Anna Selmeczi

Founder effect is responsible for the p.Leu131Phe heparin-binding-site antithrombin mutation common in Hungary: phenotype analysis in a large cohort

Essentials Antithrombin Budapest3 (ATBp3; p.Leu131Phe) causing heparin‐binding‐site defect is common. We studied the clinical and laboratory phenotype of a large Hungarian ATBp3 cohort (n = 102). Founder effect of ATBp3 was confirmed by 12 genetic markers; anti‐FXa AT assay was 100% sensitive. The spectrum of thrombotic symptoms was wide in ATBp3 patients including arterial thrombosis.

The superiority of anti-FXa assay over anti-FIIa assay in detecting heparin-binding site antithrombin deficiency.

OBJECTIVES Antithrombin is a progressive inhibitor of active factor X (FXa) and thrombin (FIIa). Its effect is 500- to 1,000-fold accelerated by heparin or heparan sulfate. Heterozygous type I (quantitative) and most type II (qualitative) antithrombin deficiencies highly increase the risk of venous thromboembolism (VTE), while homozygous mutations are lethal. The functional defect affecting the heparin-binding site confers moderate risk of VTE to heterozygous and high risk of VTE to homozygous individuals. METHODS Antithrombin activity assays based on the inhibition of FIIa and FXa were compared for their efficiency in detecting heparin-binding site defects. RESULTS With a single exception, in heterozygotes for heparin-binding site defects (n = 20), anti-FIIa activities remained in the reference interval, while anti-FXa activities were uniformly decreased. In individuals who were homozygous for heparin-binding site mutations (n = 9), anti-FIIa activities were in the range of 48% to 80%; the range of anti-FXa activities was 9% to 25%. Anti-FIIa and anti-FXa activities in type I deficiencies and type II pleiotropic deficiency did not differ significantly. CONCLUSIONS Anti-FXa antithrombin assay is recommended as a first-line test to detect type II heparin-binding site antithrombin deficiency.

Management and outcome of pregnancies in women with antithrombin deficiency: a single-center experience and review of literature.

Women with antithrombin (AT) deficiency have an increased risk for pregnancy-associated venous thromboembolism (VTE) and adverse pregnancy outcome. AT deficiency is a rare thrombophilia with heterogeneous genetic background. Owing to the few cases reported in the literature, management strategies of pregnancy with AT deficiency are inconsistent. Our aim was to examine the type of the genetic defect, management, maternal, and pregnancy outcome in patients with hereditary AT deficiency. Five expectant mothers with AT deficiency were followed in our center to evaluate thrombotic events, and maternal and pregnancy outcomes. AT gene sequencing was performed in all cases, and levels of AT and anti-activated factor X were regularly measured to guide the risk-adopted anticoagulant prophylaxis. Three mothers had homozygous type II heparin-binding site mutations and two had heterozygous type I mutations of the gene encoding AT. Two women had additional factor V Leiden heterozygous mutations. Three maternal VTEs – four healthy newborns and five pregnancy losses – were observed. The risk of patients to VTE and adverse pregnancy outcome was found to associate with the homozygous type II heparin-binding site mutation of the AT gene. High risk of maternal VTE and frequent pregnancy complications were observed to associate with AT deficiency. Our results support the need of individualized, risk-adopted anticoagulant therapy in patients with AT deficiency.

Thrombin generation and low-molecular-weight heparin prophylaxis in pregnant women with thrombophilia

