Targeted Gene Sequencing Identifies Variants in the Protein C and Endothelial Protein C Receptor Genes in Patients With Unprovoked Venous Thromboembolism

Abstract

Objective The interaction of protein C (PC) with the endothelial protein C receptor (EPCR) enhances activated PC (APC) generation. We performed targeted gene sequencing of the PC gene (PROC) and EPCR genes (PROCR) in patients with unprovoked venous thromboembolism (VTE) to determine if mutations that impair PC-EPCR interactions are associated with an increased risk of VTE. Approach and Results We sequenced exon 3 of PROC and exons 2 and 3 of PROCR (the exons that encode the protein-protein binding domains of PC and EPCR) in 653 patients with unprovoked VTE and in 627 healthy controls. Five single nucleotide variants (SNVs), each in individual patients, were identified that result in abnormal PC (Arg9Cys, Val34Met, Arg-1Cys) or abnormal EPCR proteins (Arg96Cys and Val170Leu). None of these SNVs were found in the controls. When the PC variants were expressed in human embryonic kidney (HEK) 293 cells, all exhibited decreased synthesis, and two of the variants had reduced capacity for APC generation. When expressed on the surface of HEK293 cells, the EPCR variants showed reduced affinity for fluorescently-labeled PC. In addition, the previously reported EPCR A3 haplotype, which promotes cellular “shedding” of EPCR, is overrepresented in the patient group (P=0.001). Conclusions This is the first targeted DNA sequencing analysis of PROC and PROCR in a large group of patients with unprovoked VTE. Our data suggest that mutations that impair PC-EPCR interactions may be associated with an increased risk of VTE.

Thrombophilic Gene Mutations in Relation to Different Manifestations of Venous Thromboembolism: A Single Tertiary Center Study

“…1 Factor V Leiden is the most frequent and well-studied genetic cause of VTE, followed by the PTG mutation. [10][11][12][13] The prevalence of FVL carriage is 5% among Caucasians. This condition was documented in 20% of patients with DVT and approximately 50% of patients with familial thrombophilia.…”

Multilocus Genetic Risk Scores for Venous Thromboembolism Risk Assessment

“…These findings emphasize the need to pay special attention to low‐frequency or rare variants rendering high ORs. In a recent study, the benefits of an in‐depth sequencing strategy were emphasized because it permitted the identification of a rare mutation responsible for familial cases of early‐onset VTE . The important predisposing role played by low‐frequency or rare variants in VTE was highlighted in a study based on multigenerational data by Zöller et al, who observed a high familial risk of VTE in a small number of siblings, suggesting segregation of rare but strong genetic risk factors.…”

Cross Talk Pathways Between Coagulation and Inflammation

“…Its generation relies on cleavage by thrombin, catalyzed by endothelial anchored cofactors, TM (≈1000-fold), and the endothelial protein C receptor (EPCR; ≈20-fold). 49,50 Deficiency of PC and variants of EPCR augment the risk of deep vein thrombosis and thromboembolism 51,52 and augment the response to inflammatory stimuli. There are multiple mechanisms by which the PC-TM-EPCR system modulates inflammation, innate immunity, and tissue repair ( Figure 2).…”