399 research outputs found
La terapia genica nelle terapie emorragiche e trombotiche
No full textLe possibilità di Terapia Genica sono discusse da decenni, così come le problematiche di efficacia e sicurezza di una terapia che si pone l’obiettivo di modificare, in modo prolungato se non permanente, il corredo genico presente nel nucleo cellulare di specifici organi o tessuti, senza tuttavia introdurre modificazioni genetiche della linea germinale. La Terapia Genica sta conoscendo momenti di gloria dopo aver sperimentato una stasi prolungata e qualche problema acuto. Tra gli avanzamenti di ricerca a maggiore impatto sulla Terapia Genica nell’uomo vanno senz’altro segnalati l’efficiente identificazione di geni, favorita dal progetto Genoma Umano, e di mutazioni che causano malattia, resa possibile da un impressionante miglioramento tecnologico.
Mentre la maggior parte della sperimentazioni di Terapia Genica giunte alla clinica sono dirette a patologie acquisite ed in particolare ai tumori, le patologie ereditarie della coagulazione hanno ricevuto notevole attenzione, per l’ottima definizione delle loro basi molecolari e per l’indubbio ruolo di patologia modello dell’emofilia, in particolare dell’emofilia B.
Contribuiscono a sostenere l’importanza della terapia genica nelle emofilie la gravità dei sintomi acuti emorragici e le loro conseguenze croniche invalidanti nei pazienti con bassissimi livelli dei vari fattori (VIII, IX, VII, X...) colpiti da mutazione. Inoltre, l’emivita relativamente breve o brevissima dei fattori della coagulazione comporta un’alta frequenza di infusioni nella terapia sostitutiva che, impiegando proteine purificate, risulta essere particolarmente costosa, anche in relazione alla necessità di perdurare per tutta la vita del paziente. La continua infusione della proteina per combattere un fenotipo emorragico può portare in alcune occasioni ad un effetto altrettanto indesiderato, cioè l’induzione di un fenotipo trombotico dato dalla parziale e temporanea elevata concentrazione della proteina infusa.
E’ apparso infine evidente come un ripristino anche limitato – pochi punti percentuali - del livello del fattore difettivo nel plasma potesse costituire un progresso ben misurabile e con chiaro significato clinico con notevole beneficio per il paziente.
Complessivamente, questi argomenti hanno reso le patologie ereditarie delle coagulazione oggetto di un straordinario numero di tentativi “preclinici” di Terapia Genica, che hanno favorito in modo sostanziale i recenti successi clinici. Il presente capitolo intende fornire, pur con una breve descrizione del complesso processo della ricerca, soprattutto una visione riassuntiva dello stato dell’arte relativo alla terapia genica delle patologie della coagulazione condotta nell’uomo, e delle maggiori problematiche in questo settore di avanguardia. Numerose e di notevole livello e specificità sono le “reviews” che hanno descritto il progressivo avanzamento dello stato dell’arte, sia pre-clinico che clinico, nel settore
Type 2B von Willebrand Disease : a Matter of Plasma Plus Platelet Abnormality
No full textType 2B von Willebrand disease (VWD2B) is a rare, autosomal-dominant inherited bleeding disorder, characterized by an enhanced ristocetin-induced platelet aggregation in platelet-rich plasma and often with variable degree of thrombocytopenia and loss of high-molecular-weight multimers von Willebrand factor (VWF). All these phenomena are caused by a mutant VWF, normally synthesized and assembled by endothelial cells, but with heightened affinity binding to the platelet receptor glycoprotein Ib-α (GpIb-α). When this abnormal VWF is released into the circulation and under specific clinical circumstances, in vivo platelet clumping is observed. Mutations, invariably clustered in exon 28 of the VWF gene encoding for the VWF A1 domain involved in VWF binding to GpIb-α, are responsible for VWD2B phenotype. Clinical and laboratory phenotype appears strongly related to the type of VWF-causative mutations. However, recent evidences suggest that a true platelet defect is also present in this type, with several morphological and functional abnormalities being detected in a subset of VWD2B patients
Combination of CLEC4M rs868875 G-Carriership and ABO O Genotypes May Predict Faster Decay of FVIII Infused in Hemophilia A Patients
