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Publications (18)60.32 Total impact

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    ABSTRACT: The natural history of inhibitors in patients with haemophilia A not undergoing immune tolerance induction (ITI) is largely unknown. A recent randomized controlled trial suggests that the higher the FVIII dose used for ITI, the faster the clearance and the lower the rate of bleeding, without any difference in the rate of tolerance. We aimed at assessing the rate of spontaneous inhibitor clearance in a large cohort of patients not undergoing ITI. A retrospective analysis of anti-FVIII inhibitors of long-term registry data in a single centre cohort of 524 haemophilia A patients considered for synovectomy was performed. Patients were tested for inhibitors before and 15 days after any and each surgical episode and thereafter did not undergo immune tolerance at any time. The cumulative incidence of inhibitors overall was 34% (180 out of 524) with the highest percentage of 39% (168 out of 434) in severe patients which represented 83% of the cohort. Among the 180 inhibitor patients: 63 had permanent inhibitors; 70 fulfilled current criteria for transient inhibitors but a third category of 47 additional patients cleared the alloantibody spontaneously in >6 months. At logistic regression, both the inhibitor titre and the gene mutation were shown to predict time to clearance. Spontaneous clearance of inhibitors over variable time in the absence of ITI treatment was found in up to 2/3 of the cases.
    Journal of Hematology & Oncology 08/2013; 6(1):63. · 4.46 Impact Factor
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    ABSTRACT: This article describes prenatal diagnosis (PND) of haemophilia B (HB) within the framework of Italian haemophilia centres and genetics laboratories. The study details the experience from six haemophilia genetic centres (three in the North, one in the Centre and two in the South of Italy) and summarizes the different techniques used to perform PND of HB during the last 15 years. To date, the Italian HB database includes 373 characterized unrelated patients and their genetic information has permitted the identification of 274 carriers of childbearing age. This database represents the main instrument for timely and precise PND. Sixty-six prenatal diagnoses were performed on 52 HB carriers whose average age at the time was 34 (ranging from 24 to 44 years). In 44 cases, genetic counselling for carrier status determination was performed before pregnancy, while eight were not studied prior to pregnancy. Foetal samples were obtained by chorionic villus sampling in 52 cases, by amniocentesis in 12 while two were diagnosed by analysis of free foetal DNA obtained from maternal peripheral blood. In 35 (53%) pregnancies the foetus was female. For 31 men (47%), haemophilia status was determined by analysis of previously determined informative markers or familial mutations (12 affected and 19 unaffected). There may be more than one laboratory involved in the PND diagnostic pathway (providing DNA extraction, karyotype analysis, gender determination, maternal contamination detection, molecular diagnosis and sequencing). Good communication between all the parties, coordinated by the haemophilia centre, is essential for a successful and rapid process.
    Haemophilia 06/2013; · 3.17 Impact Factor
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    ABSTRACT: We investigated the spontaneous ribosome readthrough, virtually unexplored in genes encoding secreted proteins, over coagulation F9 nonsense mutations. Expression of recombinant factor IX (FIX) in eukaryotic cells demonstrated appreciable levels of secreted FIX molecules for the mutations p.R162* (5 ± 0.3% of rFIX-wt antigen levels), p.R294* (3.1 ± 1.1%) and p.R298* (2.5 ± 0.7%), but not for the p.L103*. Western blotting revealed a large proportion of truncated molecules, which correlated with small amounts of full-length FIX (rFIX-162*, ∼0.5%; rFIX-294*; and rFIX-298*, ∼0.2%). Western blotting of plasma from FIX deficient (Hemophilia B) patients revealed traces of full-length FIX for the p.R294* and p.R298* mutations, but not for the p.L103* mutation that triggered major FIX mRNA decay. The detection of full-length proteins has clinical implication, particularly for post-therapeutic immunological complications in Hemophilia. Data in patients' plasma and in vitro, obtained in the proper protein context, support a ribosome readthrough gradient, consistent with its predicted determinants of efficiency.
