Pathogenesis of Autoimmune Cytopenias in Inborn Errors of Immunity Revealing Novel Therapeutic Targets

Autoimmune diseases are usually associated with environmental triggers and genetic predisposition. However, a few number of autoimmune diseases has a monogenic cause, mostly in children. These diseases may be the expression, isolated or associated with other symptoms, of an underlying inborn error of immunity (IEI). Autoimmune cytopenias (AICs), including immune thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA), autoimmune neutropenia (AN), and Evans' syndrome (ES) are common presentations of immunological diseases in the pediatric age, with at least 65% of cases of ES genetically determined. Autoimmune cytopenias in IEI have often a more severe, chronic, and relapsing course. Treatment refractoriness also characterizes autoimmune cytopenia with a monogenic cause, such as IEI. The mechanisms underlying autoimmune cytopenias in IEI include cellular or humoral autoimmunity, immune dysregulation in cases of hemophagocytosis or lymphoproliferation with or without splenic sequestration, bone marrow failure, myelodysplasia, or secondary myelosuppression. Genetic characterization of autoimmune cytopenias is of fundamental importance as an early diagnosis improves the outcome and allows the setting up of a targeted therapy, such as CTLA-4 IgG fusion protein (Abatacept), small molecule inhibitors (JAK-inhibitors), or gene therapy. Currently, gene therapy represents one of the most attractive targeted therapeutic approaches to treat selected inborn errors of immunity. Even in the absence of specific targeted therapies, however, whole exome genetic testing (WES) for children with chronic multilineage cytopenias should be considered as an early diagnostic tool for disease diagnosis and genetic counseling

Development and initial validation of the ONCOREUM score to differentiate childhood cancer with arthropathy from juvenile idiopathic arthritis

Objective: To develop and validate a weighted score, the ONCOREUM score, that aids physicians in differentiation of cancer with arthropathy from juvenile idiopathic arthritis (JIA). Study design: Data were extracted from the ONCOREUM Study, a multicenter, cross-sectional investigation aimed at comparing children with cancer and arthropathy to children with JIA. Three statistical approaches were applied to develop the ONCOREUM score and assess the role of each variable in the diagnosis of cancer with arthropathy, including 2 approaches based on multivariable stepwise selection (models 1 and 2) and 1 approach on a Bayesian model averaging method (model 3). The β coefficients estimated in the models were used to assign score points. Considering that not missing a child with cancer is a mandatory clinical objective, discriminating performance was assessed by fixing sensitivity at 100%. Score performance was evaluated in both developmental and validation samples (representing 80% and 20% of the study population, respectively). Results: Patients with cancer and arthropathy (49 with solid tumors and 46 with hematologic malignancies without peripheral blasts) and 677 patients with JIA were included. The highest area under the receiver operating characteristic (ROC) curve (AUC) in the validation data set was yielded by model 1, which was selected to constitute the ONCOREUM score. The score ranged from -18 to 21.8, and the optimal cutoff obtained through ROC analysis was -6. The sensitivity, specificity, and AUC of the cutoff in the validation sample were 100%, 70%, and 0.85, respectively. Conclusions: The ONCOREUM score is a powerful and easily applicable tool that may facilitate early differentiation of malignancies with articular complaints from JIA

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis

<div><p>The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.</p></div

AR binds ARSO-Sox2OT chromatin region and allows Sox2OT transcription.

