title: Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features.
abstract: Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novelFLI1variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. TheFLI1variant carriers' platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers' platelets, presumably due to a biogenesis defect. Additionally, 25-29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7-9%) and autophagosome-like structures (0-3%).In vitrostudy of megakaryocytes derived from circulating CD34+cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of theFLI1variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 inFLI1variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis ofFLI1variant-associated thrombocytopenia.
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France paul.saultier@gmail.com.
authors.forename: Paul
authors.initials: P
authors.lastname: Saultier
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.
authors.forename: Léa
authors.initials: L
authors.lastname: Vidal
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.
authors.forename: Matthias
authors.initials: M
authors.lastname: Canault
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Bernot
authors.affiliation: APHM, CHU Timone, French Reference Center on Inherited Platelet Disorders, Marseille, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Falaise
authors.affiliation: APHM, CHU Timone, French Reference Center on Inherited Platelet Disorders, Marseille, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Pouymayou
authors.affiliation: Unité d'Hémostase Biologique, Bron, France.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Bordet
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.|APHM, CHU Timone, French Reference Center on Inherited Platelet Disorders, Marseille, France.
authors.forename: Noémie
authors.initials: N
authors.lastname: Saut
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.|APHM, CHU Timone, French Reference Center on Inherited Platelet Disorders, Marseille, France.
authors.forename: Agathe
authors.initials: A
authors.lastname: Rostan
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.|APHM, CHU Timone, French Reference Center on Inherited Platelet Disorders, Marseille, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Baccini
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Peiretti
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Favier
authors.affiliation: ICAN Institute for Cardiometabolism and Nutrition, Paris, France.|Inserm, UMR_S 1166, Team Genomics and Pathophysiology of Cardiovascular Diseases, Paris, France.|Sorbonne Universités, Université Pierre et Marie Curie (UPMC Univ Paris 06), UMR_S 1166, France.
authors.forename: Pauline
authors.initials: P
authors.lastname: Lucca
authors.affiliation: Centre National de Génotypage, Institut de Génomique, CEA, Evry, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Deleuze
authors.affiliation: Centre National de Génotypage, Institut de Génomique, CEA, Evry, France.
authors.forename: Robert
authors.initials: R
authors.lastname: Olaso
authors.affiliation: Centre National de Génotypage, Institut de Génomique, CEA, Evry, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Boland
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.|APHM, CHU Timone, French Reference Center on Inherited Platelet Disorders, Marseille, France.
authors.forename: Pierre Emmanuel
authors.initials: PE
authors.lastname: Morange
authors.affiliation: UMR_S949 INSERM, Strasbourg, France.|Etablissement Français du Sang (EFS)-Alsace, Strasbourg, France.|Fédération de Médecine Translationnelle de Strasbourg (FMTS), France.|Université de Strasbourg, Marseille, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Gachet
authors.affiliation: Beckman Coulter Immunotech, Life Sciences Global Assay and Applications Development, Marseille, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Malergue
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.
authors.forename: Sixtine
authors.initials: S
authors.lastname: Fauré
authors.affiliation: UMR_S949 INSERM, Strasbourg, France.|Etablissement Français du Sang (EFS)-Alsace, Strasbourg, France.|Fédération de Médecine Translationnelle de Strasbourg (FMTS), France.|Université de Strasbourg, Marseille, France.
authors.forename: Anita
authors.initials: A
authors.lastname: Eckly
authors.affiliation: ICAN Institute for Cardiometabolism and Nutrition, Paris, France.|Inserm, UMR_S 1166, Team Genomics and Pathophysiology of Cardiovascular Diseases, Paris, France.|Sorbonne Universités, Université Pierre et Marie Curie (UPMC Univ Paris 06), UMR_S 1166, France.
authors.forename: David-Alexandre
authors.initials: DA
authors.lastname: Trégouët
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Poggi
authors.affiliation: Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France.|APHM, CHU Timone, French Reference Center on Inherited Platelet Disorders, Marseille, France.
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Alessi
mesh_terms: D000328:Adult; D001792:Blood Platelets / Q000473:pathology / Q000648:ultrastructure; D002467:Cell Nucleus / Q000737:chemistry; D003594:Cytoplasmic Granules / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D008297:Male; D008533:Megakaryocytes / Q000473:pathology; D008875:Middle Aged; D010974:Platelet Aggregation / Q000235:genetics; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics; D013921:Thrombocytopenia / Q000151:congenital / Q000209:etiology*; D014158:Transcription, Genetic; D055815:Young Adult
chemical_list: C078433:FLI1 protein, human; D051789:Proto-Oncogene Protein c-fli-1