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1.
Hum Mutat ; 43(2): 215-227, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34882887

RESUMO

Approximately 10% of von Willebrand factor (VWF) gene variants are suspected to disrupt messenger RNA (mRNA) processing, the number of which might be underestimated due to the lack of transcript assays. In the present study, we provided a detailed strategy to evaluate the effects of nine putative splice site variants (PSSVs) of VWF on mRNA processing as well as protein properties and establish their genotype-phenotype relationships. Eight of nine PSSVs affected VWF splicing: c.322A>T, c.1534-13_1551delinsCA, and c.8116-2del caused exon skipping; c.221-2A>C, c.323+1G>T, and c.2547-13T>A resulted in the activation of cryptic splice sites; c.2684A>G led to exon skipping and activation of a cryptic splice site; c.2968-14A>G created a new splice site. The remaining c.5171-9del was likely benign. The efficiency of nonsense-mediated mRNA decay (NMD) was much higher in platelets compared to leukocytes, impairing the identification of aberrant transcripts in 4 of 8 PSSVs. The nonsense variant c.322A>T partially impaired mRNA processing, leaking a small amount of correct transcripts with c.322T (p.Arg108*), while the missense variant c.2684A>G totally disrupted normal splicing of VWF, rather than produced mutant protein with the substitution of Gln895Arg. The results of this study would certainly add novel insights into the molecular events behind von Willebrand disease.


Assuntos
Sítios de Splice de RNA , Doenças de von Willebrand , Fator de von Willebrand , Humanos , Splicing de RNA , RNA Mensageiro/genética , Doenças de von Willebrand/genética , Fator de von Willebrand/genética
2.
Thromb Haemost ; 122(5): 679-691, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34256393

RESUMO

A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient's plasma clotting time indicated that both activated partial thromboplastin time (52.6 seconds) and prothrombin time (19.4 seconds) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired approximately fivefold. Thrombin generation assay using patient's plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased; however, the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with approximately 12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased by approximately 10-fold; however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated approximately 20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient's normal hemostatic phenotype.


Assuntos
Protrombina , Trombomodulina , Antitrombina III , Antitrombinas , Transtornos Herdados da Coagulação Sanguínea , Humanos , Proteína C/metabolismo , Protrombina/metabolismo , Trombina/metabolismo , Trombomodulina/metabolismo
3.
Biochim Biophys Acta Gen Subj ; 1865(6): 129892, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33722640

RESUMO

We previously demonstrated that heterozygous Gly197 to Arg mutation in PROC is associated with venous thrombosis due to the mutation abrogating both zymogenic and enzymatic activities of protein C and activated protein C (APC). In this study, we investigated the role of Gly197 on the structure and function of protein C by replacing it with Ala, Lys and Glu in separate constructs. Characterization of protein C mutants indicated their activation by thrombin is improved ~5-20-fold with the order of PC-G197K > PC-G197E > PC-G197A > PC-WT. Interestingly, the cofactor function of thrombomodulin (TM) in promoting the activation of zymogens by thrombin followed the reverse order of PC-WT > PC-G197A > PC-G197E > PC-G197K. The thrombin-generation inhibitory profiles of zymogens in a tissue factor-mediated thrombin generation assay using protein C-deficient plasma with or without supplementation with TM followed the same order of zymogen activation in the purified system. Evaluation of anticoagulant activities of APC derivatives by prothrombinase and aPTT assays revealed a normal activity for APC-G197A but dramatically impaired activity for the other two mutants. In the endothelial cell permeability assay, APC-G197A exhibited normal antiinflammatory activity, but the other two mutants were nearly inactive. These results suggest that Gly197 plays a key role in TM cofactor-dependent protein C activation by thrombin. It facilitates the recognition of protein C by thrombin in the presence of TM but impedes it in the absence of the cofactor. In APC, a small residue at this position is required for the proper folding/reactivity of the active-site pocket of the protease, a hypothesis supported by structural modeling.


