Analyses of Genetic and Clinical Parameters for Screening Patients With Inherited Thrombocytopenia with Small or Normal-Sized Platelets.

BACKGROUND: Childhood thrombocytopenias include immune thrombocytopenic purpura (ITP) and inherited thrombocytopenia; the former is caused by autoantibodies to platelets, whereas the latter can be distinguished by platelet size and underlying genetic mutations. Due to limited methods for the definite diagnosis of ITP, genetic and clinical parameters are required for diagnosing inherited thrombocytopenias with small or normal-sized platelets. PROCEDURE: In total, 32 Japanese patients with thrombocytopenia with small or normal-sized platelets from 29 families were enrolled. All the patients were under 20 years of age, with family histories of early-onset thrombocytopenia and/or poor response to conventional therapies for ITP. Genotypes and clinical parameters were retrospectively evaluated according to the disease type. RESULTS: Twelve cases of inherited thrombocytopenia were observed. We identified chromosomal deletions within the WASP gene in two patients with Wiskott-Aldrich syndrome; a missense mutation in a patient with X-linked thrombocytopenia; and mutations in the RUNX1 gene of five patients with familial platelet disorder with propensity to acute myelogenous leukemia, and in the ANKRD26 gene of four patients with autosomal dominant thrombocytopenia-2. All 12 carried germline mutations, three of which were de novo. Furthermore, we observed significantly elevated serum thrombopoietin (TPO) levels and dysplasia of megakaryocytes in patients carrying the RUNX1 and ANKRD26 mutations. CONCLUSIONS: Genetic analyses and detection of TPO levels and dysmegakaryopoiesis were clinically useful for screening patients with inherited thrombocytopenias, irrespective of the family history. We hypothesize that the WASP, RUNX1, and ANKRD26 genes are important for normal TPO signaling and the network underlying thrombopoiesis.

[Analysis of prenatal diagnosis for seven high-risk fetuses with Wiskott-Aldrich syndrome].

OBJECTIVE: To investigate the value of gene analysis of amniotic fluid exfoliated cells and WASP detection from cord blood in prenatal diagnosis of high-risk fetus with Wiskott-Aldrich syndrome. METHOD: Seven patients with Wiskott-Aldrich syndrome were diagnosed by gene analysis and WASP detected by flow cytometry from 2008 to 2010. After detailed inquiry for medical history and gene analysis of related family members, seven pedigree trees were drawn, including 15 carriers of abnormal genes. From 2008 to 2011, seven samples of amniotic cell gotten by amniocentesis were collected from seven high-risk pregnant women with abnormal gene during 18 to 20 gestational weeks. WASP gene was amplified by polymerase chain reaction (PCR) from DNA of amniotic cell gotten and sequencing was performed directly on the PCR products forward and reversely. Embryo blood sample was collected from one high-risk fetus by needle puncture of umbilical blood vessel and WASP expression was detected by flow cytometry. Karyotyping was performed in amniotic cell gotten cultivated by orthotopic slice and G band staining. Gene analysis of WASP, WASP expression detected by flow cytometry and evaluation of immune function were reexamined in high-risk fetus after delivery. RESULT: Amniocentesis and culture of amniotic cell succeeded in all the seven fetuses. Gene analysis and karyotyping showed that one male fetus and four female fetuses were normal and two female fetuses were carriers. WASP expression detected from embryo blood sample of the patient was normal. After delivery, the result of gene analysis, WASP detection and evaluation of immune function was the same as that of prenatal diagnosis. CONCLUSION: Karyotyping, gene analysis and WASP detection of cord blood can provide reliable service of prenatal diagnosis for high-risk pregnant women with Wiskott-Aldrich syndrome.

Possible application of flow cytometry for evaluation of the structure and functional status of WASP in peripheral blood mononuclear cells.

The Wiskott-Aldrich syndrome protein (WASP), which is defective in Wiskott-Aldrich syndrome (WAS) patients, is an intracellular protein expressed in non-erythroid hematopoietic cells. Previously, we have established methods to detect intracellular WASP expression in peripheral blood mononuclear cells (PBMNCs) using flow cytometric analysis (FCM-WASP) and have revealed that WAS patients showed absent or very low level intracellular WASP expression in lymphocytes and monocytes, while a significant amount of WASP was detected in those of normal individuals. We applied these methods for diagnostic screening of WAS patients and WAS carriers, as well as to the evaluation of mixed chimera in WAS patients who had previously undergone hematopoietic stem cell transplantation. During these procedures, we have noticed that lymphocytes from normal control individuals showed dual positive peaks, while their monocytes invariably showed a single sharp WASP-positive peak. To investigate the basis of the dual positive peaks (WASP(low-bright) and WASP(high-bright)), we characterized the constituent linage lymphocytes of these two WASP-positive populations. As a result, we found each WASP(low/high) population comprised different linage PBMNCs. Furthermore, we propose that the difference between the two WASP-positive peaks did not result from any difference in WASP expression in the cells, but rather from a difference in the structural and functional status of the WASP protein in the cells. It has been shown that WASP may exist in two forms; an activated or inactivated form. Thus, the structural and functional WASP status or configuration could be evaluated by flow cytometric analysis.

Rapid platelet turnover in WASP(-) mice correlates with increased ex vivo phagocytosis of opsonized WASP(-) platelets.

OBJECTIVE: Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). MATERIALS AND METHODS: Consumption rates of WAS protein (WASP)(-) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(-) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow-derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets. RESULTS: CMFDA-labeled WASP(-) platelets are consumed more rapidly than WT platelets in either WT or WASP(-) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(-) reticulated platelets. The number of reticulated platelets is reduced in WASP(-) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(-) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow-derived macrophages. In vivo consumption rates of WASP(-) platelets are more accelerated by opsonization than are those of WT platelets. CONCLUSION: Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(-) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies.

Wiskott-Aldrich syndrome protein is involved in alphaIIb beta3-mediated cell adhesion.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the alphaIIb cytoplasmic tail of platelet integrin alphaIIb beta3, which has a primary role in platelet aggregation. We also show that the WASP-CIB complex is important in alphaIIb beta3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces alphaIIb beta3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding.