ALBERT

Description: Ice flow models of the Antarctic ice sheet are commonly used to simulate its future evolution in response to different climate scenarios and assess the mass loss that would contribute to future sea level rise. However, there is currently no consensus on estimates of the future mass balance of the ice sheet, primarily because of differences in the representation of physical processes, forcings employed and initial states of ice sheet models. This study presents results from ice flow model simulations from 13 international groups focusing on the evolution of the Antarctic ice sheet during the period 2015–2100 as part of the Ice Sheet Model Intercomparison for CMIP6 (ISMIP6). They are forced with outputs from a subset of models from the Coupled Model Intercomparison Project Phase 5 (CMIP5), representative of the spread in climate model results. Simulations of the Antarctic ice sheet contribution to sea level rise in response to increased warming during this period varies between −7.8 and 30.0 cm of sea level equivalent (SLE) under Representative Concentration Pathway (RCP) 8.5 scenario forcing. These numbers are relative to a control experiment with constant climate conditions and should therefore be added to the mass loss contribution under climate conditions similar to present-day conditions over the same period. The simulated evolution of the West Antarctic ice sheet varies widely among models, with an overall mass loss, up to 18.0 cm SLE, in response to changes in oceanic conditions. East Antarctica mass change varies between −6.1 and 8.3 cm SLE in the simulations, with a significant increase in surface mass balance outweighing the increased ice discharge under most RCP 8.5 scenario forcings. The inclusion of ice shelf collapse, here assumed to be caused by large amounts of liquid water ponding at the surface of ice shelves, yields an additional simulated mass loss of 28 mm compared to simulations without ice shelf collapse. The largest sources of uncertainty come from the climate forcing, the ocean-induced melt rates, the calibration of these melt rates based on oceanic conditions taken outside of ice shelf cavities and the ice sheet dynamic response to these oceanic changes. Results under RCP 2.6 scenario based on two CMIP5 climate models show an additional mass loss of 0 and 3 cm of SLE on average compared to simulations done under present-day conditions for the two CMIP5 forcings used and display limited mass gain in East Antarctica.

Description: Abstract 4826 MYH9-related disorders (MYH9-RD) are characterized by an autosomal-dominant macrothrombocytopenia associated or not with glomerular impairment, hearing loss and/or cataracts. It is caused by mutations of the MYH9 gene (encoding for the nonmuscle myosin heavy chain IIA), present at the heterozygous state in patients. In knockout animal models, proplatelet formation (PPF) was found to be increased in certain conditions, but decreased in others (Chen et al, Blood 2007, Eckly et al, Blood 2007 and Thrombosis and Haemostasis 2010) and samples with heterozygosity did not show differences with the wild type. More recently, in a mouse model with a mutated MYH9 gene, mice with the mutation showed a phenotype similar to the one found in MYH9-RD, affecting multiple organs and, interestingly, mice heterozygous for the mutation showed an abnormal phenotype as well, although attenuated (Anderson et al, ASH Meeting 2010, Abstract 2527). However, opposite to the initial reports in mouse models with a knockout, proplatelet formation was found to be impaired in 4 patients (Pecci et al, Thrombosis and Haemostasis 2009). Therefore the question for the exact mechanism for thrombocytopenia still stands, so does the question whether the mutated protein causes disease through a dominant negative effect versus a functional haploinsufficiency. To address these questions, we performed studies on patient samples on one hand and seeked to knock down the gene expression in human megakaryocytes (MKs) on the other hand. To study PPF in these patients, we analyzed MK differentiation in CD34+ cells from 9 patients with mutations in exons encoding for the motor domain (2 patients) or for the coiled coil domain of myosin IIA (7 patients). Compared with cultured MKs derived from healthy donors, a 2 to 5 –fold decrease in PPF was observed in the patients (4.9 ± 1.2% for patients vs. 15.1 ± 1.8% for control) and proplatelet area was decreased in patients as well (threefold decrease, 6156 μm2 for patients versus 18064 μm2 for control). In confocal microscopy with immunofluorescence staining, we observed disorganization of the granules, abnormal spreading and stress fibers, meaning that actin-myosin network is unstable. We next used a shRNA strategy to knock down MYH9 expression during normal MK differentiation and compared shRNA-treated MKs (with 50% residual MYH9 expression) with those derived from patient CD34+ cells. Treatment with shRNA decreased in vitro PPF, as previously observed for cells from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as MKs from patients. Normal platelet production is dependent on the formation of branched long proplatelets. Surprisingly the defect in PPF formation seen in patients was rescued by blebbistatin, an inhibitor of class II myosin. In its presence the proplatelets increased by 2 to 5 fold, suggesting that the remaining myosin IIA might be hyperactivated and inhibiting PPF. Ultrastructural studies by electron microscopy performed on cells with blebbistatin treatment corrected abnormalities seen in the patients. Parallel to blebbistatin, ROCK (Y27632) or MLCK inhibitors (P18 or ML7) led to a threefold increase in proplatelet formation. We therefore compared by western blot the status of pMLC2 in patients and control MKs. In 3 out of 6 patients we found an increased pMLC2. In addition, we also found an increased pMLC2 in MKs treated with shRNA with 50% residual myosin expression. Altogether our results show that reducing the protein expression by half was sufficient to recreate features characteristic of the disease in megakaryocytes, while the increased PPF, relative to control, in response to rho/ROCK inhibitors and to myosin inhibitors, suggest that the defective PPF in patients may be related to an activated rho/ROCK pathway and/or increase of the contractile force. Disclosures: No relevant conflicts of interest to declare.

