The HLA–DRB1 shared epitope alleles differ in the interaction with smoking and predisposition to antibodies to cyclic citrullinated peptide

Objective

The HLA shared epitope (SE) alleles are primarily a risk factor for the presence of antibodies to cyclic citrullinated peptide (anti‐CCP antibodies) rather than for the development of rheumatoid arthritis (RA). The SE alleles interact with the environmental risk factor tobacco exposure (TE) for predisposition to anti‐CCP+ RA. The objectives of this study were to determine 1) whether different SE subtypes contribute differently to the presence of anti‐CCP antibodies, 2) whether different SE subtypes all interact with TE for the development of anti‐CCP antibodies, and 3) the effect of TE in relation to the SE alleles and anti‐CCP antibodies on the risk of progression from undifferentiated arthritis (UA) to RA.

Methods

We assessed the effect of SE subtypes and TE on the presence and level of anti‐CCP antibodies and on the risk of progression from UA to RA in 977 patients with early arthritis who were included in the Leiden Early Arthritis Clinic.

Results

The HLA–DRB1*0401, *0404, *0405, or *0408 SE alleles conferred the highest risk of developing anti‐CCP antibodies (odds ratio [OR] 5.0, compared with an OR of 2.0 for the HLA–DRB1*0101 or *0102 SE alleles and an OR of 1.7 for the HLA–DRB1*1001 SE allele). Conversely, the TE–SE allele interaction was the strongest for the HLA–DRB1*0101 or *0102 SE alleles and the HLA–DRB1*1001 SE allele. TE in SE+, anti‐CCP+ patients correlated with higher levels of anti‐CCP antibodies and with progression from UA to RA. In logistic regression analysis, only the presence and level of anti‐CCP antibodies were associated independently with RA development.

Conclusion

The HLA–DRB1 SE subtypes differ in their interaction with smoking and in their predisposition to anti‐CCP antibodies. TE contributes to the development of RA in SE+, anti‐CCP+ patients, which is explained by its effect on the level of anti‐CCP antibodies.

     

Regulation of anti–cyclic citrullinated peptide antibodies in rheumatoid arthritis: Contrasting effects of HLA–DR3 and the shared epitope alleles

Objective

To examine the association between HLA–DRB1 alleles and the production of anti–cyclic citrullinated peptide (anti‐CCP) and rheumatoid factor (RF) autoantibodies in patients with rheumatoid arthritis (RA).

Methods

We studied 1,723 Caucasian RA patients enrolled in the North American Rheumatoid Arthritis Consortium (NARAC) family cohort and the Study of New Onset Rheumatoid Arthritis (SONORA) cohort. All patients were tested for anti‐CCP antibodies (by enzyme‐linked immunosorbent assay), RF (by nephelometry), and HLA–DR genotype (by polymerase chain reaction and sequence‐specific oligonucleotide hybridization).

Results

When controlled for the presence of RF, anti‐CCP positivity was strongly associated with the HLA–DRB1 shared epitope (SE). In RF+ patients, the presence of the SE was very significantly associated with anti‐CCP positivity, with an odds ratio (OR) of 5.8 and a 95% confidence interval (95% CI) of 4.1–8.3. This relationship was also seen in RF– patients (OR 3.1 [95% CI 1.8–5.3]). In contrast, RF positivity was not significantly associated with presence of the SE independently of anti‐CCP antibodies. Strikingly, HLA–DRB1*03 was strongly associated with reduced anti‐CCP titers, even after controlling for the presence of the SE and restricting the analysis to anti‐CCP+ patients. HLA–DR3 was also associated with anti‐CCP– RA in our population.

Conclusion

The HLA–DRB1 SE is strongly associated with the production of anti‐CCP antibodies, but not RF. In contrast, HLA–DR3 alleles are associated with anti‐CCP– disease and with lower levels of anti‐CCP antibodies, even when controlling for the SE. These data emphasize the complexity of the genetic effects of the major histocompatibility complex on the RA phenotype.

     

Smoking increases rheumatoid arthritis susceptibility in individuals carrying the HLA–DRB1 shared epitope,regardless of rheumatoid factor or anti–cyclic citrullinated peptide antibody status

Objective

Smoking is associated with rheumatoid arthritis (RA) in individuals with the HLA–DRB1 shared epitope (SE). SE alleles have been shown to be predominantly associated with anti–cyclic citrullinated peptide (anti‐CCP)–positive RA. These risk factors have not been identified for anti‐CCP–negative RA. The aim of this study was to investigate whether SE‐containing HLA–DRB1 alleles, smoking, or the combination of these factors contributes to the development of RA, depending on the presence or absence of serologic markers, in a Korean population.

