A novel heterozygous missense ADAMTS13 mutation (G3368A) in a patient diagnosed with thrombotic thrombocytopenic purpura due to reduced ADAMTS13 activity and low titer of anti-ADAMTS 13 antibody

CALDERAZZO JC; KEMPFER AC; POWAZNIAK Y; SÁNCHEZ LUCEROS A; WOODS AI; PAIVA-PALOMINO J; LAZZARI MA

Amsterdam

Congreso; XXIV Congress of the International Society on Thrombosis and Haemostasis; 2013

International Society on Thrombosis and Haemostasis

Background: Thrombotic thrombocytopenic purpura (TTP) is characterized by microvascular thrombosis, associated with deficiency of ADAMTS13, induced by mutations in the gene and autoantibodies. Aims: In order to explain the phenotype of the ADAMTS13 deficiency, it was decided to explore the mechanistic effect of a mutation found by means of expression studies in mammalian cells. Methods: Patient with a history of TTP episodes that began at 13 years of age. Several plasma samples were measured for ADAMTS13 activity (ac) and antigen (ag), IgG anti-ADAMTS13 antibody and VWF multimers. The wild type (WT) and the ADAMTS13 mutant (site directed mutagenesis) construct were transiently expressed in HEK 293 cells. Culture medium and cell lysate were evaluated by ELISA kit for ADAMTS13 ac and ag levels. Results: In the plasma of patient were detected: ag lower than 0.5% (nv= 70-160%), ac= 0.6 (nv = 40-130%), 26% ULVWF multimers (normal value, nv lower than 15%) and IgG anti-ADAMTS13 antibody = 19 SD3 U/mL (nv lower than 15 U/mL). The mean SD of ag, ac and IgG anti-ADAMTS13 antibody from three samples of the patient was informed. To exclude the possibility that the novel mutation (in heterozygous state) found in our patient is not a disease-related polymorphism, we screened 50 healthy donors. The in vitro expression of the ADAMTS13 mutant led to a defect of secretion (ag =7.5, ac= 17) of protease, causing intracellular accumulation (ag = 116, ac= 83). The cotransfection of the mixtures of the mutant and the WT revealed a secretion defect of the protease (ag= 86, ac= 69). All samples were compared toWT (considered 100%). Conclusions: Mutagenesis suggests that the novel mutation G3368A (in TPS1-8 domain, exon 25) is critical for ADAMTS13 secretion. The patient had low antibody titers to ADAMTS13 although the levels of ac and ag always were lower than 1.2%. The ADAMTS13 deficiency was probably caused by a combination of the G3368A mutation (in heterozygous state) and IgG anti-ADAMTS13 antibodies, although it is unclear why the latter were present. Young Travel Award