Next-generation sequencing and in vitro expression study of ADAMTS13 single nucleotide variants in deep vein thrombosis

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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Abstract

Background: Deep vein thrombosis (DVT) genetic predisposition is partially known. Objectives: 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.

Original languageEnglish
Article numbere0165665
JournalPLoS One
Volume11
Issue number11
DOIs
Publication statusPublished - Nov 1 2016

Fingerprint

thrombosis
Venous Thrombosis
confidence interval
Nucleotides
nucleotides
Confidence Intervals
odds ratio
Odds Ratio
case-control studies
Association reactions
Case-Control Studies
Assays
HEK293 Cells
assays
Genetic Predisposition to Disease
In Vitro Techniques
Enzyme-Linked Immunosorbent Assay
enzyme-linked immunosorbent assay
antigens
Antigens

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Next-generation sequencing and in vitro expression study of ADAMTS13 single nucleotide variants in deep vein thrombosis. / Pagliari, Maria Teresa; Lotta, Luca A.; De Haan, Hugoline G.; Valsecchi, Carla; Casoli, Gloria; Pontiggia, Silvia; Martinelli, Ida; Passamonti, Serena M.; Rosendaal, Frits R.; Peyvandi, Flora.

In: PLoS One, Vol. 11, No. 11, e0165665, 01.11.2016.

Research output: Contribution to journalArticle

Pagliari, Maria Teresa ; Lotta, Luca A. ; De Haan, Hugoline G. ; Valsecchi, Carla ; Casoli, Gloria ; Pontiggia, Silvia ; Martinelli, Ida ; Passamonti, Serena M. ; Rosendaal, Frits R. ; Peyvandi, Flora. / Next-generation sequencing and in vitro expression study of ADAMTS13 single nucleotide variants in deep vein thrombosis. In: PLoS One. 2016 ; Vol. 11, No. 11.
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abstract = "Background: Deep vein thrombosis (DVT) genetic predisposition is partially known. Objectives: 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.",
author = "Pagliari, {Maria Teresa} and Lotta, {Luca A.} and {De Haan}, {Hugoline G.} and Carla Valsecchi and Gloria Casoli and Silvia Pontiggia and Ida Martinelli and Passamonti, {Serena M.} and Rosendaal, {Frits R.} and Flora Peyvandi",
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AU - Pagliari, Maria Teresa

AU - Lotta, Luca A.

AU - De Haan, Hugoline G.

AU - Valsecchi, Carla

AU - Casoli, Gloria

AU - Pontiggia, Silvia

AU - Martinelli, Ida

AU - Passamonti, Serena M.

AU - Rosendaal, Frits R.

AU - Peyvandi, Flora

PY - 2016/11/1

Y1 - 2016/11/1

N2 - Background: Deep vein thrombosis (DVT) genetic predisposition is partially known. Objectives: 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.

AB - Background: Deep vein thrombosis (DVT) genetic predisposition is partially known. Objectives: 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.

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