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Objectives: Chronic inflammation promotes cardiovascular risk in rheumatoid arthritis (RA). Biological disease-modifying antirheumatic drugs (bDMARDs) improve disease activity and cardiovascular disease outcomes. We explored whether bDMARDs influence the impact of disease activity and inflammatory markers on long-term cardiovascular risk in RA. Methods: We studied 4370 participants without cardiovascular disease in a 10-country observational cohort of patients with RA. Endpoints were (1) major adverse cardiovascular events (MACE) encompassing myocardial infarction, stroke and cardiovascular death; and (2) any ischaemic cardiovascular events (iCVE) including MACE plus revascularisation, angina, transient ischaemic attack and peripheral arterial disease. Results: Over 26 534 patient-years, 239 MACE and 362 iCVE occurred. The interaction between 28-joint Disease Activity Score with C-reactive protein (DAS28-CRP) and bDMARD use was significant for MACE (p=0.017), suggesting the effect of DAS28-CRP on MACE risk differed among bDMARD users (n=515) and non-users (n=3855). DAS28-CRP (per unit increase) is associated with MACE risk in bDMARD non-users (HR 1.21 (95% CI 1.07 to 1.37)) but not users (HR 0.69 (95% CI 0.40 to 1.20)). The interaction between CRP (per log unit increase) and bDMARD use was also significant for MACE (p=0.011). CRP associated with MACE risk in bDMARD non-users (HR 1.16 (95% CI 1.04 to 1.30)), but not users (HR 0.65 (95% CI 0.36 to 1.17)). No interaction was observed between bDMARD use and DAS28-CRP (p=0.167) or CRP (p=0.237) for iCVE risk. Conclusions: RA activity and inflammatory markers associated with risk of MACE in bDMARD non-users but not users suggesting the possibility of biological-specific benefits locally on arterial wall independently of effects on systemic inflammation.

Objective: The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are inflammatory disorders with ANCA autoantibodies recognising either proteinase 3 (PR3-AAV) or myeloperoxidase (MPO-AAV). PR3-AAV and MPO-AAV have been associated with distinct loci in the human leucocyte antigen (HLA) region. While the association between MPO-AAV and HLA has been well characterised in East Asian populations where MPO-AAV is more common, studies in populations of European descent are limited. The aim of this study was to thoroughly characterise associations to the HLA region in Scandinavian patients with PR3-AAV as well as MPO-AAV. Methods: Genotypes of single-nucleotide polymorphisms (SNPs) located in the HLA region were extracted from a targeted exome-sequencing dataset comprising Scandinavian AAV cases and controls. Classical HLA alleles were called using xHLA. After quality control, association analyses were performed of a joint SNP/classical HLA allele dataset for cases with PR3-AAV (n=411) and MPO-AAV (n=162) versus controls (n=1595). Disease-associated genetic variants were analysed for association with organ involvement, age at diagnosis and relapse, respectively. Results: PR3-AAV was significantly associated with both HLA-DPB1*04:01 and rs1042335 at the HLA-DPB1 locus, also after stepwise conditional analysis. MPO-AAV was significantly associated with HLA-DRB1*04:04. Neither carriage of HLA-DPB1*04:01 alleles in PR3-AAV nor of HLA-DRB1*04:04 alleles in MPO-AAV were associated with organ involvement, age at diagnosis or relapse. Conclusions: The association to the HLA region was distinct in Scandinavian cases with MPO-AAV compared with cases of East Asian descent. In PR3-AAV, the two separate signals of association to the HLD-DPB1 region mediate potentially different functional effects.

Objectives: To investigate the influence of genetic factors on persistence to treatment of early rheumatoid arthritis (RA) with methotrexate (MTX) monotherapy. Methods: We conducted a genome-wide association study (GWAS) in a sample of 3902 Swedish early RA patients initiating MTX in DMARD-monotherapy as their first ever DMARD. The outcome, short- and long-term persistence to this treatment, was defined as remaining on MTX at one and at three years, respectively, with no additional DMARDs added. As genetic predictors, we investigated individual SNPs, and a polygenic risk score (PRS) based on SNPs associated with RA risk. The SNP-based heritability of persistence was estimated overall and by RA serostatus. Results: No individual SNP reached genome-wide significance (p < 5e-8), neither for persistence at one nor at three years. The RA PRS was not significantly associated with persistence at one (RR = 0.98 (0.96-1.01)) nor three years (RR = 0.96 (0.93-1.00)). The heritability for persistence was estimated to be 0.45 (0.15-0.75) at one year and 0.14 (0-0.40) at three years. Results in seropositive RA were comparable to those in the analysis of RA overall, while heritability estimates and PRS RRs were attenuated towards the null in seronegative RA. Conclusions: Despite being the largest GWAS on an MTX treatment outcome to date, no genome-wide significant associations were detected. The modest heritability observed, coupled with the broad spread of suggestively associated loci, indicate that genetic influence is of polygenic nature. Nevertheless, persistence to MTX monotherapy was lower in patients with a greater genetic disposition, per the PRS, towards RA., Errata: Correction to: Genome-wide investigation of persistence with methotrexate treatment in early rheumatoid arthritis. Rheumatology, 2023; kead540. DOI: 10.1093/rheumatology/kead540