Methods

All of the patients with RA (n =1,482) and all of the control subjects (n = 1,119) were Korean. Four‐digit HLA–DRB1 typing was performed by a conventional polymerase chain reaction–sequence‐based typing method. Information about smoking history was obtained through a questionnaire. The patients with RA were tested for anti‐CCP antibodies and rheumatoid factor (RF).

Results

The SE alleles had significant effects on anti‐CCP antibody and RF formation. The DRB1*0901 allele was associated with the presence of anti‐CCP antibodies (odds ratio [OR] 2.49) and RF (OR 2.09). SE alleles and smoking were associated with both anti‐CCP–positive and anti‐CCP–negative RA. The combination of smoking and double copies of the SE allele increased the risk of anti‐CCP–positive RA 36.11‐fold and increased the risk of anti‐CCP–negative RA 12.29‐fold, compared with the risk among nonsmokers not carrying SE alleles. Interactions between SE alleles and smoking were observed for both anti‐CCP–positive and RF‐positive RA, although the associations of RF‐positive RA could be consequences of the underlying anti‐CCP antibody status.

Conclusion

We demonstrated that the combination of SE alleles and smoking is associated with RA susceptibility regardless of anti‐CCP antibody or RF status, but that the combination shows stronger effects in anti‐CCP–positive/RF‐positive patients with RA than in anti‐CCP–negative/RF‐negative patients with RA. The SE–smoking interactions were present in anti‐CCP–positive and RF‐positive RA.

     

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

Objective

The shared epitope (SE)–containing HLA–DRB1 alleles represent the most significant genetic risk factor for rheumatoid arthritis (RA). Recent studies indicate that the SE alleles are associated with only RA that is characterized by the presence of anti–cyclic citrullinated peptide (anti‐CCP) antibodies, and not with anti‐CCP–negative disease. In this study we investigated whether the SE alleles contribute to the development of anti‐CCP–positive RA, or whether they are associated solely with the presence of anti‐CCP antibodies. We therefore determined the influence of the SE alleles and anti‐CCP antibodies on the progression from recent‐onset undifferentiated arthritis (UA) to RA.

Methods

Patients with recent‐onset UA at the 2‐week visit (n = 570) were selected from the Leiden Early Arthritis Cohort. SE alleles, rheumatoid factor (RF) status, and anti‐CCP antibody levels were determined. Progression to RA or other diagnoses was monitored.

Results

One hundred seventy‐seven patients with UA developed RA during the 1‐year followup, whereas the disease in 393 patients remained unclassified or was given other diagnoses. The SE alleles correlated with the presence of anti‐CCP antibodies, but not with the presence of RF. Both in SE‐positive and in SE‐negative patients with UA, the presence of anti‐CCP antibodies was significantly associated with the development of RA. More intriguingly, however, no apparent contribution of the SE alleles to the progression to RA was found when analyses were stratified according to the presence of anti‐CCP antibodies. In patients with anti‐CCP–positive disease, the presence of SE alleles was associated with significantly higher levels of anti‐CCP antibodies, suggesting that the SE alleles act as classic immune response genes.

Conclusion

The SE alleles do not independently contribute to the progression to RA from UA, but rather contribute to the development of anti‐CCP antibodies.

     

Isotype distribution of ANTI–CYCLIC citrullinated peptide antibodies in undifferentiated arthritis and rheumatoid arthritis reflects an ongoing immune response

Objective

The evolution of the rheumatoid arthritis (RA)–specific anti–cyclic citrullinated peptide (anti‐CCP) antibody response, as measured by the isotypes of anti‐CCP, has not been described. This study was undertaken to determine anti‐CCP isotype usage in patients with undifferentiated arthritis (UA), patients with recent‐onset RA, and patients with RA of long duration.

Methods

IgA, IgM, and IgG subclasses of anti‐CCP were measured by enzyme‐linked immunosorbent assay in serum samples that were obtained from IgG anti‐CCP antibody–positive patients with UA (n = 110) and IgG anti‐CCP antibody–positive patients with RA (n = 152) early after the onset of arthritis. Patients with UA in whom RA developed within 1 year (UA→RA) were compared with patients with UA in whom RA did not develop within 1 year (UA→UA). In addition, baseline serum samples obtained from a subset of patients with RA (n = 64) were compared with sera obtained from the same patients a median of 7 years later.

Results

IgM anti‐CCP was present in early samples from both patients with UA and patients with RA and in followup samples from patients with RA. Several IgG anti‐CCP antibody–positive patients who did not have IgM anti‐CCP early after disease onset did display IgM anti‐CCP later in the course of the arthritis. A diverse pattern of isotype usage was detected in early samples, with a trend toward lower frequencies of all isotypes of anti‐CCP in patients with UA compared with patients with RA and in UA→UA patients compared with UA→RA patients. Levels of all isotypes except IgG1 had decreased after 7 years.

