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The B-lymphocyte kinase ( BLK ) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding.

Numerous loci have been found genetically associated with complex diseases, but only in a few cases has the functional variant and the molecular mechanism behind it been identified. Recently, the association of the BANK1 gene with systemic lupus erythematosus (SLE) was described. Here, we investigated the role of the associated polymorphisms on gene function and found that SNP rs17266594 located in the branch point consensus sequence has negligible effect on splicing or gene expression. The non-synonymous SNP rs10516487 located in exon 2 influenced splicing efficiency by creating an exonic splicing enhancer site for the SRp40 factor. Further, this same SNP generates protein isoforms with differential and measurable self-association properties. The full-length protein isoform containing the R61 variant forms larger protein scaffold complexes in the cell cytoplasm compared with the protective BANK1-61H variant. We also observed that, contrary to the full-length isoforms, the short Δ2 isoform of BANK1 displays a homogeneous cytoplasmic distribution, underscoring the potential role of the exon 2-coded protein domain in the scaffolding function of BANK1. We provide evidence that the non-synonymous SNP rs10516487 (G>A; R61H) shows a dual nature by first, influencing mRNA splicing and consequently the quantity of protein, and, second, by producing a risk variant-containing protein isoform with increased potential for multimerization.

The need for comprehensive published epidemiologic and clinical data from Latin American systemic lupus erythematosus (SLE) patients motivated the late Dr Alarcón-Segovia and other Latin American professionals taking care of these patients to spearhead the creation of the Grupo Latino Americano De Estudio del Lupus (GLADEL) cohort in 1997. This inception cohort recruited a total of 1480 multiethnic (Mestizo, African-Latin American (ALA), Caucasian and other) SLE patients diagnosed within two years from the time of enrollment from 34 Latin American centers with expertise in the diagnosis and management of this disease. In addition to the initial 2004 description of the cohort, GLADEL has contributed to improving our knowledge about the course and outcome of lupus in patients from this part of the Americas. The major findings from this cohort are highlighted in this review. They have had important clinical implications for the adequate care of SLE patients both in Latin America and worldwide where these patients may have emigrated.

In a genome-wide association study (GWAS) of individuals of European ancestry afflicted with systemic lupus erythematosus (SLE) the extensive utilization of imputation, step-wise multiple regression, lasso regularization and increasing study power by utilizing false discovery rate instead of a Bonferroni multiple test correction enabled us to identify 13 novel non-human leukocyte antigen (HLA) genes and confirmed the association of four genes previously reported to be associated. Novel genes associated with SLE susceptibility included two transcription factors (EHF and MED1), two components of the NF-κB pathway (RASSF2 and RNF114), one gene involved in adhesion and endothelial migration (CNTN6) and two genes involved in antigen presentation (BIN1 and SEC61G). In addition, the strongly significant association of multiple single-nucleotide polymorphisms (SNPs) in the HLA region was assigned to HLA alleles and serotypes and deconvoluted into four primary signals. The novel SLE-associated genes point to new directions for both the diagnosis and treatment of this debilitating autoimmune disease.

A recent genome-wide association study revealed a variant (rs2431697) in an intergenic region, between the pituitary tumor-transforming 1 ( PTTG1 ) and microRNA ( miR-146a ) genes, associated with systemic lupus erythematosus (SLE) susceptibility. Here, we analyzed with a case–control design this variant and other candidate polymorphisms in this region together with expression analysis in order to clarify to which gene this association is related. The single-nucleotide polymorphisms (SNPs) rs2431697, rs2910164 and rs2277920 were genotyped by TaqMan assays in 1324 SLE patients and 1453 healthy controls of European ancestry. Genetic association was statistically analyzed using Unphased. Gene expression of PTTG1 , the miRNAs miR-3142 and primary and mature forms of miR-146a in peripheral blood mononuclear cells (PBMCs) were assessed by quantitative real-time PCR. Of the three variants analyzed, only rs2431697 was genetically associated with SLE in Europeans. Gene expression analysis revealed that this SNP was not associated with PTTG1 expression levels, but with the microRNA-146a, where the risk allele correlates with lower expression of the miRNA. We replicated the genetic association of rs2341697 with SLE in a case–control study in Europeans and demonstrated that the risk allele of this SNP correlates with a downregulation of the miRNA 146a, potentially important in SLE etiology.