No full textThe C-type lectin CLEC4M binds and internalizes factor VIII (FVIII). Common CLEC4M variants have been associated with FVIII pharmacokinetic (PK) profiles in hemophilia A (HA) patients. The two-compartment PK analysis of plasma-derived (pd-) and full length recombinant FVIII concentrates was conducted in twenty-six patients (FVIII:C ≤ 2 IU/dL). F8, ABO blood-groups, and the CLEC4M rs868875A/G polymorphism were genotyped. CLEC4M genotype groups differed for the elimination rate constant K 1-0 (p < 0.001), half-life (K 1-0 HL), and the Beta rate constant. Patients treated with pd-FVIII also differed in the Alpha phase. In linear regression models, the contribution of the CLEC4M genotypes to FVIII PK parameters remained significant after correction for ABO, age, and VWF antigen levels at PK. Combined CLEC4M rs868875A/G and ABO genotypes displayed significant interaction (K 1-0, p = 0.014). Compared to other combined genotypes, the G-carriers/O genotypes showed half-reduced K 1-0 HL (p = 0.008), and faster FVIII clearance (mean 7.1 ± 2.2 mL/h/kg SE) than in the G-carriers/non-O (mean 2.4 ± 0.3 mL/h/kg SE), (p = 0.038). Comparison in HA patients recruited in several countries suggests that CLEC4M genotypes coherently influence infused FVIII half-life and clearance. Our analysis supports substantially faster FVIII decay associated with the rs868875 G-carrier/ABO O genotypes, which has potential implications for genetically tailored substitutive HA treatment
Mutations in the thrombomodulin gene are rare in patients with severe thrombophilia
No full textBecause thrombomodulin plays a key role in the protein C pathway, we evaluated the contribution of thrombomodulin gene mutations to venous thrombosis. We examined 38 patients with recurrent, documented thrombotic events at a young age and a positive family history. Twelve individuals with low levels of soluble thrombomodulin in plasma were also studied. Finally, the allelic frequency of the Ala455Val polymorphism was estimated in 192 patients with at least one thrombotic event and in 369 age- and sex-matched asymptomatic controls. Two mutations were identified; G/A)201, in a severely thrombophilic patient and G/T 1456, in a patient with low soluble thrombomodulin levels. The first mutation has been reported by some, but not others, to be associated with moderately reduced levels of thrombomodulin. The second was identified previously in a patient with low soluble thrombomodulin, but expression studies failed to show functional changes in the mutant. Thrombomodulin gene mutations thus appear to be rare even in highly selected thrombophilic patients, and possibly functionally irrelevant. The allelic frequency of the Ala455Val polymorphism was identical in patients and controls. Considering the lack of a phenotype and the costly screening procedure, we recommend that thrombomodulin defects be sought only for research purposes
A novel G-to-A mutation in the intron-N of the protein S gene leading to abnormal RNA splicing in a patient with protein S deficiency
No full textBackground and Objectives. Hereditary protein S (PS) deficiency is a rare autosomal disorder of the coagulation pathway associated with familial thrombophilia. Design and Methods. We investigated a young propositus with recurrent deep vein thrombosis, a positive family history for thrombotic episodes, and low plasma concentrations of free, but not total PS antigen (12% and 70%, respectively). Results. Sequence analysis of the PS gene showed a heterozygous G-to-A mutation at the first nucleotide of intron N. The patient's father, who had suffered from deep vein thrombosis and had reduced total and free PS antigen (59% and 28%, respectively) was a heterozygote. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant mRNA, consistent with exclusion of exon 14, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 14. Exclusion of exon 14 predicts the deletion of the amino acid sequence from residue 508 to residue 582, and the shift of the reading frame of the following 8 amino acids with a premature stop codon within exon 15 at position 591. Thus, the truncated PS gene product would not contain the terminal portion of the sex hormone binding globulin-like domain. Interpretation and Conclusions. We have identified a mutation in a highly conserved intronic region of PS gene. The mutation affects in vitro mRNA processing and efficiency of normal splicing
Molecular characterization of two novel mutations causing factor XI deficiency : a splicing defect and a missense mutation responsible for a CRM+ defect
