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Alternative splicing is a substantial contributor to the high complexity of transcriptomes of multicellular eukaryotes. In this Review, we discuss the accumulated evidence that most of this complexity is reflected at the protein level and fundamentally shapes the physiology and pathology of organisms. This notion is supported not only by genome-wide analyses but, mainly, by detailed studies showing that global and gene-specific modulations of alternative splicing regulate highly diverse processes such as tissue-specific and species-specific cell differentiation, thermal regulation, neuron self-avoidance, infrared sensing, the Warburg effect, maintenance of telomere length, cancer and autism spectrum disorders (ASD). We also discuss how mastering the control of alternative splicing paved the way to clinically approved therapies for hereditary diseases.Alternative splicing substantially contributes to proteomic complexity in multicellular eukaryotes and regulates various physiological and pathological processes, including cell differentiation, neuron self-avoidance, cancer and autism spectrum disorders. Recent advances paved the way to clinical use of alternative splicing-based therapies for hereditary diseases.

Green roofs can be an attractive strategy for adding perviousness in dense urban environments where rooftops are a high fraction of the impervious land area. As a result, green roofs are being increasingly implemented as part of urban stormwater management plans in cities around the world. In this study, three full-scale green roofs in New York City (NYC) were monitored, representing the three extensive green roof types most commonly constructed: (1) a vegetated mat system installed on a Columbia University residential building, referred to as W118; (2) a built-in-place system installed on the United States Postal Service (USPS) Morgan general mail facility; and (3) a modular tray system installed on the ConEdison (ConEd) Learning Center. Continuous rainfall and runoff data were collected from each green roof between June 2011 and June 2012, resulting in 243 storm events suitable for analysis ranging from 0.25 to 180 mm in depth. Over the monitoring period the W118, USPS, and ConEd roofs retained 36%, 47%, and 61% of the total rainfall respectively. Rainfall attenuation of individual storm events ranged from 3 to 100% for W118, 9 to 100% for USPS, and 20 to 100% for ConEd, where, generally, as total rainfall increased the per cent of rainfall attenuation decreased. Seasonal retention behavior also displayed event size dependence. For events of 10-40 mm rainfall depth, median retention was highest in the summer and lowest in the winter, whereas median retention for events of 0-10 mm and 40 +mm rainfall depth did not conform to this expectation. Given the significant influence of event size on attenuation, the total per cent retention during a given monitoring period might not be indicative of annual rooftop retention if the distribution of observed event sizes varies from characteristic annual rainfall. To account for this, the 12 months of monitoring data were used to develop a characteristic runoff equation (CRE), relating runoff depth and event size, for each green roof. When applied to Central Park, NYC precipitation records from 1971 to 2010, the CRE models estimated total rainfall retention over the 40 year period to be 45%, 53%, and 58% for the W118, USPS, and ConEd green roofs respectively. Differences between the observed and modeled rainfall retention for W118 and USPS were primarily due to an abnormally high frequency of large events, 50 mm of rainfall or more, during the monitoring period compared to historic precipitation patterns. The multi-year retention rates are a more reliable estimate of annual rainfall capture and highlight the importance of long-term evaluations when reporting green roof performance.

