Hilary Christensen

Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome

Next-generation RNA sequence analysis of platelets from an individual with autosomal recessive gray platelet syndrome (GPS, MIM139090) detected abnormal transcript reads, including intron retention, mapping to NBEAL2 (encoding neurobeachin-like 2). Genomic DNA sequencing confirmed mutations in NBEAL2 as the genetic cause of GPS. NBEAL2 encodes a protein containing a BEACH domain that is predicted to be involved in vesicular trafficking and may be critical for the development of platelet α-granules.

Myelodysplasia and acute megakaryoblastic leukemia in down's syndrome

In this report we describe the clinical and hematologic features of 23 cases of myelodysplasia (MDS) or acute megakaryoblastic leukemia (AMKL) occurring in Downs syndrome. MDS was characterized by thrombocytopenia, abnormal megakaryocytopoiesis, megakaryoblasts (< 30%) in the marrow and abnormal karyotype, the most common of which was trisomy 8, found in 7/15 patients with MDS. Three of five patients achieved a complete remission with low dose cytosine arabinoside, vincristine and retinyl palmitate. The high cure rate and the distinctive features of the leukemic process in these cases suggest that this type of MDS and AMKL are unique to patients with Downs syndrome.

Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice.

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-) platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development, and platelet production.

Platelet-associated complement factor H in healthy persons and patients with atypical HUS

Atypical hemolytic uremic syndrome (aHUS) is associated with complement system dysregulation, and more than 25% of pediatric aHUS cases are linked to mutations in complement factor H (CFH) or CFH autoantibodies. The observation of thrombocytopenia and platelet-rich thrombi in the glomerular microvasculature indicates that platelets are intimately involved in aHUS pathogenesis. It has been reported that a releasable pool of platelet CFH originates from alpha-granules. We observed that platelet CFH can arise from endogenous synthesis in megakaryocytes and that platelets constitutively lacking alpha-granules contain CFH. Electron and high-resolution laser fluorescence confocal microscopy revealed that CFH was present throughout the cytoplasm and on the surface of normal resting platelets with no evident concentration in alpha-granules, lysosomes, or dense granules. Therapeutic plasma transfusion in a CFH-null aHUS patient revealed that circulating platelets take up CFH with similar persistence of CFH in platelets and plasma in vivo. Washed normal platelets were also observed to take up labeled CFH in vitro. Exposure of washed normal platelets to plasma of an aHUS patient with CFH autoantibodies produced partial platelet aggregation or agglutination, which was prevented by preincubation of platelets with purified CFH. This CFH-dependent response did not involve P-selectin mobilization, indicating a complement-induced platelet response distinct from alpha-granule secretion.

The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis.

Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.

Interstitial deletion of 11q-implicating the KIRREL3 gene in the neurocognitive delay associated with Jacobsen syndrome†

Jacobsen syndrome (JS) is a rare contiguous gene disorder characterized by a deletion within the distal part of the long arm of chromosome 11 ranging in size from 7 to 20 Mb. The clinical findings include characteristic dysmorphic features, growth and psychomotor delays and developmental anomalies involving the brain, eyes, heart, kidneys, immune, hematologic, endocrine, and gastrointestinal systems. The majority of cases are due to a terminal deletion of 11q; however interstitial deletions have also been reported. We report on a child with clinical manifestations consistent with JS who had a 2.899 Mb interstitial deletion at 11q24.2–q24.3 which is the smallest interstitial deletion reported so far to our knowledge. This deletion includes the KIRREL3 gene, and given our patients history of neurocognitive delay and autism spectrum disorder, it raises the possibility that this gene is a candidate for the social and expressive language delay observed in our patient.

Ultrastructural Studies of the Megakaryoblastic Leukemias of Down Syndrome

The ultrastructure of the leukemic cells in transient leukemia (six cases), myelodysplasia (five cases) and acute megakaryoblastic leukemia (one case) in patients with Down syndrome were studied. The cells were identified to be of megakaryocytic lineage by virtue of the expression of platelet glycoprotein GpIIIa, detected by immunogold labelling. In all patients, some of the leukemic cells had ultrastructural features of megakaryocytes, including ectoplasmic protrusions, demarcation membranes, and alpha granules. Differentiation was greatest in the cells of patients with transient leukemia. These studies provide a detailed assessment of the ultrastructural features of the leukemic cells in the megakaryoblastic leukemias of Down syndrome.

Proteasome function is required for platelet production

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

Transient leukemia with extreme basophilia in a phenotypically normal infant with blast cells containing a pseudodiploid clone, 46,XY i(21)(q10)

PURPOSE Transient leukemia and extreme basophilia occurred in a phenotypically normal newborn with expression of isochromosome (21)(q10) in the blast population. PATIENTS AND METHODS A newborn boy was found to have an elevated white blood cell count of 120,800 with 33% blasts. The peripheral blood also contained elevated numbers of basophils and neutrophils with unusual staining properties. The blasts, evaluated by flow cytometry and light and electron microscopy, had the properties of megakaryoblasts. Cytogenetic studies revealed 46,XY karyotype in peripheral blood lymphocytes; however, analysis of the blast cells from the bone marrow showed an abnormal chromosome 21. RESULTS The blast cells in the peripheral blood disappeared by day 42 without chemotherapy. The red blood cell count and platelet count normalized by 2 months. Chromosomal analysis of skin fibroblasts and bone marrow after the disappearance of the blast cells in the peripheral blood showed a 46,XY phenotype. CONCLUSIONS The leukemic cell of transient leukemia has the potential of forming cells of basophil and megakaryocyte lineages. Trisomy of the q arm of chromosome 21 contains sufficient genetic information for the development of transient leukemia in a phenotypically normal newborn.