Conclusion

These data indicate development of the anti‐CCP isotype repertoire into full usage early in the course of arthritis. The sustained presence of IgM anti‐CCP indicates ongoing recruitment of new B cells into the anti‐CCP response, reflecting a continuous (re)activation of the RA‐specific anti‐CCP response during the course of anti‐CCP–positive arthritis.

     

Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA–DRB1 shared epitope for antibodies to citrullinated proteins

Objective

The main genetic risk factor for rheumatoid arthritis (RA), the HLA region, has been known for 25 years. Previous research has demonstrated, within the RA population, an association between HLA–DRB1 alleles carrying the shared epitope (SE) and antibodies directed against cyclic citrullinated peptides (anti‐CCP antibodies). We undertook this study to make the first comparison of SE allele frequencies in the healthy population with those in RA patients who do or do not harbor anti‐CCP antibodies.

Methods

HLA–DRB1 typing was performed in 408 RA patients from the Leiden Early Arthritis Clinic (the Leiden EAC; a Dutch population‐based inception cohort in which disease course was followed up over time), in 423 healthy Dutch controls, and in 720 affected members of 341 US multiplex (sibpair) families of Caucasian origin from the North American RA Consortium (NARAC) with well‐established disease and fulfilling the American College of Rheumatology classification criteria for RA. The presence of anti‐CCP antibodies was determined by enzyme‐linked immunosorbent assay.

Results

For the Leiden EAC, the odds ratio (OR) describing the association of 2 copies of the SE allele with anti‐CCP positivity (using no copies of the SE allele in the healthy control group as the referent) was 11.79 (P < 0.0001), while the OR for 1 SE allele was 4.37 (P < 0.0001). No association with the SE was observed in the Dutch anti‐CCP–negative RA patients. For the NARAC families, linkage and association analysis revealed the SE to be associated only with anti‐CCP–positive disease and not with anti‐CCP–negative disease. Stratified analyses indicated that anti‐CCP antibodies primarily mediated association of the SE with joint damage or disease persistence.

Conclusion

HLA–DRB1 alleles encoding the SE are specific for disease characterized by antibodies to citrullinated peptides, indicating that these alleles do not associate with RA as such, but rather with a particular phenotype.

     

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

Objective

The effect of non–shared epitope HLA–DRB1 alleles on rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate the effects of several HLA–DRB1 alleles, independent of the shared epitope, on the risk of developing anti–citrullinated protein antibody (ACPA)–positive or ACPA‐negative RA in a large case–control study.

Methods

HLA typing for the DRB1 gene was performed in 1,352 patients with RA and 922 controls from the Swedish Epidemiological Investigation of Rheumatoid Arthritis study. Relative risks (RRs) and 95% confidence intervals (95% CIs) were calculated.

Results

DRB1*13 was found to protect against ACPA‐positive RA when stratifying for the shared epitope and using a dominant genetic model (RR 0.41 [95% CI 0.26–0.64]). Furthermore, DRB1*13 neutralized the effect of the shared epitope in ACPA‐positive RA (RR 3.91 [95% CI 3.04–5.02] in patients who had the shared epitope but not DRB1*13, and RR 1.22 [95% CI 0.81–1.83] in patients with both the shared epitope and DRB1*13, as compared with patients negative for both the shared epitope and DRB1*13). However, we did not replicate the previous published risk of ACPA‐negative RA conferred by DRB1*03 when a dominant genetic model was used (RR 1.29 [95% CI 0.91–1.82]). Similarly, no significant effect of DRB1*03 on RR for ACPA‐negative RA was seen using the recessive genetic model (RR 1.18 [95% CI 0.6–2.4]). In contrast, the combination of DRB1*03 and DRB1*13 was significantly associated with increased risk of developing ACPA‐negative RA (RR 2.07 [95% CI 1.17–3.67]).

Conclusion

Our findings indicate that the DRB1*13 allele plays a dual role in the development of RA, by protecting against ACPA‐positive RA but, in combination with DRB1*03, increasing the risk of ACPA‐negative RA.

     

Association of smoking with the constitution of the anti–cyclic citrullinated peptide response in the absence of HLA–DRB1 shared epitope alleles

Objective

Smoking is a risk factor for anti–cyclic citrullinated peptide (anti‐CCP) antibody–positive rheumatoid arthritis (RA) in patients with HLA–DRB1 shared epitope (SE) alleles. It is unknown whether smoking influences not only the presence of these antibodies, but also other characteristics of the anti‐CCP response, such as isotype usage. The aim of this study was to determine the influence of smoking on anti‐CCP isotypes in RA patients, and to determine whether this influence is observed in the presence and/or absence of SE alleles.