Assuntos
Anti-Inflamatórios/farmacologia , Anticoagulantes/farmacologia , Glicina/genética , Mutação , Proteína C/química , Proteína C/metabolismo , Fator V/metabolismo , Glicina/química , Glicina/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Proteína C/genética , Conformação Proteica , Relação Estrutura-Atividade , Trombina/metabolismo , Trombomodulina/metabolismo
5.
Thromb Haemost ; 120(7): 1045-1055, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32422680

RESUMO

Antithrombin (AT) is a serine protease inhibitor that regulates the activity of coagulation proteases of both intrinsic and extrinsic pathways. We identified an AT-deficient patient with a heterozygous Thr90Ser (T90S) mutation who experiences recurrent venous thrombosis. To understand the molecular basis of the clotting defect, we expressed AT-T90S in mammalian cells, purified it to homogeneity, and characterized its properties in established kinetics, binding, and coagulation assays. The possible effect of mutation on the AT structure was also evaluated by molecular modeling. Results demonstrate the inhibitory activity of AT-T90S toward thrombin and factor Xa has been impaired three- to fivefold in both the absence and presence of heparin. The affinity of heparin for AT-T90S has been decreased by four- to fivefold. Kinetic analysis revealed the stoichiometry of AT-T90S inhibition of both thrombin and factor Xa has been elevated by three- to fourfold in both the absence and presence of heparin, suggesting that the reactivity of coagulation proteases with AT-T90S has been elevated in the substrate pathway. The anticoagulant activity of AT-T90S has been significantly impaired as analyzed in the AT-deficient plasma supplemented with AT-T90S. The anti-inflammatory effect of AT-T90S was also decreased. Structural analysis predicts the shorter side-chain of Ser in AT-T90S has a destabilizing effect on the structure of AT and/or the AT-protease complex, possibly increasing the size of an internal cavity and altering a hydrogen-bonding network that modulates conformations of the allosterically linked heparin-binding site and reactive center loop of the serpin. This mutational effect increases the reactivity of AT-T90S with coagulation proteases in the substrate pathway.


Assuntos
Deficiência de Antitrombina III/genética , Antitrombina III/genética , Coagulação Sanguínea/genética , Heterozigoto , Mutação , Trombose Venosa/genética , Adulto , Antitrombina III/metabolismo , Deficiência de Antitrombina III/sangue , Deficiência de Antitrombina III/diagnóstico , Fator Xa/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Fenótipo , Conformação Proteica , Recidiva , Relação Estrutura-Atividade , Trombina/metabolismo , Trombose Venosa/sangue , Trombose Venosa/diagnóstico
6.
J Thromb Haemost ; 18(5): 1141-1153, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32078247

RESUMO

BACKGROUND: Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein C-deficient patients both of whom carry a heterozygous Gly197 to Arg (G197R) mutation in PROC and experience venous thrombosis. OBJECTIVE: The objective of this study was to determine the molecular basis of the clotting defect in patients carrying the G197R mutation. METHODS: We expressed protein C-G197R in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems. RESULTS: The activation of protein C-G197R by thrombin was improved ~10-fold; however, its activation by thrombin was not promoted by thrombomodulin (TM). In a tissue factor-mediated thrombin generation assay, the addition of soluble TM to protein C-deficient plasma, supplemented with protein C-G197R, did not have a significant inhibitory effect on thrombin generation parameters. APC-G197R did not exhibit a significant anticoagulant activity in either purified or plasma-based assay systems. APC-G197R was essentially inactive because it showed no activity in an aPTT assay. Anti-inflammatory activity of APC-G197R was also dramatically impaired as determined by an endothelial cell permeability assay. Structural modeling predicted that the side-chain of Arg cannot be accommodated at this site of APC without a major distortion of the local structure that appears to propagate and adversely affect the reactivity/folding of the catalytic pocket. CONCLUSION: The G197R mutation in patients appears to be functionally equivalent to a heterozygous protein C knockout with half of the protein having no significant activity and thus causing thrombosis.


