RESUMO
In this study, we investigated the molecular basis of two unrelated Chinese patients with hemostatic disorders. The proband of the first family had severe hemophilia A (HA) coexisting with type 1 von Willebrand disease (VWD) and the proband of the second family had type 2N VWD. Both probands had similar phenotypes, which included joint and mucosal bleeding, very low factor VIII (FVIII) activity (FVIII:C), and moderate reductions in VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:Rco), as well as a normal multimeric pattern. One FVIII mutation and three VWF mutations were identified: FVIII p.R446* and VWF heterozygous p.E216K mutations were detected in proband 1 and compound heterozygosity of VWF mutations (p.R816W and c.1911delC) in proband 2. Transient expression studies in HEK293T cells proved that R816W mutation abolished the binding of FVIII to VWF and slightly impaired protein synthesis and secretion; 1911delC mutation mainly impaired VWF protein synthesis and secretion. These results provided insight into the possible pathogenic mechanism of type 2N VWD in Chinese patients carrying these mutations.
Assuntos
Hemofilia A/complicações , Hemofilia A/diagnóstico , Hemorragia/etiologia , Mucosa/patologia , Doença de von Willebrand Tipo 1/complicações , Doença de von Willebrand Tipo 1/diagnóstico , Doença de von Willebrand Tipo 2/complicações , Doença de von Willebrand Tipo 2/diagnóstico , Adulto , Testes de Coagulação Sanguínea , Pré-Escolar , Diagnóstico Diferencial , Fator VIII/genética , Fator VIII/metabolismo , Feminino , Genótipo , Hemartrose/etiologia , Hemofilia A/tratamento farmacológico , Hemofilia A/genética , Humanos , Masculino , Mutação , Fenótipo , Ligação Proteica , Doença de von Willebrand Tipo 1/tratamento farmacológico , Doença de von Willebrand Tipo 1/genética , Doença de von Willebrand Tipo 2/tratamento farmacológico , Doença de von Willebrand Tipo 2/genética , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Factor X (FX) deficiency is a rare autosomal-recessive bleeding disorder caused by diverse mutations in the F10 gene. To investigate the molecular basis of severe FX deficiency in a mildly hemorrhagic patient, variants of the F10 gene were detected by sequencing. A missense mutation was analysed by in vitro expression and modelling analysis, and a splice mutation using ectopic transcript analysis. The levels of activity of FX (FX:C) were <1% in both intrinsic and extrinsic pathway assays and 1.71% in chromogenic assay, the level of FX antigen (FX:Ag) was 53.36% in the proband. Two novel heterozygous mutations (IVS5+1G>A and Asp409del) were identified in the F10 gene. Ectopic transcript expression combined with informative marker (heterozygous Asp409del) analysis of the splice mutation (IVS5+1G>A) revealed and confirmed that the transcript from the mutated allele was absent, likely caused by the nonsense-mediated mRNA decay pathway. In vitro expression analysis showed that the Asp409del mutant led to a loss of enzymatic activity rather than impaired expression. Molecular modelling analysis confirmed that the Asp409del mutant dramatically altered the conformation of the 185-189 loop and impaired binding of the loop to sodium ions (Na(+) ), diminishing the enzymatic activity of FXa. This is the first report to clarify the molecular mechanisms of two naturally occurring F10 gene variants that cause severe FX deficiency.
