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MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC-IIA). MYH9-RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end-stage renal disease (ESRD). We searched for genotype–phenotype correlations in the largest series of consecutive MYH9-RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9-RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early-onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC-IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C-terminal deletions had low risk of noncongenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9-RD. Fil: Pecci, Alessandro. University of Pavia; Italia Fil: Klersy, Catherine. IRCCS Policlinico San Matteo Foundation; Italia Fil: Gresele, Paolo. Università di Perugia; Italia Fil: Lee, Kieran J. D.. University of Leeds; Reino Unido Fil: De Rocco, Daniela. Università degli Studi di Trieste; Italia Fil: Bozzi, Valeria. University of Pavia; Italia Fil: Russo, Giovanna. University of Catania; 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: Loffredo, Giuseppe. Pausilipon Hospital. Department of Oncology; Italia Fil: Ballmaier, Matthias. Hannover Medical School; Alemania Fil: Fabris, Fabrizio. Università di Padova; Italia Fil: Beggiato, Eloise. Hospital “Città della Salute e Della Scienza”; Italia Fil: Kahr, Walter H. A.. University of Toronto; Canadá. Hospital for Sick Children. Division of Hematology/Oncology; Canadá Fil: Pujol Moix, Nuria. Universitat Autònoma de Barcelona; España Fil: Platokouki, Helen. “Aghia Sophia” Children's Hospital; Grecia Fil: Van Geet, Christel. University of Leuven. Center for Molecular and Vascular Biology; Bélgica Fil: Noris, Patrizia. University of Pavia; Italia Fil: Yerram, Preethi. University of Missouri; Estados Unidos Fil: Hermans, Cedric. St-Luc University Hospital; Bélgica Fil: Gerber, Bernhard. University Hospital Zurich, Division of Hematology; Suiza Fil: Economou, Marina. Aristotle University; Grecia Fil: De Groot, Marco. University of Groningen; Países Bajos Fil: Zieger, Barbara. University Medical Center Freiburg; Alemania Fil: De Candia, Erica. Catholic University of Rome; Italia Fil: Fraticelli, Vincenzo. Giovanni Paolo II Foundation; Italia Fil: Kersseboom, Rogier. Erasmus Medical Centre; Países Bajos Fil: Piccoli, Giorgina B.. Università di Torino; Italia Fil: Zimmermann, Stefanie. Goethe Universitat Frankfurt; Alemania Fil: Fierro, Tiziana. Università di Perugia; 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: Vianello, Fabrizio. Università di Padova; Italia Fil: Zaninetti, Carlo. University of Pavia; Italia Fil: Nicchia, Elena. Università degli Studi di Trieste; Italia Fil: Güthner, Christiane. Stadtspital Triemli. Department of Medical Oncology and Hematology; Italia Fil: Baronci, Carlo. Pediatric Hospital "Bambino Gesù"; Italia Fil: Seri, Marco. Università di Bologna; Italia Fil: Knight, Peter J.. University of Leeds; Reino Unido Fil: Balduini, Carlo L.. University of Pavia; Italia Fil: Savoia, Anna. Università degli Studi di Trieste; Italia

BACKGROUND: MYH9-related disease (MYH9-RD) is a rare autosomal dominant genetic syndrome characterized by congenital thrombocytopenia associated with the risk of developing progressive nephropathy, sensorineural deafness, and presenile cataract. During the collection of a large case-series of patients with MYH9-RD we noticed several cases with unexplained elevation of liver enzymes. Our aim was to evaluate if the alteration of liver tests is a feature of the MYH9-RD and to define its clinical significance. METHODS AND FINDINGS: Data concerning liver tests, prospectively recorded in the Italian Registry for MYH9-RD, were collected and compared with those of three control populations: patients with autoimmune thrombocytopenia, patients with inherited thrombocytopenias other than MYH9-RD, and the participants to a large epidemiologic survey in an Italian geographic isolate. Thirty-eight of 75 evaluable MYH9-RD patients (50.7%) showed an elevation of ALT and/or AST, and 17 of 63 (27.0%) an increase of GGT. The increases ranged from 1.9 ± 0.7 to 2.7 ± 1.6 fold the upper normal limit. The prevalence of liver test alterations was significantly higher in MYH9-RD patients than in each of the control populations, with odds ratios ranging from 8.2 (95% CIs 2.2-44.8) to 24.7 (14.8-40.8). Clinical follow-up and more detailed liver studies of a subset of patients, including ultrasound liver scan, liver elastography and liver biopsy in one case, did not show any significant structural damage or evolution towards liver insufficiency. CONCLUSIONS: Elevation of liver enzymes is a frequent and previously unrecognized feature of the MYH9-RD syndrome; however, this defect does not appear to have poor prognostic value.