BackgroundPediatric Antiphospolipid Syndrome (APS) is an autoimmune disease characterized by venous and/or arterial thrombotic events (TE) associated with 2 consecutive positive determinations (at least 12 weeks apart) of antiphospholipid antibodies (aPL), IgG/IgM anticardiolipin (aCL), IgG/IgM β2-glycoprotein I (aβ2GPI) and/or lupus anticoagulant (LA). Recent data suggests that aPL levels may decrease over time due to the natural history of the disease or to treatments. Therefore, monitoring of aPL levels may represent a strategy to evaluate disease activity and response to therapies.ObjectivesTo investigate the trend over time of aPL titers in children with APS, comparing patients under immunomodulatory therapies and those without them.MethodsA descriptive, observational, cross-sectional study was carried out in children with APS. aPLs testing was carried out in all patients from diagnosis every 3-4 months for 2 years. Interferon Gene Signature (IGS) was assessed as described by Crow[1]. Laboratory parameters, clinical and demographic data was retrieved and analyzed. Statistical analysis was performed with software R_v. 4.0.3.ResultsSixteen children with a diagnosis of APS were included in this study. The median age at disease onset was 11.5 years (range: 6 months – 17 years) and 62.5% were female; 87.5% Caucasians. Thirteen patients (81.2%) had a diagnosis of primary APS and 3 (18.8%) out of 16 of secondary APS.Regarding clinical manifestations, 11 children developed at least one TE (7 arterial and 5 venous). Cerebral territory was the most frequently involved with 5 thrombosis, followed by 3 deep vein thrombosis, 1 pulmonary thromboembolism and 2 arterial renal thrombosis. Most of the patients received an antiaggregant and/or anticoagulant therapy (13 and 8 patients, respectively). However, 1 patient presented a new TE after the antiaggregant withdrawal.A total of 12 (75%) children developed at least one non-criterion manifestation: 7 patients (44%) cardiac (Libman-Sacks endocarditis or valvular heart disease), 7 patients (44%) neurological (chorea or white matter lesions), 6 patients (37.5%) hematological (thrombocytopenia, hemolytic anemia or Evans syndrome) and 1 patient (3.8%) a renal thrombotic microangiopathy.Related to immunological parameters, 4 (25%) were positive for only 1 aPL, 5 (42%) for 2 aPL subtypes and 7 (44%) for all 3 aPL subtypes, showing the highest rates IgG aβ2GP (87.5%) and IgG aCL (81.25%). Lower rates were identified for LA (44%), IgM aCL (18.7%) and IgM aB2GP (12.5%). Four (25%) had ANA positivity (3 secondary and 1 primary APS).Regarding treatment, 13 children (81.25%) received at least one immunomodulatory drug (13 patients mycophenolate; 4 rituximab) and 3 (18.7%) were not treated with them.During the 2-year-follow-up, 11 patients (68.8%) showed a reduction of aPL titers compared to the onset, with becoming 9 negative (56.3%) at the end of follow-up (Figure 1A). Of those who were negative, 8 children were under immunomodulatory therapies and only 1 did not receive any treatment. Five patients (31.2%) showed stable titers of aPLs during follow-up. Of them, 2 were not treated with immunomodulatory therapies and 2 were under mycophenolate but with poor compliance (reduction of titers with the restart of therapy were observed).We also evaluated the presence of the IGS in 11 patients with APS for whom a whole blood RNA sample was available: the IGS was positive in 9 (81.8%) of 11 children (7 with primary APS and 2 with secondary APS).ConclusionOur data suggest the possible effect of immunomodulatory therapies in reducing antibody titers in APS. Therefore, it may represent a strategy to control the disease activity leading to a better prognosis. Further studies are needed to confirm and expand our data.Reference[1]Rice GI, et al. Lancet Neurol 2013; 12:1159-69.Figure 1.A. Trend of IgG aB2GP during 2 years of follow-up. B. Trend of IgG aCL during 2 years of follow-up.Red line (0): children without immunomodulatory (IMM) therapies; blue line (1): children with IMM therapies.