    Human Mutation 05/2012; 33(9):1373-6. · 5.21 Impact Factor
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    ABSTRACT: It is not rare to observe in blood donors a level of haematocrit (Hct) above or close to the highest normal limit. In the case of blood donors the diagnosis and clinical evaluation of this alteration may be complicated by regular blood donations that can mask an underlying disease such as polycythaemia vera. Recently a single acquired mutation in the Janus kinase 2 gene (JAK2) on chromosome 9 was identified and it was found that the incidence of this mutation was high in patients with polycythaemia vera. From the January 1, 2006 to December 31, 2006 all consecutive donors with a Hct above 50% if males (n=84) and 46% if females (n=19) underwent JAK2 mutation analysis. Seventy-nine donors (59 males and 20 females) whose Hct was normal at their last blood donation were randomly selected and used as controls. Among the group of blood donors with a high Hct, we identified one donor who was positive for the JAK2 mutation. This man had a Hct of 50.6% at his last donation, while his average Hct in the preceding year was 51.7%. The prevalence of the JAK2 mutation could be estimated to be 1%, 0.6% or 0.02% in the three different populations considered: donors with a Hct level above the upper limit of normal, all tested donors or the entire donor cohort attending our transfusion service, respectively. The present study suggests that apparently healthy subjects with repeatedly high levels of Hct may have the acquired mutation in JAK2. Laboratory screening tests for JAK2 may be offered to blood donors at transfusion services with expertise in molecular genetics.
    Blood transfusion = Trasfusione del sangue 05/2009; 7(2):111-6. · 1.86 Impact Factor
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    ABSTRACT: To provide a National database, 1,410 unrelated hemophilia A (HA) patients were investigated using screening methods denaturing high-performance liquid chromatography (DHPLC), conformational-sensitive gel electrophoresis (CSGE)] and/or direct sequencing. F8 gene mutations were identified in 877 (81%), 146 (82%), and 133 (89%) families with severe, moderate, or mild HA, respectively. Among the 382 different mutations detected, 217 (57%) have not previously been reported in the F8 Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database. Mutations leading to a null allele accounted for 82, 15%, and less than 1% of severe, moderate, or mild HA, respectively. A missense mutation was identified in 16%, 68%, and 81% of severe, moderate, or mild HA, respectively. They included 105 missense mutations (48%), 41 small deletions (19%), 25 splice site mutations (12%), 24 nonsense mutations (11%), 18 insertions (8%), three large deletions (1%), and one deletion plus insertion. Unreported mutations were distributed throughout the F8 gene, as they affected all F8 exons but exon 20. We report a wide spectrum of mutations collected in a large National database. The type of mutation was a strong predictor of the clinical phenotype. This database is expected to considerably improve the genetic counseling and medical care of HA families in Italy.
    Journal of Human Genetics 02/2008; 53(3):275-84. · 2.53 Impact Factor
  • Haemophilia 12/2007; 13(6):772-4. · 3.17 Impact Factor
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    ABSTRACT: The Italian database of factor IX gene (F9) mutations has been built since 2001 and is, so far, the most practical instrument for comprehensive genetic counselling, carrier detection and prenatal diagnosis. Over time the haemophilia B database has been enriched by entries on a larger number of patients and molecular genetic data identifying heterogeneous mutations spanning the entire F9. Conformation sensitive gel electrophoresis is a variant of heteroduplex analysis, which has been applied for screening F9 for mutations, which are further fully characterised by direct sequencing of the amplified mutated regions. This project has involved 29 Italian haemophilia centres and provides data concerning the analysis of a cohort of 306 unrelated patients with haemophilia B (191 with severe, 67 with moderate and 48 with mild disease, including 8 patients with severe haemophilia B with inhibitors). The recorded data include levels of factor IX clotting activity, inhibitor status and clinical severity. Detailed analysis of the mutations revealed 164 different mutations, that are considered as unique molecular events (8 large deletions, 11 small deletions, 1 combined deletion/ insertion, 2 insertions, 104 missense, 20 nonsense, 14 mutations in a splicing site, 3 in the promoter and 1 silent). The data recorded in the Italian F9 mutation database provided the basis to study 85 families with haemophilia B, involving 180 females (20 obligate carriers, 106 carriers and 54 non-carriers) and enabled 14 prenatal diagnoses to be made in 12 females. Genetic analysis is required to determine female carrier status reliably. Female relatives may request carrier analysis, when a male relative is first diagnosed as having haemophilia or when they are pregnant. At present, the data collected in the Italian national register of mutations in haemophilia B provide the opportunity to perform prompt and precise determination of carrier status and prenatal diagnosis by specific mutation analysis.
