Whole-Exome Sequencing in Evaluation of Thrombophilia: Characterization of Novel Genetic Variants

Abstract

Abstract Venous thromboembolism (VTE), defined as deep venous thrombosis and pulmonary embolism, is a cause of significant morbidity and mortality worldwide, with an overall incidence of about 10,000,000 cases per year. The majority of VTE is attributable to genetic factors, yet the five major heritable thrombophilias of factor V Leiden, prothrombin gene mutation, antithrombin deficiency, protein C deficiency, and protein S deficiency comprise only a minority of VTE cases, suggesting that further genetic factors must contribute to VTE risk. We performed whole-exome sequencing (WES) in 106 patients with VTE, focusing our analysis on a 55-gene extended thrombophilia panel comprising genes previously reported in next-generation sequencing studies to be associated with VTE. In our cohort, we identified a probable disease-causing genetic variant or variant of unknown significance (VUS) in approximately 60% of study patients. We then selected several novel VUS involving genes with known roles in hemostasis for further characterization to determine their biological and pathological significance in thrombophilia. Multiple sequence alignment analysis showed that several nonsynonymous variants produced amino acid substitutions in highly conserved regions of proteins relevant to hemostasis and thrombosis, including thrombomodulin (TM), heparin cofactor II, ADAMTS13, and von Willebrand factor (vWF). To evaluate the theoretical effects of selected VUS on thrombosis susceptibility, we visualized proteins using PyMol and performed in silico mutagenesis modeling to evaluate for structural alterations that can predict functional and physiological consequences. Our analyses showed that several variants caused conformational changes predicted to either result in a deleterious effect on enzymatic activity or prevent substrate binding to the active site. For example, variants in TM (P401L and V454A) are predicted to decrease thrombin binding and protein C activation, whereas a C668R variant in the spacer domain of ADAMTS13 is predicted to prevent binding to vWF, resulting in ultra-large vWF multimers. We plan to use these results in combination with clinical laboratory-based coagulation testing and family studies to determine pathogenicity of specific variants in thrombosis and thrombophilia. Overall, these findings indicate that WES is a potentially useful tool for inherited thrombophilia and can provide insight into the genetic factors contributing to VTE.