Paris-Trousseau Syndrome Research Articles

The electron microscopy contribution to platelet structural pathology investigation

This article discusses the role of electron microscopy in the diagnosis and study of morphological changes that cause platelet structural abnormalities in a variety of congenital diseases. Morphological abnormalities can be divided into the abnormalities of the platelet cytoskeleton, of alpha and dense granules, and membrane abnormalities. Our paper describes ultrastructural platelet defects in Wiskott–Aldrich syndrome, MYH9-associated syndromes, gray platelet syndrome, Hermansky–Pudlak syndrome, Paris–Trousseau syndrome, Chediak–Higashi syndrome.

SPECTRUM OF IMMUNODEFICIENCIES IN JACOBSEN SYNDROME

Introduction Jacobsen syndrome (JS) is a rare contiguous gene deletion syndrome associated with a distal deletion of chromosome 11q. Immune deficiency in JS has not been well-established but usually involves antibody defects. Here we report 2 cases of Jacobsen syndrome with combined T and B cell defects presenting at different ages. Case Description The first case is now a 22-month-old girl with JS who was initially seen at 2 months of age. She has corneal opacities, IUGR, thrombocytopenia (Paris-Trousseau syndrome), and intracranial hemorrhages. Newborn screening (NBS) for severe combined immunodeficiency (SCID) was negative. At 2 months of age, she had slightly low T-cell, moderately low B-cell, and normal NK-cell counts. At 6 months of age, she developed progressive T-cell cytopenia and intermittently low NK cell counts. Immunoglobulin levels and IgG response to vaccines were normal. She has no history of recurrent or severe/opportunistic infections. The second case, a 22-day-old girl, was referred because of positive NBS for SCID. She has left cystic renal dysplasia, hypoplastic right hand, trigonocephaly, and coronary abnormalities (ASD, small coronary fistula). Laboratory evaluation revealed thrombocytopenia, lymphopenia, normal IgM and IgG, low IgA, and moderately low T and B cell counts and normal NK cell counts. Genetic evaluation confirmed the diagnosis of JS. Findings in these cases suggest that immune deficiency in JS can start at any age and may be progressive. Discussion Jacobsen syndrome is one of the syndromic primary immunodeficiencies where type and severity of immune deficiency vary and can be progressive.

FLI1 level during megakaryopoiesis affects thrombopoiesis and platelet biology

AbstractFriend leukemia virus integration 1 (FLI1), a critical transcription factor (TF) during megakaryocyte differentiation, is among genes hemizygously deleted in Jacobsen syndrome, resulting in a macrothrombocytopenia termed Paris-Trousseau syndrome (PTSx). Recently, heterozygote human FLI1 mutations have been ascribed to cause thrombocytopenia. We studied induced-pluripotent stem cell (iPSC)–derived megakaryocytes (iMegs) to better understand these clinical disorders, beginning with iPSCs generated from a patient with PTSx and iPSCs from a control line with a targeted heterozygous FLI1 knockout (FLI1+/−). PTSx and FLI1+/− iMegs replicate many of the described megakaryocyte/platelet features, including a decrease in iMeg yield and fewer platelets released per iMeg. Platelets released in vivo from infusion of these iMegs had poor half-lives and functionality. We noted that the closely linked E26 transformation-specific proto-oncogene 1 (ETS1) is overexpressed in these FLI1-deficient iMegs, suggesting FLI1 negatively regulates ETS1 in megakaryopoiesis. Finally, we examined whether FLI1 overexpression would affect megakaryopoiesis and thrombopoiesis. We found increased yield of noninjured, in vitro iMeg yield and increased in vivo yield, half-life, and functionality of released platelets. These studies confirm FLI1 heterozygosity results in pleiotropic defects similar to those noted with other critical megakaryocyte-specific TFs; however, unlike those TFs, FLI1 overexpression improved yield and functionality.

Jacobsen/Paris-Trousseau Syndrome: Report of a Case with Emphasis on Platelet’s Light Microscopic and Ultrastructure Findings

Jacobsen syndrome (JS) is a rare, inherited disorder, characterized by facial and skull dysmorphism, mental retardation, and platelet abnormalities. Paris-Trousseau syndrome (PTS) is a platelet function disorder that may be encountered in patients affected by JS. PTS is manifested by a mild lifelong bleeding tendency. Morphologically, the presence of large fused platelet alpha granules is characteristic. We present a case of Jacobsen syndrome in a child and highlight the typical morphologic and ultrastructure findings of platelets.

A Dominant Negative Mutation (p.P214L) in ETV6 is Associated with Megakaryocyte and Erythroid Transcript Misregulation

Background: Our group has recently described a family with autosomal dominant thrombocytopenia, high mean corpuscular volume (MCV) of red cells, and 2 occurrences of pre B-cell acute lymphoblastic leukemia (ALL) associated with a mutation in the transcription factor ETV6. ETV6 is a transcriptional repressor which functions through dimerization with itself or with other transcription factors such as FLI1, mutations in which cause a familial form of thrombocytopenia called Paris-Trousseau syndrome. The missense mutation found in this family (p.P214L) has a dominant negative effect by dimerizing with and sequestering WT ETV6 from the nucleus. This dominant negative mutation causes a phenotype in both platelets and red cells, suggesting a disruption in megakaryocyte-erythroid differentiation. Therefore, we aimed to study the effect of p.P214L ETV6 on megakaryocyte-erythroid differentiation by studying the platelet transcriptome, protein from platelet lysates, and platelet surface markers of patients with the ETV6 mutation. To investigate whether this platelet and red cell phenotype is due to interactions with FLI1, which is essential for megakaryocyte-erythroid lineage determination, we also aimed to study the interaction of p.P214L ETV6 with FLI1.Methods: RNA was isolated from leukoreduced platelets of 2 patients with the p.P214L mutation (affected), 3 unaffected family members, and 2 unrelated controls for whole transcriptome analysis. GPIX quantification from platelet lysates of 2 affected patients and 3 controls was done by Western blot. Analysis of protein dimerization was done by expression of differentially tagged ETV6 and FLI1 constructs (His or c-myc) in HEK293T cells, pull down with anti-c-myc beads, and quantification of both c-myc and His by Western blot. To determine platelet receptor levels, flow cytometry was done with antibodies against GPIIb, GPIIIa, GPIX, and GPIb on peripheral blood from one affected patient and one control.Results: Platelet transcriptome analysis of patients with the p.P214L mutation revealed significant differential expression of more than 200 transcripts between patients and controls. Seven of the significant transcripts were key erythrocyte transcripts, which were upregulated in affected patients; and 9 transcripts were key platelet transcripts, which were downregulated. Of these, the red cell marker GYPA was upregulated 25 fold in affected patients (p < 5x10-5), and the platelet markers VWF and GPIX, a target of FLI1, were downregulated 6.4 and 5.6 fold in affected patients (p < 0.05 and p < 0.07), respectively. Platelet GPIX protein expression was also downregulated in 2 affected patients as determined by Western blot. Furthermore, flow cytometry of platelets from one patient with the p.P214L mutation indicated a marginally decreased Ib-IX:IIb-IIIa ratio (0.321) when compared to a normal control (0.500). In vitro studies confirmed that the p.P214L mutation did not disrupt dimerization with WT ETV6 or FLI1, indicating that this defect could be due to dominant negative interactions with both ETV6 and FLI1.Conclusions: Several transcription factors, including FLI1, have been described to play a role in the fate determination between erythroid and megakaryocyte lineages. We report that a dominant negative mutation in ETV6, found in a family with both platelet and red blood cell abnormalities, is associated with differentially regulated red blood cell and platelet transcripts, decreased GPIX protein in platelet lysates, decreased Ib-IX:IIb-IIIa ratio, and dimerization with WT ETV6 and FLI1 in vitro. Our data indicate an important role for ETV6, either alone or through interaction with FLI1, in megakaryocyte-erythroid differentiation. DisclosuresNo relevant conflicts of interest to declare.

