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.

     

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.

     

Different patterns of associations with anti–citrullinated protein antibody–positive and anti–citrullinated protein antibody–negative rheumatoid arthritis in the extended major histocompatibility complex region

Objective

To identify additional variants in the major histocompatibility complex (MHC) region that independently contribute to risk in 2 disease subsets of rheumatoid arthritis (RA) defined according to the presence or absence of antibodies to citrullinated protein antigens (ACPAs).

Methods

In a multistep analytical strategy using unmatched as well as matched analyses to adjust for HLA–DRB1 genotype, we analyzed 2,221 single‐nucleotide polymorphisms (SNPs) spanning 10.7 Mb, from 6p22.2 to 6p21.31, across the MHC. For ACPA‐positive RA, we analyzed samples from the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) and the North American Rheumatoid Arthritis Consortium (NARAC) studies (totaling 1,255 cases and 1,719 controls). For ACPA‐negative RA, we used samples from the EIRA study (640 cases and 670 controls). Plink and SAS statistical packages were used to conduct all statistical analyses.

Results

A total of 299 SNPs reached locus‐wide significance (P < 2.3 × 10⁻⁵) for ACPA‐positive RA, whereas surprisingly, no SNPs reached this significance for ACPA‐negative RA. For ACPA‐positive RA, we adjusted for known DRB1 risk alleles and identified additional independent associations with SNPs near HLA–DPB1 (rs3117213; odds ratio 1.42 [95% confidence interval 1.17–1.73], P_combined = 0.0003 for the strongest association).

Conclusion

There are distinct genetic patterns of MHC associations in the 2 disease subsets of RA defined according to ACPA status. HLA–DPB1 is an independent risk locus for ACPA‐positive RA. We did not identify any associations with SNPs within the MHC for ACPA‐negative RA.

     

Association of HLA–DR3 with anti–cyclic citrullinated peptide antibody–negative rheumatoid arthritis

Objective

Recent data have shown that the most prominent and longest known genetic risk factors for rheumatoid arthritis (RA), HLA–DRB1 shared epitope alleles, are only associated with RA that is characterized by the presence of antibodies against cyclic citrullinated peptide (anti‐CCP antibodies) and not with anti‐CCP–negative RA. We undertook this study to investigate whether anti‐CCP–negative RA is associated with other HLA–DRB1 alleles.

Methods

HLA typing was performed for 377 patients from the Leiden Early Arthritis Clinic who were diagnosed as having RA within the first year of followup (206 anti‐CCP–positive patients and 171 anti‐CCP–negative patients), 235 patients who, after 1 year, had undifferentiated arthritis (UA) (28 anti‐CCP–positive patients and 207 anti‐CCP–negative patients), and 423 healthy control subjects. Odds ratios (ORs) with 95% confidence intervals (95% CIs) for HLA–DRB1 allele frequencies were determined for all patient groups compared with the healthy control group.

Results

HLA–DR3 was more frequently present in the anti‐CCP–negative RA group than in the control group (OR 1.84, 95% CI 1.26–2.67). This was not the case for anti‐CCP–positive RA (OR 0.92, 95% CI 0.60–1.40). HLA–DR3 was also more frequently present in anti‐CCP–negative UA patients (OR 1.59, 95% CI 1.10–2.28), but not in anti‐CCP–positive UA patients (OR 0.68, 95% CI 0.17–1.92).

Conclusion

HLA–DR3 is associated with anti‐CCP–negative arthritis and not with anti‐CCP–positive arthritis. These data show that distinct genetic risk factors are associated with the presence of anti‐CCP antibodies in RA and indicate that different pathogenetic mechanisms underlie anti‐CCP–positive and anti‐CCP–negative RA.

     

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.

     

Occupation and Risk of Developing Rheumatoid Arthritis: Results From a Population‐Based Case–Control Study

Objective

Environmental factors are of importance for the etiology of rheumatoid arthritis (RA), but much remains unknown concerning the contributions from distinct occupational hazards. We explored the association between occupation and the risk of anti–citrullinated protein antibody (ACPA)+ RA or ACPA? RA.