Objective Autoantibodies against the adrenal enzyme 21-hydroxylase is a hallmark manifestation in autoimmune Addison's disease (AAD). Steroid 21-hydroxylase is encoded by CYP21A2, which is located in the human leucocyte antigen (HLA) region together with the highly similar pseudogene CYP21A1P. A high level of copy number variation is seen for the 2 genes, and therefore, we asked whether genetic variation of the CYP21 genes is associated with AAD. Design Case-control study on patients with AAD and healthy controls. Methods Using next-generation DNA sequencing, we estimated the copy number of CYP21A2 and CYP21A1P, together with HLA alleles, in 479 Swedish patients with AAD and autoantibodies against 21-hydroxylase and in 1393 healthy controls. Results With 95% of individuals carrying 2 functional 21-hydroxylase genes, no difference in CYP21A2 copy number was found when comparing patients and controls. In contrast, we discovered a lower copy number of the pseudogene CYP21A1P among AAD patients (P = 5 x 10(-44)), together with associations of additional nucleotide variants, in the CYP21 region. However, the strongest association was found for HLA-DQB1*02:01 (P = 9 x 10(-63)), which, in combination with the DRB1*04:04-DQB1*03:02 haplotype, imposed the greatest risk of AAD. Conclusions We identified strong associations between copy number variants in the CYP21 region and risk of AAD, although these associations most likely are due to linkage disequilibrium with disease-associated HLA class II alleles.

The bile salt-stimulated lipase (BSSL) was originally recognized as a lipolytic enzyme expressed by the exocrine pancreas and in some species, notably humans, the lactating mammary gland, being secreted into the duodenum and with the mother’s milk, respectively. However, BSSL is also present in the blood and has been assigned additional functions, even beyond the gastrointestinal tract. Conventional BSSL knockout mice are protected from developing disease in animal models of arthritis, and antibodies directed towards BSSL prevent or mitigate disease in similar models. The aim of this study was to investigate the role of BSSL as a newly discovered player in inflammation and specifically in inflammatory joint disorders. As part of mechanism of action, we here show that BSSL is secreted by neutrophils, interacts with monocytes and stimulates their migration in vitro. An anti-BSSL antibody that blocks the human BSSL-monocyte interaction was shown to simultaneously prevent the signaling pathway by which BSSL induce cell migration. Moreover, in this cohort study we show that BSSL levels are significantly higher in blood samples from patients with rheumatoid arthritis and psoriatic arthritis compared to healthy controls. The BSSL levels in patients’ blood also correlated with disease activity scores and established inflammatory markers. Hence, although the mode of action is not yet fully clarified, we conclude that BSSL could be considered a proinflammatory component in the innate immune system and thus a possible novel target for treatment of chronic inflammation.

Hormonal and reproductive factors affect the risk for cardiovascular events (CVE) in the general population. Although the risk of CVE is increased in rheumatoid arthritis (RA), the knowledge about the impact of hormonal factors for CVE in RA is sparse. Female postmenopausal patients ≤80 years with early RA were consecutively included in this observational study (n = 803) between 1 January 1996 until 31 December 2017. Questionnaires regarding hormonal factors were distributed from the index date. Data regarding CVE were obtained from the Swedish National Health Register and Cause of Death Register. Associations between CVE and hormonal factors were analyzed using Cox proportional hazard regression. Of the postmenopausal women, 64 women had a CVE after RA onset. The time period from menopause to RA onset was significantly longer for CVE cases with higher proportion of postmenopausal women. In Cox proportional hazard regression models, years from last childbirth and multiparity were associated with higher CVE risk. Adjustments for traditional risk factors did not affect the results except for hypertension. RA onset after menopause and a longer duration from menopause until onset increased the CVE risk. Multiparity was associated with higher CVE risk whilst oral contraceptives decreased the risk. These results can contribute to identification of high-risk patients for CVE beyond traditional risk factors.

