ALBERT

Description: The authors provide evidence of a novel association between transcription factor IKZF5, one of the IKAROS family proteins, and thrombocytopenia with decreased alpha granules, thus significantly extending our understanding of the gene defects leading to inherited thrombocytopenia.

Description: Key Points The A1 domain of VWF contains a cryptic binding site that plays a key role in regulating macrophage binding and clearance. The N-linked glycans presented at N1515 and N1574 within the A2 domain of VWF modulate macrophage-mediated clearance.

Description: ABO blood group is an important determinant of plasma von Willebrand factor antigen (VWF:Ag) levels, with lower levels in group O. Previous reports have suggested that ABO(H) sugars affect the susceptibility of VWF to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 repeats-13) cleavage. To further test this hypothesis, we collected plasma from individuals with the rare Bombay blood group. VWF:Ag levels were significantly lower in Bombay patients (median, 0.69 IU/mL) than in groups AB, A, or B (P 〈 .05) and lower than in group O individuals (median, 0.82 IU/mL). Susceptibility of purified VWF fractions to recombinant ADAMTS13 cleavage, assessed using VWF collagen-binding assay (VWF:CB), was increased in Bombays compared with either group O or AB. Increasing urea concentration (0.5 to 2 M) increased the cleavage rate for each blood group but eliminated the differences between groups. We conclude that reduction in the number of terminal sugars on N-linked glycan increases susceptibility of globular VWF to ADAMTS13 proteolysis and is associated with reduced plasma VWF:Ag and VWF:CB levels.

Description: Investigation of three families with von Willebrand disease showed that haemorrhagic symptoms were associated with disproportionately reduced collagen binding activity whilst Ristocetin co-factor activity was commensurate with antigen and multimeric analysis was normal. Genetic analysis revealed heterozygosity for two novel mutations in two of the families: W1745C in exon 30 and S1783A in exon 31. In the third family the affected individuals were heterozygous for a previously-described mutation: S1731T in exon 30 but two unaffected individuals also carried this mutation. All three mutations lie in the A3 domain containing the main collagen binding site in VWF. In patients’ samples VWF:CB activity was measured using human type I and type III collagen. Patients heterozygous for W1745C and S1731T showed a reduction in binding to both collagens but more marked reduction in binding to type III collagen. Heterozygosity for S1731T resulted in mild impairment of type I collagen binding but normal binding to type III collagen. Site-directed mutagenesis was used to generate vectors containing the three mutations (S1731T, W1745C and S1783A) and also one containing a W1745A mutation. Mutated VWF was expressed in HEK293T cells both singly and in co-transfection with a wild-type VWF (wtVWF) vector. All VWF mutants were expressed at a similar rate to wtVWF. Multimeric analysis demonstrated that all the mutants had a similar multimeric structure compared to recombinant wtVWF. However recombinant-wtVWF (wtVWF) had a lower collagen binding to VWF antigen ratio (CB:Ag) compared to plasma VWF (0.39 type I collagen and 0.45 type III collagen vs 〉0.7 for plasma VWF). This is most likely due to the slight shift towards lower molecule weight multimers seen with recombinant VWF. CB:Ag ratios for the recombinant VWF showed the same pattern of binding to collagen type I and III as the clinical samples. The W1745A mutant demonstrated a similar CB:Ag ratio to W1745C. Kinetic analysis of binding to type I collagen demonstrated that W1745C, W1745A and S1783A did not bind and that S1731T bound with significantly less affinity compared to wtVWF (KD,app 27.1 ± 0.5nM and 7.3 ± 0.8nM respectively). Analysis of binding to type III collagen demonstrated that W1745C and W1745A both bound with ~ 8-fold reduced affinity (KD,app 16 ± 2.6nM and 21.3 ± 6.3nM) but wtVWF and S1731T bound with similar affinity, (KD,app 2.0 ± 0.1nM and 3.7 ± 0.85nM respectively). Analysis of the crystal structure of the VWF A3 domain showed that W1745 may interact with Y1780 and we noted the mutation Y1780A has also been shown to significantly reduce collagen binding. Measurement of free thiols present in VWF demonstrated that the new cysteine residue in W1745C is not involved in disulphide bond formation. These results indicate that it is the loss of W1745 rather than the creation of a new cysteine residue that is responsible for the loss of collagen binding activity. We therefore hypothesised that W1745 and Y1780 participate in an internal aromatic interaction that helps to maintain the structural configuration of A3. We sought confirmation by expressing another mutant; W1745F, replacing the tryptophan with another aromatic amino acid. As predicted this did not significantly affect collagen binding. In conclusion, our findings demonstrate that type 2 VWD may be arise from mutations in A3 causing abnormal collagen binding without other functional defects or abnormalities in multimer formation. This type of VWD may be under-recognised unless laboratories measure binding to both types I and III collagen. Mutations in A3 yield insights into the structural requirements for collagen binding may have differential effects on binding to collagen types I and III and can result in variable clinical phenotypes. Some mutations may not be consistently associated with bleeding symptoms.