Methods

We analyzed 3,522 cases and 5,580 controls from the Swedish population–based Epidemiological Investigation of Rheumatoid Arthritis case–control study. A questionnaire was used to obtain information on work history and lifestyle factors. Blood samples were drawn for serologic analyses. Unconditional logistic regression was used to calculate the odds ratio (OR) of RA associated with the last occupation before study inclusion. Analyses were performed with adjustments for known environmental exposures and lifestyle factors, including pack‐years of cigarette smoking, alcohol use, body mass index, and education.

Results

Among men, bricklayers and concrete workers (OR 2.9, 95% confidence interval [95% CI] 1.4–5.7), material handling operators (OR 2.4, 95% CI 1.3–4.4), and electrical and electronics workers (OR 2.1, 95% CI 1.1–3.8) had an increased risk of ACPA+ RA. For ACPA? RA, bricklayers and concrete workers (OR 2.4, 95% CI 1.0–5.7) and electrical and electronics workers (OR 2.6, 95% CI 1.3–5.0) had an increased risk. Among women, assistant nurses and attendants had a moderately increased risk of ACPA+ RA (OR 1.3, 95% CI 1.1–1.6). No occupations were significantly associated with ACPA? RA among women.

Conclusion

Mainly occupations related to potential noxious airborne agents were associated with an increased risk of ACPA+ or ACPA? RA, after adjustments for previously known confounders.

     

Gene–environment interaction between the DRB1 shared epitope and smoking in the risk of anti–citrullinated protein antibody–positive rheumatoid arthritis: All alleles are important

Objective

An interaction effect for developing rheumatoid arthritis (RA) was previously observed between HLA–DRB1 shared epitope (SE) alleles and smoking. We aimed to further investigate this interaction between distinct SE alleles and smoking regarding the risk of developing RA with and without anti–citrullinated protein antibodies (ACPAs).

Methods

We used data regarding smoking habits and HLA–DRB1 genotypes from 1,319 patients and 943 controls from the Epidemiological Investigation of Rheumatoid Arthritis, in which 972 patients and 488 controls were SE positive. Subsequently, 759 patients and 328 controls were subtyped for specific alleles within the DRB1*04 group. Odds ratios with 95% confidence intervals (95% CIs) were calculated by means of logistic regression. Interaction was evaluated by calculating attributable proportion due to interaction, with 95% CIs.

Results

A strong interaction between smoking and SE alleles in the development of ACPA‐positive RA was observed for all DRB1*04 SE alleles taken as a group (relative risk [RR] 8.7 [95% CI 5.7–13.1]) and for the *0401 and *0404 alleles (RR 8.9 [95% CI 5.8–13.5]) and the *01 and *10 alleles (RR 4.9 [95% CI 3.0–7.8]) as specific, separate groups, with similar strength of interaction for the different groups (attributable proportion due to interaction 0.4 [95% CI 0.2–0.6], 0.5 [95% CI 0.3–0.7], and 0.6 [95% CI 0.4–0.8], respectively).

Conclusion

There is a statistically significant interaction between distinct DRB1 SE alleles and smoking in the development of ACPA‐positive RA. Interaction occurs with the *04 group as well as the *01/*10 group, demonstrating that regardless of fine specificity, all SE alleles strongly interact with smoking in conferring an increased risk of ACPA‐positive RA.

     

Alterations of the synovial T cell repertoire in anti–citrullinated protein antibody–positive rheumatoid arthritis

Objective

The association of HLA–DRB1 alleles with anti–citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) suggests the potential involvement of T lymphocytes in ACPA‐seropositive disease. The purpose of this study was to investigate this hypothesis by systematic histologic and molecular analyses of synovial T cells in ACPA+ versus ACPA– RA patients.

Methods

Synovial biopsy samples were obtained from 158 RA patients. Inflammation was determined histologically and immunohistochemically. RNA was extracted from peripheral blood mononuclear cells and synovial tissues obtained from 11 ACPA+ RA patients, 7 ACPA– RA patients, and 10 spondylarthritis (SpA) patients (arthritis controls). T lymphocyte clonality was studied by combined quantitative and qualitative T cell receptor CDR3 length distribution (LD) analysis and direct sequencing analysis.