Assuntos
Deficiência do Fator X/genética , Fator X/genética , Mutação , Adulto , Antígenos , Sequência de Bases , Análise Mutacional de DNA , Fator X/metabolismo , Deficiência do Fator X/metabolismo , Heterozigoto , Humanos , Masculino , LinhagemRESUMO
Haemophilia A (HA) is the most common hereditary bleeding disorder caused by F8 gene mutation. Linkage analysis is an auxiliary strategy to direct mutation analysis for genetic counselling of HA. Here we characterize and validate a novel panel of six short tandem repeat (STR) loci for genetic counselling in Chinese HA pedigrees. The panel was analysed in 116 unrelated healthy female patients and 108 male patients, and verified in 169 unrelated pedigrees with HA. The six STR loci in the panel spanned a distance of 0.3 Mb from each side of the F8 gene. Three of them, F8Up226, F8Up146 and F8Down48, were first described here. Markers F8Up226, F8Up146, F8Int13, F8Int25, F8Down48 and DXS1073 exhibited the number of alleles 16, 9, 8, 6, 9 and 10, and heterozygosity rates of 74.8%, 44.8%, 60.9%, 42.6%, 61.7% and 62.0% respectively. Haplotype frequencies analysis suggested that the genotypes of haplotype provided a highly informative content (56.5%). The panel was informative in 167 of 169 unrelated haemophilic pedigrees with the combined diagnostic rate of 98.8%. In eight pedigrees could not be diagnosed by mutation detection linkage studies using the panel were informative in all the pedigrees and a reliable diagnosis was made in seven pedigrees. The novel panel of the six STR loci represents a high degree of informativeness and a low fraction of recombination. Linkage analysis using this panel provides an alternative strategy when direct mutation detection is not feasible for genetic counselling in Chinese HA families.
Assuntos
Fator VIII/genética , Triagem de Portadores Genéticos/métodos , Aconselhamento Genético , Hemofilia A/diagnóstico , Repetições de Microssatélites , Povo Asiático/genética , China/epidemiologia , Frequência do Gene , Marcadores Genéticos , Hemofilia A/epidemiologia , Hemofilia A/genética , Humanos , Diagnóstico Pré-Natal/métodos , Reprodutibilidade dos TestesRESUMO
Objective: To evaluate the effects of adeno-associated virus (AAV) carrying hFâ § by serotype 8 (AAV8/hFâ §) on hemophilia A (HA) mice by gene therapy strategy. Methods: pAAV-CB-EGFP, pH22 (serotype 2) and pfΔ6 (adenovirus helper) were used to package AAV into HEK-293 cells in different conditions (ratios of cells to plasmids). The efficiency of transfection and infection were evaluated using immunofluorescence microscope to seek an optimized package condition. pAAV-TTR-hFâ §, pH 28 (serotype 8) and pfΔ6 were applied to package AAV8/hFâ § in HEK-293 cells using the optimized package condition. The purified AAV8/hFâ § were intravenously injected into HA mice and the effects of gene therapy were estimated. Results: The efficiency of package was evaluated according to the amount and intensity of enhanced green fluorescent protein (EGFP) under immunofluorescence microscope. Four package conditions including 10 cm-dish to transfect 10 µg plasmids, 20 cm-dish to 20 µg, 30 µg and 40 µg plasmids were employed, and the condition of 20 cm-dish to transfect 20 µg plasmids reached the highest transfection efficiency at 24 h, 48 h and 72 h after transfection. The small scale AAV-EGFP was packaged using the optimized condition and an AAV crude extract was harvested by a freeze-thaw method. HEK-293 and 16095 cells were infected by the AAV crude extract, and the preferential infection efficiency was recognized in 16095 cells under immunofluorescence microscope. Then, AAV8/hFâ § was packaged and purified based on the optimized transfection condition, and the high purity of AAV8/hFâ § was detected by Western blot. Fractions of AAV8/hFâ § at the dose of 8×10(12) vg/kg were injected into HA mice through tail vein, an eye-bleeding was performed at every two weeks, and the activity of Fâ § was measured by aPTT assay. Results showed that the activity of Fâ § maintained at the therapeutic level and lasted up to 12 weeks after injection. Conclusion: The purified AAV8/hFâ § based on the optimized package condition could play a role in HA mice gene therapy, and the long-term therapeutic effects of AAV8/hFâ § were observed in vivo.
