Multiple independent second-site mutations in two siblings with somatic mosaicism for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disorder associated with microthrombocytopenia, eczema, autoimmunity and predisposition to malignant lymphoma. Although rare, few cases of somatic mosaicism have been published in WAS patients to date. We here report on two Ukrainian siblings who were referred to us at the age of 3 and 4 years, respectively. Both patients suffered from severe WAS caused by a nonsense mutation in exon 1 of the WAS gene. In both siblings, flow cytometric analysis revealed the presence of Wiskott-Aldrich syndrome protein (WASp)-positive and WASp-negative cell populations among T and B lymphocytes as well as natural killer (NK) cells. In contrast to previously described cases of revertant mosaicism in WAS, molecular analyses in both children showed that the WASp-positive T cells, B cells, and NK cells carried multiple different second-site mutations, resulting in different missense mutations. To our knowledge, this is the first report describing somatic mosaicism in WAS patients caused by several independent second-site mutations in the WAS gene.

An acquired G-CSF receptor mutation results in increased proliferation of CMML cells from a patient with severe congenital neutropenia.

Severe congenital neutropenia (CN) is characterized by a maturation arrest of myelopoiesis at the promyelocyte stage. Treatment with pharmacological doses of recombinant human granulocyte colony-stimulating factor (rh-G-CSF) stimulates neutrophil production and decreases the risk of major infectious complications. However, approximately 15% of CN patients develop myeloid malignancies that have been associated with somatic mutations in the G-CSF receptor (G-CSFR) and RAS genes as well as with acquired monosomy 7. We report a CN patient with chronic myelomonocytic leukemia (CMML) who never received rh-G-CSF. Molecular analysis demonstrated a somatic G-CSFR mutation (C2390T), which led to expression of a truncated G-CSFR protein in the CMML. Normal G-CSFR expression was unexpectedly absent in primary and cultured CMML. In addition, CMML cells showed monosomy 7 and an oncogenic NRAS mutation. In vitro culture revealed a G-CSF-dependent proliferation of CMML cells, which subsequently differentiated along the monocytic/macrophage lineage. Our results provide direct evidence for the in vivo expression of a truncated G-CSFR in leukemic cells, which emerged in the absence of rh-G-CSF treatment and transduces proliferative signals.

Thrombopoietin is essential for the maintenance of normal hematopoiesis in humans: development of aplastic anemia in patients with congenital amegakaryocytic thrombocytopenia.

Recently, we and others could define the molecular cause of the rare disease congenital amegakaryocytic thrombocytopenia (CAMT) as mutations in the c-mpl gene (Blood 97: 139, 2001). We proposed that c-mpl mutations are the cause not only for the hypomegakaryocytic thrombocytopenia, but also for the development of an aplastic anemia (AA) in patients with CAMT. The effects of thrombopoietin (TPO) on early multipotent hematopoietic progenitors were elucidated by a recent series of in vitro and in vivo studies. Like CAMT patients, mice lacking the TPO receptor c-Mpl demonstrate a major reduction of early hematopoietic progenitor cells of all lineages. However, these mice achieve a normal marrow cellularity and composition, despite the lack of megakaryocytes. On the other hand, the incidence of development of aplastic anemia in CAMT is not clear owing to difficult and not consistent diagnosis of this disease.

Concentrations of thrombopoietin and interleukin-11 in the umbilical cord blood of patients with fetal alloimmune thrombocytopenia.

The objective of this study is to determine whether the fetus compensates for fetal alloimmune thrombocytopenia (FAITP) by increasing serum concentrations of thrombopoietin (TPO) and interleukin-11 (IL-11). TPO and IL-11 concentrations were measured in cord blood sera of 12 neonates with FAITP, and 35 preterm and 25 term controls. TPO concentrations in the 12 patients (median 129 pg/mL, range 73 to 325 pg/mL) were similar to those of 35 healthy preterm neonates (median 183 pg/mL, range 71 to 290 pg/mL) and the 25 term controls (median 180 pg/mL, range 93 to 302 pg/mL), although the platelet counts were significantly lower in FAITP. TPO concentrations did not correlate with the platelet counts, platelet nadir after birth, or time to recovery of normal platelet count. IL-11 reached detectable concentrations only in four patients with FAITP (median 54 pg/mL). After birth, these patients had a more rapid recovery of the platelet count. Cord blood serum concentrations of TPO are not significantly elevated in FAITP and do not predict the severity of the thrombocytopenia. Elevated IL-11 may signal a more rapid platelet recovery in FAITP.