Description: Introduction Despite significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN) and the identification of high molecular risk (HMR) genes (i.e. ASXL1, EZH2, IDH1 and IDH2 genes), the mechanisms by which different cell types predominate in the different disease subtypes and their implications for prognosis remain uncertain. Given the recently described association of senescence and fibrosis in a number of pathologies by Menoz-Espin et al, we hypothesized that genes implicated in oncogene-induced senescence (OIS) and senescence associated secretory phenotype (SASP) may contribute to the pathogenesis of these neoplastic bone marrow disorders that frequently show evidence of fibrosis. Specifically, we were interested in the gene expression levels in different disease subtypes, at a cell-type level, and whether these patterns of differential expression were distinct from the transforming JAK-STAT pathway and the HMR genes. Aim To elucidate the role of OIS and SASP genes in the pathogenesis of MPN subtypes by determining the differential expression of the genes in specific cell types in patients with MPN. Methods We performed gene expression profiling on normal controls (NC) and patients with MPN who were diagnosed with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) according to the 2008 WHO diagnostic criteria. Two cohorts of patients, the patient and validation cohorts, from 3 tertiary-level hospitals were recruited prospectively over 3 years. Peripheral blood samples were taken and sorted into polymorphonuclear cells (PMN), mononuclear cells (MNC) and T cells. RNA was extracted from each cell population. Gene expression profiling of the human transcriptome was performed using microarray and RNA sequencing on the patient and validation cohorts respectively. Gene expression analyses (GEA) were performed on 4 sets of genes derived from publicly available or custom derived gene set enrichment analysis: 92 OIS genes, 88 SASP genes (Gil et al), 4 HMR genes, and 126 genes associated with JAK-STAT pathway. Gene expression levels for each cell type in each disease were compared with NC to obtain the differential expression of the genes. RNA-seq analysis of samples from the validation cohort was used to validate the microarray results from the patient cohort. Results Twenty-eight patients (10 ET, 11 PV and 7 PMF) and 11 NC were recruited into the patient cohort. Twelve patients (4 ET, 4 PV and 4 PMF) and 4 NC were recruited into the validation cohort. After combination of the microarray and RNA-seq datasets, GEA of the OIS genes revealed the differential expressions of MCTP1 and SULT1B1 genes by PMN in PV but of none in PMF. In contrast, the BEX1 gene was identified as differentially expressed by MNC in PMF but none in PV. GEA of the SASP genes revealed differential expression of THBS1 gene by MNC in PMF but of none in PV. None of the SASP genes were differentially expressed by PMN in either PV or PMF. No differentially expressed genes were identified by PMN or MNC in ET, or by T cells in any of the diseases. Notably, GEA of the HMR genes and genes associated with the JAK-STAT pathways did not show any differential expression in any disease subtype by any cell type. Conclusions We have found strikingly distinct patterns of differential expression of senescence associated genes by PMN (in PV) and MNC (in PMF). These results provide a novel insight into the mechanisms underlying the different phenotype of the MPN subtypes and also to the cells responsible for mediating the differences. The lack of differential expression of OIS and SASP genes in ET may reflect the milder clinical phenotype of the disease. Although mutations in the HMR genes are associated with poor prognosis in PMF, the lack of differential expression in these genes and genes associated with the JAK-STAT pathway is in keeping with their mutated status and suggests that they give rise to the disease phenotypes via altering downstream expression of genes associated in other pathways such as the senescence pathways studied here. Further studies are warranted to investigate the role of these genes and the pathways involved in senescence at a cell-type specific level in order to gain further insight into how they can potentially give rise to the various disease phenotypes in MPN and unmask potential therapeutic targets. Disclosures Aitman: Illumina: Honoraria.

