000 , 43 : 1831 - 1835 .
19. Vincent C , Nogueira L , Sebbag M , Chapuy-Regaud S , Arnaud M , Letourneur O , Rolland D , Fournie B , Cantagrel A , Jolivet M , et al.: Detection of antibodies to deiminated recombinant rat filaggrin by enzyme-linked immunosorbent assay: a highly effective test for the diagnosis of rheumatoid arthritis . Arthritis Rheum 2002 , 46 : 2051 - 2058 .
20. Meyer O , Labarre C , Dougados M , Goupille P , Cantagrel A , Dubois A , Nicaise-Roland P , Sibilia J , Combe B : Anticitrullinated protein/peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage . Ann Rheum Dis 2003 , 62 : 120 - 126 .
21. Paimela L , Gripenberg M , Kurki P , Leirisalo-Repo M : Antikeratin antibodies: diagnostic and prognostic markers for early rheumatoid arthritis . Ann Rheum Dis 1992 , 51 : 743 - 746 .
22. van Jaarsveld CH , ter Borg EJ , Jacobs JW , Schellekens GA , Gmelig-Meyling FH , van Booma-Frankfort C , de Jong BA , van Venrooij WJ , Bijlsma JW : The prognostic value of the antiperinuclear factor, anti-citrullinated peptide antibodies and rheumatoid factor in early rheumatoid arthritis . Clin Exp Rheumatol 1999 , 17 : 689 - 697 .
23. Vencovsky J , Machacek S , Sedova L , Kafkova J , Gatterova J , Pesakova V , Ruzickova S : Autoantibodies can be prognostic markers of an erosive disease in early rheumatoid arthritis . Ann Rheum Dis 2003 , 62 : 427 - 430 .
24. van der Helm-van Mil AH , Verpoort KN , Breedveld FC , Huizinga TW , Toes RE , de Vries RR : The HLA-DRB1 shared epitope alleles are primarily a risk factor for anti-cyclic citrullinated peptide antibodies and are not an independent risk factor for development of rheumatoid arthritis . Arthritis Rheum 2006 , 54 : 1117 - 1121 .
25. Newton JL , Harney SM , Wordsworth BP , Brown MA : A review of the MHC genetics of rheumatoid arthritis . Genes Immun 2004 , 5 : 151 - 157 .
26. Seldin MF , Amos CI , Ward R , Gregersen PK : The genetics revolution and the assault on rheumatoid arthritis . Arthritis Rheum 1999 , 42 : 1071 - 1079 .
27. Stastny P : Mixed lymphocyte cultures in rheumatoid arthritis . J Clin Invest 1976 , 57 : 1148 - 1157 .
28. Gregersen PK , Silver J , Winchester RJ : The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis . Arthritis Rheum 1987 , 30 : 1205 - 1213 .
29. Gorman JD , Lum RF , Chen JJ , Suarez-Almazor ME , Thomson G , Criswell LA : Impact of shared epitope genotype and ethnicity on erosive disease: a meta-analysis of 3,240 rheumatoid arthritis patients . Arthritis Rheum 2004 , 50 : 400 - 412 .
30. van Gaalen FA , van Aken J , Huizinga TW , Schreuder GM , Breedveld FC , Zanelli E , van Venrooij WJ , Verweij CL , Toes RE , de Vries RR : Association between HLA class II genes and autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis . Arthritis Rheum 2004 , 50 : 2113 - 2121 .
31. Hoppe B , Haupl T , Gruber R , Kiesewetter H , Burmester GR , Salama A , Dorner T : Detailed analysis of the variability of peptidylarginine deiminase type 4 in German patients with rheumatoid arthritis: a case-control study . Arthritis Res Ther 2006 , 8 : R34 .
32. Irigoyen P , Lee AT , Wener MH , Li W , Kern M , Batliwalla F , Lum RF , Massarotti E , Weisman M , Bombardier C , et al.: Regulation of anti-cyclic citrullinated peptide antibodies in rheumatoid arthritis: contrasting effects of HLA-DR3 and the shared epitope alleles . Arthritis Rheum 2005 , 52 : 3813 - 3818 .
