Analysis of single‐nucleotide polymorphisms in Japanese rheumatoid arthritis patients shows additional susceptibility markers besides the classic shared epitope susceptibility sequences

Objective

To examine the entire HLA region for loci (other than the DRB1 locus) associated with rheumatoid arthritis (RA) susceptibility, by typing HLA–DRB1 alleles and multiple single‐nucleotide polymorphisms (SNPs) in the Japanese population.

Methods

The HLA–DRB1 alleles and 88 SNPs distributed over the HLA gene complex were genotyped, for 828 patients with RA and 1,032 control subjects. The data were evaluated for linkage disequilibrium, and case–control associations were analyzed in 2 ways, in the presence or absence of the disease‐susceptibility DRB1 allele, to detect loci independent of the DRB1 allele.

Results

HLA–DRB1 alleles *0405, *0401, *0901, *0101, *1401, *1602, *0403, and *1405 were significantly associated with RA in the Japanese population. The smallest P value (P = 1.4 × 10⁻²⁷) was observed in association with an intronic SNP of the NOTCH4 gene, which was due to strong linkage disequilibrium with the HLA–DRB1 allele. A strong association that was independent of HLA–DRB1 shared epitope alleles was observed in 2 SNPs: one in the intron of the MICA gene, the other in the intron of the HLA–DQB2 gene. Their association with RA, independent of HLA–DRB1 shared epitope alleles, was suggestive (P = 0.0024 [corrected P (P_corr) = 0.068, and P = 0.00037 [P_corr = 0.012], respectively).

Conclusion

These findings suggest that 1 or more other loci besides the HLA–DRB1 or other DRB1 (non–shared epitope, non‐*0901) alleles are involved in RA susceptibility/protection.

     

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.

     

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.

     

Influence of shared epitope–negative HLA–DRB1 alleles on genetic susceptibility to rheumatoid arthritis

Objective

Most patients with rheumatoid arthritis (RA) express the shared epitope (SE). It is not known whether SE‐negative HLA–DRB1 alleles influence the development of RA. This study examined the influence of SE‐negative HLA–DR alleles (DRB1*X) on the development of RA in 3 different French populations.

Methods

HLA–DRB1 alleles were defined by polymerase chain reaction with sequence‐specific oligonucleotide hybridization or sequence‐specific primers. SE‐negative alleles were classified according to the electric charge of their P4 pocket. HLA–DRB1 alleles *0103, *0402, *07, *08, *11 (except *1107), *12, and *13 have a neutral or negative P4 charge and are called DRB1*XP4n. HLA–DRB1*03, *0403, *0406, *0407, *0901, *1107, *14, *15, and *16 have a positive P4 charge and are called DRB1*XP4p.

Results

Among the SE‐negative subjects, DRB1 genotypes with 1 or 2 DRB1*XP4n alleles were significantly overrepresented in the control subjects compared with the RA patients, whereas DRB1*XP4p/XP4p genotypes were equally represented in the patients and controls. In single‐dose SE–positive subjects, SE/XP4n genotypes were equally represented in the patients and controls. However, SE/XP4p genotypes were significantly overrepresented in the RA patients.

Conclusion

The DRB1*X allele polymorphism influences susceptibility to RA. Alleles that have a neutral or negative electric charge in their P4 pocket (DRB1*XP4n), such as DRB1*0103, *0402, *07, *08, *11 (except *1107), *12, and *13, protect against RA. Alleles that have a positive electric charge in their P4 pocket (DRB1*XP4p), such as DRB1*03, *0403, *0406, *0407, *0901, *1107, *14, *15, and *16, have no influence on the predisposition to RA.

     

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.

     

Particular HLA–DRB1 shared epitope genotypes are strongly associated with rheumatoid vasculitis

Objective

To examine the relationship of the HLA–DRB1 shared epitope (SE) to rheumatoid vasculitis, using individual patient data (IPD) meta‐analytic methods.

Methods

Published studies that enrolled adult patients with rheumatoid arthritis (RA) were identified by searches of Medline and Embase, and by manual searches of medical journals. All authors were contacted for IPD. Meta‐analyses were performed to assess the association of SE presence, dose, and genotype with rheumatoid vasculitis.

