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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Production of anti–citrullinated protein antibodies (ACPAs) is an important biomarker for rheumatoid arthritis (RA). We undertook this study to determine whether genetic factors (HLA—DRB1 alleles) are associated with extreme ACPA levels in individuals with ACPA-positive RA, and to ascertain whether there are any phenotypic characteristics associated with these subgroups of RA.

Methods

HLA–DRB1 allelic groups were genotyped in 1,073 ACPA-positive RA patients from the Swedish Epidemiological Investigation of Rheumatoid Arthritis study. We found that 283 patients (26.4%) had high ACPA levels (defined as >1,500 units/ml using the Euro-Diagnostica anti-CCP2 test), while the rest of the patients had moderate ACPA levels and served as the comparison group. A replication group consisted of 235 RA patients.

Results

No significant differences in baseline disease activity were observed between patients with high and those with moderate ACPA levels. However, the HLA–DRB1*15 allele was associated with high ACPA levels (P = 0.0002). A similar trend was detected in HLA–DRB1*15–positive patients in the replication cohort, with meta-analysis of the discovery and replication cohorts demonstrating an overall effect of HLA–DRB1*15 on development of high ACPA levels in both the discovery and replication cohorts (P < 0.0001 by Mantel-Haenszel test with a fixed-effects model).

Conclusion

Our data indicate that HLA–DRB1*15 may promote the production of high ACPA levels. Due to the high value of ACPA level scores in the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA, the presence of HLA–DRB1*15 may, at least in part, contribute to fulfilling the criteria for RA. This illustrates the complex nature of the genetic regulation of ACPA levels. Additional mechanistic studies of the regulation of ACPAs and ACPA-positive RA are pending.

Production of anti–citrullinated protein antibodies (ACPAs) is an important biomarker for a major subgroup of patients with rheumatoid arthritis (RA) (1). It has been demonstrated that the development of these antibodies in general is associated with HLA–DRB1 shared epitope (SE) alleles (2–5). ACPA positivity is known to be associated with more progressive, more destructive RA and extraarticular manifestations as well as cardiovascular disease (6–8). Furthermore, ACPA positivity has been reported to be associated with worse response to anti–tumor necrosis factor therapy (9).

Interestingly, the level of ACPAs in ACPA-positive RA does not correlate with disease activity (10). Nevertheless, there is a distinct group of ACPA-positive RA patients characterized by very high ACPA levels close to and even exceeding the upper detection limit of routinely available diagnostic enzyme-linked immunosorbent assays (ELISAs). Since ACPA is a very specific biomarker for RA, and since the presence or absence of ACPAs in RA influences the course of disease and depends on SE alleles, it is interesting to study the phenomenon of very high production of ACPAs from both a clinical and a genetic perspective. So far, no data are available to confirm whether there is a distinct genetic predisposition underlying very high ACPA levels. HLA–DRB1*03 has already been implicated in association with lower ACPA levels in previous studies (11, 12). However, due to the small size of these studies, the study design, and the strong influence of SE alleles on development of ACPA-positive RA overall, the question remains as to whether this is a true association (13, 14). Our aim was to determine the influence of HLA–DRB1 alleles on the production of very high levels of ACPAs in comparison with moderate levels in ACPA-positive RA and to ascertain whether there is any phenotypic characteristic associated with these subgroups of RA.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Study population.

This study was approved by ethics review boards at the Karolinska Institutet, and informed consent was obtained from all participating patients. The cohort of 1,073 ACPA-positive RA patients (764 women and 309 men) from the Swedish Epidemiological Investigation of Rheumatoid Arthritis study was included in the analysis (Table 1). RA patients with ACPA levels below the diagnostic threshold (25 units/ml) at baseline were not included in the study. All cases were newly diagnosed, and the diagnosis of RA was made according to the 1987 revised criteria of the American College of Rheumatology (ACR) (15). Data on the Disease Activity Score in 28 joints (DAS28) (16) from the time of diagnosis were available for 515 patients. Hand and foot radiographs were evaluated at baseline (i.e., time of diagnosis) and 1 and 2 years after diagnosis. Larsen scores (17) were currently available for 173 patients. Information about smoking exposure and detailed cigarette smoking history among patients was available for 979 of the 1,073 patients, obtained by questionnaire as described previously (5). The quantification of smoking was based on the smoking history before first symptoms of arthritis as described previously (18). All smokers were cigarette smokers; a minor number of subjects who had smoked pipes or cigars were excluded from the study (18).