[Congenital amegakaryocytic thrombocytopenia (CAMT) - a defect of the thrombopoietin receptor c-Mpl]. / Kongenitale amegakaryozytäre Thrombozytopenie (CAMT) - ein Defekt des Thrombopoetin-Rezeptors c-Mpl.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a very rare bone marrow failure syndrome presenting with isolated hypomegakaryocytic thrombocytopenia at birth developing into a pancytopenia during the first years of life. Bone marrow transplantation is the only curative therapy for this disease so far. Thrombopoietin (TPO) is the most important hematopoietic growth factor for the regulation of megakaryopoiesis and thrombopoiesis. We investigated TPO production and reactivity in CAMT patients. TPO plasma levels were high like in other forms of thrombocytopenia due to ineffective megakaryopoiesis. However, we found a defective reactivity to TPO: Neither hematopoietic progenitor cells in the bone marrow nor platelets from the peripheral blood did respond to TPO. Flow cytometric investigations demonstrated a lack of expression of the TPO receptor c-Mpl on the surface of platelets. Accordingly, we found mutations in the c-mpl gene, which are predicted to lead to a complete or at least partial loss of function of the TPO receptor. TPO is not only involved in the regulation of megakaryocytopoiesis but also in early hematopoiesis. This seems to be the reason for the general defect in hematopoiesis in CAMT leading to the development of pancytopenia.

Implications of mutations in hematopoietic growth factor receptor genes in congenital cytopenias.

Mutations in the genes of hematopoietic growth factor receptors as a cause of congenital cytopenia, such as congenital amegakaryocytic thrombocytopenia (CAMT) or severe congenital neutropenia (CN), are discussed. There are striking differences in the relevance of receptor mutations in these diseases. CAMT is a rare disease characterized by severe hypomegakaryocytic thrombocytopenia during the first years of life that develops into pancytopenia in later childhood. In patients with CAMT, we found inherited mutations in c-mpl, the gene coding for the thrombopoietin receptor, in 8 out of 8 cases. The type of mutation seems to correlate with the clinical course seen in the patients. Functional studies demonstrated defective thrombopoietin (TPO) reactivity in hematopoietic progenitor cells and platelets in CAMT patients. CN is a group of hematopoietic disorders characterized by profound, absolute neutropenia due to a maturation arrest of myeloid progenitor cells. About 10% of all patients develop secondary MDS/leukemia. The malignant progression is associated with acquired nonsense mutations within the G-CSF receptor gene that lead to the truncation of the carboxy-terminal cytoplasmic domain of the receptor protein involved in maturation of myeloid progenitor cells. This seems to be one important step in leukemogenesis in CN patients. CAMT is caused by inherited mutations in c-mpl, the gene for the thrombopoietin receptor, which lead to reduced or absent reactivity to TPO. In contrast, mutations in the G-CSF receptor in CN are acquired and are most probably connected with progression of the neutropenia into MDS/leukemia as a result of a loss of differentiation signaling.

c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disease presenting with isolated thrombocytopenia in infancy and developing into a pancytopenia in later childhood. Thrombopoietin (TPO) is the main regulator of thrombocytopoiesis and has also been demonstrated to be an important factor in early hematopoiesis. We analyzed 9 patients with CAMT for defects in TPO production and reactivity. We found high levels of TPO in the sera of all patients. However, platelets and hematopoietic progenitor cells of patients with CAMT did not show any reactivity to TPO, as measured by testing TPO-synergism to adenosine diphosphate in platelet activation or by megakaryocyte colony assays. Flow cytometric analysis revealed absent surface expression of the TPO receptor c-Mpl in 3 of 3 patients. Sequence analysis of the c-mpl gene revealed point mutations in 8 of 8 patients: We found frameshift or nonsense mutations that are predicted to result in a complete loss of c-Mpl function in 5 patients. Heterozygous or homozygous missense mutations predicted to lead to amino acid exchanges in the extracellular domain of the receptor were found in 3 other patients. The type of mutations correlated with the clinical course of the disease. We propose a defective c-Mpl expression due to c-mpl mutations as the cause for thrombocytopenia and progression into pancytopenia seen in patients with CAMT.

Human endothelial cells regulate survival and proliferation of human mast cells.

