Functional Analysis of Two Haplotypes of the Human Endothelial Protein C Receptor Gene

Abstract

Objective-To confirm the effect of the endothelial protein receptor gene (PROCR) haplotypes H1 and H3 on venous thromboembolism (VTE), to study their effect on endothelial protein C receptor (EPCR) expression in human umbilical vein endothelial cells, and to investigate the functionality of H1 tagging single-nucleotide polymorphisms in an in vitro model. Approach and Results-Protein C (PC), activated PC, and soluble EPCR (sEPCR) levels were measured in 702 patients with VTE and 518 healthy individuals. All subjects were genotyped for PROCR H1 and H3. Human umbilical vein endothelial cells isolated from 111 umbilical cords were used to study the relation between PROCR haplotypes, PROCR mRNA, cellular distribution of EPCR, and rate of PC activation. Finally, the functionality of the intragenic PROCR H1 singlenucleotide polymorphisms was analyzed using a luciferase-based method. We confirmed that individuals carrying H1 have reduced VTE risk, increased plasma activated PC levels, and reduced plasma sEPCR levels and that individuals with the H3H3 genotype have an increased VTE risk and increased plasma sEPCR levels. In cultured human umbilical vein endothelial cells, H1 is associated with increased membrane-bound EPCR, increased rate of PC activation, and reduced sEPCR in conditioned medium, but does not significantly influence PROCR mRNA levels. In contrast, H3 is associated with reduced membrane-bound EPCR and increased sEPCR in human umbilical vein endothelial cell-conditioned medium, higher levels of a truncated mRNA isoform, and a lower rate of PC activation. Finally, we identified the g.2132T>C singlenucleotide polymorphism in intron 1 as an intragenic H1-specific functional single-nucleotide polymorphism. Conclusions-These Medina et al Functional Analysis of PROCR Haplotypes 685H3 is tagged by the minor allele of g.4600A>G (Ser219Gly; rs867186) and has been associated with the risk of venous [8][9][10][11][12][13][14][15]18,19 and arterial thrombosis, [19][20][21][22][23] but with contradictory results. The presence of H3 results in increased plasma sEPCR levels, [8][9][10][11][12][13]18,20,22,24 which is largely explained by a Ser219Gly substitution, which renders EPCR more susceptible to cleavage by metalloproteinases such as tumor necrosis factor-α converting enzyme/ADAM17. 7 Another mechanism that could link H3 to high plasma levels of sEPCR is its association with a truncated form of PROCR mRNA lacking the transmembrane and intracellular domains. 25 Recently, PROCR H3 has also been found to be associated with increased plasma levels of PC. 20,26Therefore, we aimed to verify the effects of H1 and H3 on VTE risk in a case-control study to investigate their effects on EPCR expression in human umbilical vein endothelial cells (HUVECs) and to identify the functional SNP that mediates the protection of H1 against VTE using luciferase constructs.In the present study, we confirm previous reports on a protective effect of PROCR H1 against VTE 8,[11][12][13][14][15] and show, for the first time, that it is prob...

Endothelial Cell Protein C Receptor Gene 6936a/G, 1651c/G, 4678g/C Polymorphisms and Soluble Endothelial Protein C Receptor Levels Impact on in Vitro Fertilization Outcomes

“…There was also a relation between low sEPCR levels and EPCR polymorphism (4678G/C) genotypes, though it was not significant. These results were in agreement with Medina et al who reported that individuals with EPCR polymorphism (4678G/C) have reduced risk for venous thrombosis, elevated plasma activated PC levels and reduced plasma sEPCR levels [19].…”

“…Similarly, Yin et al reported that frequencies of the EPCR gene 6936AG and 6936GG genotypes were higher in patients with venous thromboembolism than in healthy subjects [18]. Moreover, Medina et al reported that those with homozygous mutant EPCR (6936A/G) genotype have an increased risk for venous thrombosis and increased plasma sEPCR levels [19]. Furthermore, the current results presented higher frequencies of the (1651C/G) genotype and lower soluble EPCR levels in cases than in controls.…”

Genetic Ancestry–dependent Differences in Breast Cancer–induced Field Defects in the Tumor-adjacent Normal Breast

“…These observations raised the possibility that PROCR þ /ZEB1 þ cells are naturally present at higher levels in the normal breasts of AA women and that failure to consider natural variation in gene expression pattern, influenced at least partially by genetic ancestry, could have an impact on identifying cancer-induced field effect on the adjacent normal breast. Measuring PROCR itself in the breast tissue is complicated because there are 4 haplotypes of PROCR due to SNPs and only 1 among them is a cell surface protein (20). Because ZEB1 expression is enriched in PROCR þ /EpCAM À cells, we used ZEB1 as a surrogate marker for PROCR þ /EpCAM À cells in unmanipulated breast tissues.…”

Genetic characterization of antithrombin, protein C and protein S deficiencies in Polish patients

“…It is possible that other genes on chromosome 16q23 and 1q32 influence the PC and PS levels. 16,42,43 PC activity can be modulated also by the genetic variants of EPCR 44 and the polymorphisms of the genes involved in the vitamin K -dependent γ -carboxylation reaction may also influence the activities of PC and PS. 45 It should be also noted that for the measurement of AT activity, both the bovine thrombin-and the FXa inhibition -based tests, together with an antigen method, are advisable to be performed in patients with suspected type II AT deficiency.…”