Description: Abstract 30 VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity. Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p

Description: Introduction 17% of children with sickle cell disease (SCD) between the ages of 6 and 16 could have silent infarcts1. Depending on the area of the brain affected silent infarcts can cause problems with attention, coordination, visual-motor speed and executive function. Children with SCD in the UK do not receive routine MRI scans. Subtle defects in cognition can be assessed with neuropsychometric testing which involves multiple tests assessing many areas of cognition. However, testing is limited to those with known neurological deficits due to lack of funding and shortage of specialist staff. There is a need for a robust screening tool for assessment of cognition, which could identify children for further specialist testing. The Cogstate battery is computer-based program that assesses cognition and has been used in several clinical settings, both adult and paediatric2-3. The Cogstate battery is reported to be culturally neutral and is available in several languages. Additionally the Cogstate battery is free from practice effects and so could be used as an annual assessment tool in order to identify any declines as early as possible. The Cogstate battery has not yet been used to assess cognition in children with SCD. Research Objectives The aim of this study was to assess the feasibility of using the Cogstate Battery as a tool for the assessment of cognition in children with SCD. It was hypothesised that the Cogstate battery would be easy to use within this setting and would be acceptable to patients, parents and assessors. Methods Eight clinically well children, aged 10-17 with SCD were recruited through St Mary's Hospital paediatric haematology outpatient clinics. The Cogstate software was downloaded onto a Windows laptop computer and an anonymous profile was created for each child before testing. A battery of 6 tests (Table 1) was created aiming to assess a range of cognitive domains within a reasonable amount of time. Every child completed the battery of tests once, which included a short practice before each test. After testing each patient was asked to give an opinion of how they found the tests. Upon completion of the test the patients' results were uploaded to the Cogstate website which generated a test report and a case report form. A mark was given for each test and a score of over 90 represents normal cognition in the area tested, 81-90 represents mild impairment and below 81 represents impairment.Table 1.Tests used in the Cogstate battery and corresponding cognitive domains assessedTest NameCognitive Domain TestedContinuous paired associate learning (CPAL)Paired associate learningDetection (DET)Psychomotor functionGroton maze learning test (ME)Executive functionGroton maze learning test-delayed recall (ME)Delayed recallIdentification (IDN)AttentionOne card learning (OCL)LearningOne-back memory (ONBA)Working memory Results 8 patients completed the battery, taking on average 29 minutes (Table 2). The battery was easy to carry out and although some children reported it as boring, they all finished the tests without distress. The test report generated by the Cogstate website allowed results to be analysed quickly and with ease. An overall score from each test is clearly indicated. The Continuous Paired Associate Learning test was not displayed as part of the test report as there was insufficient normal data to draw conclusions from the results within the age group tested. Table 2. Summary Report of 8 patients tested PatientID DET(Psychomotor function) IDN(Attention) OCL(Learning) ONBS(Processing speed) ONBA(Working memory) ME(Executive function & delayed recall) 0001 87 75 82 78 83 99 0002 106 105 94 90 117 99 0003 96 101 95 95 117 98 0004 76 76 94 81 94 93 0005 86 84 98 81 89 89 0006 98 104 97 97 94 111 0007 94 90 91 83 101 86 0008 91 88 108 90 96 95 Conclusion The Cogstate battery is a feasible tool for paediatric SCD patients and can be undertaken in a clinic setting. This feasibility study will help design a prospective, comparative study of cognition in children with SCD using the Cogstate battery and conventional neuropsychometric assessment and once validated, would be a useful tool to assess cognition and institute timely educational and medical intervention. References: 1. Pegelow J Pediatr. 2002 Mar;140(3):348-54. 2. Hammers, Am J Alzheimers Dis. 2011 Jun;26(4):326-33 3. Harel PLoS One. 2014 Jul 11;9(7):e101750 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1096 Introduction: ITP is an autoimmune disorder characterized by increased platelet destruction and/or decreased platelet production. In addition to risks of bleeding, an increased risk of thrombosis has been identified, despite low platelet counts. Reasons behind this remain unclear. Elevated levels of VWF and decreased levels of ADAMTS13 have been observed in patients with myocardial infarction and ischaemic stroke. Given the interaction of these plasma proteins with platelets, we hypothesized that changes in VWF/ADAMTS13 levels may contribute to the thrombotic risk in patients with ITP. Methods: Adult patients (〉18 years) with a diagnosis of ITP were included. ELISAs were used to measure the concentrations of VWF, ADAMTS13 and IL-6. Platelet count and C-Reactive protein (CRP) levels were recorded. Control samples for VWF and ADAMTS13 were taken from a previous study (n=626) (Andersson et al. Blood 2012; 119(6):1555–60). Published data from a population-based study (Marques-Vidal et al. PLoS One 2011; 6(6): e21002) enabled comparison of IL-6 levels to normal controls. Results: 93 samples were taken from 48 patients with ITP between March-June 2012. All samples were analyzed for VWF:Ag levels. Plasma concentrations of ADAMTS13 and IL-6 were analyzed in 58 and 34 plasma samples respectively. Plasma VWF antigen (VWF:Ag) levels were elevated in patients with ITP compared to control subjects (p=0.0369) (Figure 1). Levels of ADAMTS13 in patients with ITP were lower than those for control subjects (p=0.0050) (Figure 2). Plasma VWF levels did not correlate with platelet counts. Rather high VWF levels were specific to certain patients. Two patients with VWF:Ag levels 〉40 μg/mL had a past history of thrombosis; both had additional co-morbidities. Two further patients with acute disease (one recent acute intracranial bleed and one new relapse of ITP) also had high VWF levels. In these two patients, VWF, ADAMTS13 and platelets change over time (example shown in Figure 3). CRP levels did not correlate with platelet count (p=0.6091) or with VWF:Ag levels (p=0.5170). Median IL-6 levels were raised in patients with ITP (3.785 pg/mL) compared to healthy controls (1.47 pg/ml) (p=