Table 1. Baseline characteristics in the EIRA and replication cohorts of ACPA-positive patients with rheumatoid arthritis*
 EIRA cohort patientsReplication (non-EIRA) cohort patients
High ACPA levels (n = 283)Moderate ACPA levels (n = 790)High ACPA levels (n = 48)Moderate ACPA levels (n = 187)
  • *

    There were no significant differences between the groups in either cohort. EIRA = Epidemiological Investigation of Rheumatoid Arthritis; ACPA = anti–citrullinated protein antibody; NA = not available; DAS28 = Disease Activity Score in 28 joints.

  • Data were missing for some patients.

Age, mean ± SEM years51.02 ± 0.7050.83 ± 0.4446.63 ± 2.5546.07 ± 1.17
No. men/no. women87/196222/5689/3939/148
No. ever smokers/no. never smokers204/62506/207NANA
DAS28, mean ± SEM5.283 ± 0.105.217 ± 0.07NANA

The replication cohort consisted of 235 ACPA-positive RA patients from Karolinska University Hospital who fulfilled the 1987 ACR criteria and were tested for ACPA and HLA–DRB1 alleles (see below and Table 1). There was no overlap between the cohorts. For 3 of the 235 patients in the replication cohort, genetic data were not available.

HLA typing and ACPA level.

HLA–DRB1 alleles were detected by sequence-specific primer–polymerase chain reaction genotyping with the DR low-resolution kit (Olerup SSP). ACPA concentrations were measured at the time of diagnosis by Immunoscan RA (Mark 2) anti-CCP2 ELISA (Euro-Diagnostica). A level of >25 units/ml was regarded as being positive according to instructions in the kit and as confirmed by the Clinical Immunology Laboratory at Uppsala University Hospital. The cutoff between moderately and highly elevated ACPA levels was defined as 1,500 units/ml. Using a provisional cutoff of 1,500 units/ml, we found that 283 patients (26.4%) had a high ACPA level; the remaining patients were considered to have a moderate ACPA level. In the replication cohort, a similar proportion of patients (48 of 235 individuals [20.4%]) had an ACPA level of >1,500 units/ml (P = 0.06 versus the discovery cohort, by chi-square test with Yates' correction).

Statistical analysis.

The chi-square test was used to analyze the association of allele frequencies. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated to assess effect size. An unpaired t-test and Mann-Whitney U test were used to compare age at disease onset and DAS28 values. The Mantel-Haenszel test with a fixed-effects model was used for meta-analysis. The GraphPad Prism 4.0 software package and ManRev 5 were used for the analysis. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Association of HLA–DRB1 alleles with high ACPA level in RA patients.

To address the possible association of HLA–DRB1 alleles with the level of ACPA production, we first tested it in a dominant model for RA patients with moderate ACPA levels in comparison with RA patients with high ACPA levels. Among all groups of DRB1 alleles, initially we observed HLA–DRB1*03 to be negatively associated with high ACPA levels (P < 0.0001, OR 0.38 [95% CI 0.23–0.60]) and HLA–DRB1*15 to be positively associated with high ACPA levels (P = 0.0002, OR 1.82 [95% CI 1.33–2.48]) (Table 2). However, no association between high ACPA levels and SE alleles was detected in comparison with the group with moderate ACPA levels.

Table 2. Association between HLA–DRB1 allelic groups and very high ACPA levels in patients with ACPA-positive RA*
HLA–DRB1 alleleRA patients with moderate ACPA levels, no. carriers/no. noncarriers of the alleleRA patients with high ACPA levels, no. carriers/no. noncarriers of the alleleOR (95% CI)P
  • *

    ACPA = anti–citrullinated protein antibody; RA = rheumatoid arthritis; OR = odds ratio; 95% CI = 95% confidence interval; SE = shared epitope.

  • By chi-square test.

  • Including HLA–DRB1*0103.

