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Ana C Glembotsky,1,2 Geraldine De Luca,1,2 Paula G Heller1,2 1Departamento Hematología Investigación, Instituto de Investigaciones Médicas “Dr. A. Lanari”, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina; 2Departamento Hematología Investigación, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad de Buenos Aires, Instituto de Investigaciones Médicas (IDIM), Buenos Aires, ArgentinaCorrespondence: Paula G HellerDepartamento Hematología Investigación,Instituto de Investigaciones Médicas “Dr. A. Lanari”, Facultad de Medicina, Universidad de Buenos Aires, Combatientes de Malvinas 3150, Buenos Aires, 1427, ArgentinaTel +54 11 5287 3872Email paulaheller@hotmail.comAbstract: The gray platelet syndrome (GPS) is a rare platelet disorder, characterized by impaired alpha-granule biogenesis in megakaryocytes and platelets due to NBEAL2 mutations. Typical clinical features include macrothrombocytopenia, bleeding and elevated vitamin B12 levels, while bone marrow fibrosis and splenomegaly may develop during disease progression. Recently, the involvement of other blood lineages has been highlighted, revealing the role of NBEAL2 outside the megakaryocyte-platelet axis. Low leukocyte counts, decreased neutrophil granulation and impaired neutrophil extracellular trap formation represent prominent findings in GPS patients, reflecting deranged innate immunity and associated with an increased susceptibility to infection. In addition, low numbers and impaired degranulation of NK cells have been demonstrated in animal models. Autoimmune diseases involving different organs and a spectrum of autoantibodies are present in a substantial proportion of GPS patients, expanding the syndromic spectrum of this disorder and pointing to dysregulation of the adaptive immune response. Low-grade inflammation, as evidenced by elevation of liver-derived acute-phase reactants, is another previously unrecognized feature of GPS which may contribute to disease manifestations. This review will focus on the mechanisms underlying the pathogenesis of blood cell abnormalities in human GPS patients and NBEAL2-null animal models, providing insight into the effects of NBEAL2 in hemostasis, inflammation and immunity.Keywords: NBEAL2, gray platelet syndrome, α-granules, immune dysregulation, neutrophils

Gray platelet syndrome (GPS) is a rare inherited macrothrombocytopenia characterized by reduction of a-granules in platelets and megakaryocytes associated with mild-tomoderate bleeding and myelofibrosis [1]. As reported in at least 28 unrelated families [1], GPS is caused by mutations of NBEAL2, the gene encoding for the neurobeachin-like-2 protein. NBEAL2 is a member of the family containing the BEACH (BEige And Chediak Higashi) domain, a conserved region involved in vesicular trafficking that may be critical for the a-granule development [2]. Here, we report novel mutations of NBEAL2 in two affected individuals (P1 and P2), who were previously diagnosed with immune thrombocytopenia (ITP) and then suspected to have GPS because of absence of azurophilic granules on May-Grunwald-Giemsa staining. In P1, sequencing analysis identified an € homozygous missense variant (c.6212G > C; p.Arg2071Pro; Supporting Information Fig. S1). Since her parents were not available for the segregation analysis, we hypothesised that the two mutant alleles were identical by descent because of homozygosity of all the polymorphic markers at the NBEAL2 locus (data not shown). Moreover, the potential hemizygous condition was excluded using statistical analyses of NBEAL2 amplicon coverage as previously reported (Supporting Information Fig. S1C) [3]. In P2, we detected one maternal nonsense (c.3839C > T; p.Arg1280*) and one paternal missense (c.6477C > G; p.His2159Gln; Supporting Information Fig. S1). The three NBEAL2 variants are reported in SNPs databases but with a minor allele frequency

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders (IPD), however very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a multicentric, retrospective worldwide study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted and their efficacy in patients with IPDs by rating the outcome of 829 surgical procedures carried out in 423 patients with well defined forms of IPD (238 inherited platelet function disorders -IPFD- and 185 inherited platelet number disorders-IPND-). Frequency of surgical bleeding was high in patients with IPD (19.7%), with a significantly higher bleeding incidence in IPFDs (24.8%) than in IPNDs (13.4%). The frequency of bleeding varied according to the type of IPD, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%), and was predicted by a preoperative WHO bleeding score >/-2. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urologic surgery. The use of preoperative prohemostatic treatments was associated with a lower bleeding frequency in patients with IPFD but not in IPNDs. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in IPD is substantial, especially in IPFDs, and bleeding history, type of disorder, type of surgery and female gender are associated with higher bleeding frequency. Prophylactic preoperative prohemostatic treatments appear to be required and associated with a lower bleeding incidence. Fil: Orsini, Sara. Università di Perugia; Italia Fil: Noris, Patrizia. University Of Pavia. Policlinico San Matteo; Italia Fil: Bury, Loredana. Università di Perugia; Italia Fil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Santoro, Cristina. Università degli studi di Roma ; Italia Fil: Kadir RA. Royal Free Hospital; Reino Unido Fil: Butta, Nora C.. Hospital Universitario la Paz; España Fil: Falcinelli, Emanuela. Università di Perugia; Italia Fil: Cid, Ana Rosa. Hospital Universitario y Politecnico ; España Fil: Fabris, Fabrizio. Università di Padova; Italia Fil: Fouassier, Marc. Centre Hospitalier Universitaire de Nantes; Francia Fil: Miyazaki, Koji. Kitasato University. School Of Medicine; Japón Fil: Lozano, Maria Luisa. Universidad Carlos III de Madrid. Instituto de Salud; España Fil: Zuñiga, Pamela. Pontificia Universidad Católica de Chile; Chile Fil: Flaujac, Claire. Cochin Hospital; Francia Fil: Podda, Gian Marco. Università degli Studi di Milano; Italia Fil: Bermejo, Nuria. Hospital ; España Fil: Favier, Remi. French Reference Centre For Inherited Platelet Disorder; Francia Fil: Henskens, Yvonne. Maastricht University. Medical Centre; Países Bajos Fil: De Maistre, Emmanuel. Centre Hospitalier Universitaire Dijon; Francia Fil: De Candia, Erica. Universitá Cattolica Sacro. Policlinico Agostino Gemelli; Italia Fil: Mumford, Andrew. University Of Bristol. School Of Clinical Sciences; Reino Unido Fil: Ozdemir, Gul Nihal. Cerrahpasa Medical Faculty. Pediatric Hematology Depart; Turquía Fil: Eker, Ibrahim. Gülhane Military Medical Faculty. Pediatric Hematology; Turquía Fil: Nurden, Paquita. Xavier Arnozan Hospital; Francia Fil: Bayart, Sophie. Centre Régional de Traitement Des Hémophiles; Francia Fil: Lambert, Michele P. University of Pennsylvania; Estados Unidos Fil: Bussel, James. Weill Cornell Medicine; Estados Unidos Fil: Zieger, Barbara. University Medical Center Freiburg; Alemania Fil: Tosseto, Alberto. San Bortolo Hospital; Italia Fil: Melazzini, Federica. Università degli studi di Pavia. Policlinico San Matteo; Italia Fil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Pecci, Alessandro. Università degli studi di Pavia. Policlinico San Matteo; Italia Fil: Cattaneo, Marco. Università degli Studi di Milano; Italia Fil: Schlegel, Nicole. Centre Hospitalier Universitaire ; Francia Fil: Gresele, Paolo. Università di Perugia; Italia

