RESUMO
ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.
Assuntos
Megacariócitos , Complexo Glicoproteico GPIb-IX de Plaquetas , Trombocitopenia , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Citoplasma/metabolismo , Filaminas/genética , Filaminas/metabolismo , Megacariócitos/metabolismo , Morfogênese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismoRESUMO
ABSTRACT: Notch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.
Assuntos
Hepatócitos , Janus Quinase 2 , Fígado , Receptor Notch1 , Trombopoetina , Animais , Receptor Notch1/metabolismo , Receptor Notch1/genética , Trombopoetina/metabolismo , Trombopoetina/genética , Camundongos , Fígado/metabolismo , Hepatócitos/metabolismo , Janus Quinase 2/metabolismo , Janus Quinase 2/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Camundongos Knockout , Transdução de Sinais , Fosforilação , Plaquetas/metabolismo , Camundongos Endogâmicos C57BL , Trombocitopenia/metabolismo , Trombocitopenia/genética , Trombocitopenia/patologiaRESUMO
Heparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.
Assuntos
Regiões Determinantes de Complementaridade , Trombocitopenia , Humanos , Regiões Determinantes de Complementaridade/genética , Trombocitopenia/induzido quimicamente , Trombocitopenia/genética , Heparina , Anticorpos/efeitos adversos , Plaquetas/metabolismo , Fator Plaquetário 4RESUMO
Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature ß1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and ß1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.
Assuntos
Trombocitopenia , UDPglucose 4-Epimerase , Humanos , Plaquetas/metabolismo , Galactose/metabolismo , Glicosilação , Integrina beta1/metabolismo , Megacariócitos/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombopoese/genética , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismo , Difosfato de Uridina/metabolismoRESUMO
ABSTRACT: Deleterious germ line RUNX1 variants cause the autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematologic malignancies (HMs). We launched a FPDMM natural history study and, from January 2019 to December 2021, enrolled 214 participants, including 111 patients with 39 different RUNX1 variants from 45 unrelated families. Seventy of 77 patients had thrombocytopenia, 18 of 18 had abnormal platelet aggregometry, 16 of 35 had decreased platelet dense granules, and 28 of 55 had abnormal bleeding scores. Nonmalignant bone marrows showed increased numbers of megakaryocytes in 12 of 55 patients, dysmegakaryopoiesis in 42 of 55, and reduced cellularity for age in 30 of 55 adult and 17 of 21 pediatric cases. Of 111 patients, 19 were diagnosed with HMs, including myelodysplastic syndrome, acute myeloid leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and smoldering myeloma. Of those 19, 18 were relapsed or refractory to upfront therapy and referred for stem cell transplantation. In addition, 28 of 45 families had at least 1 member with HM. Moreover, 42 of 45 patients had allergic symptoms, and 24 of 30 had gastrointestinal (GI) symptoms. Our results highlight the importance of a multidisciplinary approach, early malignancy detection, and wider awareness of inherited disorders. This actively accruing, longitudinal study will genotype and phenotype more patients with FPDMM, which may lead to a better understanding of the disease pathogenesis and clinical course, which may then inform preventive and therapeutic interventions. This trial was registered at www.clinicaltrials.gov as #NCT03854318.
Assuntos
Neoplasias Hematológicas , Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Trombocitopenia , Adulto , Humanos , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Estudos Longitudinais , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/complicações , Trombocitopenia/genética , Transtornos Mieloproliferativos/complicações , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/complicaçõesRESUMO
ACTN1-related thrombocytopenia is a rare disorder caused by heterozygous variants in the ACTN1 gene characterized by macrothrombocytopenia and mild bleeding tendency. We describe for the first time two patients affected with ACTN1-RT caused by a homozygous variant in ACTN1 (c.982G>A) with mild heart valve defects unexplained by any other genetic variants investigated by WES. Within the reported family, the homozygous sisters have moderate thrombocytopenia and marked platelet macrocytosis with giant platelets, revealing a more severe haematological phenotype compared to their heterozygous relatives and highlighting a significant effect of allelic burden on platelet size. Moreover, we hypothesize that some ACTN1 variants, especially when present in the homozygous state, may also contribute to the cardiac abnormalities.