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Background:Juvenile Idiopathic Arthritis (JIA) is the most common form of chronic arthritis in childhood. The adaptive branch of the immune system plays a crucial role in the development of JIA. We have showed that switched memory B cells are expanded in patients with oligoarticular- and polyarticular-JIA.Objectives:The aim of this study is to investigate abnormalities in the switched memory B cell compartment and characterize the receptor signaling machinery in B cells of patients with oligo- and poly-JIA.Methods:We enrolled patients with a diagnosis of oligoarticular and polyarticular JIA (n=15) and age matched controls (n=15). Surface staining of lymphocytes was carried out to identify B cell and T cell subsets in peripheral blood (PB) and synovial fluid (SF). Immunoglobulins levels were retrieved from electornic records. Intracellular levels of phospho-proteins were evaluated in PB by flow cytometry. PBMC were stimulated in vitro with an anti-Ig and stained for intracellular phospho-proteins. Calcium mobilization after anti-Ig stimulation was evaluated with Indo-1 by flow cytometry.Results:The ratio of IgG to IgA switched memory B cells was higher in JIA patients than controls, indicating and expansion of IgG+ memory B cells over mucosal IgA+ memory B cells in JIA (Figure 1A). IgG+ memory B cells were the most abundant B cell subset in SF (Figure 1A). Serum levels of IgG, but not IgA, were higher in JIA patients than controls.To investigate if the hyperactivation of B cells is due to an henanced B cell receptor (BCR) signaling, we analyzed the basal phosphorylation levels of 3 key enzymes (Syk, BLNK, ERK1/2) in B cells of JIA patients. We observed a higher frequency of phosphorylation of BLNK, but not of Syk and ERK1/2 in both naïve and memory B cells compared to controls (Figure 1B). Next, we evaluated the surface expression of CD22, a negative regulator of B cell signaling in all B cell subsets. Transitional, naïve and IgM memory B cells of patients with JIA showed reduced levels of CD22 compared to controls; no significant differences were observed in switched memory B cells (Figure 1C). As BLNK and CD22 regulate the calcium mobilization following BCR stimulation, we analyzed calcium mobilization. Upon in vitro stimulation of BCR, we observed that naïve B cells of patients with JIA had a faster calcium response to BCR activation, with a shorter peak time compared to controls (Figure 1D).Conclusion:Our data confirm the expansion of IgG memory B cells and the hyperproduction of IgG in patients with JIA. We also show that naïve B cells of JIA patients exhibit a reduced expression of CD22 and an increased basal phosphorylation of BLNK. Upon activation of BCR, naïve B cells respond more promptly to BCR activation with faster calcium mobilization.Figure 1.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Emiliano Marasco: None declared, Angela Aquilani: None declared, Matteo Trevisan: None declared, Ivan Caiello: None declared, Giusyda Tarantino: None declared, Rebecca Nicolai: None declared, Silvia Magni-Manzoni: None declared, Rita Carsetti: None declared, Fabrizio De Benedetti Novartis, SOBI

Background:Methotrexate (MTX) is the most used drug for the treatment of children and adults suffering from arthritis although its intake is burdened by well-known side effects. The Methotrexate Intolerance Severity Score (MISS) was originally developed in English [1] to identify patients intolerant to MTX.Objectives:Methotrexate (MTX) is the most used drug to treat children and adults with arthritis and its use is burdened by adverse effects. The MTX intolerance severity score (MISS) was developed in English to identify patients who are intolerant to MTX. The aim of this study was to translate and validate the MISS in Italian.Methods:The Italian version of the MISS was developed following the “guidelines for process of cross-cultural adaptation of self-reported measures”. The Italian version of the MISS was validated in 125 patients with juvenile idiopathic arthritis (JIA) followed at the Rheumatology Unit of Bambino Gesù Children Hospital. We assessed the construct validity and calculated the internal consistency of the Italian MISS. We performed ROC analysis to assess the overall performance of the Italian MISS.Results:Of the study cohort (83% female), 71 patients had oligoarthritis, 43 had RF-negative polyarthritis, 4 had enthesitis-related arthritis (ERA), and 3 had psoriatic arthritis. The mean age (±IQR) at diagnosis was approximately 4 years, with a “disease duration” on the MISS questionnaire of 6 years (IQR 3.3-10.5). The mean (± SD) VAS and c-JADAS scores were 0.7 and 2.4 respectively, with 65% of patients having inactive disease. The majority (98%) of patients received MTX subcutaneously and the average duration of use of the drug was approximately 5 years. We translated and adapted the MISS to the Italian language. The Italian MISS showed a very good internal consistency as shown by a Cronbach a of 0.87 (95% CI, 0.84-0.90) and a composite reliability of 0.89 (95% CI, 0.83-0.91). A threshold of 6 to define intolerant patients, showed a sensitivity of 98.3% and specificity of 81.2%.Conclusion:We developed the Italian version of the MISS and showed its validity and reliability to identify patients intolerant to MTX in clinical practice and in a research setting.REFERENCES:[1] M. Bulatović et al., “High prevalence of methotrexate intolerance in juvenile idiopathic arthritis: development and validation of a methotrexate intolerance severity score.” Arthritis Rheum 63, 2007-2013 (2011).