The amazing genetic complexity of anucleated platelets

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F9 Missense mutations impairing factor ix activation are associated with pleiotropic plasma phenotypes

Background: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in haemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. Objectives: To investigate i) expression of a complete panel of missense mutations at FIX activation sites and ii) contribution of F9 genotypes on the FIX pharmacokinetics (PK). Methods: FIXag and activity assays in plasma and after recombinant expression of FIX variants. Analysis of infused FIX PK parameters in patients (n=30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). Results: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants result in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. PK analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 hrs, 95% CI 44.3-114.5) in patients (n=7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, p=0.004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. Conclusions: FIXag levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients’ phenotypes and substitutive treatment

Expanding the genetic spectrum of TUBB1-related thrombocytopenia

β1-Tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying 6 β1-tubulin variants, p.Cys12LeufsTer12, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size, and a minority had no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp, and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets but had a negligible effect on platelet activation, secretion, or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, PI20/00926 and CB15/00055), Fundacion Séneca (19873/ GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundacion Mutua Madrile´ña (AP172142019), and ~ Sociedad Espanola de Trombosis y Hemostasia (Premio L ~ opez Borrasca 2019 and Ayuda a Grupos de Trabajo en Patologıa Hemorragica). The authors’ research on inherited platelet disorders is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, from Grupo Espanol de Alteraciones Plaqueta- ~ rias Congenitas, which is supported by the Spanish Society of Thrombosis and Haemostasis. V.P.-B. has a predoctoral contract from CIBERER. L.B. was supported by a fellowship from Fondazione Umberto Veronesi. M.E.d.l.M.-B. holds a postdoctoral fellowship from the University of Murcia. A.M.-Q. holds a predoctoral grant from the Junta de Castilla y Leon

Outside-In Signalling Generated by a Constitutively Activated Integrin αIIbβ3 Impairs Proplatelet Formation in Human Megakaryocytes

BACKGROUND: The interaction of megakaryocytes with matrix proteins of the osteoblastic and vascular niche is essential for megakaryocyte maturation and proplatelet formation. Fibrinogen is present in the vascular niche and the fibrinogen receptor α(IIb)β(3) is abundantly expressed on megakaryocytes, however the role of the interaction between fibrinogen and α(IIb)β(3) in proplatelet formation in humans is not yet fully understood. We have recently reported a novel congenital macrothrombocytopenia associated with a heterozygous mutation of the β(3) subunit of α(IIb)β(3). The origin of thrombocytopenia in this condition remains unclear and this may represent an interesting natural model to get further insight into the role of the megakaryocyte fibrinogen receptor in megakaryopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Patients' peripheral blood CD45+ cells in culture were differentiated into primary megakaryocytes and their maturation, spreading on different extracellular matrix proteins, and proplatelet formation were analyzed. Megakaryocyte maturation was normal but proplatelet formation was severely impaired, with tips decreased in number and larger in size than those of controls. Moreover, megakaryocyte spreading on fibrinogen was abnormal, with 50% of spread cells showing disordered actin distribution and more evident focal adhesion points than stress fibres. Integrin α(IIb)β(3) expression was reduced but the receptor was constitutively activated and a sustained, and substrate-independent, activation of proteins of the outside-in signalling was observed. In addition, platelet maturation from preplatelets was impaired. CONCLUSIONS/SIGNIFICANCE: Our data show that constitutive activation of α(IIb)β(3)-mediated outside-in signalling in human megakaryocytes negatively influences proplatelet formation, leading to macrothombocytopenia

Analysis of 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia

Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 10(9)/L.Fil: Noris, Patrizia. Istituti di Ricovero e Cura a Carattere Scientifico. Policlinico San Matteo di Pavia; Italia. Università degli Studi di Pavia; ItaliaFil: Schlegel, Nicole. Université Paris Diderot - Paris 7; FranciaFil: Klersy, Catherine. Istituti di Ricovero e Cura a Carattere Scientifico. Policlinico San Matteo di Pavia; ItaliaFil: 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; ArgentinaFil: Civaschi, Elisa. Università degli Studi di Pavia; ItaliaFil: Pujol Moix, Nuria. Universitat Autònoma de Barcelona; EspañaFil: Fabris, Fabrizio. Università di Padova; ItaliaFil: Favier, Remi. Inserm; Francia. Armand Trousseau Children’s Hospital; Francia. French Reference Center for Inherited Platelet disorders; FranciaFil: Gresele, Paolo. Università di Perugia; ItaliaFil: Latger Cannard, Véronique. Centre Hospitalo-Universitaire. Service d’Hématologie Biologique; Francia. Reference French Centre. Centre de Compétence Nord-Est des Pathologies Plaquettaires; FranciaFil: Cuker, Adam. University of Pennsylvania; Estados UnidosFil: Nurden, Paquita. Hôpital Xavier Arnozan; FranciaFil: Greinacher, Andreas. Institut für Immunologie und Transfusionsmedizin; AlemaniaFil: Cattaneo, Marco. Università degli Studi di Milano; ItaliaFil: De Candia, Erica. Università Cattolica del Sacro Cuore; ItaliaFil: Pecci, Alessandro. Università degli Studi di Pavia; ItaliaFil: Hurtaud Roux, Marie Françoise. Université Paris Diderot - Paris 7; FranciaFil: 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; ArgentinaFil: Muñiz Diaz, Eduardo. Banc de Sang i Teixits de Catalunya. Immunohematology Department; EspañaFil: Randi, Maria Luigia. Università di Padova; ItaliaFil: Trillot, Nathalie. Centre Hospitalier Régional Universitaire de Lille. Pôle Biologie Pathologie Génétique. Institut d’Hématologie-Transfusion; FranciaFil: Bury, Loredana. Università di Perugia; ItaliaFil: Lecompte, Thomas. Hôpitaux Universitaires de Genève; Suiza. Université de Genève. Faculté de Médecine; SuizaFil: Marconi, Caterina. Università di Bologna; ItaliaFil: Savoia, Anna. Università degli Studi di Trieste; ItaliaFil: Balduini, Carlo L.. Istituti di Ricovero e Cura a Carattere Scientifico Burlo Garofolo. Institute for Maternal and Child Health; Italia. Università degli Studi di Pavia; ItaliaFil: European Hematology Association Scientific Working Group on Thrombocytopenias and Platelet Function Disorders. No especifica