Background:Molecules involved in antigen presentation of intra- and extracellular peptides, are encoded by genes of the human leukocyte antigen (HLA) system located within the Major Histocompatibility Complex (MHC) region in chromosome 6p21; some of these genes are among the most polymorphic in the human genome. According to the allelic divergence advantage hypothesis, an individual’s immunopeptidome is more diverse the more different their two homologous HLA allele sequences are. Although the relationship HLA to autoimmune diseases (ADs) has been widely demonstrated, the effect of evolutionary divergence in ADs is still unknown.Objectives:To investigate the immunopeptidome of patients with systemic autoimmune diseases through the study of the evolutionary divergence in the HLA region.Methods:In this study we have analyzed the HLA evolutionary divergence (HED) of the sequences of the HLA-A, B, C, DPB1, DQB1 and DRB1 genes in the European cohort of the PRECISESADS project composed of 460 controls and 1,564 ADs patients. HED value summarizes the physicochemical differences according to Graham’s distance, between the aminoacids of the peptide binding sites of the two homologous alleles of each class I and class II gene of each patient. Additionally, we employed an in silico reference proteome from the bone marrow niche to investigate the binding ability of self-generated peptides potentially implicated in the etiology of ADs.Results:In comparison to controls (HED = 12.4), patients with systemic lupus erythematosus (SLE) and Sjögren’s syndrome (13.8 and 14.4, respectively) showed a significant higher evolutionary divergence for DQB1 while a non-significant decreased HED value was observed in rheumatoid arthritis (RA) patients (11.3). DRB1 gene showed a trend towards lower HED values in ADs compared to controls. The SLE results were replicated in two independent European cohorts.The in silico analysis of the binding capacities of peptides generated form a bone marrow niche proteomic reference for the DQB1 locus was concordant with the allelic divergence advantage hypothesis, where higher divergence values correlated with larger predicted binding capabilities. SLE patients’ genotypes demonstrated greater binding capacities of the DQB1 gene alleles as well as higher HED values. Instead, DRB1 gene alleles with the highest HED value do not correlated with the greatest self-peptide binding capacities.Conclusion:This study revealed that HLA evolutionary divergence patterns as well as predicted binding capabilities are not only disease-specific but also gene specific. Whereas DQB1 results were consistent with the allelic divergence advantage hypothesis, DRB1 greater binding capacities were better explained by shared epitope hypothesis.REFERENCES:NIL.Acknowledgements:This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831434 (3TR). The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA.Disclosure of Interests:None declared.

We evaluated the roles of five single-nucleotide polymorphisms (SNPs) within PDCD1 , and haplotypes defined by these SNPs, for the development of systemic lupus erythematosus (SLE) and specific sub-phenotypes (nephritis, antiphospholipid antibody positive, arthritis and double-stranded DNA positive) within a multiethnic US cohort of 1036 patients. Family based analyses were performed using 844 simplex families from four ethnic groups (Caucasian, Asian, Hispanic and African American). Subjects were genotyped for five ‘tag’ SNPs (selected from 15) to provide complete genetic information in all main ethnic groups. We employed transmission disequilibrium testing to assess risk for SLE by allele or haplotype, and multiple logistic regression analysis of SLE cases to examine associations with specific sub-phenotypes. In family based analyses, a haplotype containing the PD1.3A allele was significantly associated with SLE susceptibility among Caucasian families ( P =0.01). Among Hispanic families, two novel SNPs were associated with SLE risk ( P =0.005 and 0.01). In multivariate logistic regression analyses, five haplotypes were associated with specific sub-phenotypes among the different ethnic groups. These results suggest that PDCD1 genetic variation influences the risk and expression of SLE and that these associations vary according to ethnic background.