Assuntos
Proteína C , Trombose , Animais , Testes de Coagulação Sanguínea , Heterozigoto , Humanos , Mutação , Proteína C/genética , Trombina , Trombose/genética
7.
Arterioscler Thromb Vasc Biol ; 40(2): 483-494, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31875702

RESUMO

OBJECTIVE: Defective PC (protein C) pathway predisposes patients to venous thromboembolism (VTE) and is mostly, but not exclusively, attributed to hereditary PC or PS (protein S) deficiencies and activated PC resistance caused by factor V Leiden mutation. Approach and Results: In a patient with acute mesenteric venous thrombosis and positive family history of VTE associated with the impaired PC pathway function determined by thrombin generation test, we identified a novel heterozygous prothrombin mutation p.Arg541Trp. Two more patients with positive family history of VTE carrying the same mutation were identified in a cohort of another 373 unrelated patients, making an overall prevalence of 0.8%. Family investigation revealed 11 individuals in the 3 pedigrees harboring the heterozygous prothrombin p.Arg541Trp mutation, and 8 of them (72%) had experienced episodes of VTE. Functional studies indicated the mutation moderately decreased procoagulant activity of prothrombin and had mild impact on the inactivation of thrombin by its inhibitor antithrombin. However, the amino acid residue substitution significantly compromised PC activation by thrombin, both in the absence and presence of soluble thrombomodulin, and thus rendered prothrombin function procoagulant biased. CONCLUSIONS: In summary, the prothrombin p.Arg541Trp mutation constitutes a new genetic risk factor of VTE by impairing function of PC pathway and tilting thrombin's procoagulant activity over anticoagulant function.


Assuntos
DNA/genética , Predisposição Genética para Doença , Isquemia Mesentérica/genética , Mutação , Proteína C/metabolismo , Protrombina/genética , Adulto , Análise Mutacional de DNA , Feminino , Heterozigoto , Humanos , Isquemia Mesentérica/sangue , Pessoa de Meia-Idade , Linhagem , Protrombina/metabolismo , Risco
8.
Haematologica ; 105(6): 1712-1722, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31399531

RESUMO

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.


Assuntos
Fator de Crescimento Epidérmico , Trombose , Glicosilação , Humanos , Mutação , Proteína C/genética , Proteína C/metabolismo , Trombina/metabolismo , Trombose/genética
9.
Haemophilia ; 25(2): 316-323, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30648777

RESUMO

INTRODUCTION: Sporadic haemophilia B (HB) without obvious familial history poses challenges for genetic diagnosis and counselling. AIM: To identify the F9 variants in sporadic HB patients and probe the origin of these de novo mutations. METHOD: A total of 294 unrelated HB pedigrees sought genetic diagnosis were analysed in this single-centre study. The F9 gene was analysed by direct sequencing, and AccuCopy technique was adopted to screen for gene copy number variations. Six short tandem repeats approximal or within F9 gene were applied for linkage analysis. Mosaicism of sequence variant was determined by ddNTP Primer Extension method. RESULTS: Sporadic HB patients constituted 36% (61/294) of cases enrolled in current study. The sporadic and familial HB patients shared similar spectrum of F9 variants, with single nucleotide substitution as predominant form of disease-causing mutation and no mutation prone hotspot sites, including CpG dinucleotide sequences, had been identified. Majority of the mothers of sporadic HB patients were F9 mutation carriers (70%, 43/61), and most of them (95%, 41/43) had the inherited bleeding trait traced back to maternal grandfathers. Although most de novo mutations occur in germ cells, 2 maternal grandfathers, who had somatic mosaic mutations of F9, were also revealed to be the source of genetic variations identified in patients. In our cohort, FIX inhibitor incidence was 1%, developed only in patients carrying null mutations. CONCLUSION: The diversity of F9 genetic variants and possible mosaicism of de novo mutation demand extensive study and more cautious in genetic counselling of sporadic HB.


