ALBERT

Description: RNA polymerase (Pol) III has a noncanonical role of viral DNA sensing in the innate immune system. This polymerase transcribes viral genomes to produce RNAs that lead to induction of type I interferons (IFNs). However, the genetic and functional links of Pol III to innate immunity in humans remain largely unknown. Here, we describe a rare homozygous mutation (D40H) in the POLR3E gene, coding for a protein subunit of Pol III, in a child with recurrent and systemic viral infections and Langerhans cell histiocytosis. Fibroblasts derived from the patient exhibit impaired induction of type I IFN and increased susceptibility to human cytomegalovirus (HCMV) infection. Cultured cell lines infected with HCMV show induction of POLR3E expression. However, induction is not restricted to DNA virus, as sindbis virus, an RNA virus, enhances the expression of this protein. Likewise, foreign nonviral DNA elevates the steady-state level of POLR3E and elicits promoter-dependent and -independent transcription by Pol III. Remarkably, the molecular mechanism underlying the D40H mutation of POLR3E involves the assembly of defective initiation complexes of Pol III. Our study links mutated POLR3E and Pol III to an innate immune deficiency state in humans.

Description: Type-1 Gaucher disease (GD1) is considered to be non- neuronopathic however recent evidence of neurological involvement continues to accumulate. There is limited evidence of retinal abnormalities in GD1. The purpose of this study was to evaluate the retinal findings of patients with GD1. Thirty GD1 individuals and 30 healthy volunteers between the ages 40–75 years were prospectively enrolled. Macular and optic nerve optical coherence tomography (OCT) scans of both eyes of each patient were performed and thickness maps were compared between groups. Patients with a known neurodegenerative disease, glaucoma, high myopia and previous intraocular surgeries were excluded. It was shown that patients with GD1 presented with higher incidence of abnormal pRNFL OCT scan and showed significantly thinner areas of pRNFL and macular ganglion cell complex (GCC) when compared to a healthy control population. Changes in retinal thickness were not associated with GD1 genotype, treatment status, disease monitoring biomarker (lyso-Gb1) and severity score index (Zimran SSI). Further investigations are needed to determine whether these findings possess functional visual implications and if retinal thinning may serve as biomarker for the development of future neurodegenerative disease in this population.

Description: The challenges in the diagnosis, prognosis, and monitoring of Gaucher disease (GD), an autosomal recessive inborn error of glycosphingolipid metabolism, can negatively impact clinical outcomes. This systematic literature review evaluated the value of glucosylsphingosine (lyso-Gb1), as the most reliable biomarker currently available for the diagnosis, prognosis, and disease/treatment monitoring of patients with GD. Literature searches were conducted using MEDLINE, Embase, PubMed, ScienceOpen, Science.gov, Biological Abstracts, and Sci-Hub to identify original research articles relevant to lyso-Gb1 and GD published before March 2019. Seventy-four articles met the inclusion criteria, encompassing 56 related to pathology and 21 related to clinical biomarkers. Evidence for lyso-Gb1 as a pathogenic mediator of GD was unequivocal, although its precise role requires further elucidation. Lyso-Gb1 was deemed a statistically reliable diagnostic and pharmacodynamic biomarker in GD. Evidence supports lyso-Gb1 as a disease-monitoring biomarker for GD, and some evidence supports lyso-Gb1 as a prognostic biomarker, but further study is required. Lyso-Gb1 meets the criteria for a biomarker as it is easily accessible and reliably quantifiable in plasma and dried blood spots, enables the elucidation of GD molecular pathogenesis, is diagnostically valuable, and reflects therapeutic responses. Evidentiary standards appropriate for verifying inter-laboratory lyso-Gb1 concentrations in plasma and in other anatomical sites are needed.