Results

ACPA+ and ACPA– RA patients were similar at both the clinical and histologic levels. At the molecular level, however, patients with ACPA+ synovitis displayed a marked elevation of qualitative CDR3 LD alterations as compared with those with ACPA– synovitis and with the SpA controls. These differences in CDR3 LD were not observed in the peripheral blood, indicating a selective recruitment and/or local expansion of T cells in the synovial compartment. The CDR3 LD alterations reflected true monoclonal or oligoclonal expansions, as confirmed by direct sequencing of the T cell receptor. The CDR3 LD alterations in RA synovium did not correlate with B cell clonal expansions but were inversely associated with synovial lymphoid neogenesis.

Conclusion

The T cell repertoire is specifically restricted in RA patients with ACPA+ synovitis. Whereas the origin and role of these clonal alterations remain to be determined, our data suggest the preferential involvement of T lymphocytes in ACPA‐seropositive RA.

     

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.

     

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.

     

Body mass index,obesity, and prevalent gout in the United States in 1988–1994 and 2007–2010

Objective

To determine the association and prevalence of gout among overweight, obese, and morbidly obese segments of the US population.

Methods

Among participants (age ≥20 years) of the National Health and Nutrition Examination Surveys in 1988–1994 and 2007–2010, gout status was ascertained by self‐report of a physician diagnosis. Body mass index (BMI) was examined in categories of <18.5 kg/m², 18.5–24.9 kg/m², 25–29.9 kg/m², 30–34.9 kg/m², and ≥35 kg/m² and as a continuous variable. The cross‐sectional association of BMI category with gout status was adjusted for demographic and obesity‐related medical disorders.

Results

In the US, the crude prevalence of gout was 1–2% among participants with a normal BMI (18.5–24.9 kg/m²), 3% among overweight participants, 4–5% with class I obesity, and 5–7% with class II or class III obesity. The adjusted prevalence ratio comparing the highest to a normal BMI category was 2.46 (95% confidence interval [95% CI] 1.44–4.21) in 1988–1994 and 2.21 (95% CI 1.50–3.26) in 2007–2010. Notably, there was a progressively greater prevalence ratio of gout associated with successively higher categories of BMI. In both survey periods, for an average American adult standing 1.76 meters (5 feet 9 inches), a 1‐unit higher BMI, corresponding to 3.1 kg (～6.8 pounds) greater weight, was associated with a 5% greater prevalence of gout, even after adjusting for serum uric acid (P < 0.001).

Conclusion

Health care providers should be aware of the elevated burden of gout among both overweight and obese adults, applicable to both women and men, and observed among non‐Hispanic whites, non‐Hispanic African Americans, and Mexican Americans in the US.     

Association between anti–cyclic citrullinated peptide antibodies and ischemic heart disease in patients with rheumatoid arthritis

Objective

Patients with rheumatoid arthritis (RA) have an increased risk of cardiovascular disease that may not always be related to the presence of traditional cardiovascular risk factors. The aim of this study was to determine if anti–cyclic citrullinated peptide (anti‐CCP) antibodies are associated with cardiovascular disease in patients with RA.

Methods

Anti‐CCP antibodies were determined by enzyme‐linked immunosorbent assay in the earliest serum sample available from 937 patients with a diagnosis of RA. We studied the relationship between anti‐CCP antibodies with traditional cardiovascular risk factors and cardiovascular events.

Results

We found positive anti‐CCP antibodies (>25 units/ml) in 672 patients (71.7%). There was no association between the anti‐CCP antibodies and cardiovascular risk factors such as smoking, hypertension, dyslipidemia, being overweight, or diabetes mellitus. However, patients who had positive anti‐CCP antibodies experienced more frequent ischemic heart disease (6.5% versus 2.6%; odds ratio [OR] 2.58, 95% confidence interval [95% CI] 1.17–5.65) and had higher mortality rates (11.2% versus 6.8%; OR 1.72, 95% CI 1.01–2.91). Similar results were obtained when we considered anti‐CCP titers 20‐fold higher (>500 units/ml). Multivariable analysis showed that ischemic heart disease is independently associated with positive anti‐CCP antibodies (OR 2.8, 95% CI 1.19–6.56; P = 0.009).

Conclusion

Anti‐CCP antibodies in patients with RA are independently associated with the development of ischemic heart disease.     