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. Patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils and might develop sensorineural deafness, presenile cataract, and/or progressive nephritis leading to end-stage renal failure. In a family with eight individuals suffering from macrothrombocytopenia and hearing impairment we identified a novel c.Ala95Asp mutation. Affecting the motor domain of the protein, the mutation is likely to be associated with a severe phenotype. Therefore, this family should be carefully monitored to follow-up the renal status even though the affected members do not seem to be at risk of early kidney disease. Fil: de Rocco, Daniela. Università degli Studi di Trieste; 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: Girotto, Giorgia. Università degli Studi di Trieste; Italia Fil: Pastore, Annalisa. National Institute for Medical Research; Reino Unido 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: Marta, Rosana Fernanda. 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: Bozzi, Valeria. Universita Degli Studi Di Pavia; Italia Fil: Pecci, Alessandro. Universita Degli Studi Di Pavia; Italia Fil: Molinas, Felisa Concepción. 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: Savoia, Anna. Università degli Studi di Trieste; Italia

Not abstract available

THC2, an autosomal-dominant thrombocytopenia described so far in only two families, has been ascribed to mutations in MASTL or ACBD5. Here, we show that ANKRD26, another gene within the THC2 locus, and neither MASTL nor ACBD5, is mutated in eight unrelated families. ANKRD26 was also found to be mutated in the family previously reported to have an ACBD5 mutation. We identified six different ANKRD26 mutations, which were clustered in a highly conserved 19 bp sequence located in the 5' untranslated region. Mutations were not detected in 500 controls and are absent from the 1000 Genomes database. Available data from an animal model and Dr. Watson's genome give evidence against haploinsufficiency as the pathogenetic mechanism for ANKRD26-mediated thrombocytopenia. The luciferase reporter assay suggests that these 5' UTR mutations might enhance ANKRD26 expression. ANKRD26 is the ancestor of a family of primate-specific genes termed POTE, which have been recently identified as a family of proapoptotic proteins. Dysregulation of apoptosis might therefore be the pathogenetic mechanism, as demonstrated for another thrombocytopenia, THC4. Further investigation is needed to provide evidence supporting this hypothesis.

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. All patients present congenital macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataract, and/or progressive nephropathy leading to end-stage renal failure. We report four families, each with a novel mutation: two missense mutations, in exons 31 and 32, and two out of frame deletions in exon 40. They were associated with no bleeding diathesis, normal, or only slightly reduced platelet count and no extra-hematological manifestations, confirming that alterations of the tail domain cause a mild form of MYH9-RD with no clinically relevant defects.