Assuntos
Dependovirus , Hemofilia A , Animais , Terapia Genética , Vetores Genéticos , Células HEK293 , Humanos , CamundongosRESUMO
INTRODUCTION: Cytogenetically normal acute myeloid leukemia (CN-AML), which accounted for nearly half of total AML patients, is a highly heterogeneous subset of AML. The specific genetic profile and the ethnic features of CN-AML are worth to be studied. METHODS: Using deep sequencing technology, we detected the mutation pattern of 39 genes in 152 Chinese CN-AML patients and analyzed their clinical features. RESULTS: A total of 503 mutations of 39 genes were identified in 145 (95.4%) patients, with the median number of 3 mutations per case. Nine genes (NPM1, CEBPA, DNMT3A, GATA2, NRAS, TET2, FLT3, IDH2, and WT1) mutated in more than 10% patients. Function groups of myeloid transcription factors, activated signaling, and DNA methylation were most affected. The distribution of variant allele frequencies (VAF) of recurrent genes was different among functional groups. High mutation rates of CEBPA and GATA2 together with the low frequency of FLT3-ITD mutation seemed to be the distinct characteristics of Chinese patients. Furthermore, CEBPAbi and GATA2 were found to mutate most in M2 subtype, while NPM1 and DNMT3A mutated more in M4 and M5. The prognostic analysis identified CEBPAmo mutation as an inferior factor. FLT3-ITD, TP53, DNMT3A, CEBPAmo, and WT1 mutations were selected as high-risk markers to identify the CN-AML patients with poor prognosis. CONCLUSION: Our study provided the valuable information of ethnic genetic characteristics and the clinical relevance of Chinese CN-AML patients.
Assuntos
Leucemia Mieloide Aguda/genética , Adulto , Idoso , Povo Asiático , Citogenética , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/epidemiologia , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Mutação , Taxa de Mutação , Nucleofosmina , Prognóstico , Análise de Sequência de DNARESUMO
In order to specifically detect the localization of thrombus in vivo, we have recently developed two monoclonal antibodies (SZ-58, SZ-63) which can specifically bind to cross-linked fibrin. The binding rates of the two monoclonal antibodies (MoAbs) to human plasma clots in vitro were 46.4 +/- 2.3% for 125I-SZ-58, 50.1 +/- 1.7% for 125I-SZ-63 and 3.4 +/- 1.6% for 125I-SZ-53 (control, MoAb against TM). It was shown that both SZ-58 and SZ-63 possess properties of inhibiting the polymerization of fibrin, and SZ-58 could also inhibit the aggregation of platelets induced by ADP. These characteristics make the two MoAbs suitable in the detection of thrombus in vivo. According to the cross reaction tests, thrombi in the jugular veins and carotid arteries in rabbits were made. After injection of the 125I-labeled MoAbs (100,000 cpm/ml of blood), the thrombi and the blood were taken and weighed at various time intervals and radioactivities were measured by an autogamma counter. The ratios of thrombus to blood radioactivity (T/B) of thrombi in jugular veins were 3.0, 5.6 and 3.0 for 125I-SZ-58, 1.5, 3.0 and 5.2 for 125I-SZ-63 and 1.2, 1.0 and 0.7 for control (125I-SZ-53) at the 3rd, 12th and 24th hour after the injection of the radiolabled MoAbs, while the radioactivities of arterial thrombi were almost the same as that in blood after the injection of the two radiotracers. Therefore, it can be concluded that both SZ-58 and SZ-63 can be used in venous thrombus imaging in vivo and the optimal times of imaging are at the 12th hour for SZ-58, 24th hour for SZ-63 after the injection of the radiolabled MoAbs.
Assuntos
Anticorpos Monoclonais , Trombose das Artérias Carótidas/diagnóstico , Fibrina/imunologia , Veias Jugulares , Trombose/diagnóstico , Animais , Anticorpos Monoclonais/biossíntese , Cães , Cobaias , Humanos , Agregação Plaquetária , Coelhos , RadioimunoensaioRESUMO
BACKGROUND: Large deletions in the F8 gene are responsible for approximately 3% of severe hemophilia A (HA) cases. However, only a few breakpoints in large deletions have been characterized. OBJECTIVES: To identify large deletions in the F8 gene and to characterize the molecular mechanisms leading to these deletions. PATIENTS AND METHODS: We used AccuCopy technology, a copy number variation (CNV) genotyping method based on multiplex competitive amplification, to confirm deletions in index patients and to screen potential female carriers in 10 HA families. Also, breakpoints of these large deletions were characterized by a primer walking strategy and genome walking technique. RESULTS: Ten large deletions and four female carriers were identified by AccuCopy. The extents of deleted regions ranged from 1.3 to 68.5 kb. Exact breakpoints of these deletions were successfully characterized. Eight of them presented microhomologies at breakpoint junctions and several recombination-associated elements (repetitive elements, non-B conformation forming motifs and sequence motifs) were also observed in close proximity to the junctions. CONCLUSIONS: AccuCopy technology is a reliable and efficient tool for detecting large deletions in the F8 gene and identifying HA female carriers. The genome walking technique is a highly specific, efficient and versatile method for characterizing the deletion breakpoints. Molecular characterization of deletion breakpoints revealed that non-homologous end joining and microhomology-mediated replication-dependent recombination were the major causative mechanisms of the 10 large deletions in the F8 gene.
