Amy E. Geddis

ANKRD26 -related thrombocytopenia and myeloid malignancies

To the editor: Since the discovery that mutations in the 5′ untranslated region (UTR) of ANKRD26 are responsible for an autosomal-dominant form of thrombocytopenia ( ANKRD26 -RT),[1][1] 21 affected families were reported.[2][2] A study analyzing this series of patients suggested that ANKRD26 -RT

Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K(553) and K(573)). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes.

Endomitotic Megakaryocytes that Form a Bipolar Spindle Exhibit Cleavage Furrow Ingression Followed by Furrow Regression

Megakaryocyte (MK) differentiation is marked by the development of progressive polyploidy, due to repeated incomplete cell cycles in which mitosis is aborted during anaphase, a process termed endomitosis. We have postulated that anaphase in endomitotic MKs diverges from diploid mitosis at a point distal to the assembly of the midzone, possibly involving impaired cleavage furrow progression. To define the extent of furrow initiation and ingression in endomitosis, we performed time-lapse imaging of MKs expressing yellow fluorescent protein (YFP)-tubulin and monitored shape change as they progressed through anaphase. We found that in early endomitotic cells that have a bipolar spindle, cleavage furrows form that can undergo significant ingression, but furrows regress to produce polyploid cells. Compared to cells that divide, cells that exhibit furrow regression have a slower rate of furrow ingression and do not furrow as deeply. More highly polyploid MKs undergoing additional endomitotic cycles also show measurable furrowing that is followed by regression, but the magnitude of the shape change is less than seen in the early MKs. This suggests that in the earliest endomitotic cycles when there is formation of a bipolar spindle, the failure of cytokinesis occurs late, following assembly and initial constriction of the actin/myosin ring, whereas in endomitotic MKs that are already polyploid there is secondary inhibition of furrow progression. This behavior of furrow ingression followed by regression may explain why midbody remnants are occasionally observed in polyploid MKs. This finding has important implications for the potential mechanisms for cytokinesis failure in endomitosis.

Inherited thrombocytopenias: toward a molecular understanding of disorders of platelet production.

Purpose of review To review the defined syndromes of inherited thrombocytopenia and discuss new genetic data for several disorders that shed light on the process of megakaryopoiesis. Recent findings The genes responsible for several inherited thrombocytopenias have been recently discovered, including congenital amegakaryocytic leukemia, amegakaryocytic thrombocytopenia with radio-ulnar synostosis, familial platelet syndrome with predisposition to acute myelogenous leukemia, Paris-Trousseau, Wiskott-Aldrich syndrome, and the May-Hegglin, Sebastian, Epstein, and Fechner syndromes. These clinical syndromes, combined with studies in mouse and in vitro models, reveal the importance of these genes for normal hematopoiesis. Summary Although inherited syndromes of thrombocytopenia are rare, characterization of mutations in these disorders has contributed greatly to our understanding of megakaryocyte and platelet development. A systematic registry of congenitally thrombocytopenic individuals would almost certainly lead to new genetic discoveries.

Congenital Amegakaryocytic Thrombocytopenia and Thrombocytopenia with Absent Radii

Thrombocytopenia is a relatively common clinical problem in hospitalized neonates, and it is critical to distinguish infants who have rare congenital thrombocytopenias from those who have acquired disorders. Two well-described inherited thrombocytopenia syndromes that present in the newborn period are congenital amegakaryocytic thrombocytopenia (CAMT) and thrombocytopenia with absent radii (TAR). Although both are characterized by severe (< 50,000/microL) thrombocytopenia at birth, the molecular mechanisms underlying these disorders and their clinical presentations and courses are distinct. CAMT is an autosomal recessive disorder caused by mutations in the thrombopoietin (TPO) receptor c-Mpl. TAR is a syndrome of variable inheritance and unclear genetic etiology consisting of thrombocytopenia in association with bilateral absent radii and frequently additional congenital abnormalities. This article summarizes the current understanding of the pathophysiology and clinical course of CAMT and TAR.

Congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is clinically characterized by thrombocytopenia presenting at birth in a child without congenital or skeletal malformations, reduced or absent bone marrow megakaryocytes, and eventual progression to bone marrow failure. Molecular studies in most cases confirm homozygous or compound heterozygous mutations in the thrombopoietin receptor c‐Mpl. In addition to the clinical importance of recognizing this disorder, characterization of mutations identified in patients with CAMT has led to insights into thrombopoietin receptor structure and function. This review will summarize the diagnosis, pathophysiology, and management of CAMT. Pediatr Blood Cancer 2011; 57: 199–203.