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable clinical evolution of the disease.We have evaluated 108 consecutive MYH9- RD patients belonging to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis.We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC-IIA (responsible for 70% of MYH9-RD cases).We concluded that mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly, those in position 702 result in severe thrombocytopenia and produce nephritis and deafness at a juvenile age, while alterations at residue 1424 or 1841 result in intermediate clinical pictures. These findings are relevant not only to patients’ clinical management but also to the elucidation of the pathogenesis of the disease. Fil: Pecci, Alessandro. Universita Degli Studi Di Pavia; Italia. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Panza, Emanuele. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. Universidad de Bologna; Italia Fil: Pujol Moix, Núria. Hospital de la Santa Creu i Sant Pau; España Fil: Klersy, Catherine. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Di Bari, Filomena. Telethon Institute of Genetics and Medicine; Italia Fil: Bozzi, Valeria. Universita Degli Studi Di Pavia; Italia. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Gresele, Paolo. Università di Perugia; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Lethagen, Stefan. Universidad de Copenhagen; Dinamarca. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Fabris, Fabrizio. Università di Padova; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Dufour, Carlo. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Granata, Antonio. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. Policlinico "Vittorio Emanuele"; Italia Fil: Doubek, Michael. University Hospital; República Checa Fil: Pecoraro, Carmine. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. ‘‘Santobono’’ Children’s Hospital; Italia Fil: Koivisto, Pasi A.. Tampere University Hospital; Finlandia 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: Iolascon, Achille. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. Università degli Studi di Napoli Federico II; Italia Fil: Alvisi, Patrizia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. Ospedale Maggiore Carlo Alberto Pizzardi; Italia Fil: Schwabe, Dirk. Goethe Universitat Frankfurt; Alemania Fil: De Candia, Erica. Università degli studi di Roma "La Sapienza"; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Rocca, Bianca. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. Università degli studi di Roma "La Sapienza"; Italia Fil: Russo, Umberto. Ospedale Luigi Sacco; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Ramenghi, Ugo. Università degli studi di Torino; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Noris, Patrizia. Universita Degli Studi Di Pavia; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia Fil: Seri, Marco. Universidad de Bologna; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia Fil: Balduini, Carlo L.. Universita Degli Studi Di Pavia; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia Fil: Savoia, Anna. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia. IRCCS Policlinico San Matteo Foundation. Italian Registry for MYH9-Related Disease; Italia

Il contributo costituisce la seconda edizione, totalmente riveduta ed aggiornata, della trattazione dell'intera disciplina legale in materia di simulazione del contratto e degli atti unilaterali, unitamente ad una compiuta analisi delle ipotesi applicative esaminate alla luce della giurisprudenza più recente della Corte di Cassazione.

The SRC gene was the first proto-oncogene to be discovered. Its product SRC is a nonreceptor protein tyrosine kinase that is the prototype, and a ubiquitously expressed member, of the SRC family kinases. SRC has been investigated for decades in mouse and in vitro models: these studies have indicated that SRC signalling has a central role in many cellular functions and in oncogenesis. 1 In platelets, SRC mediates signal activation pathways downstream different integrins and G protein-coupled receptors.2 However, much remains to be understood about SRC functions in human megakaryocytes and platelets. Recently, the first germline mutation in SRC causing human disease was reported in two families. Here we report the investigation of a new unrelated individual carrying the p.E527K variant that provides additional information on the clinical and pathogenetic features of the disorder and the role of SRC in human megakaryocytes.

Platelet transfusion is currently the primary medical treatment for reducing thrombocytopenia in patients with inherited thrombocytopenias. To evaluate whether stimulating megakaryopoiesis could increase platelet count in these conditions, we treated patients with a severe thrombocytopenia induced by MYH9 mutations (MYH9-related disease) with a nonpeptide thrombopoietin receptor agonist, eltrombopag. Twelve adult patients with MYH9-RD and platelet counts of less than 50 × 109/L received 50 mg of eltrombopag orally per day for 3 weeks. Patients who achieved a platelet count higher than 150 × 109/L stopped therapy, those with 100 to 150 platelets × 109/L continued treatment at the same eltrombopag dose for 3 additional weeks, while those with less than 100 platelets × 109/L increased the eltrombopag dose to 75 mg for 3 weeks. Major responses (platelet count of at least 100 × 109/L or 3 times the baseline value) were obtained in 8 patients, minor responses (platelet counts at least twice the baseline value) in 3. One patient did not respond. Bleeding tendency disappeared in 8 of 10 patients with bleeding symptoms at baseline. Mild adverse events were reported in 2 patients. The availability of thrombopoietin mimetics opened new prospects in the treatment of inherited thrombocytopenias. This study is registered at www.clinicaltrials.gov as NCT01133860 (European Union Drug Regulating Authorities Clinical Trials number 2008-001903-42).

