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OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.
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OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbß3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbß3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbß3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
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Codon non-sens , Intégrine alpha2 , Thrombasthénie , Animaux , Systèmes CRISPR-Cas , Modèles animaux de maladie humaine , Fibrinogène/génétique , Humains , Intégrine alpha2/génétique , Souris , Oligonucléotides , Complexe glycoprotéique IIb-IIIa de la membrane plaquettaire/génétique , , Thrombasthénie/diagnostic , Thrombasthénie/génétique , Thrombine/génétiqueRÉSUMÉ
Copper is an essential trace element for living organisms. Copper enriched by yeast of Saccharomyces cerevisiae is regarded as the biologically available organic copper supplement with great potentiality for application. However, the lower uptake ratio of copper ions makes the production of copper enriched by yeast uneconomically and environmentally unfriendly. In this study, S. cerevisiae Cu-5 with higher copper tolerance and intracellular copper accumulation was obtained by screening of our yeast strains collection. To increase the uptake ratio of copper ions, the medium composition and cultivation conditions for strain Cu-5 were optimized systematically. A medium comprised of glucose, yeast extract, (NH4)2SO4, and inorganic salts was determined, then a novel cultivation strategy including pH control at 5.5 and increasing amounts of yeast extract for a higher concentration of copper ion in the medium was developed. The uptake ratios of copper ions were more than 90% after combining 50 to 100 mg/L copper ions with 3.5 to 5.0 g/L yeast extract, which is the highest until now and is conducive to the cost-effective and environmentally friendly production of bioactive copper in yeast-enriched form.
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Cuivre , Saccharomyces cerevisiae , Transport biologique , Milieux de culture , IonsRÉSUMÉ
OBJECTIVE: To investigate whether miRNA-30a is involved in the pathogenesis of ITP by affecting the differentiation of Th17 cells, and to explore its possible mechanism of miRNA-30a involved in the pathogenesis of ITP through the verification of the target gene SOCS3 for the prediction of miRNA-30a. METHODS: Firstly, a chronic ITP mouse model was established. The expression of miRNA-30a and RORγt in the spleen mononuclear cells were detected and their correlation were analyzed. Secondly, the luciferase vector containing 3'UTR of the target gene and green fluorescent vector containing miRNA were constructed. Luciferase fluorescence detection, real-time fluorescent quantitative PCR (qPCR) and Western blot were used to verify whether SOCS3 is the target gene of miRNA-30a. RESULTS: The platelet count of mice in experimental group decreased to below 20% of normal ones after 48 hours of injection of anti-mouse platelet serum (APS), which was maintained for 14 days at least; the expression of miRNA-30a and RORγt in the spleen mononuclear cells in experimental group were higher than those in the control group(Pï¼0.05), moreover, there was a positive correlation between them (r=0.54); the activity of luciferase in PMDH-GFP-miRNA-30a and pMIR-report-UTR was significantly lower than that in PMDH-GFP empty plasmid and pMIR-report-UTR(Pï¼0.05); The expression of SOCS3 at mRNA and protein level was not different from that in the control group. CONCLUSION: Chronic ITP mouse model has been established successfully; miRNA-30a expression in spleen mononuclear cells of ITP mouse increase, and positively correlated with the expression of RORγt, which contribute to the pathogenesis of ITP by affecting the differentiation of Th17 cells; SOCS3 is able to bind to the target site of miRNA-30a, but might not be its functional target gene.
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Purpura thrombopénique idiopathique , Animaux , Différenciation cellulaire , Souris , microARN , Membre-3 du groupe F de la sous-famille-1 de récepteurs nucléaires , Cellules Th17RÉSUMÉ
BACKGROUND: Immune imbalance of regulatory T cells (Treg)/T helper 17 cells (Th17) contributes to the development of immune thrombocytopenic purpura (ITP). The dysregulation of miRNAs is important in the development of ITP. However, the role of miR-106b-5p in Treg/Th17 imbalance remains unknown in ITP. MATERIALS AND METHODS: Peripheral blood was collected from patients with ITP and healthy controls, and CD4 + T cells were further isolated. miR-106b-5p, nuclear receptor subfamily 4 group A member 3 (NR4A3), forkhead box protein 3 (Foxp3), IL-17A, and TGF-ß expressions were detected by qRT-PCR, western blot, or ELISA. The effect of miR-106b-5p on NR4A3 was detected by dual-luciferase reporter gene assay. RESULTS: Compared with healthy controls, miR-106b-5p was elevated in peripheral blood of patients with ITP, and NR4A3 expression was decreased. sh-NR4A3 significantly decreased Foxp3 and TGF-ß expressions, indicating that NR4A3 may regulate Treg differentiation via Foxp3. Additionally, NR4A3 was identified to be a target of miR-106b-5p, and miR-106b-5p was able to negatively modulate NR4A3 expression. Moreover, we found miR-106b-5p induced immune imbalance of Treg/Th17 through NR4A3. In vivo experiments revealed that silencing miR-106b-5p promoted Treg differentiation and increased the number of platelets, suggesting the relief of ITP. CONCLUSION: miR-106b-5p regulated immune imbalance of Treg/Th17 in ITP through the NR4A3/Foxp3 pathway.
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Protéines de liaison à l'ADN/métabolisme , Facteurs de transcription Forkhead/métabolisme , microARN/métabolisme , Purpura thrombopénique idiopathique/génétique , Purpura thrombopénique idiopathique/immunologie , Récepteurs aux stéroïdes/métabolisme , Récepteurs des hormones thyroïdiennes/métabolisme , Transduction du signal , Lymphocytes T régulateurs/immunologie , Cellules Th17/immunologie , Animaux , Séquence nucléotidique , Différenciation cellulaire , Enfant , Enfant d'âge préscolaire , Femelle , Extinction de l'expression des gènes , Humains , Mâle , Souris de lignée BALB C , microARN/sang , microARN/génétique , Numération des plaquettes , Purpura thrombopénique idiopathique/sangRÉSUMÉ
Hereditary platelet disorders are a heterogeneous group of disorders characterized by abnormal number or function of platelets, sometimes even involving other systems apart from blood abnormalities. The great clinical and genetic heterogeneity makes the diagnosis and treatment of hereditary platelet disorders as a huge challenge for clinicians. At present, only a small number of patients have received a clear molecular diagnosis of hereditary platelet diseases, and a lot of pathogenic genetic variations still remain unknown. The popularity of next generation sequencing (NGS) promotes the development of individualized gene sequencing. Researchers have made great progress in the field of hemostasis and thrombosis using whole genome sequencing (WGS), whole exone sequencing (WES) and target gene sequencing (TGS). The development of NGS has not only promoted the individualized molecular diagnosis of hereditary platelet diseases, but also laid a solid foundation for gene therapy in the future. In this review, the new progress of the diagnosis of platelet-related diseases by using next generation sequencing techniques, is summarized.
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Anomalies des plaquettes , Plaquettes , Séquençage nucléotidique à haut débit , HumainsRÉSUMÉ
BACKGROUND: The imbalance of Treg/Th17 cells is an important pathogenic factor for immune thrombocytopenic purpura (ITP). We previously reported miR-125a-5p targeted CXCL13 and participated in the process of ITP. In the present study, the role of miR-125a-5p in regulating Treg/Th17 ratio and its potential molecular mechanism were investigated. METHOD: A total of 30 adults with ITP and 30 healthy subjects were included. MEG3 expression in peripheral blood derived CD4+ T cells from ITP patients and healthy subjects were detected by real-time PCR. In vitro experiments, the effects of inhibiting or overexpressing MEG3 on the expression of miR-125a-5p, Foxp3 and ROTγt in CD4+ T cells were investigated. RESULTS: MEG3 expression was increased in CD4+ T cells of patients with ITP. Dexamethasone decreased MEG3 expression level of CD4+ T cells in vitro. MEG3 directly interacted with miR-125a-5p and MEG3 overexpression inhibited miR-125a-5p expression in CD4+ T cells exposed to dexamethasone. MEG3 down-regulation or miR-125a-5p overexpression promoted Foxp3 expression and inhibited RORγt expression. CONCLUSION: MEG3 interacted with miR-125a-5p and inhibited its expression, and MEG3/miR-125a-5p contributed to induce immune imbalance of Treg/Th17 in ITP.
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Régulation de l'expression des gènes , microARN/génétique , Purpura thrombopénique idiopathique/génétique , Purpura thrombopénique idiopathique/immunologie , ARN long non codant/métabolisme , Lymphocytes T régulateurs/immunologie , Cellules Th17/immunologie , Adulte , Séquence nucléotidique , Chimiokine CXCL13/pharmacologie , Dexaméthasone/pharmacologie , Régulation négative/effets des médicaments et des substances chimiques , Femelle , Facteurs de transcription Forkhead/métabolisme , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Humains , Mâle , microARN/métabolisme , Membre-3 du groupe F de la sous-famille-1 de récepteurs nucléaires/métabolisme , ARN long non codant/génétique , Lymphocytes T régulateurs/effets des médicaments et des substances chimiques , Cellules Th17/effets des médicaments et des substances chimiquesRÉSUMÉ
OBJECTIVE: To compare plasma platelet microparticles (PMPs), P-selectin, endothelial microparticles (EMPs), and von Willebrand factor (vWF) between a normal control group and patients with chronic kidney disease (CKD) and to explore the significance of PMPs and EMPs in CKD. METHODS: Levels of plasma PMPs, P-selectin, EMPs and vWF in 122 CKD patients and 20 normal controls were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Relationships between PMPs, EMPs and blood pressure, creatinine clearance rate, 24-hour urine protein, hemoglobin, and cholesterol were analyzed. RESULTS: (1) Plasma PMPs, P-selectin, EMPs and vWF levels in CKD patients were significantly higher than those of the control group. Plasma PMPs and P-selectin levels for nephrotic syndrome (NS) were significantly higher than for other CKD groups. No significant difference was found between other CKD groups. Plasma EMPs and vWF in NS, lupus nephritis (LN) and hypertensive nephropathy groups were significantly higher than that of diabetic nephropathy (DN) and chronic glomerulonephritis (CGN) groups. (2) Plasma PMPs, P-selectin, EMPs and vWF in stage I-II CKD patients were significantly higher than those of stage III-V CKD patients, no significant difference was found within stage I-II CKD patients or stage III-V CKD patients. (3) PMPs and EMPs were positively correlated with blood pressure and 24-hour urinary protein, but no significant correlation was found with the creatinine clearance rate, hemoglobin or cholesterol. P-selectin and vWF were positively correlated with PMPs and EMPs respectively. CONCLUSION: CKD patients have significant platelet activation and endothelial dysfunction, which was involved in CKD's occurrence and development; high blood pressure and proteinuria are important reasons for platelet activation and endothelial dysfunction in patients with CKD; PMPs and EMPs can be used as new markers for dysfunctional platelet activation and endothelium.
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BACKGROUND: Immune thrombocytopenia (ITP) is an acquired and autoimmune disease of adults and children characterized by decreased platelet production. CXC chemokine ligand-13 (CXCL13) participates in multiple immunological responses. However, it is still unknown the relationship between CXCL13 and ITP. METHODS: Plasma CXCL13 was detected in ITP (n = 30) children. CD4+ T cells was isolated from peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Treated CD4+ T cells with dexamethasone and/or miR-125-5p mimic/inhibitor, to observe the regulation of CXCL13. RESULTS: Compared with controls, ITP children had elevated plasma CXCL13, the concentration of which was reduced after treatment. In vitro, dexamethasone decreased CXCL13 level in in dose- dependent and in time-dependent manner. MiR-125-5p mimic decreased CXCL13 level and miR-125-5p inhibitor increased CXCL13 level in CD4+ T cells. CXCL13 was implied to be target gene of miR-125-5p. MiR-125-5p inhibitor also canceled dexamethasone induced decrease of CXCL13. CONCLUSION: CXCL13 is the target gene of miR-125-5p, which is possibly involved in the pathological process of ITP.
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We report the case of a 59-year-old Chinese man who showed an asymptomatic coagulation factor V deficiency pattern after second intravenous treatment with ceftazidime. Normal pooled plasma failed to correct the abnormalities in a mixing test, and the presence of factor V inhibitor was confirmed by the Bethesda method. The coagulopathy was not corrected by transfusion of fresh frozen plasma and prothrombin complex concentrate, but rather by treatment with prednisone and withdrawal of dubious drugs. The findings reported here should prompt clinicians to watch for drug-induced coagulation factor deficiency.