Description: Thrombocytopenia is a common condition in distressed newborns, but little is known about thrombocytopenia in an unselected cohort of neonates. In an attempt to address this issue, a multicenter prospective study was conducted in three obstetrical wards of AP-HP in Paris. We found the frequency of neonatal thrombocytopenia (

Description: Abstract 1155 MYH9-Related Diseases (RD) are inherited platelet disorders combining thrombocytopenia and giant platelets with various associations of leukocyte inclusions, deafness, nephritis and cataracts. Mutations in the MYH9 gene, that encodes the nonmuscle myosin heavy chain IIA (NMMHC-IIA), are the hallmark of these disorders. Our cohort includes a total of 161 subjects (90 women, 71 men): 76 propositi (37 isolated, 39 family index cases) with MYH9 mutations and 85 family members (45 women, 40 men) belonging to the 39 unrelated families. The cases with an identified mutation were classified according to the location of the mutation either in the motor domain (MD patients (P): “MDP”) or in the tail domain (TD patients (P): “TDP”) of NMMHC-IIA. MYH9 mutations were found in a total of 109 subjects: the 76 propositi (45 women, 31 men) and 33/85 (38.8%) family members (19 women, 14 men, from 22 families). Among the 41 different mutations detected, the majority were missense mutations (33 = 80.5%) and new (25 = 61%). Two substitutions in intron 39 were discovered for the first time: 1 in the donor splice site (corresponding to a classical MYH9-RD phenotype) and the other in position minus 3 of the acceptor splice site (moderate thrombocytopenia only without giant platelets). A total of 15 different exons were identified as having mutations, 4 exons remained unaffected. Mutations were more frequently found in exons 1, 16, 26, 30, 38 and 40, as already published. The majority of patients (102 = 93.6%) were heterozygous for one mutation but 7 patients (6.4%) were compound heterozygous for 2 mutations. Significant differences between MDP and TDP were found for bleeding symptoms (65% of MDP, 33% of TDP, p=0.0037), nephropathy (31.5% of MDP, 11.6% of TDP, p=0.015), age (years, mean) at onset for nephropathy (21 for MDP, 48 for TDP, p=0.0003), deafness (54% of MDP, 23.7% of TDP, p=0.0025), and association bleeding/extra-hematological symptoms (68.2% of MDP, 27.7% of TDP, p=0.01). Patients with bleeding had a lower platelet count than non-bleeders (mean= 42G/L versus 62G/L, p=0.0024) but there were no significant differences in the percentage of giant platelets. ITP (splenectomy for 7) was the initial misdiagnosis for 29/69 (42%), (51.7% of MDP, 35% of TDP, p= 0.165). Cataracts occurred in 4 patients only. Altogether these results showed that the clinical phenotype of MYH9-RD was more severe for MDP than TDP. New mutations were discovered and were widespread along the exons and in some cases two mutations were identified in the same patient. The sequencing of the full gene and all exons-introns boundaries should be mandatory for each patient whatever the initial result. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The inherited risk factors for venous thromboembolism differ between Caucasians and Chinese, antithrombin (AT), protein (PC) and protein (PS) deficiencies being the most common in Chinese patients. However, the age- and gender-related normal activity ranges of AT, PC, PS and the prevalence of their deficiencies in the general Chinese population remained to be determined. Methods: 3493 healthy volunteers, 1759 women and 1734 men, blood donors or individuals taking routine health checkup, were recruited in four Chinese medical centers, Beijing, Tianjin, Shanghai and Suzhou, between February and May 2006. The inclusion criteria were: informed consent, normal renal and liver functions, no anticoagulant, contraceptive or hormonal substitutive therapy, no previous thrombosis. The plasma levels of AT, PC, PS activities and APC-R were measured with the same batches of reagents and the same type of analyzer (Stago). For the individuals with low activities, mutations of PROC gene, PROS1 gene or SERPINC1 gene were determined by direct sequencing analysis. Restriction fragment length polymorphism analysis was performed in those APC-R positive samples to detect 5 single nucleotide polymorphisms (SNPs) in FV gene, including R306T, R306G, R506Q, H1299R and I359T. Results: The plasma activities (mean±SD) were: AT 109.4%±10.1%, PC 110.2%±20.4%, PS 95.4%±21.6% and APC-R 155sec±20sec. The normal ranges (mean±2SD) were: AT 89–130%, PC 69–151%, PS 52–139% and APC-R 114–195sec. Using multiple linear regression models, we identified significant (p