Background:Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a diverse pathophysiology triggered by the loss of self-tolerance, resulting in inflammation and tissue damage in multiple organs. The use of animal models has been instrumental in advancing our understanding of SLE. The elimination of the requirement for animal models in the FDA’s drug approval process prompts a reevaluation of their role.Objectives:We aim to define a molecular framework for the future design of preclinical studies in SLE, helping researchers in the selection of mouse models and appropriate time point of study based on the molecular pathways and their relationship with human disease.Methods:To address this, a 4 timepoint longitudinal study was conducted using four different spontaneous SLE mouse models: MRLlpr/lpr, NZB/W, BXSB.Yaa, and TLR7.Tg6. RNA-Seq from whole-blood, spleen, and kidney, flow-cytometry from spleen, cytokines and autoantibodies in serum were profiled. All data was investigated using both, longitudinal differential expression analysis as well as individual cross-sectional analysis of each time-point. Molecular and flow-cytometry data from blood from SLE patients from the PRECISESADS project was integrated with mouse data using the MEFISTO software, allowing us to integrate direct relationships between human and mouse along the progression of disease.Results:Mouse models were extensively characterized at the molecular level. In the spleen, lymphopenia mimicking that occurring in the human and age-associated B cells were in common between mouse models, while neutrophil and cytotoxic lymphocyte expansions differentiated the models. TLR7-involved models showed important transcriptional dysregulation in the spleen as compared to non-TLR7 models, whose dysregulation was primarily in the kidney. Molecular changes were associated with severity across all models, with splenic changes preceding kidney damage and suggesting the ideal moment to study early immune pathogenic processes.Human and mouse molecular integration showed a shared interferon-mediated pathway with MRLlpr/lpr and TLR7.Tg6 models associated with hematological and constitutional clinical domains, but at different timepoints. The neutrophil-mediated inflammatory pathway was shared between humans, MRLlpr/lpr, BXSB.Yaa and TLR7.Tg6 models and related to renal and cutaneous clinical human domains.Conclusion:The findings provide a valuable framework for future experimental design involving SLE mouse models, demonstrating their continued usefulness in investigating the elusive pathogenesis and heterogeneity of SLE.REFERENCES:NIL.Acknowledgements:This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831434 (3TR). The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under the Grant Agreement Number 115565 (PRECISESADS project), resources of which are composed of financial contribution from the European Union’s Seventh Framework Program (FP7/2007–2013) and EFPIA companies’ in-kind contribution. This work was supported in part by the Spanish Ministry of Science and Innovation under grant “Juan de la Cierva” (IJC2020-043364-I).Disclosure of Interests:Maria Rivas-Torrubia: None declared, María Morell: None declared, Zuzanna Makowska Bayer Pharma AG, Berlin, Berlin, Germany, Jorge Kageyama Bayer Pharma AG, Berlin, Berlin, Germany, Anne Buttgereit Bayer Pharma AG, Berlin, Berlin, Germany, Julius Lindblom: None declared, PRECISESADS cytometry consortium: None declared, Ioannis Parodis: None declared, Lorenzo Beretta: None declared, Ralf Lesche Bayer Pharma AG, Berlin, Berlin, Germany, Fiona McDonald Bayer Pharma AG, Berlin, Berlin, Germany, Marta Alarcon-Riquelme: None declared, Guillermo Barturen: None declared.