Assuntos
Actinina , Homozigoto , Fenótipo , Trombocitopenia , Humanos , Trombocitopenia/genética , Actinina/genética , Feminino , Masculino , Linhagem , Mutação , AdultoRESUMO
Cyclic thrombocytopenia (CTP) is characterized by periodic platelet oscillation with substantial amplitude. Most CTP cases have a thrombocytopenic background and are often misdiagnosed as immune thrombocytopenia with erratically effective treatment choices. CTP also occurs during hydroxyurea treatment in patients with myeloproliferative diseases. While the aetiology of CTP remains uncertain, here we evaluate historical, theoretical and clinical findings to provide a framework for understanding CTP pathophysiology. CTP retains the intrinsic oscillatory factors defined by the homeostatic regulation of platelet count, presenting as reciprocal platelet/thrombopoietin oscillations and stable oscillation periodicity. Moreover, CTP patients possess pathogenic factors destabilizing the platelet homeostatic system thereby creating opportunities for external perturbations to initiate and sustain the exaggerated platelet oscillations. Beyond humoral and cell-mediated autoimmunity, we propose recently uncovered germline and somatic genetic variants, such as those of MPL, STAT3 or DNMT3A, as pathogenic factors in thrombocytopenia-related CTP. Likewise, the JAK2 V617F or BCR::ABL1 translocation that drives underlying myeloproliferative diseases may also play a pathogenic role in hydroxyurea-induced CTP, where hydroxyurea treatment can serve as both a trigger and a pathogenic factor of platelet oscillation. Elucidating the pathogenic landscape of CTP provides an opportunity for targeted therapeutic approaches in the future.
Assuntos
Neoplasias da Medula Óssea , Transtornos Mieloproliferativos , Trombocitopenia , Humanos , Hidroxiureia/uso terapêutico , Trombocitopenia/etiologia , Trombocitopenia/genética , Contagem de Plaquetas , Plaquetas , Transtornos Mieloproliferativos/genéticaRESUMO
Thrombocytopenia 4 (THC4) is an autosomal-dominant thrombocytopenia caused by mutations in CYCS, the gene encoding cytochrome c (CYCS), a small haeme protein essential for electron transport in mitochondria and cell apoptosis. THC4 is considered an extremely rare condition since only a few patients have been reported so far. These subjects presented mild thrombocytopenia and no or mild bleeding tendency. In this study, we describe six Italian families with five different heterozygous missense CYCS variants: p.Gly42Ser and p.Tyr49His previously associated with THC4, and three novel variants (p.Ala52Thr, p.Arg92Gly, and p.Leu99Val), which have been classified as pathogenic by bioinformatics and segregation analyses. Moreover, we supported functional effects of p.Ala52Thr and p.Arg92Gly on oxidative growth and respiratory activity in a yeast model. The clinical characterization of the 22 affected individuals, the largest series of THC4 patients ever reported, showed that this disorder is characterized by mild-to-moderate thrombocytopenia, normal platelet size, and function, low risk of bleeding, and no additional clinical phenotypes associated with reduced platelet count. Finally, we describe a significant correlation between the region of CYCS affected by mutations and the extent of thrombocytopenia, which could reflect different degrees of impairment of CYCS functions caused by different pathogenetic variants.
Assuntos
Citocromos c , Trombocitopenia , Humanos , Trombocitopenia/genética , Feminino , Masculino , Citocromos c/genética , Adulto , Pessoa de Meia-Idade , Linhagem , Mutação de Sentido Incorreto , Idoso , Adolescente , Mutação , Adulto Jovem , CriançaRESUMO
MYH9-related disorder (MYH9-RD) is characterized by congenital macrothrombocytopenia and granulocyte inclusion bodies. MYH9-RD is often misdiagnosed as chronic immune thrombocytopenia. In this study, we investigated age at definitive diagnosis and indicative thrombocytopenia in 41 patients with MYH9-RD from the congenital thrombocytopenia registry in Japan. Our cohort comprises 54.8% adults over 18 years at confirmed diagnosis. We found a significant difference (p < 0.0001) between the median age at definitive diagnosis of 25.0 years and for indicative thrombocytopenia it was 9.0 years. Our findings strongly suggest diagnostic delay of MYH9-RD in Japan. Our registry system will continue to contribute to this issue.