Objective: Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) is a chronic relapsing condition with unknown etiology. To gain insight into the molecular processes underlying the disease, we examined biomarkers in blood samples collected prior to symptom onset. Methods: The National Patient Register and Cause of Death register were searched for AAV-related International Classification of Diseases, Ninth Revision and Tenth Revision codes and linked to the registers from 5 biobanks. Eighty-five AAV patients with samples predating symptom onset of AAV were identified. For each case of AAV, 2 matched controls were included. Proteinase 3 (PR3)–ANCA and myeloperoxidase (MPO)–ANCA expression levels were analyzed using enzyme-linked immunosorbent assays. Using an Olink Inflammation panel, 73 of 92 proteins were included after quality control. Data were replicated in a second cohort of 48 presymptomatic individuals and 96 controls. Results: Of the 20 proteins with the lowest P values in the original cohort, 7 were replicated in the second cohort and 5 proteins were found to be significant between the groups in a meta-analysis. Eleven different pathways were identified in network enrichment analyses and were found to be significant in both cohorts. Stratification of samples obtained ≤5 years before symptom onset showed significant levels of CCL23, vascular endothelial growth factor A, and hepatocyte growth factor, which were also increased at borderline significant levels in the replication cohort (interleukin-6 was found to be significantly increased in the replication cohort). In presymptomatic AAV patients, 6 proteins were associated with MPO-ANCA positivity, and 7 proteins were associated with PR3-ANCA positivity. Conclusion: To our knowledge, this is the first study to identify protein markers preceding symptom onset in AAV patients. These findings set the stage for further research into the underlying cellular and molecular mechanisms in the pathogene

Objectives: To identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA). Methods: IgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining. Results: Peptide GPI293-307 was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI293-307 epitopes, and high affinity anti-GPI293-307 IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI293-307 IgG antibodies induced arthritis in mice. Moreover, anti-GPI293-307 IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI293-307-specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab-peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI293-307 antibodies. Conclusions: We have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293-307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA.

Objective: To identify and genetically characterize subgroups of patients with ANCA-associated vasculitides (AAV) based on sex and ANCA subtype. Methods: A previously established SNP dataset derived from DNA sequencing of 1853 genes and genotyping of 1088 Scandinavian cases with AAV and 1589 controls was stratified for sex and ANCA subtype and analysed for association with five top AAV SNPs. rs9274619, a lead variant at the HLA-DQB1/HLA-DQA2 locus previously associated with AAV positive for myeloperoxidase (MPO)-ANCA, was analysed for association with the cumulative disease involvement of ten different organ systems. Results: rs9274619 showed a significantly stronger association to MPO-ANCA-positive females than males [P = 2.0 x 10(-4), OR = 2.3 (95% CI 1.5, 3.5)], whereas proteinase 3 (PR3)-ANCA-associated variants rs1042335, rs9277341 (HLA-DPB1/A1) and rs28929474 (SERPINA1) were equally associated with females and males with PR3-ANCA. In MPO-ANCA-positive cases, carriers of the rs9274619 risk allele were more prone to disease engagement of eyes [P = 0.021, OR = 11 (95% CI 2.2, 205)] but less prone to pulmonary involvement [P = 0.026, OR = 0.52 (95% CI 0.30, 0.92)]. Moreover, AAV with both MPO-ANCA and PR3-ANCA was associated with the PR3-ANCA lead SNP rs1042335 [P = 0.0015, OR = 0.091 (95% CI 0.0022, 0.55)] but not with rs9274619. Conclusions: Females and males with MPO-ANCA-positive AAV differ in genetic predisposition to disease, suggesting at least partially distinct disease mechanisms between the sexes. Double ANCA-positive AAV cases are genetically similar to PR3-ANCA-positive cases, providing clues to the clinical follow-up and treatment of these patients.

Objective. B cell function and autoantibodies are important in SLE pathogenesis. In this work, we aimed to investigate the impact of cumulative SLE B cell genetics on SLE subphenotype and autoantibody profile. Methods: Female patients with SLE (n=1248) and healthy controls (n=400) were genotyped using Illumina's Global Screening Array. Two polygenic risk scores (PRSs), one representing B cell genes and the other B cell activation genes, were calculated for each individual using risk loci for SLE in genes assigned to B cell-related pathways according to the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Reactome Databases. Results: Double-stranded DNA (dsDNA) antibodies were more prevalent among patients with a high compared with a low SLE B cell PRS (OR 1.47 (1.07 to 2.01), p=0.018), and effect sizes were augmented in patients with human leucocyte antigen (HLA) risk haplotypes HLA-DRB1∗03:01 and HLA-DRB1∗15:01 (DRB1∗03/15-/-(OR 0.99 (0.56 to 1.77), p=0.98; DRB1∗03/15 +/-or-/+ (OR 1.64 (1.06 to 2.54), p=0.028; and DRB1∗03/15 +/+ (OR 4.47 (1.21 to 16.47), p=0.024). Further, a high compared with a low B cell PRS was associated with low complement levels in DRB1∗03/15 +/+ patients (OR 3.92 (1.22 to 12.64), p=0.022). The prevalence of lupus nephritis (LN) was higher in patients with a B cell activation PRS above the third quartile compared with patients below (OR 1.32 (1.00 to 1.74), p=0.048). Conclusions: High genetic burden related to B cell function is associated with dsDNA antibody development and LN. Assessing B cell PRSs may be important in order to determine immunological pathways influencing SLE and to predict clinical phenotype.