Mast cells (MCs) are immunoregulatory and inflammatory tissue cells preferentially located around blood vessels. Since endothelial cells have been suggested to regulate MC functions, we analyzed MC-endothelial cell interactions in vitro by performing coculture experiments with purified human intestinal MCs and human umbilical vein endothelial cells (HUVECs). We found that HUVECs provide signals allowing MCs to survive for at least 3 wk and to proliferate without addition of cytokines; otherwise all MCs died. HUVEC-dependent MC proliferation was more pronounced than that induced by stem cell factor (SCF), known to act as an MC growth factor both in vitro and in vivo. After coculture with HUVECs, most MCs were of the tryptase and chymase double-positive phenotype (MC(TC)). Transwell experiments suggested that the HUVECs' effects on MCs are not mediated by soluble factors. HUVEC-dependent MC adhesion and proliferation were inhibited by neutralizing antibodies directed against SCF and vascular cell adhesion molecule (VCAM)-1 expressed on HUVECs, and c-kit and very late antigen 4 (VLA-4) on MCs. The data suggest that two mechanisms (membrane-bound SCF/c-kit and VCAM-1/VLA-4) are involved in human MC-endothelial cell interactions. In conclusion, our study provides evidence that endothelial cells regulate MC survival and preferentially support human MC(TC) development.

Thrombopoietin induces the generation of distinct Stat1, Stat3, Stat5a and Stat5b homo- and heterodimeric complexes with different kinetics in human platelets.

OBJECTIVE: Thrombopoietin (TPO) is the pivotal regulator of thrombocytopoiesis and megakaryocytopoiesis, and binding to its receptor c-Mpl leads to activation of at least two different signaling pathways: the Jak-Stat pathway and the Ras-MAPK pathway. Our aim was to elucidate which Stat-complexes are formed in TPO signal transduction in human blood platelets. MATERIALS AND METHODS: We used electrophoretic mobility shift assays (EMSA) in order to analyze the formation of distinct Stat complexes on two distinct oligonucleotide probes. Furthermore, we used immunoprecipitation and Western blotting of protein lysates from TPO-stimulated platelets. RESULTS: We found homodimers of Stat1alpha, Stat3, Stat5a, and Stat5b, as well as heterodimers of Stat1/Stat3 and Stat5a/Stat5b, but no Stat1/Stat5 or Stat3/Stat5 heterodimers are formed in platelets in response to TPO. Stat5 complexes bound to labeled DNA with a fast kinetic followed by Stat3 and Stat1. The adapter protein CrkL is present in DNA-bound Stat5 complexes and predominantly bound to Stat5b. The kinase ERK2 is also tyrosine phosphorylated after TPO-stimulation of platelets but this activation does not modulate the phosphorylation of the serine residues in the PXSP motif present in Stat1 and Stat3. CONCLUSION: Our findings thus emphasize the differential regulation of Stat1, Stat3, Stat5a, and Stat5b in platelets and may be an appropriate model of c-Mpl signaling in mega-karyopoiesis.

Serum levels of thrombopoietin, IL-11, and IL-6 in pediatric thrombocytopenias.

We measured serum levels of thrombopoietin (TPO), interleukin (IL)-11, and IL-6 in 90 different samples from 67 pediatric patients with thrombocytopenia (TP). The cytokine levels were determined by enzyme-linked immunosorbent assays (ELISA), and the biological activity of TPO was measured using a cell line transfected with human c-mpl. In patients with impaired megakaryocytopoiesis, as found in diseases such as aplastic anemia, amegakaryocytic TP, or TP with absent radii, we found TPO levels which were highly elevated compared with normal values (mean=261 AU/ml, n=52, vs. 22 AU/ml in healthy controls). In contrast, patients suffering from idiopathic thrombocytopenic purpura (mean=16 AU/ml, n=31) or platelet function defects (mean=23 AU/ml, n=7) demonstrated normal TPO levels. The biological activity tested in the bioassay correlated well with the ELISA data. However, sera of some patients with amegakaryocytic TP demonstrated a remarkably higher biological activity of TPO than expected from the ELISA data. Within the different groups there was no correlation between platelet counts and TPO levels. Only 27% of all samples had elevated levels of IL-11 (mean=450 pg/ml, n=20). Elevated IL-6 serum levels were detected in only 13% of all samples analyzed (mean=42 pg/ml, n=12). We conclude that megakaryocytopoiesis is regulated mainly by TPO, that it is dependent on the platelet and the megakaryocytic mass, and that IL-11 plays an additional role in supporting the platelet production. IL-6 does not appear to be up-regulated in children with thrombocytopenia.

Thrombopoietin acts synergistically on Ca(2+) mobilization in platelets caused by ADP or thrombin receptor agonist peptide.

Thrombopoietin (TPO) is the main regulator of megakaryopoiesis and influences also the function of mature platelets. TPO has been shown to synergize in multiple platelet activation processes induced by various agonists. Our aim was to elucidate whether TPO affects calcium signaling during platelet activation processes. TPO demonstrated a synergistic effect on the exocytosis induced by suboptimal doses of adenosine diphosphate (ADP) and the thrombin receptor agonist peptide (TRAP). We detected synergistic effects of TPO on the ADP or TRAP induced Ca(2+) mobilization in a small range of very low agonist concentrations. The TPO synergism on Ca(2+) mobilization and CD62P expression was measurable in different, nonoverlapping ranges of ADP or TRAP concentrations. Sustaining the agonist-induced calcium signal with thapsigargin led to a detectable TPO synergism in CD62P expression even in agonist concentrations in which the synergism only occurs in Ca(2+) signaling without thapsigargin.