Description: The introduction of disease specific therapy for patients with type 1 Gaucher disease (GD) was a revolution in the management of patients, but not without significant cost to the patient and to society. The management of mildly effected patients is still debated, and reviews about GD as well as chapters in textbooks fail to emphasize the fact that some patients may remain untreated for many years without any GD-related complications. Patient reported outcome measures (PROMs) were developed as a way to ascertain patients' views of their symptoms, their functional status, and their health-related quality-of-life (HRQoL). In this study, we evaluated the responses to a GD -specific PROM of untreated patients with GD1 and compared them to patients on GD-specific therapy. Methods: A PROM survey was developed for GD including 15 questions; six Point Verbal Response Scale regarding the last month and nine Visual Analogue Scales (VAS) from 0-10 regarding the last week (Elstein D, et al. Molecular Genetics and Metabolism 2019;126:S52). The PROM survey was proven to be accurate in encompassing disease-specific patient concerns. A Hebrew translated version of the GD-PROM was sent via mobile phone survey to 400 adult patients with type 1 GD followed in our Gaucher Unit. Clinical data and treatment status were extracted from the clinical charts. T-test and Mann-Whitney U test were used to compare normally and non-normally distributed data in independent samples, respectively. IBM SPSS version 25 was used for analysis. Results were considered to be statistically significant when two-tailed P-values were ≤0.01. Results: A total of 181 patients responded (45% response rate) of whom 65 (36%) were followed for at least 5 years in our unit without receiving GD specific therapy, i.e. enzyme replacement therapy (ERT) and/or substrate reduction therapy (SRT). The median (range) age of patients, 49 (20-91) years, was not significantly different between treated and untreated patients. The percentage of patients with the N370S/N370S genotype was significantly higher in untreated patients [55/65 (85%)] compared to treated patients [67/116 (57%)]. Significantly more treated patients reported that GD had restricted their education/job (38, 34%) and fun activities (29,25%) compared to untreated patients, (4, 6.5%) and (2, 3%), respectively. Compared to untreated patients, treated patients were more worried to be an emotional burden on others [27 (23%) vs. 3 (5%)], of being financial burden on others [57 (50%) vs. 16 (25%)] and more concerned regarding the risk of bone disease [82 (74%) vs. 26 (40%)], and the risk of Parkinson disease [72 (64%) vs. 27 (42%)]. Treated patients had a significantly higher score on VAS for questions on swollen abdomen, fatigue, physical weakness, severity of bone pain and worry regarding the future over the past week compared to untreated patients (Table 1). Patients concern regarding the risk for cancer (32%) and VAS score for a question on depression were similar between groups. Conclusion:The GD-specific PROM survey shows that asymptomatic or mildly affected untreated patients with GD1 have good functional status and HRQoL, supporting our practice that not all patients with GD1 require disease-specific therapy. Still, we advise a periodic (annual or bi-annual) follow-up, preferably at a referral center. Inclusion of GD-specific PROMs in the periodic assessments is important for better understanding patients' perspectives. It is important to note that mildly affected and asymptomatic patients are mainly found among Ashkenazi Jews and from this aspect our cohort reflects patients' populations in Israel, USA, UK, etc. but less relevant to non-Jewish and particularly to Asian cohorts. With the expected increase in early diagnosis via parental and/or newborn screening the understanding that not all subjects diagnosed with GD needs disease-specific therapy is all the more important. Despite the expected differences between the more severely affected treated patients and the by definition milder untreated ones, still a high percentage of the treated patients show good HRQoL parameters, reflecting the overall success of ERT/SRT. Larger cohorts and further analysis will evaluate potential predictors for differences in PROMs within the treatment group. Disclosures Revel-Vilk: Sanofi: Honoraria, Other: Travel, Research Funding; Pfizer: Honoraria, Other: Travel, Research Funding; Takeda: Honoraria, Other: Travel, Research Funding; Prevail therapeutics: Honoraria, Other: Travel, Research Funding. Zimran:Prevail Therapeutics: Consultancy; TAKEDA: Honoraria; Centogene: Other: research grant; Targeted Cell Therapies: Consultancy; Pfize: Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Bio-events: Honoraria.