Description: MYH9-related disorders are autosomal dominant giant platelet syndromes with a wide phenotypic variability known under the following names: May Hegglin Anomaly (MHA), Fechtner Syndrome (FTNS), Epstein Syndrome (EPS), Sebastian Syndrome (SBS) and Alport-like Syndrome with MT. The aim of the study was to identify MYH9-related syndromes in a series of 43 propositi with constitutional MT. In this group, 26 have a characteristic MYH9-syndrome phenotype. The 17 other patients have a constitutional MT of uncertain etiology, 8 of them presenting a partial MYH9 syndrome phenotype. The leukocyte repartition of the Non-Muscle Myosin Heavy Chain IIa (NMMHC-IIA) encoded by the MYH9 gene was explored by May-Grunwald-Giemsa staining of blood smears or by immunofluorescence, while platelets were also examined by electron microscopy (EM). The MYH9 gene was studied by genomic DNA amplification and direct sequencing of the 8 exons in which mutations have been published (Dong, 2005). The results are the following: of the 17 patients with constitutional MT of unknown etiology, no MYH9 mutations were found; in contrast, 24/26 (92.3%) patients with a characteristic MYH9-syndrome phenotype were heterozygous for a MYH9 mutation. Among their 29 family members, 5 were heterozygous for the mutated allele. In 4 families, the mutations were found only in the propositus but not in the parents, suggesting that they might be either de novo mutations or the results of somatic mosaicism, as already published ( Kunishima et al, 2005). Six mutations are novel: F1446L, K1937X, A44P, D1424E in 4 MHA patients, W33C in 1 patient with MHA/SBS, and, interestingly, D1447V was associated with 2 different phenotypes, MHA and FTNS. As already published, the majority of the mutations in the C-terminus of the NMMHC-IIA are associated with a pure hematologic disorder. In contrast, the N-terminus mutations were more generally associated with a more severe phenotype with renal manifestations. However the same mutation can be associated with different phenotypes: S96L with FTNS, EPS and SBS/MHA, D1447V with MHA and FTNS. In addition the R702C mutation, which has been identifed in 2 cases with an identical FTNS phenotype, is associated with 2 different leukocyte NMMHC-IIA distributions. No MYH9 mutation could be detected in 2 patients with FTNS. These results, as a whole, are in agreement with the hypothesis that mutations in other genes than MYH9 might be involved in defining the phenotypes of such syndromes. The genotype of the 17 patients with uncertain etiology remains to be identified. The interest to diagnose MYH9 mutations is: to avoid misdiagnosis and inadequate therapy for patients with thrombocytopenia (2 patients initially diagnosed as ITP patients underwent splenectomy); to detect as early as possible the risk and occurrence of renal failure, deafness and cataracts (1 patient initially diagnosed as a SBS patient developed a renal failure characteristic of FTNS). A long-term follow-up of the patients with MYH9 mutations is of a high interest for a better knowledge of the relationship between the mutation and the phenotypic expression.

Description: The Greenland ice sheet is one of the largest contributors to global mean sea-level rise today and is expected to continue to lose mass as the Arctic continues to warm. The two predominant mass loss mechanisms are increased surface meltwater run-off and mass loss associated with the retreat of marine-terminating outlet glaciers. In this paper we use a large ensemble of Greenland ice sheet models forced by output from a representative subset of the Coupled Model Intercomparison Project (CMIP5) global climate models to project ice sheet changes and sea-level rise contributions over the 21st century. The simulations are part of the Ice Sheet Model Intercomparison Project for CMIP6 (ISMIP6). We estimate the sea-level contribution together with uncertainties due to future climate forcing, ice sheet model formulations and ocean forcing for the two greenhouse gas concentration scenarios RCP8.5 and RCP2.6. The results indicate that the Greenland ice sheet will continue to lose mass in both scenarios until 2100, with contributions of 90±50 and 32±17 mm to sea-level rise for RCP8.5 and RCP2.6, respectively. The largest mass loss is expected from the south-west of Greenland, which is governed by surface mass balance changes, continuing what is already observed today. Because the contributions are calculated against an unforced control experiment, these numbers do not include any committed mass loss, i.e. mass loss that would occur over the coming century if the climate forcing remained constant. Under RCP8.5 forcing, ice sheet model uncertainty explains an ensemble spread of 40 mm, while climate model uncertainty and ocean forcing uncertainty account for a spread of 36 and 19 mm, respectively. Apart from those formally derived uncertainty ranges, the largest gap in our knowledge is about the physical understanding and implementation of the calving process, i.e. the interaction of the ice sheet with the ocean.

Description: The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 μg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 μg/mL. PF4 also supported the viability of CD34+ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor β1 (TGFβ1) and cytotoxic drugs. Pretreatment of CD34+ cells by PF4, but not by TGFβ1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34+ cells were preincubated with PF4 or TGFβ1 for 12 days in hematopoietic growth factor–rich medium, an increased number of remaining CD34+ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC50) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 μg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU–granulocyte/macrophage (CFU-GM), and burst-forming units–erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 μg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.