HLA–DRB1*01197/59388/1951.36 (1.01–1.82)0.0441
HLA–DRB1*03145/64522/2610.38 (0.23–0.60)<0.0001
HLA–DRB1*04540/250189/940.93 (0.70–1.24)0.6273
HLA–DRB1*0758/73228/2551.39 (0.86–2.22)0.1748
HLA–DRB1*0845/74512/2710.73 (0.38–1.41)0.3487
HLA–DRB1*0927/7634/2790.41 (0.14–1.17)0.0841
HLA–DRB1*1030/76011/2721.03 (0.51–2.07)0.9463
HLA–DRB1*1157/73327/2561.36 (0.84–2.19)0.2114
HLA–DRB1*1227/76310/2731.04 (0.49–2.17)0.9270
HLA–DRB1*1388/70219/2640.57 (0.34–0.96)0.0330
HLA–DRB1*1420/7709/2741.27 (0.57–2.81)0.5637
HLA–DRB1*15145/64582/2011.82 (1.33–2.48)0.0002
HLA–DRB1*1613/7776/2771.30 (0.49–3.44)0.6035
SE670/120243/401.09 (0.74–1.60)0.6688

Since we knew that SE alleles are strongly associated with development of ACPA-positive RA, we performed stratification for the carriage of SE alleles in DRB1*03-positive and DRB1*15-positive individuals for association with high ACPA levels. This analysis demonstrated that the protection against development of high ACPA levels by HLA–DRB1*03 was not independent from SE alleles (Table 3). In the replication cohort of 232 ACPA-positive RA patients, a similar trend was observed (data not shown).

Table 3. Test for independence from SE alleles of the association of HLA–DRB1*03 and of HLA–DRB1*15 with high levels of ACPAs in RA patients*
GroupNo. of patients with high ACPA levels/ no. of patients with moderate ACPA levelsOR (95% CI)P
  • *

    NA = not applicable (see Table 2 for other definitions).

  • †, *

    By chi-square test.

No SE/no HLA–DRB1*0330/78ReferentNA
SE/no HLA–DRB1*03231/5671.06 (0.68–1.66)0.8011
No SE/HLA–DRB1*0310/420.62 (0.28–1.39)0.2422
SE/HLA–DRB1*0312/1030.30 (0.15–0.63)0.0009
No SE/no HLA–DRB1*1514/69ReferentNA
SE/no HLA–DRB1*15187/5761.60 (0.88–2.91)0.1203
No SE/HLA–DRB1*1526/512.51 (1.19–5.29)0.0136
SE/HLA–DRB1*1556/942.94 (1.51–5.70)0.0011

On the other hand, the HLA–DRB1*15 allele was more frequent in individuals with high ACPA levels, independent of SE alleles (P = 0.0136, OR 2.51 [95% CI 1.19–5.29]) or in combination with SE alleles (P = 0.0011, OR 2.94 [95% CI 1.51–5.70]) (Table 3). Again, SE alleles without the HLA–DRB1*15 allele did not show any association with high ACPA levels (Table 3).

Since an individual who carries an SE allele and an HLA–DRB1*03 allele at the same time cannot carry an HLA–DRB1*15 allele, we wanted to clarify the possible role of these alleles and to adjust effects from DRB1*03 and DRB1*15 in one test for SE alleles' presence. The data showed that only HLA–DRB1*15, alone (P = 0.0112, OR 3.21) or in combination with the SE (P = 0.0051, OR 3.13), remained associated with high ACPA levels (Table 4). HLA–DRB1*03 or SE alleles alone or in combination with each other did not show any association with ACPA level in ACPA-positive RA patients.

Table 4. Test for independence from SE alleles of the association of HLA–DRB1*15 with high levels of ACPAs in RA patients*
GroupNo. of patients with high ACPA levels/ no. of patients with moderate ACPA levelsOR (95% CI)P
  • *

    NA = not applicable (see Table 2 for other definitions).

  • By chi-square test.

