Targeted gene sequencing identifies variants in the protein C and endothelial protein C receptor genes in patients with unprovoked venous thromboembolism.

OBJECTIVE The interaction of protein C (PC) with the endothelial PC receptor (EPCR) enhances activated PC generation. We performed targeted gene sequencing of the PC gene (PROC) and EPCR genes (PROCR) in patients with unprovoked venous thromboembolism (VTE) to determine whether 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, each in individual patients, were identified that result in abnormal PC (Arg9Cys, Val34Met, and Arg-1Cys) or abnormal EPCR proteins (Arg96Cys and Val170Leu). We did not detect any nonsynonymous coding variants in the controls. When the PC variants were expressed in human embryonic kidney 293 cells, all exhibited decreased synthesis, and 2 of the variants had reduced capacity for activated PC generation. When expressed on the surface of human embryonic kidney 293 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 over-represented 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.