[Analysis of two cases of hereditary protein C deficiency causing venous thrombosis]

Objective: To investigate the molecular pathogenic mechanism of venous thrombosis caused by heterozygous missense mutations in two protein C (PROC) genes through laboratory phenotype analysis, genetic mutation analysis, and in vitro expression experiments. Methods: Two probands presented with venous thromboembolism at the First Affiliated Hospital of Wenzhou Medical University. Clinical data and blood samples were collected from the probands and their family members to evaluate the plasma protein C (PC) activity (PC∶A), PC antigen (PC∶Ag) levels, and other relevant coagulation parameters. The anticoagulant capacity was assessed using the thrombin generation test (TGT). The mutation sites of the PROC gene were identified using direct DNA sequencing. Bioinformatics software was used to analyze the conservation and pathogenicity of the mutated gene. PyMOL software was used for the analysis of the protein three-dimensional models and interactions between mutated amino acids. Wild-type and two mutant expression vectors were constructed and HEK293T cells were transiently transfected. Total cellular RNA was extracted from positively transfected cells to investigate the transcriptional levels of the mutant PROC gene. Enzyme-linked immunosorbent assay, Western blot, and cellular immunofluorescence assays were used to investigate the translation levels of the mutant PROC protein. Results: Probands 1 and 2 exhibited PC∶A levels of 35% and 40% and PC∶Ag levels of 44% and 39%, with increasing D-dimer levels to 4.42 mg/L and 0.83 mg/L, respectively. Meanwhile, other coagulation parameters revealed no significant abnormalities. TGT demonstrated impaired anticoagulant function in both proband witnesses and their familial PC carriers. Sequencing analysis revealed heterozygous missense mutations c. 833T>C (p. Leu278Pro) in proband 1 and c. 1330T>C (p. Trp444Arg) in proband 2 within exon 9 of the PROC gene. Conservation analysis revealed that Leu278 and Trp444 were highly conserved across homologous species. Pathogenicity analysis indicated that both p. Leu278Pro and p. Trp444Arg mutations are deleterious. Protein modeling analysis demonstrated that both mutations induce structural alterations in the protein. In vitro expression experiments revealed that compared with the wild-type, both p. Leu278Pro and p. Trp444Arg mutations showed no significant differences in the mRNA expression level of the PC protein. However, both mutations caused significantly lower PC∶Ag content and protein expression levels in the cell culture supernatant compared with the wild-type, whereas higher levels were observed in the cell culture lysate. This indicates the association of both mutations with the secretion function of the PC protein. Conclusion: The heterozygous missense mutations p. Leu278Pro and p. Trp444Arg in exon 9 of the PROC gene in both probands are associated with decreased PC levels.