Assuntos
Fator IX/genética , Hemofilia B/genética , China , Códon sem Sentido , Variações do Número de Cópias de DNA , Éxons , Hemofilia B/diagnóstico , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Polimorfismo Genético , Splicing de RNA
10.
Thromb Haemost ; 117(8): 1478-1485, 2017 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-28492696

RESUMO

Haemophilia A (HA) is a common X-linked recessive bleeding disorder and almost one half of patients with severe HA are caused by intron 22 inversion (Inv22) in the F8. Inv22 is considered to be almost exclusively of meiotic origin in germ cells during spermatogenesis and only one mosaic Inv22 female carrier with the mutation possibly occurring during mitosis of the embryo has been reported so far. Previously we have identified a novel complex recombination mediated by int22h copies in a sporadic severe HA pedigree and herein we have localised the sequences flanking the breakpoint region using genome walking technique, AccuCopy technique, gene chip and real-time PCR. The disease causing genetic variant registered an 18.1 kb deletion including part of int22h-1 through the intron 23 of F8 and a 113.3 kb duplication of part of int22h-2 through the intron 1 of TMLHE inserted in the religated region of the F8. Two intrinsically linked mechanisms of recombination-dependent DNA replication: microhomology-mediated break-induced replication (MMBIR) followed by break-induced replication (BIR) might be responsible for the incident of the complex recombination during early embryogenesis of the proband's mother.


Assuntos
Fator VIII/genética , Deleção de Genes , Duplicação Gênica , Hemofilia A/genética , Íntrons , Oxigenases de Função Mista/genética , Recombinação Genética , Inversão de Sequência , Passeio de Cromossomo , Cromossomos Humanos X , Análise Mutacional de DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Predisposição Genética para Doença , Hemofilia A/sangue , Hemofilia A/diagnóstico , Hemofilia A/embriologia , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Mosaicismo , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Índice de Gravidade de Doença
11.
J Clin Pathol ; 70(2): 145-153, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27555433

RESUMO

AIMS: A novel heterozygous variant, FGA c.169_180+2 del (designated fibrinogen Shanghai), was identified in a patient with dysfibrinogenemia with antiphospholipid antibody syndrome (APS) and recurrent venous thrombosis, and in his asymptomatic father. We aimed to reveal the functional implication of structural change caused by this variant. METHODS: Transcription analysis was performed with FGA minigene transfection assay to evaluate the impact of nucleosides deletion on mRNA editing. The fibrinogen isolated from propositus' plasma was used to characterise its functional defects. Fibrin polymerization and clot lysis experiments were performed by optical measurement of turbidity. Thrombin-catalysed fibrinopeptide release was analysed by the reversed-phase, high-performance liquid chromatography. The ultrastructures of fibrin clots were visualised by scanning electron microscopy. RESULTS: FGA c.169_180+2 del led to an aberrant mRNA with exon 2 skipping and encoded an shortened Aα chain with 42 amino acids truncation at its N-terminal. The propositus' fibrinogen had an impaired release of fibrinopeptide A and abnormal polymerization with a significantly prolonged lag time, a slower maximum slope and reduced final turbidity. The fibrin clot formed with propositus' fibrinogen showed thicker fibres with looser network structure. Clot lysis was normal using the purified fibrinogen but was significantly impaired using the plasma sample from propositus, compared with that from his father. CONCLUSIONS: Fibrinogen Shanghai results in N-terminal truncation of Aα chain, which does not interfere with synthesis, assembly or secretion of fibrinogen, but compromises fibrin polymerization and clot formation. APS at least partially contributes to the development of thrombosis in the propositus.


Assuntos
Afibrinogenemia/genética , Síndrome Antifosfolipídica/genética , Fibrina/metabolismo , Fibrinogênio/genética , Mutação , Trombose Venosa/genética , Afibrinogenemia/complicações , Afibrinogenemia/metabolismo , Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/metabolismo , China , Fibrinogênio/metabolismo , Humanos , Masculino , Trombose Venosa/metabolismo , Adulto Jovem
12.
J Pathol ; 240(1): 108-19, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27319744