Methods

IgA, IgM, and IgG subclasses of anti‐CCP antibodies were measured by enzyme‐linked immunosorbent assay in serum obtained at the first visit to the Leiden Early Arthritis Clinic from 216 patients with anti‐CCP–positive RA whose smoking habits were also assessed. HLA genotyping data were available for 202 of these patients.

Results

IgA and IgM anti‐CCP were more frequent in RA patients who were smokers than in those who were nonsmokers (odds ratio 2.8 and 1.8, respectively). In addition, levels of all isotypes of anti‐CCP, except IgG3, were significantly higher (P < 0.05) in smokers. The number of anti‐CCP isotypes was higher in smokers compared with nonsmokers, both in SE‐negative RA (P = 0.04) and in SE‐positive RA (P = 0.07).

Conclusion

Patients with anti‐CCP–positive RA who have a current or former tobacco exposure display a more extensive anti‐CCP isotype usage in general, and IgA and IgM in particular, compared with patients with anti‐CCP–positive RA who have never smoked. In contrast to its influence on the incidence of anti‐CCP positivity, the influence of tobacco exposure on the constitution of the anti‐CCP response is significant in SE‐negative RA. These findings suggest a differential effect of tobacco exposure on the induction as compared with the propagation of the anti‐CCP antibody response.

     

Strong combined gene–environment effects in anti–cyclic citrullinated peptide–positive rheumatoid arthritis: A nationwide case–control study in Denmark

Objective

To study the role of shared epitope (SE) susceptibility genes, alone and in combination with tobacco smoking and other environmental risk factors, for risk of subtypes of rheumatoid arthritis (RA) defined by the presence or absence of serum antibodies against cyclic citrullinated peptides (CCPs).

Methods

To address these issues, a nationwide case–control study was conducted in Denmark during 2002–2004, comprising incident cases of RA or patients with recently diagnosed RA (309 seropositive and 136 seronegative for IgG antibodies against CCP) and 533 sex‐ and age‐matched population controls. Associations were evaluated by logistic regression analyses, in which odds ratios (ORs) served as measures of relative risk.

Results

Compared with individuals without SE susceptibility genes, SE homozygotes had an elevated risk of anti‐CCP–positive RA (OR 17.8, 95% confidence interval [95% CI] 10.8–29.4) but not anti‐CCP–negative RA (OR 1.07, 95% CI 0.53–2.18). Strong combined gene–environment effects were observed, with markedly increased risks of anti‐CCP–positive RA in SE homozygotes who were heavy smokers (OR 52.6, 95% CI 18.0–154), heavy coffee drinkers (OR 53.3, 95% CI 15.5–183), or oral contraceptive users (OR 44.6, 95% CI 15.2–131) compared with SE noncarriers who were not exposed to these environmental risk factors.

Conclusion

Persons who are homozygous for SE susceptibility genes, notably those who are also exposed to environmental risk factors, have a markedly and selectively increased risk of anti‐CCP–positive RA. A distinction between anti‐CCP–positive RA and anti‐CCP–negative RA seems warranted, because these RA subtypes most likely represent etiologically distinct disease entities.

     

The HLA–DRB1 shared epitope is associated with susceptibility to rheumatoid arthritis in African Americans through European genetic admixture

Objective

To determine whether shared epitope (SE)–containing HLA–DRB1 alleles are associated with rheumatoid arthritis (RA) in African Americans and whether their presence is associated with higher degrees of global (genome‐wide) genetic admixture from the European population.

Methods

In this multicenter cohort study, African Americans with early RA and matched control subjects were analyzed. In addition to measurement of serum anti–cyclic citrullinated peptide (anti‐CCP) antibodies and HLA–DRB1 genotyping, a panel of >1,200 ancestry‐informative markers was analyzed in patients with RA and control subjects, to estimate the proportion of European ancestry.

Results

The frequency of SE‐containing HLA–DRB1 alleles was 25.2% in African American patients with RA versus 13.6% in control subjects (P = 0.00005). Of 321 patients with RA, 42.1% had at least 1 SE‐containing allele, compared with 25.3% of 166 control subjects (P = 0.0004). The mean estimated percent European ancestry was associated with SE‐containing HLA–DRB1 alleles in African Americans, regardless of disease status (RA or control). As reported in RA patients of European ancestry, there was a significant association of the SE with the presence of the anti‐CCP antibody: 86 (48.9%) of 176 patients with anti‐CCP antibody–positive RA had at least 1 SE allele, compared with 36 (32.7%) of 110 patients with anti‐CCP antibody–negative RA (P = 0.01, by chi‐square test).