Assuntos
Deleção Cromossômica , Cromossomos/ultraestrutura , Análise Mutacional de DNA/métodos , Fator VIII/genética , Deleção de Sequência , Biologia Computacional , Variações do Número de Cópias de DNA , Primers do DNA/genética , Éxons , Feminino , Genoma , Genótipo , Hemofilia A/genética , Heterozigoto , Humanos , Recombinação GenéticaAssuntos
Proteínas de Ligação a Calmodulina/genética , Transformação Celular Neoplásica/genética , Leucemia Mielomonocítica Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Animais , Transformação Celular Neoplásica/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mielomonocítica Aguda/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismoRESUMO
A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100-solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde-fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard-Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.
Assuntos
Anticorpos Monoclonais/imunologia , Colágeno/antagonistas & inibidores , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/imunologia , Ristocetina/antagonistas & inibidores , Animais , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The effect of a low-molecular-weight heparin, faxiparin (Nadroparin), on murine megakaryocytopoiesis in vitro and in vivo was studied in comparison with unfractionated heparin. The addition of fraxiparin at 1-20 IU/ml into plasma clot cultures but not serum-free agar culture significantly enhanced MK colony growth. Furthermore, fraxiparin was found to potentiate the stimulating activity of aplastic anaemia serum (AAS) but not stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo), on MK colony growth in vitro, and to neutralize the inhibitory effect of platelet factor 4 (PF4) in vitro and in vivo. Fraxiparin also acted synergistically with heparin cofactor II and antithrombin III to promote megakaryocyte colony formation. Intraperitoneal administration of fraxiparin twice daily for 4 d at 0.1-25 IU/injection increased in mice the level of blood platelet counts and the number of single MKs and CFU-MK in bone marrow. These data demonstrate that fraxiparin is able to positively regulate megakaryocytopoiesis.
Assuntos
Megacariócitos/efeitos dos fármacos , Nadroparina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Células-TroncoRESUMO
A monoclonal antibody, SZ-2, reacts specifically on human platelets and megakaryocytes. The platelets from 10 normal donors are bound to 15,200 +/- 4,100 SZ-2 molecules/platelet. The antigen recognized by SZ-2 is chymotrypsin-sensitive but neuraminidase-insensitive, and has been identified as glycoprotein Ib (GPIb) by an affinity chromatography technique. SZ-2 is different from other monoclonal antibodies to GPIb. It inhibits not only platelet aggregation induced by ristocetin, but also platelet aggregation induced by collagen (type I) and by PAF. SZ-2 also inhibits platelet serotonin and beta-thromboglobulin release in response to these stimuli.
Assuntos
Anticorpos Monoclonais/fisiologia , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/imunologia , Colágeno , Humanos , Fator de Ativação de Plaquetas , RistocetinaRESUMO
We have previously shown that platelet factor 4 (PF 4) is a potent inhibitor of megakaryocytopoiesis and that it may protect stem cells from 5-fluorouracil (5-FU) cytotoxicity. In the present work, the effects of human PF 4 on megakaryocyte (MK) growth from human CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta 1 (TGF-beta 1). Development of MK from CD34+ cells in both plasma clot culture and liquid culture was significantly inhibited by PF 4 (5 micrograms/ml) and TGF beta 1 (1 ng/ml). Inhibition of cell growth by PF 4 was reversible judging from the fact that the CD34+ cells preincubated with PF 4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF-beta 1 pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF 4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF 4 at 40 micrograms/kg with an interval of 6 h followed by one injection of 5-FU at 150 mg/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF 4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF 4, TGF-beta 1 blocked more cells in G 1 phase. These results demonstrate that PF 4 and TFG-beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF 4, unlike TGF-beta 1, exerts its inhibitory effect on cell growth in a reversible and S phasespecific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.