GoldVariants, a resource for sharing rare genetic variants detected in bleeding, thrombotic, and platelet disorders: Communication from the ISTH SSC Subcommittee on Genomics in Thrombosis and Hemostasis.

The implementation of high-throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease-causing variants associated with specific genes, but for BTPD, such well-curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap-based interface, aimed at the community, to submit curated genetic variants for diagnostic-grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open-access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar

Analysis of 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia

65Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 10(9)/L.openopenPatrizia Noris; Nicole Schlegel; Catherine Klersy; Paula G. Heller; Elisa Civaschi; Nuria Pujol-Moix; Fabrizio Fabris; Remi Favier; Paolo Gresele; Véronique Latger-Cannard; Adam Cuker; Paquita Nurden; Andreas Greinacher; Marco Cattaneo; Erica De Candia; Alessandro Pecci; Marie-Françoise Hurtaud-Roux; Ana C. Glembotsky; Eduardo Muñiz-Diaz; Maria Luigia Randi; Nathalie Trillot; Loredana Bury; Thomas Lecompte; Caterina Marconi; Anna Savoia; Carlo L. Balduini; Sophie Bayart; Anne Bauters; Schéhérazade Benabdallah-Guedira; Françoise Boehlen; Jeanne-Yvonne Borg; Roberta Bottega; James Bussel; Daniela De Rocco; Emmanuel de Maistre; Michela Faleschini; Emanuela Falcinelli; Silvia Ferrari; Alina Ferster; Tiziana Fierro; Dominique Fleury; Pierre Fontana; Chloé James; Francois Lanza; Véronique Le Cam Duchez; Giuseppe Loffredo; Pamela Magini; Dominique Martin-Coignard; Fanny Menard; Sandra Mercier; Annamaria Mezzasoma; Pietro Minuz; Ilaria Nichele; Lucia D. Notarangelo; Tommaso Pippucci; Gian Marco Podda; Catherine Pouymayou; Agnes Rigouzzo; Bruno Royer; Pierre Sie; Virginie Siguret; Catherine Trichet; Alessandra Tucci; Béatrice Saposnik; Dino VeneriPatrizia, Noris; Nicole, Schlegel; Catherine, Klersy; Paula G., Heller; Elisa, Civaschi; Nuria Pujol, Moix; Fabrizio, Fabris; Remi, Favier; Paolo, Gresele; Véronique Latger, Cannard; Adam, Cuker; Paquita, Nurden; Andreas, Greinacher; Marco, Cattaneo; Erica De, Candia; Alessandro, Pecci; Marie Françoise Hurtaud, Roux; Ana C., Glembotsky; Eduardo Muñiz, Diaz; Maria Luigia, Randi; Nathalie, Trillot; Loredana, Bury; Thomas, Lecompte; Caterina, Marconi; Savoia, Anna; Carlo L., Balduini; Sophie, Bayart; Anne, Bauters; Schéhérazade Benabdallah, Guedira; Françoise, Boehlen; Jeanne Yvonne, Borg; Bottega, Roberta; James, Bussel; DE ROCCO, Daniela; Emmanuel de, Maistre; Faleschini, Michela; Emanuela, Falcinelli; Silvia, Ferrari; Alina, Ferster; Tiziana, Fierro; Dominique, Fleury; Pierre, Fontana; Chloé, James; Francois, Lanza; Véronique Le Cam, Duchez; Giuseppe, Loffredo; Pamela, Magini; Dominique Martin, Coignard; Fanny, Menard; Sandra, Mercier; Annamaria, Mezzasoma; Pietro, Minuz; Ilaria, Nichele; Lucia D., Notarangelo; Tommaso, Pippucci; Gian Marco, Podda; Catherine, Pouymayou; Agnes, Rigouzzo; Bruno, Royer; Pierre, Sie; Virginie, Siguret; Catherine, Trichet; Alessandra, Tucci; Béatrice, Saposnik; Dino, Vener

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.This study, including the enrollment of cases, sequencing, and analysis received support from the National Institute for Health Research (NIHR) BioResource–Rare Diseases. The NIHR BioResource is funded by the NIHR (http://www.nihr.ac.uk). Research in the Ouwehand Laboratory is also supported by grants from Bristol-Myers Squibb, the British Heart Foundation, the British Society of Haematology, the European Commission, the MRC, the NIHR, and the Wellcome Trust; the laboratory also receives funding from National Health Service Blood and Transplant (NHSBT). The clinical fellows received funding from the MRC (C.L. and S.K.W.); the NIHR–Rare Diseases Translational Research Collaboration (S. Sivapalaratnam); and the British Society for Haematology and National Health Service Blood and Transplant (T.K.B.).This is the author accepted manuscript. The final version is available from American Society of Hematology via http://dx.doi.org/10.1182/blood-2015-12-688267