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Antibactériens/effets indésirables , Ceftazidime/effets indésirables , Déficit en facteur V/induit chimiquement , Déficit en facteur V/traitement médicamenteux , Proaccélérine/antagonistes et inhibiteurs , Antibactériens/usage thérapeutique , Anti-inflammatoires/usage thérapeutique , Tests de coagulation sanguine , Tronc cérébral/imagerie diagnostique , Tronc cérébral/anatomopathologie , Ceftazidime/usage thérapeutique , Humains , Hémorragies intracrâniennes/complications , Hémorragies intracrâniennes/imagerie diagnostique , Hémorragies intracrâniennes/anatomopathologie , Mâle , Adulte d'âge moyen , Plasma sanguin , Prednisone/usage thérapeutique , Prothrombine/usage thérapeutique , TomodensitométrieRÉSUMÉ
This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded that the ADAMTS13 activity in newly diagnosed AML patients befor induction therapy has been confiremed to be lower and the vWF antigen level to be higher, especially in AML patients with infection or DIC. The ADAMTS13 and vWF antigen may play a role in the pathogenesis of AML and the formation of infection and DIC.
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Protéines ADAM/sang , Leucémie aigüe myéloïde/sang , Facteur de von Willebrand/analyse , Protéine ADAMTS13 , Coagulation intravasculaire disséminée , HumainsRÉSUMÉ
OBJECTIVE: To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia. METHODS: The plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Thrombin fibrin aggregation curve were detected in the plasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed, and then transfected into COS-7 cells. The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA. RESULTS: APTT, PT, TT were significantly longer in the proband. Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry, respectively. Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband. The proband's father, mother, brother and son were heterozygous. The polymerization curves of the patient did not show a lag phase or final turbidity, compared with the normal controls. Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions. It also could not detect the truncated Bß chain under reducing conditions. Abnormal fibrinogen molecule (molecule weight>340 000) were found in transfected COS-7 cells by Western blot, which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absent in culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47 ± 0.30) µg/ml vs (2.65±0.60) µg/ml, P=0.0889]; However, the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ± 0.01) µg/ml vs (3.80±0.80) µg/ml, P=0.0001]. CONCLUSION: Congenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.
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Afibrinogénémie/congénital , Fibrinogène/génétique , Mutagenèse par insertion , Afibrinogénémie/étiologie , Afibrinogénémie/génétique , Mutation avec décalage du cadre de lecture , Humains , Mâle , Pedigree , Jeune adulteRÉSUMÉ
A 41-year-old male had suffered from gradual hearing loss in his right ear for 2 years. Head computed tomography and magnetic resonance imaging scans showed a neoplasm in the cerebellopontine angle region, which was confirmed by the diagnosis of acoustic neurilemmoma by pathological findings after surgery. Following surgery, he routinely received valproic acid (VPA) to prevent seizures. However, the patient presented with hypofibrinogenemia and cerebral hemorrhage after taking VPA for 12 days. The hypofibrinogenemia recurred when VPA was re-administered. After withdrawal of VPA, his fibrinogen concentration rose to normal within several days. As far as we are aware, this is the first case of cerebral hemorrhage due to VPA to have been reported. Herein, as well as reporting on this case, a mini review of the relevant literature is also presented.
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Afibrinogénémie/induit chimiquement , Anticonvulsivants/effets indésirables , Hémorragie cérébrale/induit chimiquement , Acide valproïque/effets indésirables , Adulte , Anticonvulsivants/usage thérapeutique , Humains , Mâle , Crises épileptiques/prévention et contrôle , Facteurs temps , Acide valproïque/usage thérapeutiqueRÉSUMÉ
BACKGROUND: Endothelial cell protein C receptor (EPCR) is a cellular receptor for protein C and activated protein C (APC). In view of convincing evidence, it seems that EPCR, beyond its effects on coagulation and inflammation, could interfere with carcinogenesis. METHODS: In the present study, we investigated EPCR expression in 60 lung carcinoma tissues and 37 para-carcinoma tissues, and analyzed the relationship between EPCR expression and histopathological parameters, clinical parameters, and vascular density. RESULTS: In vitro, culturing lung cancer cell lines constitutively expressed EPCR at the level of mRNA and protein. The pathologic results clearly demonstrated that EPCR expression, including membranous and cytoplasmic staining, was significantly higher in carcinoma than that in the para-carcinoma tissues. The EPCR expression was therefore related to tumor size, lymph node metastasis as well as TNM stage. The expression of EPCR was also significantly correlated with microvessel density (MVD). CONCLUSIONS: These observations provide evidence that EPCR may be involved in the carcinogenesis of lung cancer. It is suggested that EPCR may be a useful biomarker for evaluating the clinical status of lung cancer.