Endomitotic Megakaryocytes form a Midzone in Anaphase but Have a Deficiency in Cleavage Furrow Formation

Megakaryocyte differentiation is marked by development of progressive polyploidy and accumulation of large nuclear mass and cytoplasmic volume. During differentiation, megakaryocytes undergo repeated incomplete cell cycles in which mitosis is aborted in late anaphase with failure of cytokinesis, termed endomitosis. Recent studies have postulated that failure of Aurora-B kinase to localize to the spindle midzone is responsible for endomitosis in megakaryocytes. In diploid cells, the translocation of Aurora-B kinase is critical for positioning of the cleavage furrow, in part through its phosphorylation of the Rho family GTPase activating protein MgcRacGAP which in turn alters activity of RhoA. However, we have previously demonstrated that Aurora-B kinase localizes to centromeres and is functional in endomitotic megakaryocytes. Here, we show that endomitotic megakaryocytes form midzone structures that recruit Aurora-B kinase and its substrate MgcRacGAP. Although many cells with polyploid anaphases showed cortical localization of Aurora-B kinase, we did not observe accumulation of RhoA in furrows or formation of an actin ring. When mitotic exit was induced by inhibition of cdk1, diploid control cells formed furrows exhibiting cortical RhoA but megakaryocytes exited endomitosis without evidence of furrowing. Therefore, localization of Aurora-B kinase to the midzone is normal in endomitotic megakaryocytes but furrowing is abnormal. These data suggest that endomitotic MKs fail to complete cytokinesis due to aberrant regulation of furrowing at a step subsequent to the localization of Aurora-B kinase, possibly involving the activation or localization of RhoA. This work explores the mechanism of a normally occurring furrowing defect in a non-malignant primary cell.

Diagnosis of immune thrombocytopenic purpura in children

Purpose of reviewThis review updates the differential diagnosis between inherited and acquired immune thrombocytopenic purpura as well as clinical practice on the initial diagnosis of children with the disease. Recent findingsA diagnosis of immune thrombocytopenic purpura may be based on an evaluation of the history, physical findings such as petechiae, bruising and mucous membrane bleeding, examination of peripheral blood films stained with Wrights or May–Grünwald–Giemsa, determination of blood counts, platelet size and appearance. Recently, diagnostic assays have been developed to detect platelet-bound antibodies. The sensitivity of these assays, however, is suboptimal, with a positive predictive value of 80–83%. If the diagnosis of immune thrombocytopenic purpura is in question due to the presence of atypical features, or if a patient with findings typical of the disease does not respond to therapy, bone marrow aspiration and biopsy are indicated to confirm the diagnosis. SummaryThe diagnosis of immune thrombocytopenic purpura is a process of elimination of other sources of thrombocytopenia. If the criteria discussed above are inconclusive and if the patient does not respond to therapy in 6–12 months (this is especially true in children) then a bone marrow aspiration is required to confirm the diagnosis, especially before initiating corticosteroid therapy.

Compound heterozygous c-Mpl mutations in a child with congenital amegakaryocytic thrombocytopenia: Functional characterization and a review of the literature

OBJECTIVE To genetically and functionally characterize mutations of c-Mpl that lead to thrombocytopenia in a child with congenital amegakaryocytic thrombocytopenia. MATERIALS AND METHODS We identified two c-Mpl mutations in a child with clinical features of congenital amegakaryocytic thrombocytopenia, one a previously described mutation in the extracellular domain (R102P) and the other a novel mutation leading to truncation of the receptor after the box 1 homology domain (541Stop). Cell line models were created to examine the ability of the mutant receptors to signal in response to thrombopoietin and thrombopoietin-like agonists. RESULTS Data from cell-line models indicate that c-Mpl R102P does not support significant signaling in response to thrombopoietin due to impaired trafficking of the mutant receptor to the cell surface. Alternative thrombopoietic agents do not circumvent this block to signaling, likely due to the inaccessibility of the receptor. In addition, previous data indicate that c-Mpl 541Stop does not support intracellular signaling due to the loss of critical intracellular domains. CONCLUSIONS This case demonstrates two different mechanisms by which c-Mpl mutations can impair thrombopoietin signaling, and suggests that mutations in the extracellular domain will not be rescued by c-Mpl agonists if they interfere with normal receptor expression.

Deletion of JAM-C, a candidate gene for heart defects in Jacobsen syndrome, results in a normal cardiac phenotype in mice†

The 11q terminal deletion disorder (11q‐) is a rare chromosomal disorder caused by a deletion in distal 11q. Fifty‐six percent of patients have clinically significant congenital heart defects. A cardiac “critical region” has been identified in distal 11q that contains over 40 annotated genes. In this study, we identify the distal breakpoint of a patient with a paracentric inversion in distal 11q who had hypoplastic left heart and congenital thrombocytopenia. The distal breakpoint mapped to JAM‐3, a gene previously identified as a candidate gene for causing HLHS in 11q‐. To determine the role of JAM‐3 in cardiac development, we performed a comprehensive cardiac phenotypic assessment in which the mouse homolog for JAM‐3, JAM‐C, has been deleted. These mice have normal cardiac structure and function, indicating that haplo‐insufficiency of JAM‐3 is unlikely to cause the congenital heart defects that occur in 11q‐ patients. Notably, we identified a previously undescribed phenotype, jitteriness, in most of the sick or dying adult JAM‐C knockout mice. These data provide further insights into the identification of the putative disease‐causing cardiac gene(s) in distal 11q, as well as the functions of JAM‐C in normal organ development.