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Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus
  1. Lina Odqvist1,
  2. Zala Jevnikar1,
  3. Rebecca Riise1,
  4. Lisa Öberg1,
  5. Magdalena Rhedin1,
  6. Dag Leonard2,
  7. Linda Yrlid1,
  8. Sonya Jackson1,
  9. Johan Mattsson1,
  10. Sambit Nanda3,
  11. Philip Cohen3,
  12. Axel Knebel3,
  13. Simon Arthur4,
  14. Kristofer Thörn1,
  15. Elisabet Svenungsson5,
  16. Andreas Jönsen6,
  17. Iva Gunnarsson5,
  18. Karolina Tandre2,
  19. Andrei Alexsson2,
  20. Alf Kastbom7,
  21. Solbritt Rantapää-Dahlqvist8,
  22. Maija-Leena Eloranta2,
  23. Ann-Christine Syvänen2,
  24. Anders Bengtsson6,
  25. Patrik Johansson9,
  26. Johanna K Sandling2,
  27. Christopher Sjöwall7,
  28. Lars Rönnblom2,
  29. Barry Collins1,
  30. Outi Vaarala1,10
  1. 1 Research and Early Development, Respiratory, Inflammation and Autoimmune, BioPharmaceuticals R&D, AstraZeneca R&D Gothenburg, Mölndal, Sweden
  2. 2 Department of Medical Sciences, Science for Life Laboratories, Uppsala University, Uppsala, Sweden
  3. 3 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK
  4. 4 Division of Immunology and Cell Signaling, School of Life Sciences, University of Dundee, Dundee, UK
  5. 5 Department of Medicine, Rheumatology Unit, Karolinska Institute, Stockholm, Sweden
  6. 6 Skåne University Hospital, Department of Clinical Science Lund, Rheumatology, Lund University, Lund, Sweden
  7. 7 Department of Rheumatology and Department of Clinical and Experimental Medicine, Linköping University, Linkoping, Sweden
  8. 8 Department of Public Health and Clinical Medicine/Rheumatology, Umeå Universitet Medicinska fakulteten, Umea, Sweden
  9. 9 Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca R&D Gothenburg, Mölndal, Sweden
  10. 10 Respiratory, Inflammation and Autoimmunity Department, MedImmune LLC, Gaithersburg, Maryland, USA
  1. Correspondence to Dr Outi Vaarala, Respiratory, Inflammation and Autoimmunity Department, MedImmune LLC, Gaithersburg, MD 20878, USA; vaaralao{at}


Objectives Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis.

Methods CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926.

Results Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes.

Conclusions We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation.

  • PAD4
  • NET
  • peptidyl arginine deiminase
  • PADI4
  • rs2230926

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  • Handling editor Josef S Smolen

  • ZJ and RR contributed equally.

  • Contributors LO, ZR, RR, MR, JM, KT, SJ, AK, SN, SA planned, performed and analysed experiments. LÖ and PJ performed bioinformatic analyses. DL performed data analysis and AA performed statistical analyses. PC and LFY were involved in data discussion and supervision of study design experiments. JKS and A-CS generated and provided genotype data. ES, AJ, IG, KT, AK, SR-D and AB collected and provided patient and/or control material and clinical characterisation. M-LE planned experiments and provided patient material. CS provided scientific and experimental input and patient material. LR planned experiments, provided patient material and clinical data and was involved in data discussion and manuscript writing. BC was involved in study design, data discussion and supervision of experiments. OV designed experiments, analysed data and supervised the study. OV, LO, BC and LR wrote the manuscript. All authors critically reviewed and approved the manuscript.

  • Funding The study was funded by DISSECT AstraZeneca-SciLifeLab collaboration, The Swedish Research Council, Knut and Alice Wallenberg Foundation, Swedish Rheumatism Foundation, King Gustaf V’s 80-year Foundation, Swedish Society of Medicine, the Ingegerd Johansson donation, and Medical Research Council Programme Grant MRC000985.

  • Competing interests LO, ZR, RR, LÖ, MR, LFY, SJ, JM, KT, PJ, BC and OV were employees at AstraZeneca Group while performing this study and may have stock/stock options in AstraZeneca. AB received research grant from DISSECT, partly funded by AstraZeneca. LR reports grants and personal fees from Astra Zeneca during the conduct of the study and personal fees from Biogen outside the submitted work. AstraZeneca provided funding to DISSECT for the conduct of this study. The remaining authors declare that they have no competing interests. There are no patents involved.

  • Patient consent for publication Not required.

  • Ethics approval The study was approved by the regional ethics review boards of Uppsala, Lund, Linköping (No. M75-08/2008), Stockholm and Umeå. Use of tissue for ex vivo functional and mechanistic studies (bone marrow and blood) were approved by the local ethics committee in Gothenburg.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available in a public, open access repository. Data are available upon reasonable request.

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