Conclusion

HLA–DRB1 alleles containing the SE are strongly associated with susceptibility to RA in African Americans. The absolute contribution is less than that reported in RA among populations of European ancestry, in which ∼50–70% of patients have at least 1 SE allele. As in Europeans with RA, the SE association was strongest in the subset of African American patients with anti‐CCP antibodies. The finding of a higher degree of European ancestry among African Americans with SE alleles suggests that a genetic risk factor for RA was introduced into the African American population through admixture, thus making these individuals more susceptible to subsequent environmental or unknown factors that trigger the disease.

     

Particular association of clinical and genetic features with autoimmunity to citrullinated α‐enolase in rheumatoid arthritis

Objective

To confirm that the presence of anti–citrullinated α‐enolase peptide 1 (anti–CEP‐1) antibodies identifies a subgroup of patients with rheumatoid arthritis (RA).

Methods

DNA and serum samples were obtained from 451 patients with RA and 279 healthy control subjects, all of whom were of Spanish ancestry. Antibodies to cyclic citrullinated peptide (CCP) and CEP‐1 were measured by enzyme‐linked immunosorbent assay. HLA–DRB1 and the R620W single‐nucleotide polymorphism of PTPN22 were genotyped.

Results

Anti–CEP‐1 and anti‐CCP antibodies were observed in 26.8% and 71.2% of the patients with RA, respectively. Most of the patients (86.6%) with anti–CEP‐1 antibodies also had anti‐CCP antibodies. Erosive arthritis, rheumatoid factor (RF) positivity, and the presence of the HLA shared epitope (especially the DRB1*04 alleles) were disproportionately associated with the group of patients with both antibodies. In addition, evidence of a significant interaction between the shared epitope and the risk allele of PTPN22 was observed only in these patients. In contrast, the association with these clinical and genetic features was weaker in patients with anti‐CCP antibodies but lacking anti–CEP‐1 antibodies. These results were obtained in patients in whom the prevalence of RA risk factors differed from that in other previously studied patients.

Conclusion

We observed that autoimmunity against citrullinated α‐enolase may identify a subset of patients with a higher frequency of joint erosions and RF positivity. In addition, we confirmed the disproportionately large effect of the susceptibility alleles of HLA–DRB1 and their interaction with PTPN22 in this subset of patients. These results extend, confirm, and generalize the evidence supporting the specificity of the anti–CEP‐1 antibody–positive subgroup of patients with RA among anti‐CCP antibody–positive patients with RA.

     

Quantitative heritability of anti–citrullinated protein antibody–positive and anti–citrullinated protein antibody–negative rheumatoid arthritis

Objective

The majority of genetic risk factors for rheumatoid arthritis (RA) are associated with anti–citrullinated protein antibody (ACPA)–positive RA, while far fewer genetic risk factors have been identified for ACPA‐negative RA. This study was undertaken to quantify the contribution of genetic risk factors in general, and of the predisposing HLA–DRB1 shared epitope (SE) alleles in particular, to the ACPA‐positive and ACPA‐negative subsets of RA, by computing their heritability and assessing the contribution of the HLA SE alleles.

Methods

One hundred forty‐eight RA twin pairs, in which at least 1 twin of each pair had RA, were tested for ACPAs and typed for HLA–DRB1 genotypes. Heritability was assessed in a logistic regression model including a bivariate, normally distributed random effect, representing the contribution of unobserved genetic factors to RA susceptibility, with the correlation of the random effects fixed according to twin zygosity. The contribution of the HLA SE alleles to genetic variance was assessed using a similar model, except that estimates were based on genotype‐specific population prevalences.

Results

The heritability of RA among the twin pairs was 66% (95% confidence interval [95% CI] 44–75%). For ACPA‐positive RA, the heritability was 68% (95% CI 55–79%), and for ACPA‐negative RA it was 66% (95% CI 21–82%). Presence of the HLA SE alleles explained 18% (95% CI 16–19%) of the genetic variance of ACPA‐positive RA but only 2.4% (95% CI 1.6–10%) of the genetic variance of ACPA‐negative RA.

Conclusion

The heritability of ACPA‐positive RA is comparable with that of ACPA‐negative RA. These data indicate that genetic predisposition plays an important role in the pathogenesis of ACPA‐negative RA, for which most individual genetic risk factors remain to be identified.

     

Differences in synovial tissue infiltrates between anti–cyclic citrullinated peptide–positive rheumatoid arthritis and anti–cyclic citrullinated peptide–negative rheumatoid arthritis

Objective

To compare synovial tissue infiltrates from patients with anti–cyclic citrullinated peptide (anti‐CCP)–positive rheumatoid arthritis (RA) with those from patients with anti‐CCP–negative RA.