Objectives: MYH9-related disease (MYH9-RD) is an autosomal-dominant disorder deriving from mutations in MYH9, the gene for the nonmuscle myosin heavy chain (NMMHC)-IIA. MYH9-RD has a complex phenotype including congenital features, such as thrombocytopenia, and noncongenital manifestations, namely sensorineural hearing loss (SNHL), nephropathy, cataract, and liver abnormalities. The disease is caused by a limited number of mutations affecting different regions of the NMMHC-IIA protein. SNHL is the most frequent noncongenital manifestation of MYH9-RD. However, only scarce and anecdotal information is currently available about the clinical and audiometric features of SNHL of MYH9-RD subjects. The objective of this study was to investigate the severity and propensity for progression of SNHL in a large series of MYH9-RD patients in relation to the causative NMMHC-IIA mutations. Design: This study included the consecutive patients diagnosed with MYH9-RD between July 2007 and March 2012 at four participating institutions. A total of 115 audiograms were analyzed from 63 patients belonging to 45 unrelated families with different NMMHCIIA mutations. Cross-sectional analyses of audiograms were performed. Regression analysis was performed, and age-related typical audiograms (ARTAs) were derived to characterize the type of SNHL associated with different mutations. Results: Severity of SNHL appeared to depend on the specific NMMHCIIA mutation. Patients carrying substitutions at the residue R702 located in the short functional SH1 helix had the most severe degree of SNHL, whereas patients with the p.E1841K substitution in the coiled-coil region or mutations at the nonhelical tailpiece presented a mild degree of SNHL even at advanced age. The authors also disclosed the effects of different amino acid changes at the same residue: for instance, individuals with the p.R702C mutation had more severe SNHL than those with the p.R702H mutation, and the p.R1165L substitution was associated with a higher degree of hearing loss than the p.R1165C. In general, mild SNHL was associated with a fairly flat audiogram configuration, whereas severe SNHL correlated with downsloping configurations. ARTA plots showed that the most progressive type of SNHL was associated with the p.R702C, the p.R702H, and the p.R1165L substitutions, whereas the p.R1165C mutation correlated with a milder, nonprogressive type of SNHL than the p.R1165L. ARTA for the p.E1841K mutation demonstrated a mild degree of SNHL with only mild progression, whereas the ARTA for the mutations at the nonhelical tailpiece did not show any substantial progression. Conclusions: These data provide useful tools to predict the progression and the expected degree of severity of SNHL in individual MYH9-RD patients, which is especially relevant in young patients. Consequences in clinical practice are important not only for appropriate patient counseling but also for development of customized, genotype-driven clinical management. The authors recently reported that cochlear implantation has a good outcome in MYH9-RD patients; thus, stricter follow-up and earlier intervention are recommended for patients with unfavorable genotypes. Fil: Verver, Eva J.J.. University of Utrecht; Países Bajos Fil: Topsakal, Vedat. University of Utrecht; Países Bajos Fil: Kunst, Henricus P. M.. Radboud University Medical Centre; Países Bajos Fil: Huygen, Patrick L. M.. Radboud University Medical Centre; Países Bajos Fil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Pujol Moix, Nuria. Universitat Autònoma de Barcelona; España Fil: Savoia, Anna. Università degli Studi di Trieste; Italia. Istituti di Ricovero e Cura a Carattere Scientifico; Italia Fil: Benazzo, Marco. Fondazione Policlinico San Matteo; Italia. Istituti di Ricovero e Cura a Carattere Scientifico; Italia Fil: Fierro, Tiziana. Università di Perugia; Italia Fil: Grolman, Wilko. University of Utrecht; Países Bajos Fil: Gresele, Paolo. Università di Perugia; Italia Fil: Pecci, Alessandro. Fondazione Policlinico San Matteo; Italia. Istituti di Ricovero e Cura a Carattere Scientifico; Italia. Università degli Studi di Pavia; Italia