Next-Generation Sequencing and In Vitro Expression Study of ADAMTS13 Single Nucleotide Variants in Deep Vein Thrombosis.
BACKGROUND
Deep vein thrombosis (DVT) genetic predisposition is partially known.


OBJECTIVE
This 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.


METHODS
Wild-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.


RESULTS
In 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).


CONCLUSIONS
Three 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.
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
Maria Teresa
MT
Pagliari
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
Luca A
LA
Lotta
Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.
Hugoline G
HG
de Haan
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
Carla
C
Valsecchi
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
Gloria
G
Casoli
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
Silvia
S
Pontiggia
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
Ida
I
Martinelli
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
Serena M
SM
Passamonti
Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.
Frits R
FR
Rosendaal
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.|Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy.
Flora
http://orcid.org/0000-0001-7423-9864
F
Peyvandi
D000071120:ADAMTS13 Protein / Q000032:analysis / Q000235:genetics*; D016022:Case-Control Studies; D005260:Female; D020022:Genetic Predisposition to Disease; D005838:Genotype; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide; D020246:Venous Thrombosis / Q000235:genetics*
D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human