Objectives: To investigate the influence of genetic factors on persistence to treatment of early rheumatoid arthritis (RA) with methotrexate (MTX) monotherapy. Methods: We conducted a genome-wide association study (GWAS) in a sample of 3902 Swedish early RA patients initiating MTX in DMARD-monotherapy as their first ever DMARD. The outcome, short- and long-term persistence to this treatment, was defined as remaining on MTX at one and at three years, respectively, with no additional DMARDs added. As genetic predictors, we investigated individual SNPs, and a polygenic risk score (PRS) based on SNPs associated with RA risk. The SNP-based heritability of persistence was estimated overall and by RA serostatus. Results: No individual SNP reached genome-wide significance (p < 5e-8), neither for persistence at one nor at three years. The RA PRS was not significantly associated with persistence at one (RR = 0.98 (0.96-1.01)) nor three years (RR = 0.96 (0.93-1.00)). The heritability for persistence was estimated to be 0.45 (0.15-0.75) at one year and 0.14 (0-0.40) at three years. Results in seropositive RA were comparable to those in the analysis of RA overall, while heritability estimates and PRS RRs were attenuated towards the null in seronegative RA. Conclusions: Despite being the largest GWAS on an MTX treatment outcome to date, no genome-wide significant associations were detected. The modest heritability observed, coupled with the broad spread of suggestively associated loci, indicate that genetic influence is of polygenic nature. Nevertheless, persistence to MTX monotherapy was lower in patients with a greater genetic disposition, per the PRS, towards RA., Errata: Correction to: Genome-wide investigation of persistence with methotrexate treatment in early rheumatoid arthritis. Rheumatology, 2023; kead540. DOI: 10.1093/rheumatology/kead540

Objective To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). Methods Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. Results PR3-ANCA+ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 x 10-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 x 10-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 x 10-10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 x 10-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 x 10-7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA+ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. Conclusion We identified a novel susceptibility locus for MPO-ANCA+ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types., Funding Agencies: Swedish Society for Medical Research; Swedish Research Council; Swedish Society of Medicine; Swedish Rheumatism Association; Knut and Alice Wallenberg Foundation; AstraZeneca-Science for Life Laboratory (SciLifeLab) Research Collaboration Grant

Objective: The autoimmune response in rheumatoid arthritis (RA) is marked by the presence of anti–citrullinated protein antibodies (ACPAs). A notable feature of IgG ACPA is the abundant expression of N-linked glycans in the variable domain. However, the presence of ACPA variable domain glycosylation (VDG) across disease stages, and its response to therapy, are poorly described. To understand its dynamics, we investigated the abundance of IgG ACPA VDG in 1,498 samples from individuals in different clinical stages. Methods: Using liquid chromatography, we analyzed IgG ACPA VDG profiles in 7 different cohorts from Japan, Canada, The Netherlands, and Sweden. We assessed 106 healthy individuals, 228 individuals with presymptomatic RA, 277 individuals with arthralgia, 307 patients with new-onset/early RA, and 117 RA patients after prespecified treatment regimens. Additionally, we measured VDG in 234 samples from patients with RA who did or did not achieve long-term drug-free remission (DFR) during up to 16 years follow-up. Results: IgG ACPA VDG significantly increased (P < 0.0001) toward disease onset and was associated with ACPA levels and epitope spreading prior to diagnosis. A slight increase in VDG was observed in patients with established RA, with a moderate influence of treatment (P = 0.007). In patients in whom DFR was later achieved, IgG ACPA VDG was already reduced at the time of RA onset. Conclusion: The abundance of IgG ACPA VDG increases toward RA onset and correlates with maturation of the ACPA response. While IgG ACPA VDG levels are fairly stable in established disease, a lower degree of VDG at RA onset correlates with DFR. Although the underlying biologic mechanisms remain elusive, our data support the concept that VDG relates to an expansion of the ACPA response in the pre-disease phase and contributes to disease development.