Description: We report a family with new autosomal recessive disorder associated with macrothrombocytopenia which is different from all reported hereditary macrothrombocytopenia. Case report: The affected family members report mild bruising and epistaxis. The females’ family members report, in addition, severe menorrhagia and recurrent episodes of hemorrhagic corpus luteum. Two family members with macrothrombocytopenia and two without macrothrombocytopenia report hearing loss and cataracts. Methods: The study protocol was approved by our local Ethics committee and written consent was obtained to prepare and store DNA. Platelet morphology, FACS analysis, protein analysis and genome-wide association studies were used to characterize the disease and locate the genetic abnormality. Results: In a consanguineous family from Arab origin we identified 5 affected individuals (3 males and 2 females from three families) with platelet count ranging around 4-20 x109/L. Their shared grandparent died of post trauma bleeding (no data on his platelet count). Their parents have normal platelet count and platelet morphology. Blood film of effected family members show macrothrombocytopenia with occasional giant platelets. By flow cytometry CD41 and CD42b expressions were around 60% in effected family members and CD62 expression was around 20-30% in resting and thrombin-activated platelets (Figure 1). Immunoflurescence of platelets shows normal distribution of non-muscular mysoin IIA, GPIbIX, dense granules and lysosome markers Lamp-1, Lamp-2 and the dense granule CD63. Platelets diffusely stained for P-selectin and stained very low for VWF and thrombospondin. GPIIbIIIa staining revealed a ring distribution at the outer cell membrane (Prof. Andreas Greinacher, Greifswald, Germany). Transmission electron microscopy imaging shows abnormal distribution of platelet granules, with suspected protein accumulation in the cytoplasm (Figure 2). Protein analysis of platelet extracts by SDS-PAGE separation shows different band patterns in the effected individuals compared to controls (Figure 3). Mass spectrometry analysis of a representative band identified five candidate proteins that their over-expression may cause the defect in platelets organelles distribution and the defect of their function. Through whole genome analysis three large homogenous regions were found. To further narrow the search SNP analysis was added between two effected members. The search was narrowed to an area in chromosome 9p and centromere 9q with 201 candidate genes. Discussion: We present a large consanguineous family with previously non reported autosomal recessive macrothrombocytopenia. The functional and morphological pattern show dual platelet population. Further work is warranted. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Patients with Wiskott-Aldrich syndrome (WAS) including X-linked thrombocytopenia (XLT) have microthrombocytopenia, and hemorrhage is a major problem. Current management options in WAS/XLT patients include splenectomy, human stem cell transplant (HSCT) and gene therapy. In this study, we asked whether eltrombopag, a thrombopoietin mimetic, would increase platelet counts, improve platelet function, and/or reduce bleeding in WAS/XLT patients. Methods In 9 WAS/XLT patients and 8 age-matched healthy control subjects, flow cytometry was used to assess platelet function by surface expression of activated GPIIb-IIIa (reported by PAC1) and P-selectin in whole blood after stimulation with low and high concentrations of ADP or thrombin receptor activating peptide (TRAP), and by annexin V binding (a measure of surface phosphatidylserine) in platelet-rich plasma after stimulation with convulxin. Eltrombopag was administered to 5 WAS and 3 XLT patients (50 mg in 2 adults, and 1 mg/kg in 6 children up to 75 mg/day) with a goal platelet count ≥50k. Results High concentration ADP- or TRAP-induced PAC1 mean fluorescence intensity (MFI) was significantly reduced in WAS/XLT patients compared to healthy controls (Figure). Platelet surface P-selectin MFI in response to TRAP was also significantly reduced. In contrast, annexin V binding to platelets was not different between WAS/XLT and controls. As expected, platelet size of WAS/XLT patients was smaller than controls. WAS protein (which is deficient in WAS/XLT), is important for cytoskeletal movement and could therefore be involved in trafficking of surface proteins. However, surface expression of activated GPIIb-IIIa and P-selectin were no longer different in WAS/XLT patients vs. controls when corrected for size by platelet surface CD41 MFI. In 3 WAS/XLT patients whose platelet count improved on eltrombopag, platelet function did not improve. The table summarizes the results of eltrombopag treatment in 5 responders (2 WAS, 3 XLT patients) and 3 non-responders (3 WAS patients). Comparison of baseline, peak and change in immature platelet fraction in 5 WAS/XLT responders to eltrombopag vs. 7 pediatric chronic immune thrombocytopenia (ITP) patients responding to eltrombopag showed a significant decrease in all three measures, suggesting that platelet production in WAS/XLT patients is more difficult to increase than in ITP patients. Long term eltrombopag use in WAS/XLT patients showed no tachyphylaxis, transaminitis or induction of malignancy. Conclusions 1) Baseline platelet function in WAS/XLT is reduced compared to healthy age-matched controls, as measured by agonist-induced platelet surface activated GPIIb-IIIa and P-selectin. 2) This reduction is proportional to the reduced platelet size in WAS/XLT compared to controls. 3) In contrast, annexin V binding (a measure of platelet procoagulant activity) showed no differences between WAS/XLT and controls. 4) Eltrombopag has beneficial effects on the thrombocytopenia and bleeding, but not platelet function, in the majority of WAS/XLT patients. 5) This eltrombopag-induced reduction in bleeding is presumably primarily the result of the increased platelet count, but it was also observed in 2 eltrombopag “non-responders” (i.e. patients whose platelet counts did not increase after eltrombopag). 6) The production of new platelets with eltrombopag is less in WAS/XLT than in ITP. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Michelson:Sysmex: Honoraria. Bussel:GlaxoSmithKline: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

