Symptomatic type 1 protein C deficiency caused by a de novo Ser270Leu mutation in the catalytic domain

Abstract

Summary. Heterozygosity for a C8524T transition in the protein C gene converting Ser270(TCG) to Leu(TTG) in the protease domain was identified in a family with venous thrombosis. The mutation was associated with parallel reduction in plasma levels of protein C anticoagulant activity and protein C antigen, which is consistent with a type 1 deficiency. Transient expression of mutant protein C cDNA in human kidney 293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein compared with wild-type protein was reduced by at least 97% while the intracellular content of mutant and wild-type protein was similar. Northern blot analysis of total mRNA from transfected cells showed no reduction of the mutant protein C mRNA compared with wild-type protein C mRNA. Collectively, these results indicate that the Ser270Leu mutation in the affected family caused the plasma protein C deficiency and are consistent with a disease mechanism that involves synthesis of mutant protein followed by intracellular degradation before its secretion into the extracellular space. The mutation was not present in the parents of the proband, suggesting a de novo mutation. Non-paternity was excluded after the analysis of three intragenic protein C polymorphisms and six dinucleotide repeat allele sets located in five different chromosomes.

Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport

“…In vitro studies of various mutations in the PROC gene have provided insight into how these mutations can cause PC deficiency [7-15,20,28]. Most of these studies revealed that the PC deficiency was due to impaired secretion caused by intracellular degradation of the mutated proteins [8,10,11,15,20,28]. Enhanced proteolytic degradation of mutated proteins is a common molecular pathological mechanism in many diseases [30].…”

“…Results from these studies indicated that mutated PC variants were secreted inefficiently from transfected cells compared to wild-type (WT) PC [7-15]. Some of the studies also demonstrated that the intracellular levels of the mutated PC were decreased compared to WT PC, suggesting increased intracellular degradation of the mutated PC to be a dominant pathway behind the impaired secretion [8,10,11,15]. …”

Hereditary protein C deficiency caused by compound heterozygous mutants in two independent Chinese families

“…16 Cell culture, transfection and phenotype assays COS-7 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ mL penicillin G and 100 mg/mL streptomycin in a 5% CO 2 atmosphere at 378C. 16 Cell culture, transfection and phenotype assays COS-7 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ mL penicillin G and 100 mg/mL streptomycin in a 5% CO 2 atmosphere at 378C.…”

Protein C Deficiency caused by Homozygosity for a Novel PROC D180G Mutation – in vitro Expression and Structural Analysis of the Mutation

“…The Protein C activity and antigen concentration in plasma were analysed by Stachrom™ Protein C and Asserachrom™ Protein C, respectively (both Diagnostica Stago, Asnieres-Sur-Seine, France). Protein C antigen in cell lysate and media were analysed by ELISA using polyclonal antibodies as described (18,19). Determination of antithrombin, activated protein C resistance and total protein S in plasma was performed using COAMATIC ® Antithrombin (Chromogenix, Mölndal, Sweden), COATEST ® APC™ Resistance V (Chromogenix) and Assera™-plate protein S (Diagnostica Stago), respectively.…”

“…Isolation of genomic DNA from peripheral blood cells, analysis of the protein C gene and assay of the Arg506Gln Leiden mutation in the coagulation factor V gene and the G20210A transition in the prothrombin gene was performed as described (19).…”