Introduction Crohn’s disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application. Methods T-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors’ MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes. Results A significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4+CD25+ subset and increase of the CD3+CD69+ population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interferon-γ, interleukin-17A and -21, whereas that of the transforming growth factor-β and interleukin-6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used. Conclusion MSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use.

MYH9 -related disease ( MYH9 -RD) is an autosomal dominant thrombocytopenia with giant platelets variably associated with young-adult onset of progressive sensorineural hearing loss, presenile cataract, and renal damage. MYH9 -RD is caused by mutations of MYH9 , the gene encoding for non-muscle heavy-chain myosin-9. Wild-type and mutant myosin-9 aggregate as cytoplasmic inclusions in patients’ leukocytes, the identification of which by immunofluorescence has been proposed as a suitable tool for the diagnosis of MYH9 -RD. Since the predictive value of this assay, in terms of sensitivity and specificity, is unknown, we investigated 118 consecutive unrelated patients with a clinical presentation strongly consistent with MYH9 -RD. All patients prospectively underwent both the immunofluorescence assay for myosin-9 aggregate detection and molecular genetic analysis of the MYH9 gene. Myosin-9 aggregates were identified in 82 patients, 80 of which (98%) had also a MYH9 mutation. In the remaining 36 patients neither myosin-9 aggregates nor MYH9 mutations were found. Sensitivity and specificity of the immunofluorescence assay was evaluated to be 100% and 95%, respectively. Except for the presence of aggregates, we did not find any other significant difference between patients with or without aggregates, demonstrating that the myosin-9 inclusions in neutrophils are a pathognomonic sign of the disease. However, the identification of the specific MYH9 mutation is still of importance for prognostic aspects of MYH9 -RD. Fil: Savoia, Anna. Universita Degli Studi Di Trieste; Italia. Italian Registry for MYH9-Related Disease; Italia Fil: de Rocco, Daniela. Universita Degli Studi Di Trieste; Italia. Italian Registry for MYH9-Related Disease; Italia Fil: Panza, Emanuele. Italian Registry for MYH9-Related Disease; Italia. Universita Di Bologna; Italia Fil: Bozzi, Valeria. Italian Registry for MYH9-Related Disease; Italia. University of Pavia; Italia Fil: Scandellari, Raffaella. Italian Registry for MYH9-Related Disease; Italia. Universita Di Padova; Italia Fil: Loffredo, Giuseppe. Italian Registry for MYH9-Related Disease; Italia. Pausilipon Hospital; Italia Fil: Mumford, Andrew. University of Bristol; Reino Unido Fil: Heller, Paula Graciela. Italian Registry for MYH9-Related Disease; Italia. 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: Noris, Patrizia. Italian Registry for MYH9-Related Disease; Italia Fil: de Groot, Marco R.. University of Bristol; Reino Unido Fil: Giani, Marisa. Italian Registry for MYH9-Related Disease; Italia. Istituto di Ricovero e Cura a Carattere Scientifico; Italia Fil: Freddi, Paolo. Italian Registry for MYH9-Related Disease; Italia Fil: Scognamiglio, Francesca. Italian Registry for MYH9-Related Disease; Italia Fil: Riondino, Silvia. Italian Registry for MYH9-Related Disease; Italia. Istituto di Ricovero e Cura a Carattere Scientifico; Italia Fil: Pujol Moix, Núria. Universitat Autonoma de Barcelona; España Fil: Fabris, Fabrizio. Italian Registry for MYH9-Related Disease; Italia. Universita Di Padova; Italia Fil: Seri, Marco. Italian Registry for MYH9-Related Disease; Italia. Universita Di Bologna; Italia Fil: Balduini, Carlo L.. Italian Registry for MYH9-Related Disease; Italia. University of Pavia; Italia Fil: Pecci, Alessandro. Italian Registry for MYH9-Related Disease; Italia. University of Pavia; Italia

