Next-Generation Sequencing and In Vitro Expression Study of ADAMTS13 Single Nucleotide Variants in Deep Vein Thrombosis |
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| Authors: | Maria Teresa Pagliari Luca A Lotta Hugoline G de Haan Carla Valsecchi Gloria Casoli Silvia Pontiggia Ida Martinelli Serena M Passamonti Frits R Rosendaal Flora Peyvandi |
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| Institution: | 1Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca'' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy;2Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands;3Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy;National Cerebral and Cardiovascular Center, JAPAN |
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| Abstract: | BackgroundDeep vein thrombosis (DVT) genetic predisposition is partially known.ObjectivesThis study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies.MethodsWild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity of recombinant ADAMTS13 were measured by ELISA and FRETS-VWF73 assays, respectively. The replication studies were performed in an Italian case-control study (Milan study; 298/298 patients/controls) using a next-generation sequencing approach and in a Dutch case-control study (MEGA study; 4306/4887 patients/controls) by TaqMan assays.ResultsIn vitro results showed reduced ADAMTS13 activity for three SNVs (p.Val154Ile 15%; 95% confidence interval CI] 14–16], p.Asp187His 19%; 95%CI] 17–21], p.Arg421Cys 24%; 95%CI] 22–26]) similar to reduced plasma ADAMTS13 levels of patients carriers for these SNVs. Therefore these three SNVs were interrogated for risk association. The first replication study identified 3 heterozygous carriers (2 cases, 1 control) of p.Arg421Cys (odds ratio OR] 2, 95%CI] 0.18–22.25). The second replication study identified 2 heterozygous carriers (1 case, 1 control) of p.Asp187His (OR] 1.14, 95%CI] 0.07–18.15) and 10 heterozygous carriers (4 cases, 6 controls) of p.Arg421Cys (OR] 0.76, 95%CI] 0.21–2.68).ConclusionsThree SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced ex vivo and in vitro ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT. |
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