[2] D. Wild et al., “Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes (PRO) Measures: report of the ISPOR Task Force for Translation and Cultural Adaptation.” Value Health 8, 94-104 (2005).Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Juvenile Idiopathic Arthritis (JIA) is the most common form of chronic arthritis in childhood. The adaptive branch of the immune system plays a crucial role in the development of JIA. We have showed that switched memory B cells are expanded in patients with oligoarticular- and polyarticular-JIA.Objectives:To characterize the antigen-experienced immunoglobulin repertoire, we performed deep sequencing of the switched repertoire in peripheral blood (PB) of oligo-/ploy-JIA patients(n=14) and age matched controls(n=4), and in matched synovial fluid (SF) of oligo-/ploy-JIA patients(n=7).Methods:We employed a cDNA-based-5′RACE approach with unique molecular identifiers. De-multiplexing, UMI extraction, and UMI-based consensus assembling were performed using the MIGEC software. Clonotype assembly were performed using MiXCR. SHM analysis was performed with SHazaM. B cell tolerance checkpoints were analyzed with a validated flow cytometry-based system (Malkiel S, Arthritis Rheumatol. 2016).Results:Immunoglobulin heavy chain variable gene segment usage frequency was similar between patients and controls, and PB and SF. Thus, we did not observe clustering of patients from controls. Analysis of averaged complementarity-determining region 3 (CDR3) repertoire characteristics revealed that CDR3 length was similar among the three groups, whereas physico-chemical characteristics of amino acid residues were significantly different in the IgG repertoire, but not in the IgA repertoire. IgG from SF showed higher charge and polarity and an increased hydrophobicity index compared to PB of controls and patients. We then analyzed the frequency of somatic hypermutation (SHM) and the selection pressure with SHazaM. We found a significant reduction in the frequency of SHM and a higher frequency of sequences with less than 10 mutations per Ig molecule in patients (in both PB and SF) than controls. The IgG repertoire in SF exhibited decreased positive selection in CDRs. To further characterize the immunoglobulin repertoire, we performed a flow cytometry-based staining to assess the frequency of autoreactive B cells in the transitional, naïve and memory B cell subsets in PB and also of memory B cells in SF. We found that central tolerance checkpoints are effective in both JIA patients and controls: the frequency of autoreactive B cells decreased from transitional, to naïve B cells in both JIA patients and controls. However, the frequency of autoreactive B cells was higher in memory B cells of JIA patients compared to controls, with the highest frequency observed in memory B cells of SF.Conclusion:Altogether, the observed changes in the IgG repertoire in PB and SF establish an altered peripheral selection process of IgG+ B cells in oligo-/ploy-JIA with a decreased load of SHM. Our data show for the first time that an altered GC response, leading to the accumulation of autoreactive B cells in the memory compartment, is a feature of oligo-/ploy-JIA.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Emiliano Marasco: None declared, Angela Aquilani: None declared, Ivan Caiello: None declared, Rebecca Nicolai: None declared, Giusyda Tarantino: None declared, Silvia Magni-Manzoni: None declared, Rita Carsetti: None declared, Fabrizio De Benedetti Novartis, SOBI.