Assuntos
Diagnóstico Tardio , Cadeias Pesadas de Miosina , Trombocitopenia , Humanos , Japão/epidemiologia , Adulto , Trombocitopenia/diagnóstico , Trombocitopenia/genética , Trombocitopenia/congênito , Masculino , Feminino , Criança , Adolescente , Cadeias Pesadas de Miosina/genética , Pessoa de Meia-Idade , Pré-Escolar , Adulto Jovem , Lactente , Proteínas Motores Moleculares/genética , Sistema de Registros , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , IdosoRESUMO
Kabuki syndrome (KS) is now listed in the Human Inborn Errors of Immunity (IEI) Classification. It is a rare disease caused by KMT2D and KDM6A variants, dominated by intellectual disability and characteristic facial features. Recurrently, pathogenic variants are identified in those genes in patients examined for autoimmune cytopenia (AIC), but interpretation remains challenging. This study aims to describe the genetic diagnosis and the clinical management of patients with paediatric-onset AIC and KS. Among 11 patients with AIC and KS, all had chronic immune thrombocytopenic purpura, and seven had Evans syndrome. All had other associated immunopathological manifestations, mainly symptomatic hypogammaglobinaemia. They had a median of 8 (5-10) KS-associated manifestations. Pathogenic variants were detected in KMT2D gene without clustering, during the immunological work-up of AIC in three cases, and the clinical strategy to validate them is emphasized. Eight patients received second-line treatments, mainly rituximab and mycophenolate mofetil. With a median follow-up of 17 (2-31) years, 8/10 alive patients still needed treatment for AIC. First-line paediatricians should be able to recognize and confirm KS in children with ITP or multiple AIC, to provide early appropriate clinical management and specific long-term follow-up. The epigenetic immune dysregulation in KS opens exciting new perspectives.
Assuntos
Anormalidades Múltiplas , Proteínas de Ligação a DNA , Face , Doenças Hematológicas , Histona Desmetilases , Proteínas de Neoplasias , Doenças Vestibulares , Humanos , Doenças Vestibulares/genética , Doenças Vestibulares/diagnóstico , Criança , Face/anormalidades , Feminino , Masculino , Pré-Escolar , Anormalidades Múltiplas/genética , Adolescente , Histona Desmetilases/genética , Proteínas de Neoplasias/genética , Doenças Hematológicas/genética , Proteínas de Ligação a DNA/genética , Púrpura Trombocitopênica Idiopática/genética , Púrpura Trombocitopênica Idiopática/terapia , Púrpura Trombocitopênica Idiopática/diagnóstico , Lactente , Trombocitopenia/genética , Trombocitopenia/diagnóstico , Trombocitopenia/etiologia , Trombocitopenia/terapia , Anemia Hemolítica Autoimune/genética , Anemia Hemolítica Autoimune/diagnóstico , Anemia Hemolítica Autoimune/terapia , Doenças Autoimunes/genética , Doenças Autoimunes/diagnóstico , Rituximab/uso terapêutico , Mutação , CitopeniaRESUMO
Megakaryopoiesis and platelet production is a complex process that is underpotential regulation at multiple stages. Many long non-coding RNAs (lncRNAs) are distributed in hematopoietic stem cells and platelets. lncRNAs may play important roles as key epigenetic regulators in megakaryocyte differentiation and proplatelet formation. lncRNA NORAD can affect cell ploidy by sequestering PUMILIO proteins, although its direct effect on megakaryocyte differentiation and thrombopoiesis is still unknown. In this study, we demonstrate NORAD RNA is highly expressed in the cytoplasm during megakaryocyte differentiation. Interestingly, we identified for the first time that NORAD has a strong inhibitory effect on megakaryocyte differentiation and proplatelet formation from cultured megakaryocytes. DUSP6/ERK1/2 pathway is activated in response to NORAD knockdown during megakaryocytopoiesis, which is achieved by sequestering PUM2 proteins. Finally, compared with the wild-type control mice, NORAD knockout mice show a faster platelet recovery after severe thrombocytopenia induced by 6 Gy total body irradiation. These findings demonstrate lncRNA NORAD has a key role in regulating megakaryocyte differentiation and thrombopoiesis, which provides a promising molecular target for the treatment of platelet-related diseases such as severe thrombocytopenia.