Results

A total of 14 studies and 1,568 patients (129 with vasculitis) were included in the analysis. RA patients with vasculitis were significantly more likely to have rheumatoid nodules (odds ratio [OR] 2.5, 95% confidence interval [95% CI] 1.5–3.9], but there was no significant association with male sex, rheumatoid factor positivity, or erosive disease. No significant association was observed between the presence of the SE (i.e., 1 or 2 alleles versus 0 alleles) and rheumatoid vasculitis (summary OR 1.4, 95% CI 0.7–2.7). Analysis by SE genotype, however, demonstrated a striking relationship of vasculitis to 3 genotypes containing a double dose of the SE, specifically HLA–DRB1*0401/*0401 (OR 6.2, 95% CI 1.01–37.9), *0401/*0404 (OR 4.1, 95% CI 1.1–16.2), and *0101/*0401 (OR 4.0, 95% CI 1.4–11.6).

Conclusion

The HLA–DRB1 SE genotypes *0401/*0401, *0401/*0404, and *0101/*0401 may be of particular importance to rheumatoid vasculitis. It is hoped that additional investigation of these and other SE genotypes will lead to improved insight into the mechanisms influencing the clinical expression of RA.

     

Interaction between heat‐shock protein 73 and HLA–DRB1 alleles associated or not with rheumatoid arthritis

Objective

HLA–DRB1 alleles whose third hypervariable region contains a QKRAA/QRRAA/RRRAA motif are associated with rheumatoid arthritis (RA) through unknown mechanisms. We previously demonstrated that the QKRAA motif was also expressed on the Escherichia coli 40‐kd heat‐shock protein (HSP) DnaJ. The QKRAA motif helps DnaJ bind its partner chaperone, the E coli 70‐kd HSP DnaK. Furthermore, we observed that in lymphoblastoid cells, Hsp73, the constitutive 70‐kd HSP, associates with HLA–DRB1*0401 (an allele with a QKRAA motif) and targets it to lysosomes. In this study, we sought to classify different HLA–DRB1 alleles according to their ability to bind Hsp73.

Methods

To evaluate how well different HLA–DRB1 alleles could bind Hsp73, we developed a quantitative precipitation assay and a direct binding assay.

Results

Quantitative precipitation assay from total cellular proteins and from lysosomal extracts demonstrated that RA‐associated HLA–DRB1 alleles bound Hsp73 better than did HLA–DRB1 alleles that were not associated with RA. HLA–DRB1*0401 was the best Hsp73 binder. These findings were confirmed by direct binding assay between purified proteins.

Conclusion

HLA–DRB1*0401 was the best Hsp73 binder among the 8 different HLA–DRB1 alleles that were tested.

     

Transfer of the shared epitope through microchimerism in women with rheumatoid arthritis

Objective

Rheumatoid arthritis (RA) is an autoimmune disease that affects mostly women and is associated with HLA–DRB1 genes having in common a shared epitope sequence. In parallel, cells and/or DNA originating from pregnancy (microchimerism) persist for decades and could contribute to autoimmunity. The aim of this study was to examine whether microchimerism may be a source of the shared epitope among women with RA.

Methods

Women with RA and healthy women who lacked RA‐associated genes such as HLA–DRB1*01 (n = 33 and n = 46, respectively) and/or HLA–DRB1*04 (n = 48 and n = 64, respectively), were tested for DRB1*01 or DRB1*04 microchimerism by HLA‐specific quantitative polymerase chain reaction assays. As controls, alleles not associated with RA (DQB1*02 and DRB1*15/16) were also analyzed.

Results

Compared with healthy women, women (42% with RA had a higher frequency and higher levels of DRB1*04 microchimerism versus 8%; P = 0.00002) as well as DRB1*01 microchimerism (30% versus 4%; P = 0.0015). Moreover, no difference in microchimerism was observed for alleles not associated with RA.

Conclusion

Women with RA had microchimerism with RA‐associated HLA alleles, but not with non–RA‐associated HLA alleles, more often and at higher levels compared with healthy women. These observations are the first to indicate that microchimerism can contribute to the risk of an autoimmune disease by providing HLA susceptibility alleles.

     

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.