MYH10 protein expression in platelets as a biomarker of RUNX1 and FLI1 alterations

AbstractRUNX1 gene alterations are associated with acquired and inherited hematologic malignancies that include familial platelet disorder/acute myeloid leukemia, primary or secondary acute myeloid leukemia, and chronic myelomonocytic leukemia. Recently, we reported that RUNX1-mediated silencing of nonmuscle myosin heavy chain IIB (MYH10) was required for megakaryocyte ploidization and maturation. Here we demonstrate that runx1 deletion in mice induces the persistence of MYH10 in platelets, and a similar persistence was observed in platelets of patients with constitutional (familial platelet disorder/acute myeloid leukemia) or acquired (chronic myelomonocytic leukemia) RUNX1 mutations. MYH10 was also detected in platelets of patients with the Paris-Trousseau syndrome, a thrombocytopenia related to the deletion of the transcription factor FLI1 that forms a complex with RUNX1 to regulate megakaryopoiesis, whereas MYH10 persistence was not observed in other inherited forms of thrombocytopenia. We propose MYH10 detection as a new and simple tool to identify inherited platelet disorders and myeloid neoplasms with abnormalities in RUNX1 and its associated proteins.

Role of Platelet Electron Microscopy in the Diagnosis of Platelet Disorders

The electron microscope has proved to be a useful tool to study and understand the biology of platelets and to classify many platelet disorders. After a technical overview, this article reviews syndromes originating from platelet organelle, cytoskeleton, and membrane defects for which electron microscopy plays a role in the diagnostic process, such as gray platelet syndrome, Paris-Trousseau syndrome, storage pool diseases, MYH9-related thrombocytopenias, or Wiskott-Aldrich syndrome. Particular focus is given to the ultrastructural aspect of platelets in these disorders.

Transcriptional Regulation of Thrombopoiesis.

Abstract Over the past two decades, a number of key transcription factors have been identified that play essential roles in megakaryocyte development. These include GATA-1, GATA-2, Friend of GATA-1 (FOG-1), Runx-1, Cbf-β, Fli-1, GABPα, TEL, NF-E2 p45, Gfi-1b, and SCL/TAL. Importantly, mutations in genes encoding several of these have been linked to human disorders of thrombopoieisis. Germline GATA-1 mutations that disrupt binding to FOG-1 cause X-linked macrothrombocytopenia and dyserythropoietic anemia. Acquired GATA-1 mutations that lead to exclusive production of a short isoform (GATA-1s) play initiating roles in Down Syndrome Transient Myeloproliferative Disorder (DS-TMD) and subsequent Acute Megakaryoblastic Leukemia (DS-AMKL). Haploinsufficiency of Runx-1 causes Familial Platelet Disorder with Propensity to Develop AML (FPD/ AML). Heterozygous loss of the Fli-1 gene leads to the macrothrombocytopenia seen in Jacobsen’s (Paris-Trousseau) syndrome. Important outstanding questions include: how these transcription factors act together to control megakaryocyte terminal maturation; how they differentially act as activators or repressors depending on gene context; how they intersect with cell signaling pathways; how they may coordinate terminal megakaryocyte maturation with spatial location within the bone marrow; how they may control cell fate decisions of bipotential erythroid/megakaryocytic progenitor cells; and whether additional key transcription factors exist. Application of proteomic approaches involving multi-protein complex purification has provided novel insights into some of these questions. We have isolated GATA-1 containing complexes from megakaryocytic cells and identified the Krüppel-type zinc finger transcription factor ZBP-89 as a novel regulator of megakaryocyte and erythroid development. Knockdown of ZBP-89 expression in zebrafish embryos and mice results in blocked early megakaryopoiesis and definitive erythropoiesis, phenocopying aspects of GATA-1- and FOG-1-deficient animals. We have also found that the focal adhesion component Kindlin-3 co-localizes to the nucleus and interacts with FOG-1, suggesting a possible link between integrin signaling and megakaryocyte transcriptional control. Runx-1 multi-protein complex purifications have led to the identification of Fli-1 as a direct binding partner. This interaction results in synergistic transcriptional activation of megakaryocyte-specific genes. Interestingly, the interaction between Runx-1 and Fli-1 occurs preferentially in cells that are differentiating, even though both proteins are expressed abundantly in undifferentiated megakaryoblastic cells. This binding event correlates with assembly of a large complex containing Runx-1/ Fli-1/GATA-1/FOG-1 based on gel filtration chromatography experiments. These factors may, therefore, act as a megakaryocyte-specific enhancesome. Key future directions are aimed at elucidating the molecular mechanisms that regulate these protein-protein interactions and how cell signaling pathways may modulate them.