Assuntos
Plaquetas , Diferenciação Celular , Fosfatase 6 de Especificidade Dupla , Megacariócitos , RNA Longo não Codificante , Trombopoese , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Fosfatase 6 de Especificidade Dupla/metabolismo , Fosfatase 6 de Especificidade Dupla/genética , Sistema de Sinalização das MAP Quinases , Megacariócitos/metabolismo , Megacariócitos/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Trombopoese/genéticaRESUMO
Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without megakaryocyte-like precursors but derived from erythrocyte thrombocyte bi-functional precursor cells. One unique feature of thrombocyte differentiation involves the maturation of young thrombocytes in circulation. In this study, we investigated the role of hox genes in zebrafish thrombopoiesis to model platelet production. We selected hoxa10b, hoxb2a, hoxc5a, hoxd3a, and hoxc11b from thrombocyte RNA expression data, and checked whether they are expressed in young or mature thrombocytes. We found hoxa10b, hoxb2a, hoxc5a, and hoxd3a were expressed in both young and mature thrombocytes and hoxc11b was expressed in only young thrombocytes. We then performed knockdowns of these 5 hox genes and found hoxc11b knockdown resulted in thrombocytosis and the rest showed thrombocytopenia. To identify hox genes that could have been missed by the above datasets, we performed knockdowns 47 hox genes in the zebrafish genome and found hoxa9a, and hoxb1a knockdowns resulted in thrombocytopenia and they were expressed in both young and mature thrombocytes. In conclusion, our comprehensive knockdown study identified Hoxa10b, Hoxb2a, Hoxc5a, Hoxd3a, Hoxa9a, and Hoxb1a, as positive regulators and Hoxc11b, as a negative regulator for thrombocyte development.
Assuntos
Trombocitopenia , Trombopoese , Animais , Trombopoese/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Genes Homeobox , Plaquetas/metabolismo , Megacariócitos , Trombocitopenia/genética , Mamíferos/genéticaRESUMO
Syndromic constitutive thrombocytopenia encompasses a heterogeneous group of disorders characterised by quantitative and qualitative defects of platelets while featuring other malformations. Recently, heterozygous, de novo variants in RAP1B were reported in three cases of syndromic thrombocytopenia. Here, we report two additional, unrelated individuals identified retrospectively in our data repository with heterozygous variants in RAP1B: NM_001010942.2(RAP1B):c.35G>A, p.(Gly12Glu) (de novo) and NM_001010942.2(RAP1B):c.178G>A, p.(Gly60Arg). Both individuals had thrombocytopenia, as well as congenital malformations, and neurological, behavioural, and dysmorphic features, in line with previous reports. Our data supports the causal role of monoallelic RAP1B variants that disrupt RAP1B GTPase activity in syndromic congenital thrombocytopenia.
Assuntos
Plaquetas , Trombocitopenia , Humanos , Estudos Retrospectivos , Plaquetas/metabolismo , Trombocitopenia/genética , Proteínas rap de Ligação ao GTPRESUMO
Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.
Assuntos
Regiões 5' não Traduzidas , Proteína Proto-Oncogênica c-fli-1 , Trombocitopenia , Humanos , Trombocitopenia/genética , Trombocitopenia/patologia , Regiões 5' não Traduzidas/genética , Sítios de Ligação , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Masculino , Feminino , Linhagem , Megacariócitos/metabolismo , Megacariócitos/patologia , Plaquetas/metabolismo , Plaquetas/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Ligação Proteica , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
The inherited thrombocytopenia syndromes are a group of disorders characterized primarily by quantitative defects in platelet number, though with a variety demonstrating qualitative defects and/or extrahematopoietic findings. Through collaborative international efforts applying next-generation sequencing approaches, the list of genetic syndromes that cause thrombocytopenia has expanded significantly in recent years, now with over 40 genes implicated. In this review, we focus on what is known about the genetic etiology of inherited thrombocytopenia syndromes and how the field has worked to validate new genetic discoveries. We highlight the important role for the clinician in identifying a germline genetic diagnosis and strategies for identifying novel causes through research-based endeavors.
