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Objective: The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are heterogeneous disorders. The aim of this study was to identify and characterize subgroups of patients based on sex, ANCA, age at diagnosis, and organ involvement.

Methods: In total, 1,167 patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) were retrospectively recruited to the study. Data including cumulative involvement of 10 different organ systems, end-stage kidney disease (ESKD), sex, proteinase (PR) 3–ANCA, myeloperoxidase (MPO)-ANCA, age at diagnosis, disease duration, and relapse were obtained from medical records. Clinical variables were analyzed for associations with sex, age at diagnosis, and relapse using logistic regression analysis. Thirteen clinical variables were included in hierarchical cluster analyses using the Ward method.

Results: In patients with GPA, PR3-ANCA, renal and pulmonary involvement, and ESKD were significantly associated with male sex, whereas MPO-ANCA was associated with female sex. Patients with GPA who were younger than 32 years of age at diagnosis were significantly more often females and had more ear–nose–throat involvement than patients older than 32 years. In patients with MPA, female patients were significantly younger at diagnosis than male patients. Relapse was significantly associated with young age at diagnosis and pulmonary involvement in GPA and with musculoskeletal involvement in MPA. Hierarchical cluster analyses identified five and seven patient clusters among individuals with GPA and MPA, respectively. PR3-/MPO-ANCA defined the largest clusters, whereas heart, gastrointestinal, and central nervous system involvement were hallmarks for three clusters for both patients with GPA and MPA.

Conclusion: Sex, age at diagnosis, and specific organ involvements define clinically relevant subgroups among patients with ANCA-associated vasculitides.

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 HLADPB1 locus, also after stepwise conditional analysis. MPO-AAV was significantly associated with HLADRB1*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.

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 pathogenesis of AAV and the diversification of patients into PR3-ANCA+ and MPO-ANCA+ subphenotypes. (Figure presented.).

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 × 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: Increased soluble levels of complement effectors have been demonstrated in active anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but the timing of complement activation in the autoimmune inflammation remains elusive. This study investigated whether the complement system is activated before onset of symptoms in AAV.

Method: The Swedish National Patient Register and Cause of Death register were linked to registers of five biobanks to identify individuals sampled before AAV symptom onset. Diagnosis of AAV and time-point for symptom onset were confirmed by reviewing medical records. We identified 64 presymptomatic individuals with serum samples > 1 month < 10 years from AAV symptom onset and 122 matched controls. Complement factors (C2, C5) and activation markers (C5a, C4b) were measured using Luminex technology.

Results: Presymptomatic individuals had higher levels of C5 up to 6.5 years before symptom onset, compared with controls [median (IQR) 80.7 (131.9) vs 46.6 (63.4) µg/mL, p = 0.05]. Levels of C5a increased significantly during the pre-dating time (p = 0.033) until symptom onset. The complement levels were significantly higher in presymptomatic myeloperoxidase (MPO)-ANCA⁺ individuals versus MPO-ANCA⁻ and proteinase-3-ANCA⁺ individuals. C5 was significantly increased in cases with renal involvement at diagnosis versus controls (p = 0.022), whereas levels of both C5 and C5a were significantly increased in presymptomatic individuals diagnosed with microscopic polyangiitis after onset compared with controls (C5: p = 0.027; C5a: p = 0.027).

Conclusion: Activation of the complement system is an early event in the pathogenesis of AAV and is mainly associated with MPO-ANCA⁺ AAV and with microscopic polyangiitis.

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 × 10-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 × 10-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 × 10-10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 × 10-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 × 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.

Microbiota has been associated with autoimmune diseases, with nasal Staphylococcus aureus being implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody–associated vasculitis (AAV). Little is known about the role of oral microbiota in AAV. In this study, levels of IgG antibodies to 53 oral bacterial species/subspecies were screened using immunoblotting in plasma/serum in pre-symptomatic AAV-individuals (n = 85), matched controls, and established AAV-patients (n = 78). Saliva microbiota from acute-AAV and controls was sequenced from 16s rDNA amplicons. Information on dental status was extracted from a national register. IgG levels against oral bacteria were lower in established AAV versus pre-AAV and controls. Specifically, pre-AAV samples had, compared to controls, a higher abundance of periodontitis-associated species paralleling more signs of periodontitis in established AAV-patients than controls. Saliva microbiota in acute-AAV showed higher within-sample diversity but fewer detectable amplicon-sequence variants and taxa in their core microbiota than controls. Acute-AAV was not associated with increased abundance of periodontal bacteria but species in, e.g., Arthrospira, Staphylococcus, Lactobacillus, and Scardovia. In conclusion, the IgG profiles against oral bacteria differed between pre-AAV, established AAV, and controls, and microbiota profiles between acute AAV and controls. The IgG shift from a pre-symptomatic stage to established disease cooccurred with treatment of immunosuppression and/or antibiotics.