RESUMO

Mutations of vacuolar protein sorting-associated protein 33b (VPS33B) cause arthrogryposis, renal dysfunction, and cholestasis syndrome, and a lack of platelet α-granules in the affected patients. Conditional Vps33b knockout mice were developed to investigate the function(s) of Vps33b in platelet α-granule formation. We found that early embryonic deletion of Vps33b was lethal. PF4-Cre-driven megakaryocyte-targeted Vps33b gene deletion greatly diminished Vps33b expression in platelets, but had no effect on platelet α-granule formation and protein content. Tamoxifen-induced, haematopoietic stem cell (HSC)-specific Vps33b deletion completely depleted Vps33b in platelets, caused the absence of α-granules, and increased the number of vacuoles in platelets and megakaryocytes. VPS33B association with VIPAS39, α-tubulin, and SEC22B was identified by co-immunoprecipitation, mass spectra, and immunoblotting in human embryonic kidney 293T (HEK293T) cells. Also, pull-down experiments revealed that VIPAS39 bound to intact VPS33B; in contrast, α-tubulin and SEC22B separately interacted with the sec1-like domains of VPS33B. Vps33b deficiency in megakaryocytes disturbs the redistribution of Vipas39 and Sec22b to proplatelets, and interrupted the co-localization of Sec22b with Vwf-positive vesicles. The data presented in this study suggest that Vps33b is involved in α-granule formation possibly by facilitating the Vwf-positive vesicular trafficking to α-granule-related vacuoles in megakaryocytes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Megacariócitos/metabolismo , Transporte Proteico/genética , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fator de von Willebrand/metabolismo , Animais , Plaquetas/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteínas de Transporte Vesicular/genética
13.
Clin Chim Acta ; 458: 78-83, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27101812

RESUMO

BACKGROUND: To develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A. METHODS: The design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products. RESULTS: PLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 8.3% and >20%, respectively and negative results were obtained in pedigree 2. CONCLUSIONS: AccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection.


Assuntos
Inversão Cromossômica/genética , Hemofilia A/genética , Íntrons/genética , Reação em Cadeia da Polimerase , Feminino , Genótipo , Hemofilia A/diagnóstico , Humanos , Masculino
14.
Blood Cells Mol Dis ; 58: 29-34, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27067486

RESUMO

Congenital factor XI (FXI) deficiency is a rare bleeding disorder with unpredictable bleeding tendency. Few studies in a large cohort have been reported regarding associations between FXI activity (FXI:C) or genotypes and bleeding symptoms currently. This study characterized clinical manifestations and mutation spectrum of 57 subjects with FXI deficiency in China. Clinical data were collected and mutations were identified by direct sequencing and determined by mRNA analysis. The result revealed bleeding symptoms were only found in 12 patients (12/57, 21.1%) with severely reduced FXI:C, and prolonged bleeding post injury/surgery as well as easy bruising were the commonest bleeding manifestations presented in respective 5 cases (5/12, 41.7%). A total number of 37 mutations were identified including 19 missense mutations, 9 nonsense mutations, 6 splice site mutations and 3 small deletions. Among them, 4 missense mutations, 5 splice mutations, 3 small deletions and a nonsense mutation were newly detected. W228*, G400V, Q263* and c.1136-4delGTTG with a total frequency of 48.3% were the most four common mutations in Chinese patients. RT-PCR analysis was carried out and confirmed that both c.596-8T>A and c.1136-4delGTTG were pathogenic due to frameshift resulting in respective truncated proteins. Our findings suggested clinical manifestations had little to do with FXI:C or genotypes, which required further study. This study, the largest investigation of FXI deficiency in China revealed that the F11 mutation spectrum of Chinese population was distinct from those of other populations earlier established.