Prenatal diagnosis of mosaicism for 11q terminal deletion

The phenotype of 11q terminal deletion also known as Jacobsen syndrome is a clinically well known entity whose diagnosis in infancy and childhood is based on clinical examination, hematological and cytogenetic findings. Hematological features in Jacobsen syndrome are very similar to those reported in Paris–Trousseau syndrome (PTS) which is also associated with11q terminal deletion. Karyotype analysis shows a variable terminal deletion from 11q23 sub-band extending to the telomere. Most often in patients with Jacobsen syndrome, this chromosomal deletion is present in all metaphases. We report on the identification of a distal 11q deletion in mosaic (20% of deleted cells) in a fetus ascertained after amniocentesis for maternal serum screening test indicative for Down syndrome. The present case is the third prenatal diagnosis of a mosaic for a distal 11q deletion with the lowest mosaicism rate. The 2D-ultrasound examination and cord blood hematological studies were useful to estimate the prognosis at term, considering the contribution of the mosaicism rate to the phenotypic variability in Jacobsen syndrome. The identification of mosaicism for distal 11q deletion is a very rare event in prenatal diagnosis. This case illustrates the complexity in genetic counselling for prenatally ascertained partial monosomy 11qter in mosaic.

Platelet storage pool deficiency in Jacobsen syndrome

Jacobsen syndrome and Paris-Trousseau Syndrome share similar congenital anomalies, thrombocytopenia, giant platelet alpha granules resulting from fusion of smaller organelles, and an 11q terminal deletion at 11q23.3. Similarities in the two cohorts have suggested that the Paris-Trousseau Syndrome is a variant of Jacobsen syndrome, or the same disorder. The present study has pointed out a significant difference between the two syndromes. Platelets from six patients with Jacobsen syndrome were markedly diminished in serotonin adenine nucleotide rich dense bodies, indicating the presence of platelet storage pool deficiency. Since platelet dense bodies are reported to be normal in size, number and distribution in the Paris-Trousseau Syndrome, the presence of platelet storage pool deficiency in six patients evaluated in the present study may distinguish the two disorders.

Molecular characterization of an 11q interstitial deletion in a patient with the clinical features of Jacobsen syndrome

The 11q terminal deletion disorder or Jacobsen syndrome is a contiguous gene disorder. It is characterized by psychomotor retardation, cardiac defects, blood dyscrasias (Paris-Trousseau syndrome) and craniofacial anomalies. We report on a female patient with an approximately 10 Mb interstitial deletion with many of the features of Jacobsen syndrome: A congenital heart defect, dysmorphic features, developmental delay, and Paris-Trousseau syndrome. The karyotype of the patient is 46,XX,del(11)(q24.1q24.3). The interstitial deletion was confirmed using FISH probes for distal 11q, and the breakpoints were characterized by microarray analysis. This is the first molecularly characterized interstitial deletion in a patient with the clinical features of Jacobsen syndrome. The deletion includes FLI-1, but not JAM-3, which will help to determine the critical genes involved in this syndrome.

FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia

Paris-Trousseau syndrome (PTS; also known as Jacobsen syndrome) is characterized by several congenital anomalies including a dysmegakaryopoiesis with two morphologically distinct populations of megakaryocytes (MKs). PTS patients harbor deletions on the long arm of chromosome 11, including the FLI1 gene, which encodes a transcription factor essential for megakaryopoiesis. We show here that lentivirus-mediated overexpression of FLI1 in patient CD34+ cells restores the megakaryopoiesis in vitro, indicating that FLI1 hemizygous deletion contributes to the PTS hematopoietic defects. FISH analysis on pre-mRNA and single-cell RT-PCR revealed that FLI1 expression is mainly monoallelic in CD41+CD42– progenitors, while it is predominantly biallelic in the other stages of megakaryopoiesis. In PTS cells, the hemizygous deletion of FLI1 generates a subpopulation of CD41+CD42– cells completely lacking FLI1 transcription. We propose that the absence of FLI1 expression in these CD41+CD42– cells might prevent their differentiation, which could explain the segregation of the PTS MKs into two subpopulations: one normal and one composed of small immature MKs undergoing a massive lysis, presumably originating from either FLI1+ or FLI1– CD41+CD42– cells, respectively. Thus, we point to the role of transient monoallelic expression of a gene essential for differentiation in the genesis of human haploinsufficiency-associated disease and suggest that such a mechanism may be involved in the pathogenesis of other congenital or acquired genetic diseases.

FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia.

Paris-Trousseau syndrome (PTS; also known as Jacobsen syndrome) is characterized by several congenital anomalies including a dysmegakaryopoiesis with two morphologically distinct populations of megakaryocytes (MKs). PTS patients harbor deletions on the long arm of chromosome 11, including the FLI1 gene, which encodes a transcription factor essential for megakaryopoiesis. We show here that lentivirus-mediated overexpression of FLI1 in patient CD34(+) cells restores the megakaryopoiesis in vitro, indicating that FLI1 hemizygous deletion contributes to the PTS hematopoietic defects. FISH analysis on pre-mRNA and single-cell RT-PCR revealed that FLI1 expression is mainly monoallelic in CD41(+)CD42(-) progenitors, while it is predominantly biallelic in the other stages of megakaryopoiesis. In PTS cells, the hemizygous deletion of FLI1 generates a subpopulation of CD41(+)CD42(-) cells completely lacking FLI1 transcription. We propose that the absence of FLI1 expression in these CD41(+)CD42(-) cells might prevent their differentiation, which could explain the segregation of the PTS MKs into two subpopulations: one normal and one composed of small immature MKs undergoing a massive lysis, presumably originating from either FLI1(+) or FLI1(-) CD41(+)CD42(-) cells, respectively. Thus, we point to the role of transient monoallelic expression of a gene essential for differentiation in the genesis of human haploinsufficiency-associated disease and suggest that such a mechanism may be involved in the pathogenesis of other congenital or acquired genetic diseases.

Paris-Trousseau syndrome : clinical, hematological, molecular data of ten new cases.

Paris-Trousseau syndrome (PTS) is an inherited disorder characterized by mild hemorragic tendency associated with 11q chromosome deletion. Here we report ten new patients (5 boys, 5 girls) with complete clinical history, biological data, ultra-structural and molecular investigations. Thrombocytopenia is chronic in all the patients except two boys in whom it disappeared during the two first years of life. On Romanovsky stained peripheral blood smears, abnormal platelets with giant granules were detected in all the children and confirmed by electron microscopy (EM). On bone marrow smears, dysmegakaryopoiesis with many micromegakaryocytes was constantly observed. Abnormal alpha-granules were virtually absent from bone marrow and cultured megakaryocytes, while EM detected numerous images of granule fusion within blood platelets. Molecular analyses evidenced that the fli-1 gene is deleted in all the patients except one confirming the crucial role of the transcription factor FLI-1 in megakaryopoiesis. In summary, this study documents ten new cases of PTS with characteristic alpha-granule abnormalities, and shows the putative pathogenic role of fli-1 gene in the pathophysiology of this syndrome.

Fli-1 Is Required for Murine Vascular and Megakaryocytic Development and Is Hemizygously Deleted in Patients with Thrombocytopenia

The ETS gene Fli-1 is involved in the induction of erythroleukemia in mice by Friend murine leukemia virus and Ewings sarcoma in children. Mice with a targeted null mutation in the Fli-1 locus die at day 11.5 of embryogenesis with loss of vascular integrity leading to bleeding within the vascular plexus of the cerebral meninges and specific downregulation of Tek/Tie-2, the receptor for angiopoietin-1. We also show that dysmegakaryopoiesis in Fli-1 null embryos resembles that frequently seen in patients with terminal deletions of 11q (Jacobsen or Paris-Trousseau Syndrome). We map the megakaryocytic defects in 14 Jacobsen patients to a minimal region on 11q that includes the Fli-1 gene and suggest that dysmegakaryopoiesis in these patients may be caused by hemizygous loss of Fli-1.