33. de Vries N , Tijssen H , van Riel PL , van de Putte LB : Reshaping the shared epitope hypothesis: HLA-associated risk for rheumatoid arthritis is encoded by amino acid substitutions at positions 67-74 of the HLA-DRB1 molecule . Arthritis Rheum 2002 , 46 : 921 - 928 .
34. Reviron D , Perdriger A , Toussirot E , Wendling D , Balandraud N , Guis S , Semana G , Tiberghien P , Mercier P , Roudier J : Influence of shared epitope-negative HLA-DRB1 alleles on genetic susceptibility to rheumatoid arthritis . Arthritis Rheum 2001 , 44 : 535 - 540 .
35. du Montcel ST , Michou L , Petit-Teixeira E , Osorio J , Lemaire I , Lasbleiz S , Pierlot C , Quillet P , Bardin T , Prum B , et al.: New classification of HLA-DRB1 alleles supports the shared epitope hypothesis of rheumatoid arthritis susceptibility . Arthritis Rheum 2005 , 52 : 1063 - 1068 .
36. Gourraud PA , Boyer JF , Barnetche T , Abbal M , Cambon-Thomsen A , Cantagrel A , Constantin A : A new classification of HLA-DRB1 alleles differentiates predisposing and protective alleles for rheumatoid arthritis structural severity . Arthritis Rheum 2006 , 54 : 593 - 599 .
37. Michou L , Croiseau P , Petit-Teixeira E , du Montcel ST , Lemaire I , Pierlot C , Osorio J , Frigui W , Lasbleiz S , Quillet P , et al.: Validation of the reshaped shared epitope HLA-DRB1 classification in rheumatoid arthritis . Arthritis Res Ther 2006 , 8 : R79 .
38. Arnett FC , Edworthy SM , Bloch DA , McShane DJ , Fries JF , Cooper NS , Healey LA , Kaplan SR , Liang MH , Luthra HS , et al.: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis . Arthritis Rheum 1988 , 31 : 315 - 324 .
39. Zanelli E , Breedveld FC , de Vries RR : HLA class II association with rheumatoid arthritis: facts and interpretations . Hum Immunol 2000 , 61 : 1254 - 1261 .
40. Forslind K , Ahlmen M , Eberhardt K , Hafstrom I , Svensson B : Prediction of radiological outcome in early rheumatoid arthritis in clinical practice: role of antibodies to citrullinated peptides (anti-CCP) . Ann Rheum Dis 2004 , 63 : 1090 - 1095 .
41. Huizinga TW , Amos CI , van der Helm-van Mil AH , Chen W , van Gaalen FA , Jawaheer D , Schreuder GM , Wener M , Breedveld FC , Ahmad N , et al.: Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA-DRB1 shared epitope for antibodies to citrullinated proteins . Arthritis Rheum 2005 , 52 : 3433 - 3438 .
42. Bas S , Perneger TV , Mikhnevitch E , Seitz M , Tiercy JM , Roux-Lombard P , Guerne PA : Association of rheumatoid factors and antifilaggrin antibodies with severity of erosions in rheumatoid arthritis . Rheumatology (Oxford) 2000 , 39 : 1082 - 1088 .
43. Boki KA , Kurki P , Holthofer H , Tzioufas AG , Drosos AA , Moutsopoulos HM : Prevalence of antikeratin antibodies in Greek patients with rheumatoid arthritis. A clinical, serologic, and immunogenetic study . J Rheumatol 1995 , 22 : 2046 - 2048 .
44. Goldbach-Mansky R , Lee J , McCoy A , Hoxworth J , Yarboro C , Smolen JS , Steiner G , Rosen A , Zhang C , Menard HA , et al.: Rheumatoid arthritis associated autoantibodies in patients with synovitis of recent onset . Arthritis Res 2000 , 2 : 236 - 243 .
45. Verpoort KN , van Gaalen FA , van der Helm-van Mil AH , Schreuder GM , Breedveld FC , Huizinga TW , de Vries RR , Toes RE : Association of HLA-DR3 with anti-cyclic citrullinated peptide antibody-negative rheumatoid arthritis . Arthritis Rheum 2005 , 52 : 3058 - 3062 .
46. Hill JA , Southwood S , Sette A , Jevnikar AM , Bell DA , Cairns E : Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule . J Immunol 2003 , 171 : 538 - 541 .