Association of the PTPN22 R620W polymorphism with anti–topoisomerase I– and anticentromere antibody–positive systemic sclerosis

Objective

To determine any associations of the PTPN22 R620W single‐nucleotide polymorphism (SNP) with systemic sclerosis (SSc) or with anticentromere antibody (ACA)–positive or anti–topoisomerase I (anti–topo I) antibody–positive SSc, in a case–control study of US white, black, Hispanic, and Choctaw Indian individuals.

Methods

A total of 850 white, 130 black, 120 Hispanic, and 20 Choctaw Indian patients with SSc were compared with 430 white, 164 black, 146 Hispanic, and 76 Choctaw Indian control subjects, respectively. All subjects were living in the US. PTPN22 SNP (rs2476601) genotyping was performed by TaqMan 5′ allelic discrimination assay and pyrosequencing.

Results

The PTPN22 CT/TT genotype showed significant association with anti–topo I antibody–positive SSc in white patients (odds ratio [OR] 2.21, 95% confidence interval [95% CI] 1.3–3.7) and with ACA‐positive white patients with SSc (OR 1.70, 95% CI 1.1–2.7). Frequency of the PTPN22*T allele also showed significant association with anti–topo I antibody–positive SSc in white patients (OR 2.03, 95% CI 1.3–3.2). When data for patients in the 3 ethnic groups (black, white, and Hispanic) were combined, a significant association with both genotype and allele frequencies was observed, suggesting a trend toward association in ACA‐positive and anti–topo I antibody–positive SSc. Stepwise logistic regression analysis (controlled for the confounding effects of sex and race) showed that the PTPN22 CT/TT genotype was associated with a significantly higher risk of SSc compared with the CC genotype (for patients with SSc, OR 1.64, 95% CI 1.2–2.2; for ACA‐positive patients with SSc, OR 1.63, 95% CI 1.0–2.6; for anti–topo I antibody–positive SSc, OR 2.33, 95% CI 1.5–3.7).

Conclusion

Our results indicate that the PTPN22 R620W polymorphism is associated with ACA‐positive and anti–topo I antibody–positive subsets of SSc and represents a risk factor in both white patients and black patients. The association of subsets of SSc with the PTPN22 R620W polymorphism further strengthens the classification of SSc within the spectrum of autoimmune diseases and strongly suggests the involvement of common susceptibility genes and similarly disordered immunoregulatory pathways.

     

Development of the anti–citrullinated protein antibody repertoire prior to the onset of rheumatoid arthritis

Objective

To examine how anti–citrullinated protein antibody (ACPA) epitope spreading takes place prior to the onset of clinical rheumatoid arthritis (RA), and to analyze the pattern of autoantigen reactivity at the beginning of the immune response.

Methods

Multiple consecutive serum samples from 79 RA patients who had donated blood before disease onset were available for analysis. Fifty‐three patients tested positive for ACPAs prior to the onset of clinical RA. For these patients, a median of 6 (interquartile range 4–9) sequential pre‐RA serum samples obtained 1–2 years apart were tested. Reactivity to 5 distinct citrullinated peptides was measured by enzyme‐linked immunosorbent assay. Two peptides were derived from fibrinogen, 1 from vimentin, 1 from α‐enolase, and 1 from filaggrin.

Results

In 25 of 53 ACPA‐positive patients, seroconversion from ACPA absence to ACPA presence was observed. In 72% of these patients, the immune response started with reactivity to 1 peptide, without preference for a particular peptide. The number of peptides recognized increased over time, without a dominant epitope‐spreading pattern. ACPAs appeared in low levels several years prior to the diagnosis of RA. Antibody titers increased markedly ∼2–4 years before diagnosis.

Conclusion

Our findings indicate that ACPA epitope spreading occurs over several years prior to the onset of clinical RA. The initial autoimmune response is mostly directed toward only 1 autoantigen, but this is not always the same antigen. The marked increase in ACPA titers a few years prior to the diagnosis of RA suggests a second stage in disease development, which might be due to a variety of factors.

     

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.

     

An unexpectedly high frequency of MEFV mutations in patients with anti–citrullinated protein antibody–negative palindromic rheumatism

Objective

To investigate whether the MEFV gene, which is involved in the regulation of the inflammatory response and has been associated with familial Mediterranean fever (FMF) and intermittent hydrarthrosis, is implicated in the pathogenesis of palindromic rheumatism (PR) and to examine its clinical presentation and its evolution in a Spanish cohort of PR patients.