Objectives: Anti-neutrophil cytoplasmic autoantibodies [ANCA) are important for diagnosis of ANCA-associated vasculitides (AAV). The timing of antibody development is not well established. To investigate the development of proteinase 3 (PR3)- and myeloperoxidase (MPO)-ANCA, blood samples collected before onset of symptoms of AAV were analysed.

Methods: To identify AAV patients with blood samples predating symptoms, the National Patient Register and Cause of Death register were scrutinized for ICD codes for AAV and linked to the registers of five biobanks. Diagnoses of AAV and time point for symptom onset were confirmed by reviewing 504 case-record. Eighty-five AAV cases (34 males, 51 females) with samples >1 month < 10 years from AAV symptom onset and two controls matched for sex, age, and sampling time for each case were included. Samples were screened using ELISAs for ANCA and further analysed for PR3-or MPO- specificities.

Results: In ANCA-screen 35.7% of the pre-symptomatic cases and 3.5% of controls tested positive (p < 0.01). 26.2% of the cases were PR3-ANCA+ and 10.7% MPO-ANCA+. Median (Q1-Q3) predating time for PR3-ANCA+ was 2.7 (0.3–7.7) years and MPO-ANCA+ 2.0 (0.9–3.5) years. PR3-ANCA was demonstrated in samples up to nine years before symptom onset. At symptom onset predating PR3-ANCA+ cases were younger than PR3-ANCA- (P < 0.01), and MPO-ANCA+ were older than MPO-ANCA- (p < 0.05). Predating MPO-ANCA+ cases vs. MPO-ANCA- and vs. PR3-ANCA+ cases had more often at symptoms onset manifestations from lungs, kidneys or peripheral nervous system (p < 0.01 and p < 0.05, respectively).

Conclusion: The PR3-and MPO-ANCAs are present years before AAV symptom onset and represent distinct diseases.

Objective: The discovery of anti-citrullinated protein antibodies (ACPAs) and the introduction of new therapeutic options have had profound impacts on early rheumatoid arthritis (RA) care. Since ACPA status, most widely assessed as reactivity to cyclic citrullinated peptides (CCPs), influences treatment decisions in early RA, we aimed to determine whether anti-CCP remains a predictor of disease activity and radiographic joint damage in more recent 'real-world' early RA.

Method: Two observational early RA cohorts from Sweden enrolled patients in 1996-1999 (TIRA-1, n = 239) and 2006-2009 (TIRA-2, n = 444). Clinical and radiographic data and ongoing treatment were prospectively collected up to 3 years. Two other cohorts served as confirmation cohorts (TRAM-1, with enrolment 1996-2000, n = 249; and TRAM-2, 2006-2011, n = 528). Baseline anti-CCP status was related to disease activity, pharmacotherapy, and radiographic joint damage according to Larsen score.

Results: In the TIRA-1 cohort, anti-CCP-positive patients had significantly higher 28-joint Disease Activity Score, swollen joint count, C-reactive protein level, and erythrocyte sedimentation rate during follow-up compared with anti-CCP-negative patients. In TIRA-2, no such differences were found, but baseline anti-CCP positivity was associated with higher 3 year Larsen score (5.4 vs 3.5, p = 0.039). In TRAM-2, anti-CCP also predicted radiographic damage (8.9 vs 6.7, p = 0.027), with no significant differences in disease activity.

Conclusion: In the early RA cohorts recruiting patients in 2006-2011, baseline anti-CCP positivity was not associated with disease activity over time, but was associated with increased radiographic damage at follow-up. Hence, close radiographic monitoring is warranted in early anti-CCP-positive RA regardless of disease activity.