Abnormalities of platelet size are one of the distinguishing features of inherited thrombocytopenias (ITs), and evaluation of blood films is recommended as an essential step for differential diagnosis of these disorders. Nevertheless, what we presently know about this subject is derived mainly from anecdotal evidence. To improve knowledge in this field, we evaluated platelet size on blood films obtained from 376 patients with all 19 forms of IT identified so far and found that these conditions differ not only in mean platelet diameter, but also in platelet diameter distribution width and the percentage of platelets with increased or reduced diameters. On the basis of these findings, we propose a new classification of ITs according to platelet size. It distinguishes forms with giant platelets, with large platelets, with normal or slightly increased platelet size, and with normal or slightly decreased platelet size. We also measured platelet diameters in 87 patients with immune thrombocytopenia and identified cutoff values for mean platelet diameter and the percentage of platelets with increased or reduced size that have good diagnostic accuracy in differentiating ITs with giant platelets and with normal or slightly decreased platelet size from immune thrombocytopenia and all other forms of IT. Fil: Noris, Patrizia. University of Pavia; Italia Fil: Biino, Ginevra. Consiglio Nazionale Delle Ricerche. Istituto di Genetica Molecolare; Italia Fil: Pecci, Alessandro. University of Pavia; Italia Fil: Civaschi, Elisa. University of Pavia; Italia Fil: Savoia, Anna. Università degli Studi di Trieste; Italia. Istituto Di Ricovero e Cura a Carattere Scientifico Burlo Garofolo; Italia Fil: Seri, Marco. Università di Bologna; Italia Fil: Melazzini, Federica. University of Pavia; Italia Fil: Loffreddo, Giuseppe. Pausilipon Hospital; Italia Fil: Russo, Giovana. Università degli Studi di Catania; Italia Fil: Bozzi, Valeria. University of Pavia; Italia Fil: Notarangelo, Lucia Dora. Spedali Civili. Ospedale dei Bambini; Italia Fil: Gresele, Paolo. 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: Pujol Moix, Nuria. Universitat Autònoma de Barcelona; España Fil: Kunishima, Shinji. National Hospital Organization Nagoya Medical Center; Japón Fil: Cattaneo, Marco. Università degli Studi di Milano; Italia Fil: Bussel, James. Cornell University; Estados Unidos Fil: de Candia, Erica. Universita Cattolica del Sacro Cuore; Italia Fil: Cagioni, Claudia. University of Pavia; Italia Fil: Ramenghi, Ugo. Università di Torino; Italia Fil: Barozzi, Serena. University of Pavia; Italia Fil: Fabris, Fabrizio. Università di Padova; Italia Fil: Balduini, Carlo L.. University of Pavia; Italia

SummaryMYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for the heavy chain of nonmuscle myosin-IIA (NMMHC-IIA). Recent in vitro studies led to the hypothesis that thrombocytopenia of MYH9-RD derives from an ectopic platelet release by megakaryocytes in the osteoblastic areas of bone marrow (BM), which are enriched in type I collagen, rather than in vascular spaces. SDF-1-driven migration of megakaryocytes within BM to reach the vascular spaces is a key mechanism for platelet biogenesis. Since myosin-IIA is implicated in polarised migration of different cell types, we hypothesised that MYH9 mutations could interfere with this mechanism. We therefore investigated the SDF-1-driven migration of a megakaryoblastic cell line, Dami cells, on type I collagen or fibrinogen by a modified transwell assay. Inhibition of myosin-IIA ATPase activity suppressed the SDF-1-driven migration of Dami cells, while over-expression of NMMHC-IIA increased the efficiency of chemotaxis, indicat- ing a role for NMMHC-IIA in this mechanism. Transfection of cells with three MYH9 mutations frequently responsible for MYH9-RD (p.R702C, p.D1424H, or p.R1933X) resulted in a defective SDF-1-driven migration with respect to the wild-type counterpart and in increased cell spreading onto collagen. Analysis of differential localisation of wild-type and mutant proteins suggested that mutant NMMHC-IIAs had an impaired cytoplasmic re-organisation in functional cytoskeletal structures after cell adhesion to collagen. These findings support the hypothesis that a defect of SDF-1-driven migration of megakaryocytes induced by MYH9 mutations contributes to ectopic platelet release in the BM osteoblastic areas, resulting in ineffective platelet production.