Pregnancy is associated with increased risk of venous thromboembolism, especially in the presence of thrombophilia. However, there is no consensus on the optimal approach for thromboprophylaxis in this population. Recent evidence suggests that thrombin generation correlates with the overall procoagulant state of the plasma. Our aim was to evaluate thrombin generation in a prospective cohort of thrombophilic pregnant women, and investigate the effectiveness of low-molecular-weight heparin (LMWH) prophylaxis in pregnancy. Women with severe (n=8), mild (n=47) and no (n=15) thrombophilia were followed throughout their pregnancies. Thrombin generation was evaluated in each trimester as well as five days and eight weeks postpartum (as a reference category). In women undergoing LMWH prophylaxis, thrombin generation and anti-Factor-Xa activity were measured just before and 4 hours after administration (peak effect). Thrombin generation was determined using Technothrombin TGA assay system. For the analysis, median peak thrombin and endogenous thrombin potential were used. Peak thrombin and endogenous thrombin potential were increased during pregnancy compared to the non-pregnant state with the highest results in the severe thrombophilia group. In women receiving LMWH prophylaxis a decrease was observed in thrombin generation at peak effect but over the progression of pregnancy the extent of this decrease reduced in a stepwise fashion. Our results show that thrombin generation demonstrates the hypercoagulable state in thrombophilic pregnancies. In addition, we found the effect of LMWH prophylaxis to progressively decrease with advancing stages of pregnancy.

Progressive chromogenic anti-factor Xa assay and its use in the classification of antithrombin deficiencies.

Abstract Background: Antithrombin (AT) is a slow-acting progressive inhibitor of activated clotting factors, particularly thrombin and activated factor X (FXa). However, the presence of heparin or heparan sulfate accelerates its effect by several magnitudes. AT deficiency, a severe thrombophilia, is classified as type I (quantitative) and type II (qualitative) deficiency. In the latter case mutations may influence the reactive site, the heparin binding-site (HBS) and exert pleiotropic effect. Heterozygous type II-HBS deficiency is a less severe thrombophilia than other heterozygous subtypes. However, as opposed to other subtypes, it also exists in homozygous form which represents a very high risk of venous thromboembolism. Methods: A modified anti-FXa chromogenic AT assay was developed which determines both the progressive (p) and the heparin cofactor (hc) activities, in parallel. The method was evaluated and reference intervals were established. The usefulness of the assay in detecting type II-HBS AT deficiency was tested on 78 AT deficient patients including 51 type II-HBS heterozygotes and 18 homozygotes. Results: Both p-anti-FXa and hc-anti-FXa assays showed excellent reproducibility and were not influenced by high concentrations of triglyceride, bilirubin and hemoglobin. Reference intervals for p-anti-FXa and hc-anti-FXa AT activities were 84%–117% and 81%–117%, respectively. Type II-HBS deficient patients demonstrated low (heterozygotes) or very low (homozygotes) hc-anti-FXa activity with normal or slightly decreased p-anti-FXa activity. The p/hc ratio clearly distinguished wild type controls, type II-HBS heterozygotes and homozygotes. Conclusions: Concomitant determination of p-anti-FXa and hc-anti-FXa activities provides a reliable, clinically important diagnosis of type II-HBS AT deficiency and distinguishes between homozygotes and heterozygotes.

Antithrombin Debrecen (p.Leu205Pro) – Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency

INTRODUCTION Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. MATERIALS AND METHODS Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. RESULTS Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans‑Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. CONCLUSIONS AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

Clinical and laboratory characteristics of antithrombin deficiencies: A large cohort study from a single diagnostic center

INTRODUCTION Inherited antithrombin (AT) deficiency is a heterogeneous disease. Due to low prevalence, only a few studies are available concerning genotype-phenotype associations. The aim was to describe the clinical, laboratory and genetic characteristics of AT deficiency in a large cohort including children and to add further laboratory data on the different sensitivity of functional AT assays. PATIENTS AND METHODS Non-related AT deficient patients (n=156) and their family members (total n=246) were recruited. Clinical and laboratory data were collected, the mutation spectrum of SERPINC1 was described. Three different AT functional assays were explored. RESULTS Thirty-one SERPINC1 mutations including 11 novel ones and high mutation detection rate (98%) were detected. Heparin binding site deficiency (type IIHBS) was the most frequent (75.6%) including AT Budapest3 (ATBp3), AT Padua I and AT Basel (86%, 9% and 4% of type IIHBS, respectively). Clinical and laboratory phenotypes of IIHBS were heterogeneous and dependent on the specific mutation. Arterial thrombosis and pregnancy complications were the most frequent in AT Basel and AT Padua I, respectively. Median age at the time of thrombosis was the lowest in ATBp3 homozygotes. The functional assay with high heparin concentration and pH7.4 as assay conditions had low (44%) sensitivity for ATBp3 and it was insensitive for AT Basel and Padua I. CONCLUSION Type IIHBS deficiencies behave differently in clinical and laboratory phenotypes from each other and from other AT deficiencies. Heparin concentration and pH seem to be the key factors influencing the sensitivity of AT functional assays to IIHBS.