     

Reshaping the shared epitope hypothesis: HLA‐associated risk for rheumatoid arthritis is encoded by amino acid substitutions at positions 67–74 of the HLA–DRB1 molecule

Objective

To further analyze the association of HLA–DRB1 alleles with disease susceptibility in recent‐onset rheumatoid arthritis (RA).

Methods

One hundred sixty‐seven Caucasian RA patients and 166 healthy controls were typed for HLA–DRB1.

Results

The association of susceptibility to RA with the group of alleles encoding the shared epitope susceptibility sequences (SESSs) was confirmed in recent‐onset RA. Among non‐SESS alleles, DRB1*07, *1201, *1301, and *1501 showed significant protective effects. Even after correction for the influence of SESS alleles, significant independent protective effects of DRB1 alleles were observed. Protective alleles shared a third hypervariable region motif. Independent homozygosity effects were observed both for susceptibility and for protective alleles.

Conclusion

Nonsusceptibility alleles differ significantly with regard to RA risk. Protective alleles show clear homology at positions 67–74, often encoding isoleucine at position 67 or aspartic acid at position 70. Susceptibility and protective alleles both show homozygosity effects. Based on these results and on data reported in the literature, in order to incorporate the finding of differential risks among nonsusceptibility alleles, we propose to reshape the shared epitope hypothesis as follows: HLA‐associated risk for RA is encoded by amino acid substitutions at positions 67–74 of the HLA–DRB1 molecule.

     

The effect of HLA–DR on susceptibility to rheumatoid arthritis is influenced by the associated lymphotoxin α–tumor necrosis factor haplotype

Objective

HLA–DRB1, a major genetic determinant of susceptibility to rheumatoid arthritis (RA), is located within 1,000 kb of the gene encoding tumor necrosis factor (TNF). Because certain HLA–DRB1*04 subtypes increase susceptibility to RA, investigation of the role of the TNF gene is complicated by linkage disequilibrium (LD) between TNF and DRB1 alleles. By adequately controlling for this LD, we aimed to investigate the presence of additional major histocompatibility complex (MHC) susceptibility genes.

Methods

We identified 274 HLA–DRB1*04–positive cases of RA and 271 HLA–DRB1*04–positive population controls. Each subject was typed for 6 single‐nucleotide polymorphisms within a 4.5‐kb region encompassing TNF and lymphotoxin α (LTA). LTA–TNF haplotypes in these unrelated individuals were determined using a combination of family data and the PHASE software program.

Results

Significant differences in LTA–TNF haplotype frequencies were observed between different subtypes of HLA–DRB1*04. The LTA–TNF haplotypes observed were very restricted, with only 4 haplotypes constituting 81% of all haplotypes present. Among individuals carrying DRB1*0401, the LTA–TNF 2 haplotype was significantly underrepresented in cases compared with controls (odds ratio 0.5 [95% confidence interval 0.3–0.8], P = 0.007), while in those with DRB1*0404, the opposite effect was observed (P = 0.007).

Conclusion

These findings suggest that the MHC contains genetic elements outside the LTA–TNF region that modify the effect of HLA–DRB1 on susceptibility to RA.

     

Contribution of a haplotype in the HLA region to anti–cyclic citrullinated peptide antibody positivity in rheumatoid arthritis,independently of HLA–DRB1

Objective

To examine the risk of anti–cyclic citrullinated peptide (anti‐CCP) antibody positivity in rheumatoid arthritis (RA) patients carrying certain haplotypes in the HLA region.

Methods

A total of 1,389 Japanese patients with RA were genotyped for 30 single‐nucleotide polymorphisms (SNPs) in the HLA region using commercial oligonucleotide arrays (from Perlegen or Affymetrix) as well as for HLA–DRB1 alleles using a sequence‐specific polymerase chain reaction method. Stepwise logistic regression was used to select from among the 30 SNPs the ones that represented a risk of anti‐CCP antibody positivity. Haplotypes of the selected SNPs were inferred using an expectation‐maximization algorithm. Associations of individual SNPs were evaluated with the Cochran‐Armitage test for trend. DRB1 alleles and haplotypes were evaluated with the chi‐square test. Heterogeneities of risks among the shared epitope (SE) and non‐SE HLA–DRB1 alleles were examined using the exact test. Haplotype associations that were independent of individual HLA–DRB1 alleles were evaluated using the likelihood ratio test.