Background Bernard-Soulier syndrome is a severe bleeding disease due to a defect of GPIb/IX/V, a platelet complex that binds the von Willebrand factor. Due to the rarity of the disease, there are reports only on a few cases compromising any attempt to establish correlations between genotype and phenotype. In order to identify any associations, we describe the largest case series ever reported, which was evaluated systematically at the same center. Design and Methods Thirteen patients with the disease and seven obligate carriers were enrolled. We collected clinical aspects and determined platelet features, including number and size, expression of membrane glycoproteins, and ristocetin induced platelet aggregation. Mutations were identified by direct sequencing of the GP1BA, GP1BB, and GP9 genes and their effect was shown by molecular modeling analyses. Results Patients all had a moderate thrombocytopenia with giant platelets and a bleeding tendency whose severity varied among individuals. Consistent with expression levels of GPIb alpha always lower than 10% of control values, platelet aggregation was absent or severely reduced. Homozygous mutations were identified in the GP1BA, GP1BB and GP9 genes; six were novel alterations expected to destabilize the conformation of the respective protein. Except for obligate carriers of a GP9 mutation with a reduced GPIb/IX/V expression and defective aggregation, all the other carriers had no obvious anomalies. Conclusions Regardless of mutations identified, the patients' bleeding diathesis did not correlate with thrombocytopenia, which was always moderate, and platelet GPIba expression, which was always severely impaired. Obligate carriers had features similar to controls though their GPIb/IX/V expression showed discrepancies. Aware of the limitations of our cohort, we cannot define any correlations. However, further investigations should be encouraged to better understand the causes of this rare and underestimated disease.

MYH9-related disease (MYH9RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. All patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. The spectrum of mutations so far identified is peculiar, consisting of mostly missense mutations. Others are nonsense and frameshift mutations, all localized in the COOH terminus of the protein, or in-frame deletions. We report a family with three affected members carrying a novel mutation, the first duplication (p.E1066_A1072dup), of MYH9. The mutation was localized within exon 24, where the presence of a 16 nucleotide repeat was likely to be responsible for unequal crossing-over. Of note, a deletion of the same amino acids 1066_1072 was also identified in another MHY9RD family. Since two of the four patients with the duplication or the deletion in exon 24 were affected with bilateral neonatal cataracts, we speculate that these mutations might correlate with the ocular defect, which is reported only in 16% of MYH9RD patients.

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Background Heterozygous mutations of MYH9, encoding the Non-Muscular Myosin Heavy Chain-IIA (NMMHC-IIA), cause a complex disorder named MYH9-related disease, characterized by a combination of different phenotypic features. At birth, patients present platelet macrocytosis, thrombocytopenia and leukocyte inclusions containing NMMHC-IIA. Moreover, later in life some of them develop the additional features of sensorineural hearing loss, cataracts and/or glomerulonephritis that sometimes leads to end stage renal failure. Results To clarify the mechanism by which the mutant NMMHC-IIA could cause phenotypic anomalies at the cellular level, we examined the effect of transfection of the full-length mutated D1424H MYH9 cDNAs. We have observed, by confocal microscopy, abnormal distribution of the protein and formation of rod-like aggregates reminiscent of the leukocyte inclusions found in patients. Co-transfection of differently labeled wild-type and mutant full-length cDNAs showed the simultaneous presence of both forms of the protein in the intracellular aggregates. Conclusion These findings suggest that the NMMHC-IIA mutated in position 1424 is able to interact with the WT form in living cells, despite part of the mutant protein precipitates in non-functional aggregates. Transfection of the entire WT or mutant MYH9 in cell lines represents a powerful experimental model to investigate consequences of MYH9 mutations.