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease causing significant morbidity and mortality. B cells play a central role in SLE pathogenesis. T-bet, is a transcription factor promoting T helper-1 development. B cells expressing T-bet are expanded in aging, chronically infected individuals, and murine models of autoimmunity [1, 2].ObjectivesThe aim of our work is to analyze the expression and regulatory mechanisms of the transcription factor T-bet in B cells of patients with systemic lupus erythematosus (SLE).MethodsThe intracellular expression of T-bet was evaluated by flow cytometry in the different subsets of B lymphocytes of SLE patients and controls (HD). Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with IFNγ and IFNα and the expression of T-bet was evaluated by flow cytometry. The clinical characteristics of patients were analyzed by Multiple Factor Analysis (MFA). The analysis was carried out with the software R.ResultsWe performed B cell staining and assessed the expression of T-bet in B cell subsets in patients with SLE and HD. We observed a significant expansion of naïve B cells and double negative (DN) B cells expressing T-bet in patients with SLE compared to HD (Figure 1A-B). An expansion of T-bet+ naïve B cells above the 99th percentile of HD (>1% of total naïve B cells) was evident in 68% of patients; 47% of patients showed an expansion of T-bet+ DN B cells above the 99th percentile of HD (>22% of total DN B cells). We assessed if patients with expanded T-bet+ naïve or DN B cells showed significant clinical differences from patients without the expanded subpopulations. To this end we collected clinical and laboratory parameters (SLEDAI, organ involvement as classified in the BILAG, damage with SDI, autoantibody profile, treatment and disease duration) and performed a multiple factor analysis (MFA). The aim of this analysis was to reduce the dimensionality of the complex data to few variables without losing information. The first 4 dimensions of MFA explained 58% of the total variance. The first dimension accounted for 23.6% of variance, and it was mostly driven by therapy and BILAG scores; the second dimension accounted for 17.7% of the variance and it was mostly driven by SLEDAI, SDI and serology. Patients with expanded T-bet+ naïve B cells, distributed differently from patients with no expansion of T-bet+ naïve B cells (Figure 1C). The first dimension was the most informative in separating the two groups of patients: significantly higher values were reported for patients with expanded T-bet+ naïve B cells. No differences were observed for patients with and without expanded T-bet+ DN B cells (Figure 1C). We investigated in vitro if the expression of T-bet in B cells was driven by interferons. We stimulated PBMCs of HD with IFNγ or IFNα and assessed the frequency of T-bet+ B cells: both interferons induced the expression of T-bet in naïve (CD19+CD27-) and memory (CD19+CD27+) B cells (Figure 1D). Interestingly, when cells were stimulated with IFNα in the presence of IFNγ-blocking antibodies the expression of T-bet was not induced.ConclusionAltogether, T-bet+ naïve B cells are expanded in patients with SLE and define a group of patients with clinically different disease from patients without expanded T-bet+ Naïve B cells. The expression of T-bet is induced specifically by IFNγ and not by IFNα.References[1]Karnell JL, Cellular immunology 2017[2]Knox JJ, JCI insight 2017Figure 1.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinuclear antibodies (ANA). Monitoring of anti-DNA antibody levels may reflect disease activity, by contrast a single anti-RBP antibody determination is thought to suffice for clinical purposes. Recent data suggests that ANA levels may decrease over time secondary to the natural history or to treatments.ObjectivesTo investigate the trend of ANA and anti-dsDNA titers over time in children with a diagnosis of SLE.MethodsWe enrolled 15 children with SLE. ANA and anti-dsDNA testing were carried out in all patients from diagnosis every 3-4 months for 2 years. ANA were defined as negative for titers < 1:80. Laboratory parameters, clinical and demographic data was retrieved and analyzed. Interferon Gene Signature (IGS was assessed by the expression of 6 interferon-induced genes (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) in whole blood RNA. Statistical analysis was performed with SW R_v. 4.0.3.ResultsFollowing 2 years of follow-up, all patients had ANA titers significantly lower than at time of the onset (MWW, p=0.0002) (Figure 1A). After two years of follow-up, 11 patients (73%) remained ANA positive (group 1), while 4 patients (26%) became negative (group 0). At time of diagnosis no significant differences in ANA titers (MWW, p=0.74) nor in disease activity, measured by SLEDAI, (MWW, p=0.88) were observed (table 1; Figure 1E). No significant differences in organ involvement were observed (Table 1). Assessing the change over time in ANA titers, the 2 groups of patients showed 2 different patterns: in group 0, ANA titers quickly declined and disappeared in the first 6 months after diagnosis; in group 1, ANA titers declined more slowly, remaining positive at 2 years (Figure 1C). ANA pattern (by IFA) was also