Assuntos
Deficiência do Fator XI/genética , Fator XI/genética , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , China/epidemiologia , Deficiência do Fator XI/complicações , Deficiência do Fator XI/epidemiologia , Feminino , Genótipo , Hemorragia/epidemiologia , Hemorragia/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , RNA Mensageiro/genética , Adulto Jovem
16.
Clin Exp Pharmacol Physiol ; 43(2): 149-56, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26510121

RESUMO

Congenital (hypo)dysfibrinogenemia patients may have obstetric complications during their pregnancies. This study aimed to evaluate thromboelastography (TEG) as a potential tool for assessing the tendency for obstetric complications in those patients in a non-pregnant state. A total of 22 female subjects with congenital (hypo)dysfibrinogenemia were recruited. Nine subjects had histories of obstetric complications and the other 13 subjects had at least one uneventful pregnancy without obstetric complications as yet. Detailed clinical investigation and phenotype/genotype detection were carried out, and both kaolin-activated TEG and functional fibrinogen TEG (FF-TEG) were applied in all subjects. Significant differences were identified in all TEG parameters except for R and angle between these two groups (P < 0.05) by covariance analysis. Receiver operating characteristic (ROC) analysis of discrimination between these two groups of patients was performed for TEG parameters. Significantly high odds ratio (OR) of obstetric complications occurrence were demonstrated in K ≥ 3.8 min, maximum amplitude (MA) ≤ 54.2 mm, comprehensive index (CI) ≤ -3 (11.67, 95% CI 1.527-89.121, P < 0.05 in all), and MA-CFF ≤ 12.1 mm (20.00, 95% confidence interval (95% CI) 1.967-203.322, P = 0.002). Moreover, MA-CFF had better prognostic performance, with a corresponding area under the receiver operating curve of 0.923 (range 0.815-1.031, P = 0.001). This study suggests that (hypo)dysfibrinogenemia patients with values outside of the cut-off values of TEG assays under non-pregnant state may have a higher risk of obstetric complications occurring when they are pregnant. No parameters under non-pregnant state in clinical laboratory have ever been reported to be risk factors for obstetric complication occurrence in (hypo)dysfibrinogenemia patients. This study explored such parameters in TEG assays and found that parameters of TEG assays under non-pregnant status might predict the occurrence of obstetric complications, which could provide physicians with important information about whether fibrinogen replacement therapy is required, so as to prevent the occurrence of obstetric complications, especially for patients who are asymptomatic in daily life.


Assuntos
Afibrinogenemia/diagnóstico , Complicações na Gravidez/diagnóstico , Tromboelastografia , Adulto , Afibrinogenemia/complicações , Afibrinogenemia/metabolismo , Feminino , Fibrinogênio/metabolismo , Humanos , Caulim/farmacologia , Gravidez , Complicações na Gravidez/metabolismo , Risco
17.
Blood Cells Mol Dis ; 55(4): 308-15, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26460252

RESUMO

INTRODUCTION: Congenital dysfibrinogenemia (CD) is a rare qualitative disorder of fibrinogen (Fg) with heterogeneous clinical manifestations. We aimed to analyze clinical phenotype and molecular basis of 102 Chinese CD patients and to evaluate the application of thromboelastography (TEG). MATERIALS AND METHODS: Clinical manifestations were recorded and quantified using the consensus ISTH bleeding assessment tool. Kaolin activated TEG and functional Fg TEG were applied in 30 patients. Genetic analysis of Fg genes were performed by direct sequencing. RESULTS: 27.5% patients experienced bleeding, 3.9% had thrombosis and 68.6% were asymptomatic. Females were more prone to experience bleeding (P=0.01). Significant difference (P<0.05) in TEG results were found between patients with hot-spot mutations at AαArg35(16) and γArg301(275), but were not identified between patients with and without bleeding. Normal TEG results were found in patients with mutations at AαArg35(16), AαPro37(18) or AαArg38(19). Six novel mutations were identified, including AαGly33(14)del, AαAsp57(38)_Trp60(41)delIVS2+1_+2GTdel, AαPhe742(723)Tyr, γAsn334(308)Thr, γGly335(309)Cys and γTrp395(369)Leu. CONCLUSIONS: CD patients have similar clinical manifestations and hot-spot mutations worldwide with no ethnic difference. TEG results could not indicate the bleeding risk in patients, but priority of mutation screening at thrombin cleavage site or polymerization site on Aа chain may be given if TEG results are normal.


