Quantile-Quantile (QQ) plot of p-value distributions for A. common and low-frequency variants (MAF > 1%) and B. rare variants (MAF ≤ 1%).

<p>An arrow indicates the rs2232710 variant.</p

Association results of the 18 variants identified in the NGS and replicated in up to 585 idiopathic DVT patients and 550 healthy controls from the DVT-Milan study.

<p>Analysis was performed using chi-squared test.</p

Replication analysis of the rs2232710 (<i>SERPINA10/ZPI</i> gene) single nucleotide variant in patients with idiopathic deep vein thrombosis of lower extremities and heathy controls belonging to three independent cohorts: DVT-Milan, LETS and MEGA.

<p>Analysis was performed using chi-squared test.</p

Next-Generation Sequencing and <i>In Vitro</i> Expression Study of <i>ADAMTS13</i> Single Nucleotide Variants in Deep Vein Thrombosis

<div><p>Background</p><p>Deep vein thrombosis (DVT) genetic predisposition is partially known.</p><p>Objectives</p><p>This study aimed at assessing the functional impact of nine <i>ADAMTS13</i> 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.</p><p>Methods</p><p>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.</p><p>Results</p><p><i>In vitro</i> 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).</p><p>Conclusions</p><p>Three SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced <i>ex vivo</i> and <i>in vitro</i> ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT.</p></div

<i>In vitro</i> expression studies results.

<p><i>In vitro</i> expression studies results.</p

Western blot of WT and mutant p.V154I, p.D187H and p.R421C recombinant ADAMTS13 expressed in HEK293 cells.

<p>WT and mutant recombinant proteins were detected in the conditioned media (A) and cell lysates (B). Cellular alpha-tubulin was used as control to verify equal total protein loading and detected using anti-alpha-tubulin monoclonal antibody (bottom). (M) Marker. The amount of each mutant rADAMTS13 contained in cell lysates was normalized using the respective band of alpha-tubulin (loading control), quantified by densitometry analysis and referred to the WT taken as 100%.</p

Western blot of WT and mutant p.Y603C recombinant ADAMTS13 expressed in HEK293 cells.

<p>WT and mutant recombinant protein were detected in the conditioned media (left) and cell lysates (right). Cellular alpha-tubulin was used as control to verify equal total protein loading and detected using anti-alpha-tubulin monoclonal antibody (bottom). (M) Marker; C-, medium and lysate of untransfected cells used as a negative control. The amount of each mutant rADAMTS13 contained in cell lysates was normalized using the respective band of alpha-tubulin (loading control), quantified by densitometry analysis and referred to the WT taken as 100%.</p

Rare previously identified <i>ADAMTS13</i> SNVs associated with DVT.

<p>Rare previously identified <i>ADAMTS13</i> SNVs associated with DVT.</p

Characteristics of genotyped single nucleotide variants (SNVs) in Italian and Dutch populations.

<p>Characteristics of genotyped single nucleotide variants (SNVs) in Italian and Dutch populations.</p