Description: Introduction The discoveries of JAK2 V617F and CALR mutations have significantly improved our understanding of MPN and its subtypes. Somatic JAK2 V617F mutation is present in almost all patients with polycythemia vera (PV) and in 50% to 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF).Somatic CALR mutations are generally absent in PV but their frequencies are approximately 20-25% in ET and PMF. Initial reports suggested that these variants were mutually exclusive. However subsequently co-occurrence has been reported and it is suggested but not yet confirmed that this may confer a distinct phenotype. The role of germline variation is unknown. We report here the first occurrence of a germline CALR variant in association with a somatic JAK2 V617F mutation. Case report A 73-year old Malay gentleman was noted to have an abnormal cardiac rhythm and function during admission for herniorrhaphy. On further questioning, he reported a 2 month history of reduced cognitive function, short term memory loss, occasional urinary incontinence and involuntary movements. Splenomegaly was noted and blood test showed Hb 205 g/L, WCC 33.5 x 109/L and platelet 304 x 109/L. Cardiac investigations revealed an anteroseptal myocardial infarction requiring an angioplasty and stent insertion. Targeted Ion Torrent high-throughput sequencing and fragment analysis showed a somatic JAK2 V617F mutation and a germline 3bp in-frame deletion c.1213_1215delGAG (p.Glu405del) in exon 9 of CALR. Discussion Although somatic CALR exon 9 mutations are mostly found in patients with ET and PMF who are JAK2-negative, coexistence of somatic JAK2 V617F and CALR exon 9 alterations have been reported previously, mostly in patients with ET. To the best of our knowledge, this is the first case of a patient with PV presenting with a somatic JAK2 V617F mutation and a germline CALR variant, Glu405del, comprising a 3 bp in-frame deletion in exon 9. According to existing databases, this CALR variant is a previously undescribed germline variant not found in the 60,000 exomes on the ExAC database; but one instance of an overlapping p.Glu405_Asp408del was reported (frequency of 2 x 10-5). Although it is at present of unknown clinical significance, it is predicted by in silico modeling as being likely pathogenic: MutationTaster predicted both splice-site and protein feature changes, including loss of an endoplasmic reticulum export motif. Unlike the "classical" CALR-positive patients who present at a younger age with a higher platelet count, this patient presented incidentally at the age of 73 with a normal platelet count but a rather severe clinical phenotype with significant thrombosis requiring surgical intervention. Further studies are required to examine in detail the molecular signatures in this group of patients and whether they represent a distinct group of patients with clinical phenotypes which are different from the "classical" cases. Disclosures Aitman: Illumina: Honoraria. Laffan:CSL: Other: Travel support; Octapharma: Speakers Bureau.