Description: Key Points Deficiency of TPP2 is associated with Evans syndrome and viral infection susceptibility. TPP2 deficiency links premature immunosenescence of T and B cells with severe autoimmunity.

Description: Background Studies on pediatric norms to be used for upper extremity post thrombotic syndrome (PTS) assessment have been conducted in North America, but these norms have never been evaluated in non-North American children. A validation study to assess the validity and reliability of the two currently available pediatric PTS assessment instruments, i.e. the Modified Villalta Score (MVS) and the Manco-Johnson Instrument (MJI), on a non-North American convenience sample of healthy children is necessary in order to account for differences that may exist across cultural boundaries. Aim To determine pediatric norms for upper extremity PTS assessment in a non-North American cohort and to determine normal discrepancies in arm circumference, which in American children have been found to be ≤1 cm in the MJI (1) and 1.0 cm is a more applicable and reliable measurement than a 3% cut-off. The presence of signs and symptoms of PTS in the upper extremity of healthy children questions the specificity of the current available PTS assessment tools for upper extremity in children. 1. Goldenberg NA, Pounder E, Knapp-Clevenger R, Manco-Johnson MJ. Validation of Upper Extremity Post-Thrombotic Syndrome Outcome Measurement in Children. J Pediatr. 2010 Nov; 157(5):852–5. 2. Boulden BM, Crary SE, Buchanan GR, Journeycake JM. Determination of pediatric norms for assessment of upper venous system post-thrombotic syndrome. J Thromb Haemost. 2007 May; 5(5): 1077–9. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Anemia is a global public health problem affecting both developing and developed countries. Most literature on severe anemia in children is reported from developing countries, where malaria infection and malnutrition are the main causative factors. According to the world health organization (WHO) report, in Israel, as in many western countries, anemia is considered a mild public health problem (1). The aim of this study it to analyze the causative factors and management of severe anemia in children living in a country where anemia is not considered a major health problem. Methods: The electronic hospital records of all children (≤16 years) with hemoglobin 〈 7.0 g/dl presenting to two largest Emergency Department (ED) in Jerusalem from 2008 to 2013 were retrospectively reviewed. Data including demographic characteristics, presenting signs and symptoms, laboratory findings, causal factors and management were extracted from medical records and analyzed. Cases secondary to surgical bleeding, chemotherapy, known chronic anemia or developing during prolonged admission were excluded. The diagnosis of iron deficiency anemia was based on low plasma iron and ferritin levels with compatible blood smear, when available. If unavailable, the diagnosis was based on the findings of low MCV and MCH for age with increased RDW. Results: A total of 227 children (female 123, 54%) at a median (range) age at of 4.5 years (1 day-15.5 years) were included. The median (range) hemoglobin at presentation was 6.3 (2.6-6.9) g/dl. In the entire cohort the most common etiologies for severe anemia were iron deficiency anemia (IDA) (38%) and acute hemolysis; autoimmune (AIHA) (8.5%) or G6PD deficiency (8%). The distribution of etiologies for severe anemia differed between infants, pre-school age, school age and adolescents (Figure 1). While the diagnosis of IDA was the most common in all age groups, some diagnoses such as G6PD deficiency and congenital anemia were more common in the younger age groups (up to 6 years) compared to the older ages. Children diagnosed with IDA were significantly older; median (range) 9.5 years (4 months-15.5 years), compared to children with other diagnoses, 3 years (1 day-15.5 years) (p