    Blood transfusion = Trasfusione del sangue 08/2007; 5(3):158-63. · 1.86 Impact Factor
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    ABSTRACT: Anti factor VIII (FVIII) antibodies represent the main complication of replacement therapy in severe cases of haemophilia and most patients with inhibitor have gross gene rearrangements or point mutations that hamper the production of normal circulating FVIII. In this study we have investigated 82 haemophilia A patients with inhibitors. Seventy six were severe, three were moderate and three were mild. We screened the patients for the causative mutations using long range PCR for the recurrent intron 22 inversion (invint22), multiplex PCR for intron 1 inversion (invint1) and conformation sensitive gel electrophoresis followed by DNA sequencing for all other mutation types in the F8 gene. Diverse genetic defects were detected in the severe cases (with a predominance of severe molecular defects): F8 gene inversions, large deletions and non-sense mutations account for 71% of the mutations. Only missense and splicing mutations were identified in the non-severe patients and we confirmed that the presence of inhibitors correlates well with the presence of severe mutations, but a proportion of severe patients develops inhibitors despite the presence of diverse less severe mutations. When we have analysed the subgroup of patients who underwent immunetolerance, we have found that F8 gene large deletions are likely to be a high risk factor also for immunetolerance therapy unresponsiveness, while no clear evidence has been demonstrated for other mutation types.
    Haemophilia 08/2007; 13(4):361-72. · 3.17 Impact Factor
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    ABSTRACT: A relevant aspect in the treatment of patients with hemophilia A (HA) presenting inhibitor against factor VIII (FVIII) is the different antigenicity of FVIII used for replacement therapy. The aim of the study was to assess the effect of different products, with variable von Willebrand factor (vWF) concentration, in preventing the binding of inhibitor to FVIII. The reactivity of inhibitors from plasma of 18 patients with HA versus three commercial concentrates containing different amounts of vWF was compared. The results show that increasing amounts of vWF might have a protective effect on the transfused FVIII inactivation.
    American Journal of Hematology 07/2007; 82(6):460-2. · 4.00 Impact Factor
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    ABSTRACT: The aim of the study, funded by the Italian Ministry of Health, was to identify the causative mutation in all known patients with hemophilia B in Italy. Overall, 269 patients followed by 25 regional centers were considered in the study; after exclusion of the related individuals, 238 unrelated patients were analyzed (153 with severe, 59 with moderate and 26 with mild hemophilia B). Screening of the factor IX gene was performed using conformation sensitive gel electrophoresis (CSGE) followed by denaturing high performance liquid chromatography (dHPLC) or direct sequencing in negative cases, or by dHPLC/sequencing (36 cases). A mutation was identified in 236 of the 238 patients: 6 had large gene deletions (4 total and 2 partial), 14 small deletions, 1 combined deletion/insertion and 215 single nucleotide substitutions. A correlation was observed between the type of mutation and severity of hemophilia; however, a number of patients with the same genotype had varying severities of the disease. Eight of the 169 patients with severe hemophilia B (4.7%) developed inhibitors: 2 of these had a complete gene deletion, 1 had a large partial deletion (from exon A to part of exon H) while 5 had 3 different nonsense mutations. One patient with a nonsense mutation developed anaphylaxis. We also studied 65 families with hemophilia B involving 144 females (14 obligatory carriers, 85 carriers and 45 non-carriers) and performed 12 antenatal diagnoses. The data have been used to build the Italian mutation database to provide each family with knowledge of the disease-causing defect for genetic counseling. This Italian study confirms the marked heterogeneity of factor IX mutations in the population and the presence of a degree of genotype/phenotype discordance. The identification of the mutation can also be used to predict risk of inhibitor development.
