UBE2L3 polymorphism amplifies NF-kB activation and promotes B cell development linking linear ubiquitination to multiple autoimmune diseases. M. J. Lewis1, S. Vyse1, A. M. Shields2, S. Boeltz2, D. Leirer2, P. A. Gordon6, T. D. Spector3, P. J. Lehner5, H. Walczak4, T. J. Vyse2 1) Experimental Medicine and Rheumatology, Queen Mary University of London, London, United Kingdom; 2) Medical and Molecular Genetics, King's College London, London, United Kingdom; 3) Twin Research Unit, King's College London, London, United Kingdom; 4) Centre for Cell Death, Cancer and Inflammation, UCL Cancer Institute, University College London, London, United Kingdom; 5) Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; 6) Rheumatology Dept, King's College Hospital, London, United Kingdom.

   Background: Genome-wide association studies have identified a strong association between a single risk haplotype of the UBE2L3 gene and Systemic Lupus Erythematosus (SLE), as well as multiple autoimmune diseases (rheumatoid arthritis, juvenile idiopathic arthritis, ulcerative colitis, Crohns disease, coeliac disease, psoriasis). UBE2L3 is a highly specific E2 ubiquitin-conjugating enzyme. Linear ubiquitination is a newly described form of ubiquitination, whose only known function is controlling activation of NF-kB, mediated by the linear ubiquitination chain assembly complex (LUBAC). Results: Data from SLE GWAS, imputed to 1000 Genomes level identified rs140490 as the most strongly associated UBE2L3 SNP, located at -270bp of the promoter region (P=8.6x10-14; OR 1.30, 95%CI: 1.21-1.39). Microarray /western blot studies found that the rs140490 risk allele increased UBE2L3 expression in B cells and monocytes from PBMC. Overexpression of UBE2L3 in combination with LUBAC in HEK293-NF-kB reporter cell line led to a marked upregulation in NF-kB activity, which was abolished by dominant-negative mutant UBE2L3[C86S]. RNAi blockade of UBE2L3 antagonised TNF signalling by inhibiting phosphorylation and degradation of the NF-kB sequestration protein IB. Ex vivo human B cells and monocytes were isolated from genotyped healthy twins stimulated with CD40L or TNF respectively and NF-kB translocation quantified by Imagestream analysis. rs140490 genotype was correlated with both basal NF-kB activation in healthy human individuals, as well as the sensitivity of NF-kB to CD40 stimulation in B cells and TNF stimulation in monocytes. Consistent with this functional effect of UBE2L3 on CD40 signalling in B cells, rs140490 genotype correlated with increased plasmablast and plasma cell differentiation in SLE patients (P<0.001). Conclusion: This is the first study to show that the UBE2L3 risk haplotype exerts a critical rate-limiting effect on TNF and CD40 signalling in primary human cells. Our functional data show the critical importance of UBE2L3 in regulation of LUBAC and NF-kB activation. By tracking NF-kB nuclear translocation in B cells and monocytes from genotyped individuals, this is the first demonstration that a complex trait variant at UBE2L3 regulates both basal NF-kB activation and sensitivity of NF-kB to stimulation in ex vivo cells, resulting in accelerated B cell differentiation in SLE.

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