Assuntos
Trombocitopenia , Plaquetas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Contagem de Plaquetas , Síndrome , Trombocitopenia/diagnóstico , Trombocitopenia/genéticaRESUMO
RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine-rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , RNA Mensageiro , Fatores de Processamento de Serina-Arginina , Trombocitopenia , Trombopoese/genética , Animais , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismoRESUMO
Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. We performed a genome-wide association study (GWAS) with positive functional assay as the outcome in a large discovery cohort of patients divided into 3 groups: (1) functional assay-positive cases (n = 1269), (2) antibody-positive (functional assay-negative) controls (n = 1131), and (3) antibody-negative controls (n = 1766). Significant associations (α = 5 × 10-8) were investigated in a replication cohort (α = 0.05) of functional assay-confirmed HIT cases (n = 177), antibody-positive (function assay-negative) controls (n = 258), and antibody-negative controls (n = 351). We observed a strong association for positive functional assay with increasing PF4/heparin immunoglobulin-G (IgG) level (odds ratio [OR], 16.53; 95% confidence interval [CI], 13.83-19.74; P = 1.51 × 10-209) and female sex (OR, 1.15; 95% CI, 1.01-1.32; P = .034). The rs8176719 C insertion variant in ABO was significantly associated with positive functional assay status in the discovery cohort (frequency = 0.41; OR, 0.751; 95% CI, 0.682-0.828; P = 7.80 × 10-9) and in the replication cohort (OR, 0.467; 95% CI, 0.228-0.954; P = .0367). The rs8176719 C insertion, which encodes all non-O blood group alleles, had a protective effect, indicating that the rs8176719 C deletion and the O blood group were risk factors for HIT (O blood group OR, 1.42; 95% CI, 1.26-1.61; P = 3.09 × 10-8). Meta-analyses indicated that the ABO association was independent of PF4/heparin IgG levels and was stronger when functional assay-positive cases were compared with antibody-positive (functional assay-negative) controls than with antibody-negative controls. Sequencing and fine-mapping of ABO demonstrated that rs8176719 was the causal single nucleotide polymorphism (SNP). Our results clarify the biology underlying HIT pathogenesis with ramifications for prediction and may have important implications for related conditions, such as vaccine-induced thrombotic thrombocytopenia.
Assuntos
Estudo de Associação Genômica Ampla , Trombocitopenia , Sistema ABO de Grupos Sanguíneos/genética , Anticoagulantes/efeitos adversos , Feminino , Heparina/efeitos adversos , Humanos , Imunoglobulina G , Masculino , Fator Plaquetário 4/genética , Fatores de Risco , Trombocitopenia/induzido quimicamente , Trombocitopenia/genéticaRESUMO
The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage-specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1-/- mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1-/- mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1-/- MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.
Assuntos
Megacariócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Trombocitopenia , Actinas/metabolismo , Animais , Plaquetas/metabolismo , Cálcio , Camundongos , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/genética , Trombocitopenia/genética , Trombopoese/fisiologiaRESUMO
Thrombocytopenia can be inherited or acquired from a variety of causes. While hereditary causes of thrombocytopenia are rare, several genes have been associated with the condition. In this report, we describe an 18-year-old man and his mother, both of whom have congenital thrombocytopenia. Exome sequencing in the man revealed a 1006 kb maternally inherited deletion in the 10p12.1 region (arr[GRCh37] 10p12.1(27378928_28384564)x1) of uncertain clinical significance. This deletion in the THC2 locus includes genes ANKRD26, known to be involved in normal megakaryocyte differentiation, and MASTL, which some studies suggest is linked to autosomal dominant thrombocytopenia. In the family presented here, the deletion segregated with the congenital thrombocytopenia phenotype, suggesting that haploinsufficiency of one or both genes may be the cause. To our knowledge, this is the first report of a deletion of the THC2 locus associated with thrombocytopenia. Future functional studies of deletions of the THC2 locus may elucidate the mechanism for this phenotype observed clinically.
Assuntos
Transtornos Cromossômicos , Trombocitopenia , Humanos , Adolescente , Trombocitopenia/genética , Trombocitopenia/congênito , Transtornos Cromossômicos/genética , Quebra Cromossômica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Casitas B-lineage lymphoma (CBL) encodes an adaptor protein with E3-ligase activity negatively controlling intracellular signaling downstream of receptor tyrosine kinases. Somatic CBL mutations play a driver role in a variety of cancers, particularly myeloid malignancies, whereas germline defects in the same gene underlie a RASopathy having clinical overlap with Noonan syndrome (NS) and predisposing to juvenile myelomonocytic leukemia and vasculitis. Other features of the disorder include cardiac defects, postnatal growth delay, cryptorchidism, facial dysmorphisms, and predisposition to develop autoimmune disorders. Here we report a novel CBL variant (c.1202G>T; p.Cys401Phe) occurring de novo in a subject with café-au-lait macules, feeding difficulties, mild dysmorphic features, psychomotor delay, autism spectrum disorder, thrombocytopenia, hepatosplenomegaly, and recurrent hypertransaminasemia. The identified variant affects an evolutionarily conserved residue located in the RING finger domain, a known mutational hot spot of both germline and somatic mutations. Functional studies documented enhanced EGF-induced ERK phosphorylation in transiently transfected COS1 cells. The present findings further support the association of pathogenic CBL variants with immunological and hematological manifestations in the context of a presentation with only minor findings reminiscent of NS or a clinically related RASopathy.