evaluated, changes from homogenous pattern to speckled was observed during follow up (Figure 1B). Both C3 and C4 increased, with no different patterns between the 2 groups (Figure 1D). Similarly, anti-dsDNA antibodies titers declined over time with no clear different patterns between the groups (Figure 1C). We also analyzed the levels of IGS at last of follow-up, observing significant differences between the groups (MWW, p=0.018) with higher levels of IGS in ANA+ patients (Figure 1F).ConclusionOur analysis showed 2 different patterns in the reduction of ANA titers over time in children with SLE, with 26% of them becoming ANA negative after 6 months from diagnosis and remaining persistently negative during follow-up. Our data have important implications, specifically for the recruitment of patients into clinical trials, where the latest classification criteria of SLE require ANA positivity as entry criterion. A seronegative state may represent a different subcategory of patients with SLE with specific pathogenetic pathways, possibly independently from autoantibodies. Therefore, further studies are needed to confirm our data.Reference[1] Pisetsky DS, Nat Rev Rheumatol. 2020.Table 1.ANA- at 2yANA+ at 2yPatients, n411Female, n (%)3 (25)10 (9)Age, mean ± SD (years)13.80 ± 1.9113.45 ± 2.72Disease duration, mean ± SD (years)6.47 ± 4.226.17 ± 2.71EULAR/ACR 2019 criteriaFever, n (%)3 (75)8 (73)Acute cutaneous, n (%)4 (100)6 (55)Chronic cutaneous, n (%)0 (0)0 (0)Non-scarring alopecia, n (%)0 (0)1 (9)Oral/nasal ulcers, n (%)2 (50)7 (64)Joint involvement, n, (%)3 (75)4 (36)Serositis, n (%)0 (0)2 (18)Renal, n (%)1 (25)2 (18)Neurological, n (%)0 (0)0 (0)Hemolytic anemia, n (%)1 (25)4 (36)Leukopenia, n (%)4 (100)9 (82)Thrombocytopenia, n (%)3 (75)5 (45)Anti-dsDNA, n (%)4 (100)11 (100)Anti-Sm, n (%)1 (25)2 (18)LA, n (%)0 (0)1 (9.1)aCL, n (%)2 (50)2 (18)aB2GPI, n (%)2(50)2 (18)Low complement, n (%)4 (100)11 (100)C3, mean ± SD (mg/dl)40.25 ± 5.5152.73 ± 20.81C4, mean ± SD (mg/dl)3.67 ± 2.894.60 ± 2.72SLEDAI at diagnosis, mean ± SD12.75 ± 4.5012.18 ± 7.14SLEDAI at last follow-up, mean ± SD0.00 ± 0.001.36 ± 1.43Damage (SDI at last follow-up >0), n (%)0 (100)0 (100)TreatmentPDN, n (%)3 (75)11 (100)HCQ, n (%)4 (100)11 (100)MMF, n (%)4 (100)11 (100)RTX, n (%)0 (0)2 (18)Figure 1.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

BackgroundPediatric Sjögren’s syndrome (pSS) is a rare disorder that is often diagnosed late due to the lack of validated diagnostic criteria and validated biomarkers. The pathogenesis is largely unknown, but there is evidence of involvement of both the innate and adaptive branch of the immune system. Immunological overactivity is central in the pathogenesis of pSS. Several studies showed the presence of B cells abnormalities in patients with SS with an expansion of naïve B cells and a decrease in the frequency of memory B cells.ObjectivesWe set out to investigate the distribution of B cell subsets and B cell cytokines in patients with pSS at disease onset and at follow up visits.MethodsA monocentric retrospective cohort study was conducted on 23 patients with pSS enrolled at the department of Rheumatology of Bambino Gesù Children’s Hospital. Serum levels of CXCL13 and BAFF were analyzed by ELISA. B cell phenotype was assessed on peripheral blood mononuclear cells (PBMCs) by flow cytometry. Systemic disease activity was evaluated by ESSDAI (EULAR Sjogren’s syndrome disease activity index) and Clinical-ESSDAI score (Clin-ESSDAI), according to 2020 EULAR recommendations; active disease was defined by ClinESSDAI ≥1 and remission by ClinESSDAI=0. As controls we selected age-matched people with no diagnosis of pSS or any other systemic autoimmune disease.ResultsSerum levels of CXCL13 and BAFF were significantly higher in patients with pSS than the control group (p<0.05) (Figure 1A). We correlated levels of biomarkers with clinical and laboratory parameters: we observed a positive correlation between hypergammaglobulinemia and BAFF (rho=+0,80). Analysis of B-cell subsets at disease onset revealed the expansion of a population of atypical memory B cells (p=0,00049) and a reduction in lgM memory B cells (p=0,0034) compared to the control group (Figure 1B). We then compared the distribution of B cell subpopulations at disease onset (pre) with samples obtained at follow-up (post) for each patient no significant differences were observed (Figure 1B). We also investigated the distribution of Tfh cells in patients with pSS and we observed a significant expansion of CXCR5-PD1hi Tfh (Figure 1B).ConclusionPatients with pSS showed high levels of CXCL13 and BAFF at disease onset. Alteration in B cell subsets are present in patients with pSS compared to controls, with an expansion of atypical memory B cells and Tfh. The B cell abnormalities are not affected by treatment. Our data confirm an hyperactivation of the humoral immune system in patients with pSS and provide evidence for their development as biomarkers and to develop new therapeutic strategies aimed at controlling B cell hyperactivation in pediatric patients with SS.Figure 1.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