<p><b>(a)</b> AR consensus sites in the chromosome 3 and in Sox2OT ncRNA. Grey arrows indicate DNA region tested by ChIP with ChIP-grade antibodies (ChIP site); vertical red lines indicate AR consensus site (ARE); green arrows indicate the sequence on Sox2OT RNA tested by RIP (RIP site); <b>(b)</b> Representative images showing Chromatin Immuno Precipitation of AR and RNA Polymerase II. The images show PCR of antibody–precipitated E12.0 forebrains and eNSCs chromatins with primers amplifying ARSO-Sox2ot region. The lower panels show the quantification of AR and RNA Pol ChIPs. Values are mean±SEM of ratios between PCR signal intensity of the AR and RNA Pol II antibodies–precipitated sample and input chromatin (IN). Notably, RNA pol II binds ARSO-Sox2ot sequence in an AR-dependent manner. For RNA pol II in E12.0 forebrains: control = 0.51±0.06, Bicalutamide = -0.18±0.06, t-test p = 0.0023. For AR in E12.0 forebrains: control = 0.51±0.13, Bicalutamide = -0.26±0.08, t-test p = 0.0003. For RNA pol II in embryonic NSCs: control = 1.36±0.21, Bicalutamide = 1.28±0.07, t-test p = 0.0002. For AR in embryonic NSCs: control = 1.28±0.07, Bicalutamide = -0.15±0.09, t-test p = 0.000017. Results derived from four independent experiments. Data were analyzed by two-tailed t-test. <b>(c)</b> RNA Immunoprecipitation (RIP) assay of eNSCs and E12.0 forebrains. RNA was subjected to IP assays with anti-AR and anti-Rpb1 CTD phosphorylated at Serine 2 and Serine 5 (RNA Pol II) antibodies or normal rabbit IgG as described in Materials and Methods section. RNA immunoprecipitates and input lysate RNAs were reverse transcription-PCR (RT-PCR)–amplified to measure the abundance of Sox2OT RNA present in the eNSCs and forebrains (control). Agarose gel of RT-PCR products from RIP and input (upper panel). Molecular weight marker sizes (base pair lengths; bps) are shown at the right. Values are mean± SEM of ratios between PCR signal intensity of the AR and RNA Pol II antibodies–precipitated sample and input (lower panel). For RNA pol II in E12.0 forebrains: control = 1.66±0.26, Bicalutamide = -0.60±0.27, t-test p = 0.0084. For AR in E12.0 forebrains: control = 0.51±0.09, Bicalutamide = -0.20±0.16, t-test p = 0,016. For RNA pol II in embryonic NSCs: control = 0.75±0.18, Bicalutamide = 0.16±0.11, t-test p = 0.032. For AR in embryonic NSCs: control = 0.85±0.07, Bicalutamide = 0.13±0.06, t-test p = 0.0002. Results are average of four independent experiments. Data were analyzed by two-tailed t-test. Lane RT+ contains an aliquot of the PCR sample. In the RT- lane, the reverse transcriptase was omitted from the RT reaction. Input are 20% of total RNA. IgG: rabbit IgG as negative control. Bicalutamide treatment were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180579#pone.0180579.g001" target="_blank">Fig 1</a>.</p

AR antagonist Bicalutamide reduces AR and Sox2OT levels in mouse E12.0.

<p>AR antagonist Bicalutamide reduces AR and Sox2OT levels in mouse E12.0.</p

Table2_“CHildren with Inherited Platelet disorders Surveillance” (CHIPS) retrospective and prospective observational cohort study by Italian Association of Pediatric Hematology and Oncology (AIEOP).xls

AbstractBackgroundInherited thrombocytopenias (ITs) are rare congenital bleeding disorders characterized by different clinical expression and variable prognosis. ITs are poorly known by clinicians and often misdiagnosed with most common forms of thrombocytopenia.Material and methods“CHildren with Inherited Platelet disorders Surveillance” study (CHIPS) is a retrospective – prospective observational cohort study conducted between January 2003 and January 2022 in 17 centers affiliated to the Italian Association of Pediatric Hematology and Oncology (AIEOP). The primary objective of this study was to collect clinical and laboratory data on Italian pediatric patients with inherited thrombocytopenias. Secondary objectives were to calculate prevalence of ITs in Italian pediatric population and to assess frequency and genotype–phenotype correlation of different types of mutations in our study cohort.ResultsA total of 139 children, with ITs (82 male - 57 female) were enrolled. ITs prevalence in Italy ranged from 0.7 per 100,000 children during 2010 to 2 per 100,000 children during 2022. The median time between the onset of thrombocytopenia and the diagnosis of ITs was 1 years (range 0 - 18 years). A family history of thrombocytopenia has been reported in 90 patients (65%). Among 139 children with ITs, in 73 (53%) children almost one defective gene has been identified. In 61 patients a pathogenic mutation has been identified. Among them, 2 patients also carry a variant of uncertain significance (VUS), and 4 others harbour 2 VUS variants. VUS variants were identified in further 8 patients (6%), 4 of which carry more than one variant VUS. Three patients (2%) had a likely pathogenic variant while in 1 patient (1%) a variant was identified that was initially given an uncertain significance but was later classified as benign. In addition, in 17 patients the genetic diagnosis is not available, but their family history and clinical/laboratory features strongly suggest the presence of a specific genetic cause. In 49 children (35%) no genetic defect were identified. In ninetyseven patients (70%), thrombocytopenia was not associated with other clinically apparent disorders. However, 42 children (30%) had one or more additional clinical alterations.ConclusionOur study provides a descriptive collection of ITs in the pediatric Italian population.</p