Methods

Family histories, demographic clinical data, and laboratory characteristics of 75 patients diagnosed as having PR were collected from medical records and personal interviews. The healthy control group included 325 blood bank donors. The FMF control group was made up of 84 Spanish FMF patients. Genomic DNA was isolated, and MEFV gene mutation analysis was performed by polymerase chain reaction amplification and sequence analysis.

Results

Sixty‐five unrelated PR patients were finally included in the study. MEFV gene mutation analysis identified 8 of these 65 patients (12.3%) as carriers of at least 1 mutated MEFV allele. Patients with MEFV mutations had higher mean age and age at disease onset, but lower mean serum levels of anti–citrullinated protein antibodies (ACPAs). No other significant differences were observed between patients with and those without mutations. The frequency of MEFV mutations in ACPA‐negative PR patients was 22.2%, compared with 5.3% in ACPA‐positive PR patients (P = 0.058).

Conclusion

This study shows a previously unreported high prevalence of mutations of the MEFV gene in patients with ACPA‐negative PR. This supports the hypothesis that it might be a susceptibility gene. Our findings also support the hypothesis that the MEFV gene might participate in the pathogenesis of other undifferentiated relapsing inflammatory rheumatic disorders.

     

Effect of body mass index on mortality and clinical status in rheumatoid arthritis

Objective

To study the relative risk (RR) of all‐cause and cause‐specific mortality in rheumatoid arthritis (RA) associated with body mass index (BMI), and to quantify the clinical and outcome consequences of abnormal BMI.

Methods

We studied mortality in 24,535 patients over 12.3 years, dividing patients into 3 age groups, <50, 50–70, and >70 years and fit Cox regression models separately within each age stratum. We used BMI categories of <18.5 kg/m² (underweight), 18.5 to <25 kg/m² (normal weight, reference category), 25 to <30 kg/m² (overweight), and ≥30 kg/m² (obesity).

Results

BMI ≥30 kg/m² was seen in 63–68% and underweight in ～2%. Reduction in the RR (95% confidence interval [95% CI]) for all‐cause (AC) and cardiovascular mortality was seen for overweight (AC 0.8 [95% CI 0.8, 0.9]) and obese groups (AC 0.8 [95% CI 0.7, 0.8]), with and without comorbidity adjustment. Underweight was associated with increased mortality risk (AC 1.9 [95% CI 1.7, 2.3]). By contrast, obesity produced profound changes in clinical variables. Compared with normal weight, the odds ratio in the obese group was 4.8 for diabetes mellitus, 3.4 for hypertension, 1.3 for myocardial infarction, 1.4 for joint replacement, and 1.9 for work disability. Total semiannual direct medical costs were $1,683 greater, annual household income $6,481 less, pain scores 1.1 units higher, Health Assessment Questionnaire 0.28 higher, and EuroQol utility 0.7 units lower in the obese.

Conclusion

Overweight and obesity reduce the RR of all‐cause and cardiovascular mortality across different age groups and durations of RA. By contrast, overweight and obesity are associated with substantial increased risks of comorbidity, total joint replacement, greater pain, medical costs, and decreased quality of life.     

Synovial intracellular citrullinated proteins colocalizing with peptidyl arginine deiminase as pathophysiologically relevant antigenic determinants of rheumatoid arthritis–specific humoral autoimmunity

Objective

To address the ongoing debate concerning the specificity of synovial citrullinated proteins for rheumatoid arthritis (RA) and to analyze their pathophysiologic relevance to the induction or perpetuation of the RA‐specific anti–citrullinated protein antibodies (ACPAs).

Methods

Synovium of 19 RA patients and 19 non‐RA controls was immunostained for the presence of citrullinated proteins with a mouse monoclonal antibody (F95), for the citrullinating enzyme peptidyl arginine deiminase type 2 (PAD‐2), and for the free citrulline–producing enzyme inducible nitric oxide synthase (iNOS). Extending the RA cohort to 61 patients, the findings of anticitrulline staining in synovium were related to serum and synovial fluid ACPA levels, as measured by enzyme‐linked immunosorbent assay.