Results

Significant associations were found for 9 SNPs (smallest P value being 2.4 × 10⁻⁸) and in 4 HLA–DRB1 alleles (smallest P value being 2.0 × 10⁻¹⁰ in DRB1*0405). Stepwise logistic regression selected 4 SNPs (rs9262638, rs7775228, rs4713580, and rs9277359). Among the 16 inferred haplotypes of these 4 SNPs, 6 indicated significant associations (smallest P value being 1.9 × 10⁻¹¹). Risks among SE and non‐SE alleles were significantly heterogeneous (P = 0.0095 and P = 9.8 × 10⁻⁹, respectively), indicating the importance of stratification with individual DRB1 alleles rather than SE alleles. Conditional analysis of the risk associated with individual DRB1 alleles identified a risk haplotype that was independent of DRB1 (odds ratio 2.00 [95% confidence interval 1.44–2.79], P = 2.6 × 10⁻⁵).

Conclusion

Heterogeneous risks of anti‐CCP antibody positivity were confirmed among SE and non‐SE alleles in our patient population. A risk haplotype in the HLA region that is independent of HLA–DRB1 was confirmed.

     

Increased frequency of DRB1*11:01 in anti–hydroxymethylglutaryl‐coenzyme A reductase–associated autoimmune myopathy

Objective

To investigate the association of anti–hydroxymethylglutaryl‐coenzyme A reductase (anti‐HMGCR) myopathy with HLA class I and II antigens.

Methods

HLA antigens were determined in 1) 20 white and 8 African American anti‐HMGCR patients, 2) 487 white and 167 African American controls, and 3) 51 white subjects with mild self‐limited statin intolerance.

Results

White anti‐HMGCR patients had a higher frequency of the combination HLA–DR11, DQA5, and DQB7 than controls or statin‐intolerant subjects (70% versus 17%; odds ratio [OR] 11.7 [95% confidence interval (95% CI) 4.0–35.3], P = 4.1 × 10^?7 and 70% versus 21%; OR 8.3 [95% CI 2.2–33.9], P = 5.4 × 10^?4, respectively). This combination was not increased in African American anti‐HMGCR subjects compared to controls (13% versus 3%; OR 4.6 [95% CI 0.2–53.3], P = 0.2). However, DR11 was increased in African American anti‐HMGCR patients compared to controls (88% versus 21%; OR 26.4 [95% CI 3.1–590.3], P = 0.0002). High‐resolution mapping showed that 95% with DR11 had DRB1*11:01. DQA1 and DQB6 were less frequent in white anti‐HMGCR–positive patients compared to controls (25% versus 65%; OR 0.2 [95% CI 0.1–0.5], P = 5.5 × 10^?4 and 0% versus 45%; OR 0.0 [95% CI 0.0–0.3], P = 2.1 × 10^?5, respectively). DRB11 was not associated with particular disease features.

Conclusion

DRB1*11:01 is associated with an increased risk of anti‐HMGCR myopathy in whites and African Americans. These findings suggest a mechanistic link between statin exposure, increased HMGCR expression, and the possible presentation of HMGCR‐derived peptide(s) by DRB1*11:01.

     

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.

     

The influence of genetic variation in the HLA–DRB1 and LTA–TNF regions on the response to treatment of early rheumatoid arthritis with methotrexate or etanercept

Objective

To examine the roles of specific genetic polymorphisms as predictors of response to treatment of early rheumatoid arthritis (RA).

Methods

Subjects included 457 patients with early RA (duration of ≤3 years) who participated in a randomized controlled trial comparing weekly methotrexate and 2 dosages of etanercept (10 mg twice weekly and 25 mg twice weekly). Our primary outcome measure was achievement of 50% improvement in disease activity according to the criteria of the American College of Rheumatology (ACR50 response) after 12 months of treatment. Subjects were genotyped for HLA–DRB1 alleles and polymorphisms in the following genes: TNF, LTA, TNFRSF1A, TNFRSF1B, FCGR2A, FCGR3A, and FCGR3B. Univariate and multivariate analyses were performed to define the impact of specific polymorphisms and haplotypes on response to treatment. Covariates for the multivariate analyses included sex, ethnicity, age, disease duration, and baseline values for rheumatoid factor and the tender and swollen joint counts.