Assuntos
Afibrinogenemia/diagnóstico , Afibrinogenemia/genética , Adolescente , Adulto , Afibrinogenemia/sangue , Idoso , Alelos , Substituição de Aminoácidos , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Criança , Pré-Escolar , China , Feminino , Fibrinogênio/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Tromboelastografia , Adulto Jovem
18.
Thromb Haemost ; 113(3): 585-92, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25503412

RESUMO

Mutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %, 14.2 %, 5.2 %, 19.2 %, 1.8 % and 2.5 % of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.


Assuntos
Coagulação Sanguínea/genética , Fator VIII/genética , Hemofilia A/genética , Mutação , Sítios de Splice de RNA , RNA Mensageiro/genética , Simulação por Computador , Análise Mutacional de DNA , Éxons , Regulação da Expressão Gênica , Predisposição Genética para Doença , Hemofilia A/sangue , Humanos , Linhagem , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Pathology ; 46(7): 630-5, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25393254

RESUMO

We report two compound heterozygous mutants that caused severe type I protein C (PC) deficiency in two independent Chinese families.PC antigen was determined by enzyme-linked immunosorbent assay (ELISA), and PC activity was measured by chromogenic assay. Genetic mutations were screened with polymerase chain reaction (PCR) followed by direct sequencing. PC mutants were transiently expressed in COS-7 cells for the evaluation of PC secretory activity and function. The subcellular location was visualised by immunofluorescence assay. The structural analysis of mutation was performed as well.Compound heterozygous mutations of Arg178Trp and Asp255His with reduced PC activity and antigen levels were identified in Proband 1, a 28-year-old male with deep vein thrombosis (DVT) and pulmonary embolism. The other mutations of Leu-34Pro and Thr295Ile with reduced PC activity and antigen levels were identified in Proband 2, a 19-year-old male with DVT. The PC activities with Arg178Trp, Asp255His, Leu-34Pro and Thr295Ile mutations decreased significantly. Immunofluorescence assay demonstrated that only trace amount of PC with novel Thr295Ile mutation was transported to the Golgi apparatus. Subsequent structural analysis indicated severe impairments of intracellular folding and secretion.The two rare compound heterozygous mutations could cause type I PC deficiency via impairment of secretory activity of PC.


Assuntos
/genética , Heterozigoto , Mutação/genética , Deficiência de Proteína C/genética , Adulto , Animais , Células COS , Linhagem Celular , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Masculino , Linhagem , Embolia Pulmonar/genética , Trombose Venosa/genética , Adulto Jovem
20.
Zhonghua Xue Ye Xue Za Zhi ; 35(11): 995-9, 2014 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-25417877

RESUMO

OBJECTIVE: To disclose the impact of Trp1707Ser mutation on the binding mechanism of rFVIII light chain (rFVIII LC) with VWF. METHODS: Using long-chain PCR technique, we constructed rFVIII LC plasmids of both wild type and Trp1707Ser mutant type. BL21 competent cells were used for protein expression. Gradient renaturation was employed to refold protein. SDS-PAGE and Western blot were performed to identify the molecular weight of expressed protein. GST-Sefinose was used for protein purification and surface plasmon resonance (SPR) was employed to detect binding of B-domain-deleted rFVIII (BDD-rFVIII), wild and mutant rFVIII LC with VWF, respectively. RESULTS: The results of SDS-PAGE and Western blot showed a molecular weight of 110×10(3) of expressed proteins, which were consistent with objective proteins. The expression quantity of wild type was higher than that of mutant type. A concentration-dependent combination of the 3 testing proteins with VWF was found. The KD value of BDDrFVIII (12.2) was lower than that of both rFVIII LCs (wild type 48.9 and mutant type 46.3), whereas there was no discrepancy between wild rFVIII LC and mutant rFVIII LC. CONCLUSION: Trp1707Ser mutation didn't impact the binding of rFVIII LC expressed by BL21 competent cells with VWF. The heavy chain played a more important role in impacting the binding of FVIII with VWF.


Assuntos
Mutação , Fator de von Willebrand/genética
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