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Antithrombin deficiency and pediatric thrombosis
known risk factor for pediatric thrombosis. A meta-analy- sis of these studies and a recent nation-wide survey showed that children with first-onset VTE were more likely to have severe inherited thrombophilia, like defi- ciencies of natural anticoagulants (antithrombin, protein C and protein S), than controls.5,6
Antithrombin deficiency, an autosomal dominant disor- der, was the first thrombophilia to be described 50 years ago and so far is associated with the highest risk of throm- bosis.7 The key hemostatic role of this anticoagulant serine protease inhibitor (serpin) explains why heterozygous mutations in SERPINC1, the gene encoding for antithrom- bin, significantly increase the risk of VTE (OR: 20-40)8 and why the complete absence of antithrombin causes embry- onic lethality in mice.9 However, there is a significant clin- ical variability among patients with antithrombin defi- ciency. Patients with quantitative type I deficiency, where the genetic defect disturbs the production or secretion of the variant protein, have a higher incidence of thrombosis compared to patients with qualitative type II deficiency, where the genetic defect allows the production of a vari- ant antithrombin with impaired anticoagulant activity.10,11 Three different subgroups of type II deficiency can be dis- tinguished: Reactive Site (RS), when the binding of the substrate to the reactive site is affected; Heparin Binding Site (HBS) when the heparin binding domain is altered; and Pleiotropic Effect (PE), with both effects on the pro- tein.10 Homozygotes have only been described for type II deficiency.8 Age is an additional risk factor for patients with antithrombin deficiency, as up to 60% of patients develop a thrombotic event before the age of 65.12 In con- trast to adults, less data are available for young subjects as pediatric studies on antithrombin deficiency are mainly restricted to case reports or small patient cohorts, due to the rarity of the disorder.13-16
The objective of this study was to investigate the preva- lence and clinical characteristics of pediatric thrombosis in a large cohort of subjects with inherited antithrombin deficiency recruited in two countries.
Methods
Ethics
This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Hospital Universitario Reina Sofia (8/2013). Written informed consent was provided.
Patients
During a period of 21 years in Spain (from 1996 to 2017) and 27 years in Belgium (from January 1990 to December 2017), two ref- erence centers for antithrombin deficiency recruited 441 index patients. Initial diagnosis could have been made in another center but was always confirmed by measurements of antithrombin activity (anti-FXa activity <80%) and genetic analysis. In 206 of the index patients, family studies were performed and 527 first and second degree affected relatives were identified and enrolled in the study, generating a final cohort of 968 patients with antithrombin deficiency.
The patient's history was evaluated to record for thrombotic events and possible provoking risk factors such as oral contracep- tives, pregnancy, complicated delivery, obesity, immobilization, infection, surgery, and trauma. Information about antithrombotic therapy and family history of thromboembolism was also collect-
ed. Results from additional thrombophilic parameters (protein C activity, free protein S antigen, resistance to activated protein C, Factor V Leiden and prothrombin G20210A mutation) were col- lected when available. Thrombotic events were objectively diag- nosed by experienced radiologists through established imaging procedures such as Doppler-ultrasonography, computed or mag- netic resonance tomography for venous thrombosis and spiral computed pulmonary angiography or lung perfusion scintigraphy for pulmonary embolism.
Definitions
Pediatric thrombosis was defined as any objectively diagnosed thrombotic event during childhood (≤ 18 years). Pediatric patients were divided into age groups according to the proposed World Health Organization (WHO) classification: neonates from birth to 30 days, infants from one month up to 2 years, children from 2 up to 12 years, and adolescents from 12 to 18 years.17
Genetic analysis
Genetic analysis was performed in every patient with reduced antithrombin anti-FXa activity. Genetic variants in SERPINC1 were identified by sequencing the 7 exons and flanking regions. Gross rearrangements were assessed by multiplex ligation-depen- dent probe amplification using the SALSA MLPA Kit P227 SerpinC1 (MRC-Holland). Mutations were described following the Human Genome Variation Society Guidelines (http://varnomen.hgvs.org/recommendations/). The GenBank NM_000488.3 cDNA sequence was used as reference sequence. Where available, HGMD accession numbers were mentioned.
Biochemical and functional characterization
Antithrombin anti-FXa activity was determined in citrated plas- ma by chromogenic methods following the manufacturer's instructions (HemosIL Antithrombin, Werfen, Barcelona, Spain and Innovance Antithrombin, Siemens, Marburg, Germany). Antigen levels were measured by rocket immunoelectrophoresis and/or ELISA.
Analysis of plasma antithrombin forms included crossed immu- noelectrophoresis and polyacrylamide gel electrophoresis.
The reported results were performed in samples collected long after the acute event and in absence of any anticoagulant treat- ment. For the neonatal patients, reported results were performed after the first six months of life, except in one patient who died as a consequence of the thrombotic event (Online Supplementary Table S1).
Statistical analysis
Continuous variables were expressed as means and standard deviations and categorical data as counts and percentages. Relative risks and 95% confidence intervals (CI) were calculated using pre- viously published formulas.18 The significance of differences in continuous variables was tested by Mann-Withney test. Kaplan- Meier survival curves were used to illustrate the difference in thrombosis-free survival among different groups. P<0.05 was con- sidered statistically significant. Statistical and graphical analysis were performed with GraphPad Prism version 7.03 (GraphPad Software, San Diego CA, USA) and SPSS, version 21 (Chicago, IL, USA).
Results
Seventy-three patients (37 from Spain and 36 from Belgium) out of 968 subjects with congenital antithrombin deficiency developed a first thrombotic event before the age of 19 (Table 1 and Figure 1) corresponding to a fre-
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