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Antigènes CD/métabolisme , Tumeurs du poumon/métabolisme , Récepteurs de surface cellulaire/métabolisme , Sujet âgé , Antigènes CD/génétique , Lignée cellulaire tumorale , Récepteur endothélial de la protéine C , Femelle , Cytométrie en flux , Humains , Immunohistochimie , Tumeurs du poumon/anatomopathologie , Métastase lymphatique , Mâle , Adulte d'âge moyen , ARN messager/génétique , Récepteurs de surface cellulaire/génétique , RT-PCRRÉSUMÉ
OBJECTIVE: To identify and characterize a missence mutation Ser250 Phe underlying coagulation factor â ¦ (Fâ ¦) deficiency in a Chinese patient and his family. METHODS: The Fâ ¦ gene (F7) was analyzed by DNA sequencing, and the Fâ ¦ levels (including antigen and activity) in patient's plasma were determined with enzyme-linked immunoabsorbent assay (ELISA) and one stage prothrombin time based method. In addition, a Fâ ¦-250 Phe mutant corresponding to the identified mutation was expressed in HEK293 cells, and a subcellular localization experiment in CHO cells was performed to clarify the molecular mechanism of Fâ ¦ deficiency caused by the Fâ ¦-250 Phe mutation. RESULTS: The patient had a prolonged prothrombin time (PT: 36.5 s) and low levels of both Fâ ¦ antigen and activity (130.2 ng/mL and 4.0%, respectively). Two heterozygous mutations were identified in the F7 gene (NG-009262.1), which included a g.15975 G>A mutation at the splice receptor site of intron 6 (IVS6-1G>A) and a novel g.16750 C>T mutation in exon 8, which resulted in replacement of Ser (TCC) 250 with Phe (TTC)250 in the vicinity of a charge-stablizing system. By gene expression experiments, the antigen and activity levels of Fâ ¦-250 Ser and Fâ ¦-250 Phe in the culture medium were (37.77 ± 2.30) ng/mL and (4.02 ± 0.52) ng/mL, respectively. ELISA and Western blotting analyses indicated that expression of the mutant Fâ ¦-250 Phe and wild type Fâ ¦-250 Ser was (130.51 ± 2.32) ng/mL and (172.45 ± 2.25) ng/mL, respectively. Fâ ¦-250 Phe was found in endoplasmic reticulum and Golgi apparatus, suggesting that the mutant Fâ ¦-250 Phe could be normally synthesized in the cells but was inefficiently secreted. CONCLUSION: Compound heterozygous mutations in F7 gene (g.15975G>A and g.16750C>T) may be responsible for the Fâ ¦ deficiency in this patient. The novel Fâ ¦ 250 Phe can be transported from endoplasmic reticulum to Golgi apparatus, but may be degraded or inefficient.
Sujet(s)
Déficit en facteur VII/génétique , Facteur VII/génétique , Mutation faux-sens , Adulte , Animaux , Cellules CHO , Cricetinae , Cricetulus , Test ELISA , Facteur VII/physiologie , Cellules HEK293 , Humains , MâleRÉSUMÉ
MYH9-related disease (MYH9-RD) is an autosomal dominant disorder caused by mutations in the MYH9 gene. It is characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like granulocyte inclusions. In this study we report 10 unrelated patients with MYH9-RD in whom the following seven MYH9 gene mutations were found: W33R, p.Q1443_K1445dup, R702H, D1424N, E1841K, R1933X, and E1945X (the first two were novel mutations). The region of the MYH9 mutation determines in some regards the phenotype, but clinical expression can vary between individuals with the same mutation. The neutrophil inclusion bodies of two patients were too small to be detected, but could be found with immunofluorescence staining. Immunoblotting analysis revealed that the calculated NMMHC-IIA/ß-actin ratio for MYH9-RD neutrophils was 39% of normal controls. Kidney biopsy showed segmental glomerulosclerosis and NMMHC-IIA expression was decreased in podocytes. This disease is not as rare as originally thought. In any individual with persistent macrothrombocytopenia and no response to corticosteroids and immunosuppressive agents, even if neutrophil inclusions were inconspicuous in routine staining, MYH9-RD should be suspected.