HLA–DRB1*158/42ReferentNA
SE/no HLA–DRB1*03/no HLA–DRB1*15175/4731.94 (0.891–4.22)0.0882
No SE/HLA–DRB1*03/no HLA–DRB1*156/271.17 (0.36–3.74)0.7950
No SE/no HLA–DRB1*03/HLA–DRB1*1522/363.21 (1.27–8.08)0.0112
SE/HLA–DRB1*0312/1030.61 (0.23–1.60)0.3141
SE/HLA–DRB1*1556/943.13 (1.37–7.14)0.0051
HLA–DRB1*03/HLA–DRB1*154/151.40 (0.37–5.33)0.6209

Since the effect was truly significant only for association with HLA–DRB1*15, we decided to limit replication analysis to this specific finding. A similar tendency was observed in the replication cohort. There were 43 patients carrying the HLA–DRB1*15 allele (30 with moderate ACPA levels and 13 with high ACPA levels), while there were 189 noncarriers of the HLA–DRB1*15 allele (155 with moderate ACPA levels and 34 with high ACPA levels) (P = 0.0714 by chi-square test, OR 1.98 [95% CI 0.93–4.18]). There were 186 patients carrying the SE allele (146 with moderate ACPA levels and 40 with high ACPA levels), while there were 46 noncarriers of the SE allele (39 with moderate ACPA levels and 7 with high ACPA levels) (P = 0.3421 by chi-square test, OR 1.53 [95% CI 0.63–3.67]). (P values represent carriers versus noncarriers for both the HLA–DRB1*15 and the SE alleles.) A meta-analysis of the discovery and replication cohorts demonstrated a strong effect of HLA–DRB1*15 on the development of ACPAs (Figure 1), with no heterogeneity between the studies.

thumbnail image

Figure 1. Meta-analysis of association of HLA–DRB1*15 with high levels of anti–citrullinated protein antibodies (ACPAs) in ACPA-positive patients with rheumatoid arthritis. This meta-analysis between the discovery and replication cohorts demonstrates the strong effect of HLA–DRB1*15 on development of ACPAs with no heterogeneity between the studies. Events = HLA–DRB1*15 positivity; M-H = Mantel- Haenszel test; Fixed = fixed-effects model; 95% CI = 95% confidence interval.

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Smoking.

To address smoking status as a possible risk factor for the development of high levels of ACPAs in RA patients, we compared the proportion of ever smokers between those with high and those with moderate ACPA levels. Our data did not show a significant association of smoking with ACPA level (Table 1), although smoking was slightly more common in the group of RA patients with a high ACPA level (OR 1.35 [95% CI 0.97–1.87]).

However, when patients were stratified for smoking status, the carriage of the HLA–DRB1*15 allele was found to be more strongly associated with a high ACPA level (>1,500 units/ml) only in ever smokers. Among ever smokers who carried the HLA–DRB1*15 allele, we observed 64 patients with high ACPA levels and 97 patients with moderate ACPA levels. This contrasts with the finding of 50 patients with high ACPA levels and 176 patients with moderate ACPA levels in the reference group (never smokers who did not carry the HLA–DRB1*15 allele) (P = 0.0002, OR 2.32 [95% CI 1.49–3.63]). No other combinations differed from the reference group. Any conclusions should be made with caution, however, since no data about smoking status were available in the replication cohort and we were unable to repeat the analysis. In a similar analysis of SE alleles and smoking, no association of either single or combined factors with high ACPA levels was detected in comparison with the group with moderate ACPA levels (data not shown).

Age at onset of the disease.

We could not detect any significant difference in the age at onset of the disease between patients with high ACPA levels and those with moderate ACPA levels (Table 1). However, according to our observations in the discovery cohort, ever smokers who carried the SE allele, the HLA–DRB1*15 allele, or both alleles developed RA significantly later, at the mean ± SEM ages of 52.49 ± 0.48 years, 53.71 ± 1.48 years, and 52.74 ± 1.00 years, respectively (P = 0.0008, P = 0.0008, and P = 0.0013, respectively), than never smokers who carried neither the SE allele nor the HLA–DRB1*15 allele (mean ± SEM age 44.46 ± 2.36 years). However, in the replication cohort, the SE and HLA–DRB1*15 alleles alone or in combination were not associated with delayed onset of disease (data not shown).

Testing of disease activity markers.