    Haematologica 06/2005; 90(5):635-42. · 5.94 Impact Factor
  • Haemophilia 04/2004; 10(2):194-6. · 3.17 Impact Factor
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    ABSTRACT: Patients with haemophilia show high prevalence of hepatitis C infection but low rate of progressive liver disease when they are not co-infected with HIV. The balance between host immune system and hepatitis C virus (HCV) variability seems to play a major role in the evolution of the HCV-related disease. To address this point we have studied, in a group of selected patients with haemophilia, the composition and in some cases the evolution, of the highest variable envelope gene within the hypervariable region 1 (HVR1) of the HCV, which is the region more directly exposed to the host immune response. Five of 12 patients show a very high homogeneity of the HVR1 and four of those had severe progressive liver disease. These results seem to confirm the major role of the immunity in driving the variability of the HCV rather than the high degree of different HCV strains to which haemophiliacs have been in touch with, during their long-term replacement therapy. Our results seem in keeping with other studies on different type of patients, where a low degree of quasispecies variability has been demonstrated in relationship with the progression and the severity of their liver disease.
    Haemophilia 02/2004; 10(1):81-6. · 3.17 Impact Factor
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    ABSTRACT: Haemophilia A displays a broad heterogeneity of genetic defects and of clinical severity. Inhibitor development is the main complication of replacement therapy in severe cases and most patients with inhibitors have gross gene rearrangement or point mutations, which hamper the production of normal circulating factor VIII (FVIII). We have investigated three related severe haemophilia A patients, all of whom have high titre inhibitors. By using long-range polymerase chain reaction (PCR) for FVIII gene inversion, we observed an unusual pattern in these patients. We therefore decided to screen the whole FVIII gene by conformation-sensitive gel electrophoresis. A large FVIII gene deletion spanning exon 2 to exon 25 was identified and we were able to obtain a 18.5 kb PCR product, which is specific for this mutation and useful for determining the carrier state in this family. All three haemophiliacs carrying this very large gene deletion show similar clinical history and very high-titre inhibitors, supporting the observation that inhibitor development seems to be an inherited characteristic. On the basis of our observations we think that this subgroup of patients at very high risk of inhibitor development should be identified by mutation analysis whenever possible, before the beginning of replacement therapy.
    Haemophilia 02/2002; 8(1):17-21. · 3.17 Impact Factor
  • Haemophilia 12/2001; 7(6):603-4. · 3.17 Impact Factor
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    ABSTRACT: Haemophilia B patients with factor IX (FIX) activity < 1% are usually characterized by severe bleeding episodes early in life. We report a case of sporadic severe haemophilia B, clinically characterized by mild bleeding diathesis. The presence of anamnestic thrombophlebitis in the patient's mother prompted us to investigate a possible associated hypercoagulable condition. Resistance to activated protein C due to factor V R506Q mutation was present in the mother and in the propositus, in the homozygous and heterozygous form, respectively. Molecular analysis of the FIX gene led to the identification of a nonsense mutation resulting in a stop codon at position 50, previously described and usually responsible for a severe pattern of haemophilia B. The implications of this unusual association are discussed.
    Haemophilia 09/2001; 7(5):511-4. · 3.17 Impact Factor
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    ABSTRACT: Haemophilia B patients with factor IX (FIX) activity Keywords: factor V Leiden; haemophilia B; hypercoagulable states Document Type: Miscellaneous Publication date: September 1, 2001 $(document).ready(function() { var shortdescription = $(".originaldescription").text().replace(/\\&/g, '&').replace(/\\, '<').replace(/\\>/g, '>').replace(/\\t/g, ' ').replace(/\\n/g, ''); if (shortdescription.length > 350){ shortdescription = "" + shortdescription.substring(0,250) + "... more"; } $(".descriptionitem").prepend(shortdescription); $(".shortdescription a").click(function() { $(".shortdescription").hide(); $(".originaldescription").slideDown(); return false; }); }); Related content In this: publication By this: publisher In this Subject: Anatomy & Physiology , Internal Medicine By this author: Vianello, F. ; Belvini, D. ; Dal Bello, F. ; Tagariello, G. ; Zanon, E. ; Lombardi, A-M. ; Zerbinati, P. ; Girolami, A. GA_googleFillSlot("Horizontal_banner_bottom");
    Haemophilia 01/2001; 7(5). · 3.17 Impact Factor
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    ABSTRACT: We describe our three year experience in genetic counseling at the Castelfranco Veneto Hemophilia Center, Italy. A total of 258 individuals were involved in the study of 142 females. These formed 40 families with hemophilia A and 6 families with hemophilia B. Following pedigree analysis, the FVIII inversion was first examined in severe hemophilia A patients by polymerase chain reaction (PCR) analysis. DNA polymorphisms were used to track the affected gene through the remaining families. In uninformative cases, we initiated analysis of the FVIII or FIX gene coding region by conformation sensitive gel electrophoresis and DNA sequencing to identify the mutation responsible for the disease. The FVIII gene inversion was present in 16 of the 32 patients (50%) affected by severe hemophilia A and was informative for 44 females. For hemophilia A, 45 cases (55%) were informative by linkage analysis, however 37 (45%) were uninformative because of lack of key individuals, homozygosity, or sporadic disease. Information from extragenic linked polymorphisms alone was present in 9 cases (6%). For hemophilia B, linkage analysis was informative in only 50% of females (8 out of 16). To date, nine mutations have been identified in patients with hemophilia A and three in patients with hemophilia B. Six novel missense mutations in hemophilia A are discussed briefly. Using this approach we are now able to offer accurate genetic analysis to virtually all families with hemophilia.