[Treatment of acute myeloid leukemia -- a single center experience (2007-2013)].

INTRODUCTION Mortality of acute myeloid leukemia is still 60-70% in young (<60 years) adults and 90% in elderly (≥60 years) patients. AIM The aim of the authors was to analyse the outcome of treatment in their patients with acute myeloid leukemia. METHOD From 2007 to 2013, 173 patients with acute myeloid leukemia were treated. Patients were classified according to the European LeukemiaNet prognostic guideline. Association between mortality and the type of acute myeloid leukemia (secondary or primary), dose of daunoblastin at induction of treatment, and the rate of minimal residual disease were investigated. RESULTS The 5-year survival probability was 25% in young adults and 2% in the elderly. The survival was significantly influenced by these prognostic factors. The 5-year survival rate was 50% in the young, favorable prognostic group. The 90 mg/m2 daunoblastin dose was found to be beneficial. Addition of bortezomib to the standard induction protocol had an additional beneficial effect. CONCLUSIONS The speed and depth of the response to induction therapy, and the initial white blood cell count had an apparent effect on survival.

Heveny myeloid leukaemiás betegeink kezelésével szerzett tapasztalataink (2007–2013)@@@Treatment of acute myeloid leukemia – a single center experience (2007–2013)

INTRODUCTION Mortality of acute myeloid leukemia is still 60-70% in young (<60 years) adults and 90% in elderly (≥60 years) patients. AIM The aim of the authors was to analyse the outcome of treatment in their patients with acute myeloid leukemia. METHOD From 2007 to 2013, 173 patients with acute myeloid leukemia were treated. Patients were classified according to the European LeukemiaNet prognostic guideline. Association between mortality and the type of acute myeloid leukemia (secondary or primary), dose of daunoblastin at induction of treatment, and the rate of minimal residual disease were investigated. RESULTS The 5-year survival probability was 25% in young adults and 2% in the elderly. The survival was significantly influenced by these prognostic factors. The 5-year survival rate was 50% in the young, favorable prognostic group. The 90 mg/m2 daunoblastin dose was found to be beneficial. Addition of bortezomib to the standard induction protocol had an additional beneficial effect. CONCLUSIONS The speed and depth of the response to induction therapy, and the initial white blood cell count had an apparent effect on survival.

Heveny myeloid leukaemiás betegeink kezelésével szerzett tapasztalataink (2007-2013)

INTRODUCTION Mortality of acute myeloid leukemia is still 60-70% in young (<60 years) adults and 90% in elderly (≥60 years) patients. AIM The aim of the authors was to analyse the outcome of treatment in their patients with acute myeloid leukemia. METHOD From 2007 to 2013, 173 patients with acute myeloid leukemia were treated. Patients were classified according to the European LeukemiaNet prognostic guideline. Association between mortality and the type of acute myeloid leukemia (secondary or primary), dose of daunoblastin at induction of treatment, and the rate of minimal residual disease were investigated. RESULTS The 5-year survival probability was 25% in young adults and 2% in the elderly. The survival was significantly influenced by these prognostic factors. The 5-year survival rate was 50% in the young, favorable prognostic group. The 90 mg/m2 daunoblastin dose was found to be beneficial. Addition of bortezomib to the standard induction protocol had an additional beneficial effect. CONCLUSIONS The speed and depth of the response to induction therapy, and the initial white blood cell count had an apparent effect on survival.