No full textSevere factor XI (FXI) deficiency is a bleeding disorder generally inherited as an autosomal recessive trait and characterized by haemorrhagic symptoms mainly associated with injury or surgery. So far, more than 150 causative molecular defects have been identified throughout the F11 gene. In the present study, we investigated the molecular basis of FXI deficiency in two Italian patients. Mutational screening of the F11 gene disclosed a novel missense substitution (Arg184Gly) in exon 7 and two splicing mutations: a novel G>A transition affecting the last nucleotide of exon 4 (325G>A), and the already known IVS6+3A>G. RT-PCR assays were performed on total RNA extracted from platelets and lymphocytes of each patient. Sequencing of RT-PCR products demonstrated that both 325G>A and IVS6+3A>G mutations abolish the corresponding donor splice site, causing the skipping of the affected exon; this in turn results in a frameshift introducing a premature termination codon. Expression of recombinant FXI-Arg184Gly revealed a 70% reduction in FXI activity, suggesting that the Arg184Gly mutation might cause a cross-reactive material positive (CRM+) deficiency. In conclusion, the functional consequences of two splicing mutations leading to FXI deficiency have been elucidated. Moreover, we report a novel missense mutation in the FIX-binding region of the FXI A3 domain leading to a CRM+ deficiency
Bleeding tendency and efficacy of anti-haemorrhagic treatments in patients with type 1 von Willebrand disease and increased von Willebrand factor clearance
No full textAccelerated clearance of von Willebrand factor (VWF) has been recently identified as a major pathophysiologic mechanism inducing low VWF in some patients with von Willebrand disease (VWD). The frequency of bleeding and the best treatment of these patients have never been evaluated prospectively in large series of patients. It was the aim of the present study to prospectively evaluate clinical events of 60 heterozygous patients with VWD Vicenza (VWD-VI) carrying R1205H VWF mutation and 23 with C1130F mutation, both characterised by markedly increased VWF clearance. During 71 months of follow-up, 65% of patients with VWD-VI and 61% with C1130F required treatment. The rate of spontaneous bleeding requiring consultation/treatment was 7.5/100 patients-year in patients with C1130F mutation vs. 1.9/100 patients-year in those with R1205H (p=0.004). This difference persisted also by multivariate analysis adjusted for sex, age and blood group (hazard ratio [HR]=3.3 for C1130F, 95% con fidence interval [CI] 1.16-9.27) and females were at greater risk of bleeding (HR=3, 95%CI 1.01-9.93) because of menorrhagia. Only 3/15 (20 %) women in fertile age with VWD-VI compared to 8/9 (89 %) with C1130F mutation required consultation/treatment for menorrhagia (iron supplementation, combined oral contraceptives, tranexamic acid). Almost all dental extractions, minor surgeries and deliveries occurring during followup were successfully managed with desmopressin. Major surgery required factor VIII/VWF concentrates, but a few cases benefited from desmopressin. In conclusion, similar to patients with type 1 VWD, also in patients with increased VWF clearance desmopressin maintains a major therapeutic role
A Sampling-Based Tree Planner for Navigation Among Movable Obstacles
No full textThis paper proposes a planner that solves Navigation Among Movable Obstacles problems giving robots the ability to reason about the environment and choose when manipulating obstacles. It finds a path from a robot start configuration S to a goal configuration G taking into consideration the possibility of moving objects if G cannot be reached or if moving objects may significantly shorten the path. The planner combines the A*-Search and the exploration strategy of the Kinodynamic Motion Planning by Interior-Exterior Cell Exploration algorithm. It is locally optimal and independent from the size of the map and from the number, shape, and position of obstacles. It assumes full world knowledge but it can be easily extended in order to explore unknown environments
A sampling-based tree planner for navigation among movable obstacles
No full textThis paper proposes a planner that solves Navigation Among Movable Obstacles problems giving robots the ability to reason about the environment and choose when manipulating obstacles. It finds a path from a robot start configuration S to a goal configuration G taking into consideration the possibility of moving objects if G cannot be reached or if moving objects may significantly shorten the path. The planner combines the A*-Search and the exploration strategy of the Kinodynamic Motion Planning by Interior-Exterior Cell Exploration algorithm. It is locally optimal and independent from the size of the map and from the number, shape, and position of obstacles. It assumes full world knowledge but it can be easily extended in order to explore unknown environments
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