When addressing possible differences in baseline disease activity and in bone erosion in relation to ACPA level, we could not find any significant difference in baseline DAS28 values between patients with high and those with moderate ACPA levels. When we compared RA patients with moderate and those with high ACPA levels, we found that smoking, the carriage of the SE allele, the carriage of the HLA–DRB1*15 allele, or any combination of these factors did not affect baseline DAS28 or Larsen scores significantly in our study (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

The novel observation of our study was that a non-SE HLA–DRB1 allele, namely, HLA–DRB1*15, is associated with production of high levels of ACPAs in RA patients. It is important to differentiate this issue from association with production of ACPAs in the general population. All previously reported studies have shown association of HLA alleles with production of ACPAs (12), while we report association with high production of ACPAs.

HLA–DRB1*15 has been previously reported to be associated with anti-SSA antibody positivity in primary Sjögren's syndrome (19, 20) and with a high level of natural antibodies in healthy individuals (21). HLA–DRB1*15 was also found to be associated with renal involvement (22) and with secondary Sjögren's syndrome (23) in RA. These observations together with ours support the hypothesis that HLA–DRB1*15 may play a specific role in the production of high levels of autoantibodies. In the latter study, HLA–DRB1*15 was also found to be associated with RA in RA patients lacking the SE (23), although we did not replicate this finding in our own previous work (13).

Cigarette smoking is the strongest proven environmental risk factor for the development of RA (24–26). Although interaction of smoking and HLA–DRB1 SE alleles has been shown to confer high risk for the development of rheumatoid factor–positive or ACPA-positive RA (5, 27, 28), we did not find this gene–environment combination to have any effect on ACPA levels. We may speculate that HLA–DRB1*15 serves as a second trigger that may aggravate autoimmune response in individuals with predisposition to RA caused by the SE, other genetic factors, or early life exposure to an environmental risk factor, when the first trigger (smoking, the SE, or another) breaks the tolerance to citrullinated protein antigens. However, we did not have a large enough cohort to confirm these findings in an independent study, and inferences about the influence of smoking on the level of ACPAs in ACPA-positive RA patients should be made with caution.

Irigoyen et al found HLA–DRB1*03 to be associated with a lower level of anti–cyclic citrullinated peptide (anti-CCP) antibodies, even when adjusted for the presence of the SE (11). Cui et al reported that a single-nucleotide polymorphism in linkage disequilibrium with HLA–DRB1*03 was associated with anti-CCP levels below the diagnostic threshold (12). They also considered HLA–DRB1*03 a factor that could potentially influence anti-CCP levels independent of HLA SE alleles (12). In fact, all of these studies compared anti-CCP–negative with anti-CCP–positive disease, but they did not address the question of ACPA levels in seropositive RA. Our data show that neither HLA–DRB1*03 nor the SE alone affected ACPA production in ACPA-positive RA. It is difficult to determine association with ACPA levels outside the range (upper and lower thresholds) of the ELISA kits due to their inaccuracy outside this range.

An interesting association was observed in our study. The SE allele and/or HLA–DRB1*15 allele together with smoking were significantly associated with late RA onset in the discovery cohort, but this finding was not replicated.

One may speculate that the relatively lower ACPA level in individuals who have earlier disease onset may be explained by more active “consumption” of autoantibodies in joints and more active disease and severe joint damage. However, our analyses failed to detect any associations of ACPA level, the presence of the SE allele and/or the HLA–DRB1*15 allele, and smoking with disease severity or radiographic progression. In a relatively small study, it was found that serial anti-CCP measurements during the first 3 years of RA may be better predictors of radiographic progression than baseline anti-CCP level (29). In accordance with our findings, however, Mattey et al failed to demonstrate any association between HLA–DRB1*15 and erosive disease or nodule formation (23).

A high ACPA level contributes 3 points toward the 2010 ACR/European League Against Rheumatism (EULAR) classification criteria for RA (30). In this clinical context, “highly positive” refers to detected values >3 times the upper limit of normal laboratory test results in healthy individuals (i.e., >75 units/ml). In our cohorts the definition of high ACPA levels meant remarkably higher ACPA levels (i.e., >1,500 units/ml). Nevertheless, the group of patients with high ACPA levels associated with the HLA–DRB1*15 allele is included in the group of patients who score the highest in the serology part of the new ACR/EULAR classification criteria for RA. Therefore, the presence of HLA–DRB1*15 may, at least in part, contribute to fulfilling the criteria for RA, which emphasizes the clinical significance of our observation. Our study shows that this contribution is independent of SE alleles, while there is a possible protective effect against development of high ACPA levels conferred by HLA–DRB1*03 linked to SE alleles.