Methods

Synovial tissue samples were obtained arthroscopically from the inflamed knee joints of 57 patients with RA (34 of whom were anti‐CCP positive) and examined for several histologic features along with immunohistologic expression of cell markers. Joint damage was assessed using the Kellgren/Lawrence (K/L) scale (range 0–4) on standard anteroposterior radiographs. In 31 patients (18 of whom were anti‐CCP positive), synovial tissue was available from an earlier time point, allowing analysis of temporal changes.

Results

Synovial tissue from anti‐CCP–positive patients was characterized by a higher mean number of infiltrating lymphocytes (61.6 versus 31.4/high‐power field [hpf] [400×]; P = 0.01), less extensive fibrosis (mean score of 1.2 versus 2.0; P = 0.04), and a thinner synovial lining layer (mean score of 2.1 versus 3.3; P = 0.002) compared with synovial tissue from anti‐CCP–negative patients. Anti‐CCP–positive patients expressed more CD3, CD8, CD45RO, and CXCL12. More anti‐CCP–positive patients had a K/L score >1 compared with anti‐CCP–negative patients. The difference in the mean lymphocyte counts was already present a mean of 3.8 years before the index biopsy (76.7 lymphocytes/hpf and 26.7 lymphocytes/hpf in anti‐CCP–positive patients and anti‐CCP–negative patients, respectively; P = 0.008) and was independent of disease duration and K/L score.

Conclusion

Synovitis in patients with anti‐CCP–positive RA differs from that in patients with anti‐CCP– negative RA, notably with respect to infiltrating lymphocytes, and is associated with a higher rate of local joint destruction.

     

Association of anti–citrullinated vimentin and anti–citrullinated α‐enolase antibodies with subsets of rheumatoid arthritis

Objective

To determine whether the anti–citrullinated vimentin peptide 60–75 (anti–Cit‐vimentin) and the immunodominant anti–citrullinated α‐enolase peptide 1 (anti–CEP‐1) antibodies are associated with subsets of patients with rheumatoid arthritis (RA) independently of the associations between anti–cyclic citrullinated peptide (anti‐CCP) antibodies and clinical features of RA.

Methods

The 3 antibody types were quantified by enzyme‐linked immunosorbent assay (ELISA) in serum samples from 521 patients with RA and 173 healthy controls of Spanish ancestry. Genotypes for HLA–DRB1 alleles and rs2476601 in PTPN22 were available for these patients and controls plus an additional 106 healthy controls. A combined analysis of the 3 antibodies was conducted using stratified contingency tables and logistic regression models.

Results

A differential, particularly strong, and independent association was observed between the presence of anti–Cit‐vimentin antibodies and the presence of shared epitope (SE) alleles, specifically in patients carrying 2 SE alleles, and between the presence of anti– Cit‐vimentin antibodies and the prevalence of joint erosion. Associations were observed between anti–CEP‐1 positivity and the presence of HLA–DRB1 and PTPN22 risk alleles and their additive interaction. These associations were not accounted for by the anti‐CCP status.

Conclusion

Our results indicate that the 2 antibodies against citrullinated peptides analyzed in this study add specific information beyond that obtained with the anti‐CCP status. They define subgroups of patients with RA in which genetic factors have different weight and there is an observed difference in the prevalence of erosions.

     

Brief Report: Anti–Carbamylated Protein Antibodies Are Present in Arthralgia Patients and Predict the Development of Rheumatoid Arthritis

Objective

Recently, we discovered a new autoantibody system in rheumatoid arthritis (RA): anti–carbamylated protein (anti‐CarP) antibodies. These antibodies have value in predicting joint destruction; however, it is not clear whether they are present before the diagnosis of RA and whether they have value as predictors of RA development. Therefore, we studied whether anti‐CarP antibodies are present in patients with arthralgia and whether their presence is associated with the development of RA.

Methods

Sera from 340 arthralgia patients who did not have clinical signs of arthritis but who were positive for IgM rheumatoid factor (IgM‐RF) and/or anti–cyclic citrullinated peptide 2 (anti–CCP‐2) and 32 healthy controls were tested for anti‐CarP IgG antibodies. Of the patients with arthralgia, 111 were IgM‐RF positive/anti–CCP‐2 antibody negative and 229 were anti–CCP‐2 antibody positive. Patients were observed for the development of RA (based on the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria) during a median followup period of 36 months. Cox proportional hazards regression analysis was performed to compare the risk of developing RA between arthralgia patients who were positive for anti‐CarP antibodies and those who were negative for anti‐CarP antibodies during followup.

