Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled. Our extended thrombophilia panel identified a probable disease-causing genetic variant or variant of unknown significance in 39 of 64 study patients (60.9%), compared with 6 of 237 control patients without VTE (2.5%) ( < .0001). Clinical laboratory-based thrombophilia testing identified a heritable thrombophilia in only 14 of 54 study patients (25.9%). The majority of WES variants were either associated with thrombosis based on prior reports in the literature or predicted to affect protein structure based on protein modeling performed as part of this study. Variants were found in major thrombophilia genes, various genes, and highly conserved areas of other genes with established or potential roles in coagulation or fibrinolysis. Ten patients (15.6%) had>1 variant. Sanger sequencing performed in family members of 4 study patients with and without VTE showed generally concordant results with thrombotic history. WES and extended thrombophilia testing are promising tools for improving our understanding of VTE pathogenesis and identifying inherited thrombophilias.
© F e r r a t a S t o r t i F o u n d a t i o nTTP. Sixty-eight patients were tested during the acute phase, with 48 patients experiencing their first acute episode and 20 a relapse. For 18 of these patients, corresponding samples in clinical remission were also available. Ten patients were analyzed only during remission, giving a total of 28 patients tested in remission. The patients' demographics and clinical features are summarized in Table 1. The inclusion criteria for patients with acute acquired TTP were: presence of severe ADAMTS13 deficiency (<10%), thrombocytopenia (platelet count <150x10 9 /L), microangiopathic hemolytic anemia (hemoglobin <12 g/dL) with presence of schistocytes on the peripheral blood smear, and elevated lactate dehydrogenase levels (>450 IU/L). Fever, neurological symptoms or renal failure were not mandatory. Remission was defined as a normal platelet count (>150x10 9 /L) and no plasma exchange treatment for ≥30 consecutive days. Relapse was defined as the reappearance of clinical manifestation and/or laboratory data compatible with TTP after remission.Frozen citrated plasma samples were obtained from four international centers. The study was approved by the ethic committees of the University Hospital of Berne, Switzerland; Medical University of Vienna, Austria; Lille University Hospital, France; and Icahn School of Medicine, New York, USA. ADAMTS13 assaysADAMTS13 activity (ADAMTS13:Ac) and ADAMTS13 functional inhibitor titers were measured using fluorometric FRETS-VWF73 assay as described elsewhere. 24,25 The limit of quantification of ADAMTS13:Ac was 0.05 U/mL (5%); values <0.10 U/mL (<10%) were considered severely reduced; levels of 0.10-0.50 U/mL as reduced and levels >0.5 U/mL as normal. An inhibitor titer <0.7 BU/mL was considered negative. Plasma ADAMTS13 antigen (ADAMTS13:Ag) levels were determined by ELISA as described previously. 20 The reference range of the assay was 403 -907 ng/mL; the limit of quantification was 10 ng/mL. Levels of 100 -403 ng/mL were considered reduced, levels <100 ng/mL as severely reduced, and values <10 ng/mL as undetectable. Detection of free anti-ADAMTS13 antibodies by enzyme-linked immunosorbent assayFree antibodies were detected as described elsewhere 25 with modifications (Online Supplementary Methods). Detection of ADAMTS13-specific immune complexesMicrotiter plates were coated with a polyclonal rabbit antihuman ADAMTS13 antibody. After blocking the non-specific sites, diluted patient's plasma samples and controls were added and incubated overnight at 4°C. The next day, the immunoglobulin fraction of the bound immune complexes was detected with horseradish peroxidase-conjugated class-specific secondary antibodies followed by detection with an appropriate chromogenic substrate. For details, see Online Supplementary Methods and Online Supplementary Figure S1.An ELISA to detect total IgG-immune complexes was not established, however, the total amount of IgG-immune complexes was investigated in selected patients by co-immunoprecipitation,...
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