In summary, we have found and confirmed that HLA–DRB1*15 alleles are increased in RA patients with high ACPA levels in comparison with patients with moderate ACPA levels. Our finding of a relationship between high ACPA levels and HLA–DRB1*15 further emphasizes the importance of genetic background and adaptive immunity in development of autoantibody-positive RA.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

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. Laki 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. Laki, Lundström, Snir, Bengtsson, Alfredsson, Klareskog, Padyukov.

Acquisition of data. Lundström, Snir, Rönnelid, Ganji, Bengtsson, Wick, Alfredsson, Klareskog, Padyukov.

Analysis and interpretation of data. Laki, Snir, Rönnelid, Catrina, Saevarsdottir, Wick, Padyukov.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  • 1
    Klareskog L, Catrina AI, Paget S. Rheumatoid arthritis. Lancet 2009; 373: 65972.
  • 2
    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: 111721.
  • 3
    Berglin E, Padyukov L, Sundin U, Hallmans G, Stenlund H, van Venrooij WJ, et al. A combination of autoantibodies to cyclic citrullinated peptide (CCP) and HLA-DRB1 locus antigens is strongly associated with future onset of rheumatoid arthritis. Arthritis Res Ther 2004; 6: R3038.
  • 4
    Huizinga TW, Amos CI, van der Helm-van Mil AH, Chen W, van Gaalen FA, Jawaheer D, 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: 34338.
  • 5
    Klareskog L, Stolt P, Lundberg K, Kallberg H, Bengtsson C, Grunewald J, et al and the Epidemiological Investigation of Rheumatoid Arthritis Study Group. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA–DR (shared epitope)–restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum 2006; 54: 3846.
  • 6
    De Rycke L, Peene I, Hoffman IE, Kruithof E, Union A, Meheus L, et al. Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations. Ann Rheum Dis 2004; 63: 158793.
  • 7
    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 arthritis-specific autoantibodies. J Clin Invest 1998; 101: 27381.
  • 8
    Visser K, Goekoop-Ruiterman YP, de Vries-Bouwstra JK, Ronday HK, Seys PE, Kerstens PJ, et al. A matrix risk model for the prediction of rapid radiographic progression in patients with rheumatoid arthritis receiving different dynamic treatment strategies: post hoc analyses from the BeSt study. Ann Rheum Dis 2010; 69: 13337.
  • 9
    Potter C, Hyrich KL, Tracey A, Lunt M, Plant D, Symmons DP, et al. Association of rheumatoid factor and anti-cyclic citrullinated peptide positivity, but not carriage of shared epitope or PTPN22 susceptibility variants, with anti-tumour necrosis factor response in rheumatoid arthritis. Ann Rheum Dis 2009; 68: 6974.
  • 10
    Ronnelid J, Wick MC, Lampa J, Lindblad S, Nordmark B, Klareskog L, et al. Longitudinal analysis of citrullinated protein/peptide antibodies (anti-CP) during 5 year follow up in early rheumatoid arthritis: anti-CP status predicts worse disease activity and greater radiological progression. Ann Rheum Dis 2005; 64: 17449.
  • 11
    Irigoyen P, Lee AT, Wener MH, Li W, Kern M, Batliwalla F, 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: 38138.
  • 12
    Cui J, Taylor KE, Destefano AL, Criswell LA, Izmailova ES, Parker A, et al. Genome-wide association study of determinants of anti-cyclic citrullinated peptide antibody titer in adults with rheumatoid arthritis. Mol Med 2009; 15: 13643.
  • 13
    Lundstrom E, Kallberg H, Smolnikova M, Ding B, Ronnelid J, Alfredsson L, et al. Opposing effects of HLA–DRB1*13 alleles on the risk of developing anti–citrullinated protein antibody–positive and anti–citrullinated protein antibody–negative rheumatoid arthritis. Arthritis Rheum 2009; 60: 92430.
  • 14