Results

Anti‐CarP antibodies were present in the sera of 39% of the patients. One hundred twenty patients developed RA, after a median of 12 months (interquartile range [IQR] 6–24). The presence of anti‐CarP antibodies was associated with the development of RA in the entire arthralgia cohort after correction for RF and anti–CCP‐2 antibody status (hazard ratio 1.56 [95% confidence interval 1.06–2.29], P = 0.023), as well as in the anti–CCP‐2 antibody–positive subgroup (odds ratio 2.231 [95% confidence interval 1.31–3.79], P = 0.003).

Conclusion

Anti‐CarP antibodies are present in patients with arthralgia, and their presence predicts the development of RA independent of anti–CCP‐2 antibodies.

     

Fine specificity of the anti–citrullinated protein antibody response is influenced by the shared epitope alleles

Objective

In classic studies on the genetic background of antibody production, the major histocompatibility complex (MHC) has been shown to act as the most prominent immune response gene that controls the magnitude and the specificity of antibody production. The strongest genetic risk factor for rheumatoid arthritis (RA), the human MHC HLA–DRB1 shared epitope (SE) alleles, predisposes for antibodies against citrullinated proteins (ACPAs). ACPA levels are higher in SE‐positive patients with RA than in SE‐negative patients with RA. The aim of the present study was to determine whether SE influences not only the magnitude but also the specificity of the ACPA response.

Methods

In 2 cohorts of anti–citrullinated peptide 2–positive patients with RA, one from a study of recent‐onset arthritis (n = 206) and the other from a treatment strategy study (n = 141), serum antibodies against a citrullinated peptide derived from vimentin (cVim) and antibodies against a citrullinated fibrinogen peptide (cFibr) were determined by enzyme‐linked immunosorbent assay. HLA–DRB1 genotyping was performed.

Results

In the first cohort, SE alleles were significantly associated with the presence of antibodies against cVim (odds ratio [OR] 4.95, 95% confidence interval [95% CI] 1.87–15.3) and were not significantly associated with the presence of antibodies against cFibr (OR 1.71, 95% CI 0.70–4.14). These results were replicated in the second cohort (OR 5.05, 95% CI 1.92–13.6 and OR 1.19, 95% CI 0.30–3.97, respectively).

Conclusion

In 2 cohorts of ACPA‐positive patients with RA, SE alleles predisposed for the development of antibodies against cVim but not for the development of antibodies against cFibr. These data indicate that SE alleles act as “classic” immune response genes in the ACPA response, because they influence both the magnitude and the specificity of this RA‐specific antibody response.

     

Antibodies against cyclic citrullinated peptide are associated with HLA–DR4 in simplex and multiplex polyarticular‐onset juvenile rheumatoid arthritis

Objective

Anti–cyclic citrullinated peptide (anti‐CCP) antibodies have been detected in patients with juvenile rheumatoid arthritis (JRA), particularly in those with polyarticular, rheumatoid factor (RF)‐positive JRA. Our objectives were to determine whether anti‐CCP antibodies are associated with HLA–DR4 in children with polyarticular JRA, whether anti‐CCP antibodies are associated with clinical features of disease, and whether affected sibling pairs (ASPs) with JRA are concordant for this antibody.

Methods

Stored serum samples obtained from 230 HLA‐typed patients with JRA (77 with polyarticular‐onset disease and 153 with pauciarticular‐ or systemic‐onset disease), 100 JRA ASPs, and 688 healthy children were tested for anti‐CCP antibodies and RF.

Results

Thirteen percent of the patients with polyarticular‐onset JRA and 2% of the other JRA patients exhibited anti‐CCP antibodies, compared with only 0.6% of the controls. Fifty‐seven percent of RF‐positive patients with polyarticular‐onset JRA had anti‐CCP antibodies. HLA–DR4–positive patients with polyarticular‐onset JRA were more likely to have anti‐CCP antibodies than were those without HLA–DR4 alleles (odds ratio [OR] 5.20, 95% confidence interval [95% CI] 1.30–20.9). Anti‐CCP antibodies were associated with polyarticular onset (OR 7.46, 95% CI 1.99–28.0), a polyarticular disease course (OR 9.78, 95% CI 1.25–76.7), and erosive disease (OR 14.3, 95% CI 3.01–67.9). Concordance rates for anti‐CCP antibodies among ASPs were statistically significant.

Conclusion

These data demonstrate increased anti‐CCP antibody formation in HLA–DR4–positive patients with polyarticular‐onset JRA. The overall prevalence of anti‐CCP antibodies in JRA is low, but a substantial proportion of RF‐positive patients with polyarticular‐onset JRA have these antibodies. Anti‐CCP antibodies in JRA are associated with polyarticular onset, a polyarticular course, and erosive disease.

