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. 2017 Jun 29;1(16):1224-1237.
doi: 10.1182/bloodadvances.2017005249. eCollection 2017 Jul 11.

Whole-exome sequencing in evaluation of patients with venous thromboembolism

Eun-Ju Lee  1 , Daniel J Dykas  2 , Andrew D Leavitt  3   4 , Rodney M Camire  5 , Eduard Ebberink  6 , Pablo García de Frutos  7 , Kavitha Gnanasambandan  8 , Sean X Gu  9 , James A Huntington  10 , Steven R Lentz  9 , Koen Mertens  6 , Christopher R Parish  11 , Alireza R Rezaie  12 , Peter P Sayeski  8 , Caroline Cromwell  13 , Noffar Bar  14 , Stephanie Halene  14 , Natalia Neparidze  14 , Terri L Parker  14 , Adrienne J Burns  15 , Anne Dumont  15 , Xiaopan Yao  16 , Cassius Iyad Ochoa Chaar  17 , Jean M Connors  18 , Allen E Bale  2 , Alfred Ian Lee  14
Affiliations

Whole-exome sequencing in evaluation of patients with venous thromboembolism

Eun-Ju Lee et al. Blood Adv. .

Abstract

Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled. Our extended thrombophilia panel identified a probable disease-causing genetic variant or variant of unknown significance in 39 of 64 study patients (60.9%), compared with 6 of 237 control patients without VTE (2.5%) (P < .0001). Clinical laboratory-based thrombophilia testing identified a heritable thrombophilia in only 14 of 54 study patients (25.9%). The majority of WES variants were either associated with thrombosis based on prior reports in the literature or predicted to affect protein structure based on protein modeling performed as part of this study. Variants were found in major thrombophilia genes, various SERPIN genes, and highly conserved areas of other genes with established or potential roles in coagulation or fibrinolysis. Ten patients (15.6%) had >1 variant. Sanger sequencing performed in family members of 4 study patients with and without VTE showed generally concordant results with thrombotic history. WES and extended thrombophilia testing are promising tools for improving our understanding of VTE pathogenesis and identifying inherited thrombophilias.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Extended thrombophilia panel. For each gene, the standard gene abbreviation is followed in parentheses by the common name.
Figure 2.
Figure 2.
Summary of WES and clinical laboratory-based thrombophilia testing.
Figure 3.
Figure 3.
Variants in combination. Each line represents variants identified on extended thrombophilia testing from an individual patient.
Figure 4.
Figure 4.
Representative structure of the protein Z–dependent protease inhibitor (SERPINA10) showing the 3-dimensional orientation of residue Q384 (in red) and the reactive center loop (RCL, in pink). The protein model was created using PyMOL (PDB codes: 3H5C for native and 4AFX for RCL-inserted forms). (A) The native protein. (B) Binding of a target protease to the RCL results in cleavage and insertion of the RCL into the central β-sheet.
Figure 5.
Figure 5.
Structure of the HABP2 protein and locations of C533, E393, and the active site residues. The protein model of HABP2 was constructed using MODELER 9v7, with the crystal structure of the homologous hepatocyte growth factor activator (PDB code: 1YC0) as a template. (A) The active site of wild-type HABP2 contains a catalytic triad of 3 residues (E411, H362, and S509, shown in blue and red). (B) C533 forms a cysteine bridge with C505, adjacent to G534 (the site of the Marburg I polymorphism, G534E). These 3 residues are located on the same functionally important surface loop, near the active site residue S509 and the N terminus of the protease domain. The C533F mutation is predicted to break this cysteine bridge, destabilizing these interactions and presumably reducing protein activity. (C) E393 interacts with nearby lysine residues K416 and K418, located on the same β-strand as D411 (part of the catalytic triad). The Marburg II polymorphism (E393Q) is predicted to disrupt this interaction.
Figure 6.
Figure 6.
Characterization of the THBD P401L mutation. (A) Schematic representation of thrombomodulin epidermal growth factor-like domains 4 and 5 (EGF4, EGF5). The P401 (blue), M406 (red), and C390 and C404 (yellow) residues are highlighted. Disulfide bonds are shown by yellow lines. P401 is located in the C-loop of EGF4 at the turn of a β-hairpin motif, near a critical oxidation-sensitive M406 amino acid in the linker region between EGF4 and EGF5 essential for normal thrombomodulin function. The P401L mutation is predicted to disrupt the C-loop β-turn and destabilize a disulfide bond between C390 and C404. (B) Structure of thrombomodulin EGF-like domains 4, 5, and 6 in complex with thrombin. The crystal structure was downloaded from the RCSB PDB database and visualized by PyMOL (PBD ID: 1DX5). Amino acids of interest in the EGF4 C-loop are highlighted.
Figure 7.
Figure 7.
Projected changes in JAK2 protein due to R1063H. Models were created by homology modeling on the SWISS-MODEL server and visualized using PyMOL (PDB 2B7A). (A) The JAK2-JH1 domain, with the catalytic loop highlighted in cyan, the αC loop in yellow, and phosphotyrosines within the activation loop in green. (B) In the wild-type protein, R1063 forms a salt bridge with E1060, which is exposed on the surface of the JH1 domain. (C) In the R1063H mutant protein, substitution of the charged arginine to a polar histidine results in the loss of the native salt bridge with El 060.
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