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Infarctus cérébral/génétique , Maladies chromosomiques/génétique , Surdité neurosensorielle/génétique , Moteurs moléculaires/génétique , Chaînes lourdes de myosine/génétique , Néphropathie familiale avec surdité/génétique , Thrombopénie/génétique , Adolescent , Adulte , Plaquettes/ultrastructure , Enfant , Enfant d'âge préscolaire , Femelle , Technique d'immunofluorescence , Humains , Rein/anatomopathologie , Rein/ultrastructure , Mâle , Microscopie électronique , Adulte d'âge moyen , Mutation , Phénotype , Polymorphisme génétique , Syndrome , Thrombopénie/anatomopathologieRÉSUMÉ
OBJECTIVE: To investigate the relationship between the efficacy and safety of different doses of thalidomide (Thal) plus dexamethasone (Dex) as the initial therapy in elderly patients with newly diagnosed multiple myeloma (MM). METHODS: Clinical data of 28 elderly patients with newly diagnosed MM who underwent the TD regimen as the initial therapy were analyzed retrospectively. The patients were divided into two groups according to the maximal sustained dose of Thal: lower dose (group A) and higher dose (group B). The overall response rate (ORR), progression free survival (PFS), overall survival (OS), and adverse events (AES) were compared between the two groups. RESULTS: A total of 28 patients were followed up with a median of 18 months. The ORR was 60.1%. The median response time and PFS were 2.0 and 17.0 months, respectively. The mean sustained dose of Thal in group B was significantly higher than group A (292.9 mg v 180.4 mg, P=0.01). There was no significantly difference in ORR (57.1% v 64.3%, P=1.00) and PFS (9.63months v 17.66 months, P=0.73) between groups A and B. During the follow up, only five patients died (<40%) and, therefore, median OS values were not available. It is estimated, however, that the mean survival time in the two groups was 35.6 and 33.4 months (P>0.05), respectively. All of the patients tolerated the treatment well. The incidence of AES in patients with a grading above 3 in group B was significantly higher than in group A (P=0.033). CONCLUSIONS: The TD regimen results in a high response rate and manageable AES as the initial therapy in elderly patients with MM. TD should be considered as the front line regimen for the treatment of elderly patients with MM in areas with financial constraints. The clinical response can be achieved at a low dose Thal with minimal toxicity.
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Protocoles de polychimiothérapie antinéoplasique/usage thérapeutique , Myélome multiple/traitement médicamenteux , Sujet âgé , Sujet âgé de 80 ans ou plus , Protocoles de polychimiothérapie antinéoplasique/administration et posologie , Protocoles de polychimiothérapie antinéoplasique/effets indésirables , Dexaméthasone/administration et posologie , Survie sans rechute , Femelle , Études de suivi , Humains , Estimation de Kaplan-Meier , Mâle , Adulte d'âge moyen , Études rétrospectives , Thalidomide/administration et posologie , Résultat thérapeutiqueRÉSUMÉ
OBJECTIVE: To investigate clinical features and to identify gene mutations in six patients with nonmuscle myosin heavy chain 9 gene (MYH9)-related disease. METHODS: The platelet counts were measured using automated complete blood cell counter and manual manner. The size of platelets and inclusion bodies were observed under light microscopy. All the 40 exons and exon-intron boundaries of MYH9 gene were amplified by PCR and then DNA sequencing was performed. Restriction endonuclease analysis and polyacrylamide gel electrophoresis (PAGE) were used for polymorphism analysis. RESULTS: Six patients all shared the common features of thrombocytopenia with giant platelets and granulocyte inclusions. Four MYH9 gene mutations were found in the six patients: T97C (W33R) in exon 1, 4335Insert CAGAAGAAG (1445InsQKK) and G4269A (D1424N) in exon 30 and G5833T (E1945Stop) in exon 40. The former two were novel mutations which have not been reported in the literature. The results of restriction endonuclease analysis and PAGE could exclude the possibility of nucleotide polymorphisms. CONCLUSIONS: The MYH9 gene mutations were identified in six patients with MYH9 related disorders, and T97C (W33R) and 4335InsCAGAAGAAG (1445InsQKK) were novel mutations. MYH9 related disease should be considered in individuals with persistent thrombocytopenia which is non-responsive to corticosteroids and immuno-repressive agents.
Sujet(s)
Moteurs moléculaires/génétique , Chaînes lourdes de myosine/génétique , Thrombopénie/étiologie , Thrombopénie/génétique , Adolescent , Adulte , Séquence nucléotidique , Enfant , Femelle , Humains , Corps d'inclusion , Mâle , Adulte d'âge moyen , Phénotype , Analyse de séquence d'ADNRÉSUMÉ
Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.