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Objective Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjogrens syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. Methods The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. Results Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 x 10(-9)) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. Conclusion We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS. Funding Agencies|Swedish Research Council for Medicine and Health; Swedish Rheumatism Association; Swedish Heart-Lung Foundation; Swedish Society for Medical Research; Swedish Society of Medicine; Gustafsson Family Foundation; Wallenberg Scholarship; King Gustaf Vs 80-Year Foundation; Stockholm County and Region Ostergotland

Objectives: Clinical presentation of primary Sjögren's syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. Methods: We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. Results: We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10-62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. Conclusion: Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.

Objectives Clinical presentation of primary Sjögren’s syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. Methods We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. Results We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10−62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. Conclusion Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups. publishedVersion

Supplemental material, Suppl_Table_2_17.04.2019 for Fatigue in primary Sjögren’s syndrome: A proteomic pilot study of cerebrospinal fluid by Eivind Larssen, Cato Brede, Anne Hjelle, Anne Bolette Tjensvoll, Katrine Brække Norheim, Kjetil Bårdsen, Kristin Jonsdottir, Peter Ruoff, Roald Omdal and Mari Mæland Nilsen in SAGE Open Medicine

Supplemental material, Supplementary_table_1_fVAS_09.11-2018_(1) for Fatigue in primary Sjögren’s syndrome: A proteomic pilot study of cerebrospinal fluid by Eivind Larssen, Cato Brede, Anne Hjelle, Anne Bolette Tjensvoll, Katrine Brække Norheim, Kjetil Bårdsen, Kristin Jonsdottir, Peter Ruoff, Roald Omdal and Mari Mæland Nilsen in SAGE Open Medicine

Background: Primary Sjøgren`s syndrome (pSS) is a chronic autoimmune disease, characterised by lymphocytic infiltration of exocrine glands and autoantibody production. Fatigue is a frequent phenomenon in pSS, associated with reduced health-related quality of life. Fatigue is influenced by depressed mood, sleep disorder and autonomic dysfunction, but also occurs without these co-factors. Evidence from animal and human studies indicates that immune activation may directly influence fatigue in chronic inflammatory disorders. Sickness behaviour in animals is characterized by decreased activity, social withdrawal and a reduction in the intake of food and water. This behaviour is hypothesized to increase survival by shielding the sick animal from predators, and occurs automatically as a response to infection and inflammation. The proinflammatory cytokine interleukin (IL)-1β is crucial for this behaviour. Fatigue in humans can be considered an element of sickness behaviour, and we hypothesized that inhibition of IL-1 would lead to a reduction in fatigue in pSS. We tested this in a randomized clinical trial by giving anakinra, a recombinant IL-1 receptor antagonist (IL-1Ra) - or placebo - to pSS patients with a high level of fatigue. Inflammation is closely connected with oxidative stress, and generation of reactive oxygen species is an important mechanism for killing of pathogens. Increased oxidative stress has been reported in relation to fatigue in human diseases, but has never been investigated in relation to fatigue in pSS. We hypothesized that pSS patients would have higher levels of oxidative stress than healthy controls, and that oxidative stress would be associated with fatigue. Taking oxidative stress and pro-inflammatory cytokines into consideration, some pSS patients are still more affected by fatigue than otherwise comparable individuals. Part of the explanation for this might be found in the genetic makeup of each individual patient, and several recent studies point to both genetic and epigenetic factors that may be important for fatigue generation. Based on this, we aimed to investigate genetic variation in relation to fatigue in pSS. Main objectives: 􀁸 Write a review article of the current knowledge of biological mechanisms of fatigue in inflammatory and non-inflammatory conditions. 􀁸 Investigate the efficacy and safety of IL-1 inhibition on fatigue in pSS. 􀁸 Investigate the plasma levels of oxidative stress markers in pSS as compared to healthy individuals, and further explore any association of oxidative stress with fatigue in pSS. 􀁸 Investigate genetic variation, i.e., single nucleotide polymorphisms (SNP) in relation to fatigue in pSS. Subjects and methods: All patients included in this dissertation were recruited from the pSS patient pools at Stavanger University Hospital (SUS) and Haukeland University Hospital (HUS). SUS is the only hospital in the southern part of Rogaland County and HUS is the main hospital in Hordaland County, Norway. For the double-blind, randomised treatment trial all pSS patients in the southern part of Rogaland County were identified and invited to the study, and a total of 26 patients were eligible and agreed to participate. The patients were randomly allocated to treatment with either an IL-1Ra or placebo (0.9% NaCl in identical syringes), and self-administered the drug or the placebo by a daily subcutaneous injection. Neither patients nor investigators were aware of the treatment allocation. The study ran over four weeks. Blood was sampled and a visual analogue scale (VAS) and the Fatigue Severity Scale (FSS) were used to asses fatigue at the start of the study (week 0), at week 2, at the end of the study (week 4) and at week 5. The same 26 patients were included for the plasma measures of oxidative stress. Two markers of protein oxidation; advanced oxidation protein products (AOPP) and protein carbonyl (PC), were measured in blood samples collected at week 0, before any interventions took place. For the genetic analysis we used whole blood samples from 207 pSS patients and 376 healthy controls. We investigated the associations of fatigue and minor allele frequencies in 85 SNPs in 12 genes, half of which are related to mitochondrial function. The genes were selected based on previous studies of gene expression in the chronic fatigue syndrome. Results: We found that: 􀁸 IL-1 inhibition influences fatigue in pSS as compared to placebo. We were not able to show this in the primary endpoint, but ad hoc analysis points to a strong positive effect of IL-1 inhibition on fatigue. 􀁸 IL-1 inhibition appears to be safe in pSS. 􀁸 Markers of protein oxidation are increased in pSS as compared to healthy controls. There is no association between fatigue and plasma protein oxidation in pSS. 􀁸 Genetic variation in SLC25A40 and PKN1 show signals of association with fatigue in pSS. Conclusions: This dissertation strengthens the view that at least some part of fatigue has a biological fundament, related to inflammation. The IL-1 system is crucial in the development of fatigue in this setting, and IL-inhibition seems to reduce fatigue generation. There is a trend for association between genetic variation and fatigue in pSS. Fatigue is not associated with the amount of oxidised plasma proteins in pSS.