    Van der Woude D, Lie BA, Lundstrom E, Balsa A, Feitsma AL, Houwing-Duistermaat JJ, et al. Protection against anti– citrullinated protein antibody–positive rheumatoid arthritis is predominantly associated with HLA–DRB1*1301: a meta-analysis of HLA–DRB1 associations with anti–citrullinated protein antibody–positive and anti–citrullinated protein antibody–negative rheumatoid arthritis in four European populations. Arthritis Rheum 2010; 62: 123645.
  • 15
    Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31: 31524.
  • 16
    Prevoo ML, van 't Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight–joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995; 38: 448.
  • 17
    Larsen A, Dale K, Eek M. Radiographic evaluation of rheumatoid arthritis and related conditions by standard reference films. Acta Radiol Diagn (Stockh) 1977; 18: 48191.
  • 18
    Stolt P, Bengtsson C, Nordmark B, Lindblad S, Lundberg I, Klareskog L, et al. Quantification of the influence of cigarette smoking on rheumatoid arthritis: results from a population based case-control study, using incident cases. Ann Rheum Dis 2003; 62: 83541.
  • 19
    Gottenberg JE, Busson M, Loiseau P, Cohen-Solal J, Lepage V, Charron D, et al. In primary Sjögren's syndrome, HLA class II is associated exclusively with autoantibody production and spreading of the autoimmune response. Arthritis Rheum 2003; 48: 22405.
  • 20
    Harley JB, Reichlin M, Arnett FC, Alexander EL, Bias WB, Provost TT. Gene interaction at HLA-DQ enhances autoantibody production in primary Sjögren's syndrome. Science 1986; 232: 11457.
  • 21
    Pozsonyi E, Gyorgy B, Berki T, Banlaki Z, Buzas E, Rajczy K, et al. HLA-association of serum levels of natural antibodies. Mol Immunol 2009; 46: 141623.
  • 22
    Tokunaga NK, Noda R, Kaneoka H, Ogahara S, Murata T, Hiratsuka T, et al. Association between HLA-DRB1*15 and Japanese patients with rheumatoid arthritis complicated by renal involvement. Nephron 1999; 81: 16571.
  • 23
    Mattey DL, Gonzalez-Gay MA, Hajeer AH, Dababneh A, Thomson W, Garcia-Porrua C, et al. Association between HLA-DRB1*15 and secondary Sjögren's syndrome in patients with rheumatoid arthritis. J Rheumatol 2000; 27: 26116.
  • 24
    Symmons DP, Bankhead CR, Harrison BJ, Brennan P, Barrett EM, Scott DG, et al. Blood transfusion, smoking, and obesity as risk factors for the development of rheumatoid arthritis: results from a primary care–based incident case–control study in Norfolk, England. Arthritis Rheum 1997; 40: 195561.
  • 25
    Vessey MP, Villard-Mackintosh L, Yeates D. Oral contraceptives, cigarette smoking and other factors in relation to arthritis. Contraception 1987; 35: 45764.
  • 26
    Karlson EW, Lee IM, Cook NR, Manson JE, Buring JE, Hennekens CH. A retrospective cohort study of cigarette smoking and risk of rheumatoid arthritis in female health professionals. Arthritis Rheum 1999; 42: 9107.
  • 27
    Linn-Rasker SP, van der Helm-van Mil AH, van Gaalen FA, Kloppenburg M, de Vries RR, le Cessie S, et al. Smoking is a risk factor for anti-CCP antibodies only in rheumatoid arthritis patients who carry HLA-DRB1 shared epitope alleles. Ann Rheum Dis 2006; 65: 36671.
  • 28
    Pedersen M, Jacobsen S, Garred P, Madsen HO, Klarlund M, Svejgaard A, et al. Strong combined gene–environment effects in anti–cyclic citrullinated peptide–positive rheumatoid arthritis: a nationwide case–control study in Denmark. Arthritis Rheum 2007; 56: 144653.
  • 29
    Meyer O, Nicaise-Roland P, Santos MD, Labarre C, Dougados M, Goupille P, et al. Serial determination of cyclic citrullinated peptide autoantibodies predicted five-year radiological outcomes in a prospective cohort of patients with early rheumatoid arthritis. Arthritis Res Ther 2006; 8: R40.
  • 30
    Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO III, et al. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62: 256981.