Results

The presence of 2 HLA–DRB1 alleles encoding the shared epitope (SE) (compared with 1 or 0 copies) was associated with response to treatment with standard‐dose etanercept (odds ratio [OR] 4.3, 95% confidence interval [95% CI] 1.8–10.3). Among Caucasian patients, 2 extended haplotypes that included the HLA–DRB1 alleles *0404 and *0101 (both of which encode the SE) and 6 single‐nucleotide polymorphisms in the LTA–TNF region were associated with response to treatment. In a multivariate model that included treatment received and the aforementioned covariates, the ORs for the association of these haplotypes with achievement of an ACR50 response at 12 months were 2.5 (95% CI 0.8–7.3) and 4.9 (95% CI 1.5–16.1) for the *0404‐ and *0101‐containing haplotypes, respectively.

Conclusion

Genetic variation in the HLA–DRB1 and the LTA–TNF regions is significantly associated with response to treatment of early RA. These findings may have clinical application through the identification of patients who are most likely to benefit from treatment with methotrexate or etanercept.

     

HLA–DRB4 as a genetic risk factor for Churg‐Strauss syndrome

Objective

To explore the association between HLA alleles and Churg‐Strauss syndrome (CSS), and to investigate the potential influence of HLA alleles on the clinical spectrum of the disease.

Methods

Low‐resolution genotyping of HLA–A, HLA–B, and HLA–DR loci and genotyping of TNFA −238A/G and TNFA −308A/G single‐nucleotide polymorphisms were performed in 48 consecutive CSS patients and 350 healthy controls.

Results

The frequency of the HLA–DRB1*07 allele was higher in the CSS patients than in controls (27.1% versus 13.3%; χ² = 12.64, P = 0.0003, corrected P [P_corr] = 0.0042, odds ratio [OR] 2.42, 95% confidence interval [95% CI] 1.47–3.99). The HLA–DRB4 gene, present in subjects carrying either HLA–DRB1*04, HLA–DRB1*07, or HLA–DRB1*09 alleles, was also far more frequent in patients than in controls (38.5% versus 20.1%; χ² = 16.46, P = 0.000058, P_corr = 0.000232, OR 2.49, 95% CI 1.58–3.09). Conversely, the frequency of the HLA–DRB3 gene was lower in patients than in controls (35.4% versus 50.4%; χ² = 7.62, P = 0.0057, P_corr = 0.0228, OR 0.54, 95% CI 0.35–0.84). CSS has 2 major clinical subsets, antineutrophil cytoplasmic antibody (ANCA)–positive, with features of small‐vessel vasculitis, and ANCA‐negative, in which organ damage is mainly mediated by tissue eosinophilic infiltration; analysis of HLA–DRB4 in patients categorized by different numbers of vasculitic manifestations (purpura, alveolar hemorrhage, mononeuritis multiplex, rapidly progressive glomerulonephritis, and constitutional symptoms) showed that its frequency strongly correlated with the number of vasculitis symptoms (P for trend = 0.001).

Conclusion

These findings indicate that HLA–DRB4 is a genetic risk factor for the development of CSS and increases the likelihood of development of vasculitic manifestations of the disease.

     

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.

     

Genetic association of the major histocompatibility complex with rheumatoid arthritis implicates two non‐DRB1 loci

Objective

The HLA–DRB1 locus within the major histocompatibility complex (MHC) at 6p21.3 has been identified as a susceptibility gene for rheumatoid arthritis (RA); however, there is increasing evidence of additional susceptibility genes in the MHC region. The aim of this study was to estimate their number and location.

Methods

A case–control study was performed involving 977 control subjects and 855 RA patients. The HLA–DRB1 locus was genotyped together with 2,360 single‐nucleotide polymorphisms in the MHC region. Logistic regression was used to detect DRB1‐independent effects.

Results

After adjusting for the effect of HLA–DRB1, 18 markers in 14 genes were strongly associated with RA (P < 10⁻⁴). Multivariate logistic regression analysis of these markers and DRB1 led to a model containing DRB1 plus the following 3 markers: rs4678, a nonsynonymous change in the VARS2L locus, ∼1.7 Mb telomeric of DRB1; rs2442728, upstream of HLA–B, ∼1.2 Mb telomeric of DRB1; and rs17499655, located in the 5′‐untranslated region of DQA2, only 0.1 Mb centromeric of DRB1. In‐depth investigation of the DQA2 association, however, suggested that it arose through cryptic linkage disequilibrium with an allele of DRB1. Two non–shared epitope alleles were also strongly associated with RA (P < 10⁻⁴): *0301 with anti– cyclic citrullinated peptide–negative RA and *0701 independently of autoantibody status.