Results

F95 staining indicated the presence of synovial intracellular citrullinated proteins in 53% of RA samples versus 5% of control samples, whereas extracellular staining was not RA specific. Immunoblotting and inhibition experiments confirmed that the antibody recognized citrullinated proteins but not free citrulline. Accordingly, iNOS was equally found in RA and control synovium and in intracellular citrullinated protein–positive and intracellular citrullinated protein–negative samples. In contrast, intracellular citrullinated proteins colocalized with PAD‐2, which was found in 59% of RA samples versus 17% of control samples. Independent of local disease activity, the presence of the RA‐specific synovial intracellular citrullinated proteins was associated with significantly higher systemic and local ACPA levels and with local ACPA production in the joint.

Conclusion

These data confirm the presence of RA‐specific intracellular citrullinated proteins in synovium. The link with PAD‐2 and local and systemic ACPA levels emphasizes their pathophysiologic relevance for RA‐specific humoral autoimmunity.

     

Risk factors and metabolic abnormality of patients with non-alcoholic fatty liver disease: Either non-obese or obese Chinese population

Background: Non-alcoholic fatty liver disease(NAFLD) occurs not only in obese individuals but also in non-obese ones. The aim of this study was to focus on the association between NAFLD and metabolic events in a non-obese or obese Chinese population.Methods: Data collected from subjects registered at Taichung Veterans General Hospital from January to December 2009 were analyzed. The exclusion criteria were alcoholics, chronic hepatitis B or C. Patients included in analyses were assigned to four groups according to sonography of their liver(normal or NAFLD), and body mass index(BMI) levels(non-obese if BMI 25 kg/m~2 or obese if BMI ≥ 25 kg/m~2).Results: There were 745, 208, 770 and 285 patients enrolled in four groups labeled non-obese normal liver(group A), non-obese NAFLD(group B), obese normal liver(group C) and obese NAFLD(group D),respectively. The highest ratio of metabolic syndrome existed in the group B(26.9%), followed by group A(11.7%), group D(10.9%) and finally the group C(5.2%). The positive association with NAFLD in non-obese individuals was significant in triglyceride(OR = 1.01; 95% CI: 1.01–1.02) and glucose(OR = 1.02; 95% CI:1.01–1.03), while the positive association with NAFLD in obese subjects was only significant in triglyceride(OR = 1.01; 95% CI: 1.01–1.02). The positive association was most significant in all cases(adjusted OR = 2.41; 95% CI: 1.78–3.24), especially in non-obese individuals(OR = 2.81; 95% CI: 1.92–4.12).Conclusions: Non-obese NAFLD subjects displayed a higher proportion of metabolic abnormality. Hyperlipidemia and hyperglycemia had the most positive strength association with NAFLD.     

Glycan profiling of anti–citrullinated protein antibodies isolated from human serum and synovial fluid

Objective

Anti–citrullinated protein antibodies (ACPA) exhibit unique specificity for rheumatoid arthritis. However, it is incompletely understood whether and how ACPA contribute to disease pathogenesis. The Fc part of human IgG carries 2 N‐linked glycan moieties that are crucial for the structural stability of the antibody and that modulate both its binding affinity to Fcγ receptors and its ability to activate complement. We undertook this study to analyze Fc glycosylation of IgG1 ACPA in serum and synovial fluid (SF) in order to further characterize the immune response to citrullinated antigens.

Methods

ACPA were isolated by affinity purification using cyclic citrullinated peptides as antigen. IgG1 Fc glycosylation was analyzed by mass spectrometry. ACPA IgG1 glycan profiles were compared with glycan profiles of total serum IgG1 obtained from 85 well‐characterized patients. Glycan profiles of paired SF and serum samples were available from 11 additional patients.

Results

Compared with the pool of serum IgG1, ACPA IgG1 lacked terminal sialic acid residues. In SF, ACPA were highly agalactosylated and lacked sialic acid residues, a feature that was not detected for total SF IgG1. Moreover, differential ACPA glycan profiles were detected in rheumatoid factor (RF)–positive and RF‐negative patients.

Conclusion

ACPA IgG1 exhibit a specific Fc‐linked glycan profile that is distinct from that of total serum IgG1. Moreover, Fc glycosylation of ACPA differs markedly between SF and serum. Since Fc glycosylation directly affects the recruitment of Fc‐mediated effector mechanisms, these data could further our understanding of the contribution of ACPA to disease pathogenesis.