High resolution melting for F9 gene mutation analysis in patients with haemophilia B.

Background: Since 2001, we have used conformation sensitive gel electrophoresis (CSGE) as our first choice for F9 gene mutation screening, leading to the identification of about 300 mutations causing haemophilia B (HB). To circumvent the disadvantages of CSGE, we recently evaluated high-resolution melting analysis (HRM), which represents the next-generation mutation scanning technology.

Materials And Methods: In order to explore and validate HRM as a new screening method, we analysed 26 HB patients with previous CSGE-detected mutations, 22 patients with CSGE-undetectable mutations and 13 HB patients who had not been previously investigated.

Multiplex ligation-dependent probe amplification as first mutation screening for large deletions and duplications in haemophilia.

Introduction: Molecular characterization has shown a wide mutational spectrum underlying haemophilia A (HA) and haemophilia B (HB). Different molecular assays have allowed laboratories to perform genetic testing for F8 and F9 mutations.

Aim: Recently, multiplex ligation-dependent probe amplification (MLPA), a simple technique for relative quantitation of targeted genomic regions, has been introduced in HA and HB for detection of large deletions and duplications.

Clinical relevance of isolated prolongation of the activated partial thromboplastin time in a cohort of adults undergoing surgical procedures.

Background: Coagulation screening prior to surgery is performed routinely worldwide to identify patients at risk of bleeding during the procedure. Evidence from medical and surgical literature suggests that the activated partial thromboplastin time (aPTT) alone is suitable for predicting individual bleeding risk during surgery and it is current practice in our hospital to measure this parameter.

Materials And Methods: We retrospectively reviewed aPTT ratio results in 8,069 consecutive adult subjects undergoing elective surgery from January 1 to December 31, 2014 to confirm the validity of this approach.

High rate of spontaneous inhibitor clearance during the long term observation study of a single cohort of 524 haemophilia A patients not undergoing immunotolerance.

Background: 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.

Prenatal diagnosis of haemophilia B: the Italian experience.

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.

The JAK2(V617F) tyrosine kinase mutation in blood donors with upper-limit haematocrit levels.

Background: 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.

Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A.

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.

F8 gene mutation profile and ITT response in a cohort of Italian haemophilia A patients with inhibitors.

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.

The Italian haemophilia B mutation database: a tool for genetic counselling, carrier detection and prenatal diagnosis.

Introduction: 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.

Methods: 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.

In vitro reactivity of factor VIII inhibitors with von Willebrand factor in different commercial factor VIII concentrates.

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.

Molecular genotyping of the Italian cohort of patients with hemophilia B.

Background And Objectives: 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.

Design And Methods: 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).

Hepatitis C virus quasispecies in the natural course of HCV-related disease in patients with haemophilia.

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.

Large FVIII gene deletion confers very high risk of inhibitor development in three related severe haemophiliacs.

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.

Experience of a single Italian center in genetic counseling for hemophilia: from linkage analysis to molecular diagnosis.

Background And Objective: We describe our three year experience in genetic counseling at the Castelfranco Veneto Hemophilia Center, Italy.

Design And Methods: 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.