BackgroundSystemic lupus erythematosus (SLE) is a systemic autoimmune disease which leads to inflammation and organ damage caused by immune complex deposition. Classically, childhood SLE has been considered as a polygenic autoimmune disease; however, a pediatric monogenic lupus-like phenotype (LL) is emerging due to the recent recognition of several related novel high-penetrance gene variants. This fact associated to the high degree of concordance among monozygotic twins, supports the importance of genetic background in cSLE pathogenesis.ObjectivesTo identify the presence of variants in gene related to monogenic lupus and their relationship with clinical manifestations in cSLE or LL.MethodsA descriptive, observational, cross-sectional study was carried out in children with cSLE or LL. The genetic analysis (Sanger/Clinical Exome Sequencing) was performed from isolated DNA obtained from blood sample.ResultsForty-five children were included in the study. The genetic analysis detected at least one variant in 14 (31.1%) children, 5 (35.7%) with cSLE and 9 (64.3%) with LL. Of those who carry a genetic variant, the median age at disease onset was 11 years (range: 2-16) and 85.7% were female; most of them Caucasians (85.7%). Seven (50%) and 3 (21.4%) out of 14 patients had a positive family and/or a personal history for autoimmune diseases, respectively. Regarding clinical manifestations at onset, musculoskeletal were the most frequent (10 patients, 71.4%), followed by hematological (9 patients, 64.3%), cutaneous (9 patients, 64.3%), constitutional with fever (8 patients, 57.1%), neurological (7 patients, 50%), renal (5 patients, 35.7%), cardiac (3 patients, 21.4%) and pulmonary (2 patients, 14.3%) manifestations. Related to immunological parameters, 13 (92.8%) were ANA positive, 7 (50%) anti-dsDNA, 4 (28.6%) ENA and 3 (21.4%) aPL positive. Both C3 and C4 were low in 8 (57.1.4%) children and isolated C3 levels were low in 4 (28.6%) patients. Among the variants, we found that only two patients who carry a TREX variant showed normal C3 and C4 levels; one of them presented with lupus pernio as reported in literature. Also, we identified the same RNASEH2B (c.868G>A) variant in two siblings with similar phenotypes and TLR7 (c.1520T>C) variant in two siblings of other family. The patient who carried the SHOC2 variant presented polyarthritis and serositis, while the patient with the TNFRSF13B variant onset with a glomerulonephritis. Those manifestations have already been described related to these genes. Variants and phenotypes are detailed in Table 1.ConclusionAround 30% pediatric patients with cSLE or LL showed at least one variant in gene related to monogenic-lupus and some of them with similar phenotypes to those already described. This fact may suggest the genetics potential contribution to the cSLE pathogenesis. Further studies are necessary to confirm these data.Reference[1]Alperin JM, Ortiz-Fernández L, Sawalha AH. Monogenic Lupus: A Developing Paradigm of Disease. Front Immunol. 2018;9:2496.Table 1.GeneDiagnSkin N=6MSK N=8Hematological N=6Fever N=7Lung N=2Cardiac N=3NRL N=7Renal N=5C3/C4 N=12ANA N= 13 DNA N=7ADAR c.16-8 T>CSLExArthralgia↓ Hb, L, N+xxxx↓/↓+/+TNFAIP3 c.2170A>CSLERashx↓ Hb, L, N+xConduction disorderxLN↓/↓+/+RNASEH2Bc.105 107delAATSLExArthralgia↓ PLT, L+xSerositisxx↓/N+/-SHOC2SLERashArthritis↓ Hb, PLT, L+SerositisSerositisxLN type III↓/↓+/+IFIH1 c.2807 + 1G>ASLEOral ulcersArthralgia↓ PLT-xxxx↓/↓+/+TREX c.797A>G; TNFAIP3 c.1405C>GLLRash, oral ulcers, lupus pernioArthralgiax-xxHeadachexN/N+/-DNASE1 c.105G>CLLRashArthritis↓ Hb, L, N, PLT-xxxx↓/N+/-RNASEH2B c.868G>A;C1S c.619C>TLLxArthralgiax-xxHeadachex↓/N-/-RNASEH2B c.868G>A;TLR7 c.3094G>A; STAT5A c.1248C>GLLxArthralgiax+xxHeadachex↓/N+/-TREX1c.341G>ALLxxx-ILDxWMIxN/N+/-TNFRSF13B c.41G>ALLRashxx+xxxMP GMN↓/↓+/+TLR7 c.1520T>CLLRashArthritis↓ PLT+xxTEMP GMN↓/↓++TLR7 c.1520T>CLLRashx↓ N+xxSNC calcificationsx↓/↓+/-C2 c.719G>TPEPD c.703G>ACYBB c.593G>ALLRashArthritis↓ Hb, L+xxSNC calcificationsMP GMN↓/↓+/+Acknowledgements:NIL.Disclosure of InterestsNone Declared.