Defective c-Mpl signaling in the syndrome of thrombocytopenia with absent radii.

Thrombocytopenia with absent radii (TAR) syndrome is a rare congenital defect with severe hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. To elucidate a possible relationship between thrombocytopenia in TAR and defects in the thrombopoietin (TPO)/c-Mpl system, we examined TPO activity in sera from six patients and in vitro reactivity of the patients' platelets to recombinant human TPO. We found elevated TPO serum levels in all patients, excluding a TPO production defect as a pathomechanism for the thrombocytopenia. In contrast to healthy controls, however, platelets of TAR patients failed to respond to recombinant TPO as measured by testing TPO synergism to suboptimal concentration of platelet activators. Most interestingly, TPO-induced tyrosine phosphorylation of platelet proteins was completely absent (four out of five) or markedly decreased (one out of five). More detailed investigations of the signal cascades of c-Mpl demonstrated the absence of Jak2 phosphorylation after TPO stimulation in a TAR patient's platelets. A defect in the early events of c-Mpl signal transduction might be the reason for impaired megakaryocytopoiesis in TAR syndrome.

Thrombopoietin in patients with congenital thrombocytopenia and absent radii: elevated serum levels, normal receptor expression, but defective reactivity to thrombopoietin.

The pathophysiology of thrombocytopenia in the syndrome of thrombocytopenia with absent radii (TAR) is not yet understood. We examined thrombopoietin (TPO) serum levels and the in vitro reactivity of platelets to TPO in five patients affected with TAR syndrome. We found elevated TPO serum levels in all patients tested, excluding a TPO production defect as cause for thrombocytopenia in TAR syndrome. In addition, we found similar expression of the TPO receptor c-Mpl on the surface of platelets from TAR patients (5 of 5) and a similar molecular weight of the receptor as compared with healthy controls (4 of 4). Platelet response to adenosine diphosphate or thrombin receptor agonist peptide SFLLRN (TRAP) was normal in TAR patients. However, in contrast to results with healthy controls we could show absence of in vitro reactivity of platelets from TAR patients to recombinant TPO as measured by testing TPO synergism to adenine diphosphate and TRAP in platelet activation. TPO induced tyrosine phosphorylation of platelet proteins was completely absent (3 of 4) or markedly decreased (1 of 4). Our results indicate that defective megakaryocytopoiesis/thrombocytopoiesis in TAR syndrome is not caused by a defect in TPO production but a lack of response to TPO in the signal transduction pathway of c-Mpl.

Downregulation of c-kit expression in human endothelial cells by inflammatory stimuli.

In recent studies we have shown that the expression of stem cell factor (SCF) in human endothelial cells is regulated by inflammatory processes. Gram-negative bacteria, interleukin-1 (IL-1), and lipopolysaccharide were able to upregulate the expression of SCF in human umbilical vein endothelial cells (HUVEC) (Blood 83:2836, 1994). Interestingly enough c-kit, the receptor of SCF, is coexpressed on HUVEC, suggesting an autoregulatory mechanism. To investigate the relation of c-kit and inflammatory processes we stimulated HUVEC with IL-1alpha and we established an in vitro model of inflammation. Binding experiments with 125I-SCF were performed to study the c-kit receptor expression on HUVEC. Scatchard analysis revealed both high-affinity receptors (K(d) approximately 0.36 nmol/L) and low-affinity receptors (K(d) approximately 2.9 nmol/L). Exposure to IL-1alpha led to a significant 50% reduction of c-kit high-affinity receptors, whereas the number of low-affinity receptors was not affected, in comparison to a control group of untreated HUVEC. Furthermore, using Northern blot analysis we studied the regulation c-kit mRNA expression in HUVEC after stimulation with IL-1alpha. Kinetic experiments showed a time-dependent downregulation of c-kit specific transcripts. In addition, we cocultured HUVEC with diverse bacterial strains. Experiments were performed over time with 1 x 10(6) bacteria/mL. Our data showed that, in contrary to the previously reported upregulation of SCF mRNA expression, stimulation with Yersinia enterocolitica or with Neisseria meningitidis led to a significant time-dependent downregulation of c-kit mRNA within 3 hours. These data indicate that inflammatory stimuli such as IL-1 or living bacteria activate a mechanism that downregulates c-kit receptor expression in human endothelial cells during the state of inflammation.