Background:Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by persistent inflammation affecting multiple organs, including the intestine. A new feature that seems decisive in autoimmune pathogenesis is the gut microbiota composition. Its role, however, remains elusive. The B-cell scaffold protein with ankyrin repeats (Bank1) regulates Toll-like receptor-7 (TLR7) signalling in B cells and it has been genetically associated with human lupus and its absence correlated with a less severe disease in mice. In the context of autoimmunity, IL-10+ B cells are being characterised in recent years for their immunosuppressive role and their feasible relationship with gut bacterial composition.Objectives:To investigate the involvement of BANK1 signalling in the gut mucosal B cell response to commensals during lupus pathogenesis.Methods:Lupus derived gut affectation was characterised by using two TLR7-mediated models of the disease by topical application of the TLR7 agonist Imiquimod (IMQ) or the transgenic expression of the Tlr7 gene. We assessed splenomegaly and anti-dsDNA autoantibodies by ELISA and the IL-10+ B cell response by flow cytometry. IgA response in the gut and the faeces were assessed by flow cytometry and ELISA, and the IgA antigenic targets were evaluated by sorting and sequencing of IgA+ and IgA- faecal bacteria. Presence of Parabacteroides distasonis was assessed by qPCR. Bacterial composition was analysed by V4 16S rRNA sequencing.Results:In steady-state, Bank1 deficiency (B1KO mice) resulted in reduced frequency of CD19+B220+ B cells and IgA+ plasma cells in the small intestine lamina propria, which correlated with reduced levels of faecal-free IgA antibodies. Additionally, B1KO mice presented differential microbial composition and bacterial specificity. While the TLR7-mediated intestinal inflammation induced enlarged germinal centers (GC) in the Peyer’s patches and accumulation of GC B cells, these disease manifestations were reduced in the absence of Bank1. B1KO developed a milder form of the disease characterised by lower serum anti-dsDNA IgG2a levels, and reduced splenomegaly and gut inflammation. Concomitantly, upon TLR7-induced inflammation, levels of IgA+ plasma cells, concentration of faecal-free IgA antibodies and percentage of IgA-coated bacteria were comparable between B1KO and WT mice. However, analysis of IgA coating pattern of faecal bacteria showed that the specificities of gut-secreted IgA antibodies (antigenic targets) were significantly different between both genotypes, targeting different bacteria at genus and species level. Amelioration of lupus symptoms in mice lacking Bank1 was associated with an increased proportion of P. distasonis that, when transferred onto Bank1-sufficient WT mice, reduced the autoimmune manifestations. The effect of these bacteria was evaluated through two independent experiments. First, littermates carrying the B1KO-deficient-associated microbiota were generated and grown in shared cages before and during lupus induction. In the second, P. distasonis was given orally to assess its effect as a probiotic. Both experiments showed reduced gut inflammation, germinal center formation, and splenomegaly and lower levels of autoantibodies, regardless of genotype. As a mechanistic insight, we evaluated the induction of IL-10-producung B cells. Bank1 deficiency promoted the accumulation of IL-10-producing B cells in Peyer’s patches of lupus mice upon lupus development. Moreover, colonization of gut microbiota with P. distasonis led to increased numbers of IL-10+ B cells in WT mice, which correlated with lower splenomegaly and reduced levels of serum anti-dsDNA IgG2a.Conclusion:The absence of Bank1 predisposed to differential microbiota and differences in the numbers of IL-10+ B cells in the gut, together with reduced autoimmune inflammation. Changes in the humoral and cellular IgA response, may pave the way for the gut-activated IgA+ plasma cells to act peripherally and regulate autoimmunity. These results highlight the complex crosstalk between genes and environmental factors that modulate autoimmune inflammation.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:The management of neuropsychiatric (NP) systemic lupus erythematosus (SLE) is poorly optimised and specific treatment is lacking.Objectives:The aim of this study was to perform an in-depth investigation of the transcriptome of SLE patients with active central nervous system (CNS) involvement to gain insights into underlying molecular mechanisms and identify new potential drug targets for CNS lupus.Methods:We analysed differentially expressed genes in peripheral blood from patients with active CNS lupus (n=26) and active non-NP SLE (n=43) versus healthy controls (n=497) from the European PRECISESADS project (NTC02890121), as well as dysregulated gene modules. Gene modules were subjected to correlation analyses with serological markers, and regulatory network and druggability analysis.Results:Unsupervised co-expression network analysis revealed 23 dysregulated gene modules. Four showed differential dysregulation between two distinct subgroups of CNS lupus patients. The interferon module was upregulated in both subgroups. The “B cell”, “T cell”, “cytotoxic/NK cell”, and “mitochondrial cluster” gene modules were all found to be more downregulated in one subgroup, while the other subgroup showed varied dysregulation patterns. Drugs annotated to the cytotoxic/NK cell network included pegaptanib, a selective vascular endothelial growth factor (VEGF) antagonist, while many anticonvulsants such as zonisamide, lamotrigine, and oxcarbazepine showed potential for counteracting the transcriptomic signature associated with the B cell module. Druggability analysis for the mitochondrial cluster module revealed potential for the centrally acting angiotensin-converting enzyme inhibitor captopril, the mammalian target of rapamycin (mTOR) inhibitor everolimus, the proteasome inhibitor bortezomib, the toll-like receptor 5 (TLR5) agonist entolimod, and the spleen tyrosine kinase (SYK) inhibitor fostamatinib. In silico prediction algorithms demonstrated a greater anticipated response to anifrolumab and calcineurin inhibitors for the active CNS subgroup with B cell, T cell, cytotoxic/NK cell, and mitochondrial gene downregulation compared with the patient subgroup of mixed dysregulation patterns.Conclusion:In this cohort of SLE patients of European origin, B cell, T cell, cytotoxic/NK cell, and mitochondrial gene dysregulation patterns separated active CNS lupus patients into two distinct subgroups with differential anticipated response to type I interferon and calcineurin inhibition. Our study provides a conceptual framework for precision medicine in CNS lupus.REFERENCES:NIL.Acknowledgements:PRECISESADS Clinical Consortium.Disclosure of Interests:Julius Lindblom: None declared, Dionysis Nikolopoulos: None declared, Daniel Toro-Domínguez: None declared, Elena Carnero-Montoro: None declared, Maria Orietta Borghi: None declared, Jessica Castillo: None declared, Ellen Iacobaeus: None declared, Yvonne Enman: None declared, Chandra Mohan: None declared, Lorenzo Beretta: None declared, Marta Alarcon-Riquelme: None declared, Guillermo Barturen: None declared, Ioannis Parodis I have received research funding and/or honoraria from Amgen, AstraZeneca, Aurinia, Bristol Myers Squibb, Elli Lilly, Gilead, GlaxoSmithKline, Janssen, Novartis, Otsuka, and Roche.