     

Specific association of type 1 diabetes mellitus with anti–cyclic citrullinated peptide–positive rheumatoid arthritis

Objective

The co‐occurrence of autoimmune diseases such as rheumatoid arthritis (RA) and type 1 diabetes mellitus (DM) has been reported in individuals and families. In this study, the strength and nature of this association were investigated at the population level in a Swedish case–control cohort.

Methods

For this case–control study, 1,419 patients with incident RA diagnosed between 1996 and 2003 were recruited from university, public, and private rheumatology units throughout Sweden; 1,674 matched control subjects were recruited from the Swedish national population registry. Sera from the subjects were tested for the presence of antibodies to cyclic citrullinated peptide (anti‐CCP), rheumatoid factor (RF), and the 620W PTPN22 allele. Information on a history of diabetes was obtained by questionnaire, telephone interview, and/or medical record review. The prevalence of type 1 DM and type 2 DM was compared between patients with incident RA and control subjects and further stratified for the presence of anti‐CCP, RF, and the PTPN22 risk allele.

Results

Type 1 DM was associated with an increased risk of RA (odds ratio [OR] 4.9, 95% confidence interval [95% CI] 1.8–13.1), and this association was specific for anti‐CCP–positive RA (OR 7.3, 95% CI 2.7–20.0), but not anti‐CCP–negative RA. Further adjustment for the presence of PTPN22 attenuated the risk of anti‐CCP–positive RA in patients with type 1 DM to an OR of 5.3 (95% CI 1.5–18.7). No association between RA and type 2 DM was observed.

Conclusion

The association between type 1 DM and RA is specific for a particular RA subset, anti‐CCP–positive RA. The risk of developing RA later in life in patients with type 1 DM may be attributed, in part, to the presence of the 620W PTPN22 allele, suggesting that this risk factor may represent a common pathway for the pathogenesis of these 2 diseases.

     

Influence of HLA–DR genes on the production of rheumatoid arthritis–specific autoantibodies to citrullinated fibrinogen

Objective

Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA‐associated HLA–DR alleles are associated with anti–citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA–DR molecules and their recognition by T cells.

Methods

Antikeratin, antifilaggrin, and anti–citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA–DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire α‐ and β‐chains of fibrinogen) to purified HLA–DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls.

Results

HLA–DRB1*0404 was associated with anti–citrullinated fibrinogen in RA sera (P = 0.002). For the RA‐associated alleles HLA–DRB1*0401 and HLA–DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the α‐ and β‐chains of fibrinogen bound many HLA–DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA–DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls.

Conclusion

The RA‐associated HLA–DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti–citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide–DR–T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.

     

Brief Report: Association of HLA–DRB1*0101/*0405 with susceptibility to anti–melanoma differentiation–associated gene 5 antibody–positive dermatomyositis in the Japanese population

Objective

The complication of interstitial lung disease (ILD) in polymyositis/dermatomyositis (PM/DM) is associated with anti–aminoacyl–transfer RNA synthetase (anti‐aaRS) antibody or anti–melanoma differentiation–associated gene 5 (anti–MDA‐5) antibody positivity. Anti–MDA‐5 antibody is associated with clinically amyopathic DM and fatal outcome due to rapidly progressive ILD in Asian populations. The association between genetic factors and anti–MDA‐5 antibody–positive DM is unclear. This study was undertaken to investigate the HLA–DRB1 genotype in patients with anti–MDA‐5 antibody–positive DM.

Methods

We examined genetic differences among 17 patients with anti–MDA‐5 antibody–positive DM, 33 patients with anti‐aaRS antibody–positive PM/DM, 33 patients with PM/DM without anti‐aaRS antibody or ILD, and 265 healthy controls.

Results

The frequencies of HLA–DRB1*0101 and DRB1*0405 were 29% and 71%, respectively, in patients with anti–MDA‐5 antibody–positive DM, which were higher than the frequencies in healthy controls (10% and 25%, respectively). Among the 17 patients with anti–MDA‐5 antibody–positive DM, 16 (94%) harbored either the DRB1*0101 or DRB1*0405 allele. The combined frequency of the DRB1*0101 allele and the DRB1*0405 allele was significantly higher in patients with anti–MDA‐5 antibody–positive DM than in patients with PM/DM without anti‐aaRS antibody or ILD, with an odds ratio (OR) of 42.7 (95% confidence interval [95% CI] 4.9–370.2) (P = 1.1 × 10⁻⁵), or in patients with anti‐aaRS antibody–positive PM/DM (OR 13.3 [95% CI 1.6–112.6], P = 4.5 × 10⁻³).

Conclusion

Our findings indicate that HLA–DRB1*0101/*0405 is associated with susceptibility to anti–MDA‐5 antibody–positive DM in the Japanese population.