Conclusion

These results confirm the polygenic contribution of the MHC to RA and implicate 2 additional non‐DRB1 susceptibility loci. The role of the HLA–DQ locus in RA has been a subject of controversy, but in our data, it appears to be spurious.

     

Lack of association of the HLA–DRB1 shared epitope with rheumatoid nodules: An individual patient data meta‐analysis of 3,272 Caucasian patients with rheumatoid arthritis

Objective

The objective of this individual patient data (IPD) meta‐analysis was to examine the relationship of rheumatoid nodules to the HLA–DRB1 shared epitope (SE) and to individual SE genotypes.

Methods

English‐language studies that enrolled adult non‐Hispanic Caucasian patients with rheumatoid arthritis (RA) were identified by searches of Medline and Embase, and by manual searches of medical journals. All authors were contacted for IPD. Metaanalysis was performed to assess the association of SE presence, dose, and genotype with rheumatoid nodules. Meta‐analyses adjusted for disease duration and cumulative meta‐analyses were also performed to assess the influence of RA duration and year of study publication on the results.

Results

A total of 24 studies and 3,272 patients were available for analysis. IPD were obtained for 22 of the studies. There was a nonsignificant association between the presence of the SE (i.e., 1 or 2 alleles versus 0 alleles) and rheumatoid nodules (summary odds ratio [OR] 1.3, 95% confidence interval [95% CI] 0.97–1.6). Analysis by SE genotype, however, demonstrated a weak relationship with inheritance of a single DRB1*0401 SE allele (OR 1.4, 95% CI 1.1–1.8). No other genotypes achieved statistical significance in the adjusted or unadjusted analyses.

Conclusion

The presence of the HLA–DRB1 SE does not appear to significantly increase the risk of rheumatoid nodules among Caucasian patients with RA. Analysis by DRB1 SE genotype was uninformative, suggesting only a potential (and at most modest) role of the DRB1*0401 SE allele. Results from this IPD meta‐analysis implicate other genetic, stochastic, and/or environmental factors in the susceptibility to rheumatoid nodules.

     

HLA–DRB1*0407 and *1304 are risk factors for scleroderma renal crisis

Objective

To examine the predictive role of HLA genetic markers in scleroderma renal crisis (SRC), beyond the known clinical correlates, in a large population of patients with systemic sclerosis (SSc).

Methods

SSc patients from the Scleroderma Family Registry and DNA Repository, the Genetics versus Environment in Scleroderma Outcomes Study, and the rheumatology division registry at the University of Texas Health Science Center at Houston were included in the study. Relevant clinical data were obtained by chart review, and autoantibodies were detected utilizing commercially available kits. HLA class II genotyping was performed on extracted and purified genomic DNA.

Results

Overall, 1,519 SSc patients were included in the study, of whom 90 (6%) had developed SRC. Among the 90 patients with SRC, the diffuse cutaneous disease subtype was found in 76%, antitopoisomerase antibodies (antitopo) in 9%, anticentromere antibodies (ACAs) in 2%, and anti–RNA polymerase III (anti–RNAP III) in 50% of patients. In multivariate analyses of clinical and demographic parameters, diffuse disease type and anti–RNAP III were strong risk factors for the presence of SRC, whereas ACAs and antitopo were protective. In the final multivariate analysis, which included HLA alleles, HLA–DRB1*0407 (odds ratio [OR] 3.21, 95% confidence interval [95% CI] 1.27–8.08; P = 0.013) and DRB1*1304 (OR 4.51, 95% CI 1.30–15.65; P = 0.018) were identified as independent risk factors for SRC. Only 3 clinical characteristics, diffuse disease type, anti–RNAP III, and ACAs, remained significantly associated with SRC in the final model.

Conclusion

The results of this study suggest that DRB1*0407 and *1304 are independent risk factors, beyond the known clinical correlates, for the development of SRC.