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. RESULTS: All 61 investigated samples, including the previously investigated and not previously investigated samples, proved to be HRM-positive, with the new screening method showing good efficiency and higher sensitivity than the previously used method. Mixing normal and unknown DNA to generate heterozygote conditions proved an excellent strategy to push the detection performance to its maximum. DISCUSSION: Mutation scanning by HRM analysis seems to be ideal in our context because it is rapid, cheap and capable of detecting the vast majority of mutations in HB patients. Nevertheless, to improve the detection ability of this scanning technology, it is recommended to start with a good strategy, based on good quality samples and optimised polymerase chain reaction amplification parameters, especially regarding primers and length of the amplicons.

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. RESULTS: In 7,606 patients (94.2%) the aPTT ratio was within the normal range while it was abnormal in 463 (5.8%). Out of these 463, 223 aPTT ratios were between 1.2 and 1.3 and we considered these results not worthy enough of further investigations. In 240 patients the aPTT ratio was higher than 1.3; in the vast majority of these cases (201/240; 83%) this abnormality was associated with oral anticoagulant treatment. Seventeen of the other 39 cases underwent detailed investigations which revealed lupus anticoagulant (n=7), decompensated chronic liver disease (n=4), factor XII deficiency (n=3), mild combined reduction of FXI and FXII (n=1) and mild haemophilia A (n=2). The other 22 patients underwent successful surgery without further investigation. DISCUSSION: Our results from a pre-surgical setting seem to confirm the low prevalence of coagulation defects in the general population. Increased aPTT ratios were mainly attributable to oral anticoagulant therapy, with a few cases caused by mild, clinically irrelevant clotting factor deficiencies. A carefully taken personal history, including medications (i.e. oral anticoagulants) and/or previous bleeding symptoms seem more useful than coagulation screening tests to predict the risk of bleeding.

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). RESULTS: 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. INTERPRETATION AND CONCLUSIONS: 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.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Spontaneous clearance of inhibitors over variable time in the absence of ITI treatment was found in up to 2/3 of the cases.

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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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. The results show that increasing amounts of vWF might have a protective effect on the transfused FVIII inactivation.

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. 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. RESULTS: 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. CONCLUSIONS: 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.