    Haematologica 06/2000; 85(5):525-9. · 5.94 Impact Factor
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    ABSTRACT: Haemophilia A (HA) and haemophilia B (HB) are X-linked bleeding disorders caused by diverse mutations throughout the factor VIII and factor IX genes. To date, genetic analysis is one of the most important support for patients with haemophilia and their families. Mutation type identification represents the main risk factor for inhibitor development and it is crucial in approaching the genetic therapy. The molecular biology and in particular the molecular diagnosis of haemophilia, including the identification of the specific defect responsible for the disease, provides the best possible genetic counselling for patients with haemophilia and their families. Aim of this paper is to describe the state of the art of genetics in the field of haemophilia as well as the experience in the last few years in our Haemophilia Centre. Parole chiave: consulenza genetica, emofilia A, emofilia B, inversione FVIII, CSGE. Introduzione Le emofilie A (HA) e B (HB) sono difetti congeniti della coagulazione caratterizzati dall'assenza o dalla ridotta produzione dei fattori della coagulazione VIII (FVIII) o IX (FIX). Attualmente, la conoscenza del difetto molecolare alla base dell'emofilia rappresenta uno dei più importanti ausili nella gestione dei pazienti affetti da queste coagulopatie 32,38 . L'identificazione della mutazione che determina la malattia permette, infatti, una definizione precisa del difetto in ciascun paziente. Inoltre, offre chiarimenti alle più importanti problematiche relative all'emofilia quali la gravità della malattia, il rapporto fenotipo/ genotipo o lo sviluppo di inibitori anti-FVIII o IX. L'unico presidio terapeutico in grado di correggere tali difetti è rappresentato dall'utilizzo di concentrati del fattore mancante, di origine plasmatica o ricombinante, somministrati in occasione di episodi emorragici o a scopo profilattico: questi hanno raggiunto oggi standard di elevata purezza e sicurezza in termini di trasmissione di malattie di origine virale. La complicanza più grave del trattamento sostitutivo è lo sviluppo di anticorpi "inibitori" diretti contro il FVIII o FIX infusi. Le cause di tale fenomeno non sono ancora del tutto chiarite, ma numerose evidenze dimostrano come il tipo di difetto molecolare alla base della malattia sia il più importante fattore di rischio per lo sviluppo di tale risposta anticorpale 37 . Ma l'aspetto che ha subito maggiori trasformazioni dall'avvento della biologia molecolare nell'emofilia è stato senz'altro la consulenza genetica alle famiglie dei pazienti emofilici 30 , dapprima con l'analisi in linkage, più recentemente con l'identificazione precisa del difetto. Ciò permette di definire con sicurezza la possibilità o meno di trasmettere il gene alterato. Tali conoscenze possono venire applicate anche sul prodotto del concepimento, consentendo di rilevare la presenza della mutazione e, quindi, della malattia nel feto 28 . Di seguito vengono esposte le basi molecolari dell'HA e HB, le procedure e le problematiche in tema di studi familiari applicati a tali malattie genetiche con particolare attenzione all'esperienza maturata presso la nostra istituzione.