Homozygous protein C deficiency with late onset venous thrombosis: identification and in vitro expression study of a novel Pro275Ser mutation.

AIMS

To identify the mutation and study the molecular mechanism of inherited protein C (PC) deficiency in a Chinese pedigree.

METHODS

The plasma levels of PC activity (PC:A) and antigen (PC:Ag) were measured by chromogenic assay and ELISA, respectively. The PROC gene was amplified and sequenced for mutational screening. Wild type and Pro275Ser mutant PC cDNA expression plasmids were constructed and transfected into HEK 293T cells and COS 7 cells, respectively. The expression and transcription of PC were investigated by ELISA, Western blot and real time RT-PCR. Immunofluorescence staining was utilised to analyse the intracellular distribution of PC, and pulse-chase experiments were used to detect the intracellular stability of the mutant PC.

RESULTS

The proband's plasma PC:A and PC:Ag were 5% and 13.9%, respectively. A missense mutation (p.Pro275Ser) was identified in exon 9 of PROC gene. In vitro expression study showed that Pro275Ser variant was present at 22.6% and 78.9% of wild type levels in culture supernatants and cell lysates, respectively. No significant differences in the molecular weights, mRNA levels or intracellular stability were observed between the mutant and wild type PC. Immunofluorescence staining revealed that the mutant protein was mainly located in the endoplasmic reticulum.

CONCLUSIONS

A homozygous Pro275Ser mutation was identified in a Chinese pedigree of PC deficiency. Impaired secretion of the mutant PC might be the molecular mechanism of PC deficiency caused by Pro275Ser mutation.