Molecular and clinical characterization of two independent Chinese families with protein C deficiency

This study aims to investigate the clinical characterization and molecular pathogenic basis of hereditary protein C (PC) deficiency in two independent Chinese families, and conduct in vitro expression studies on the newly discovered p.Trp444Arg mutation. The PC activity (PC: A) was tested using the chromogenic substrate, and PC antigen (PC: Ag) was detected via enzyme-linked immunosorbent assay (ELISA). To identify the mutation sites, nine exons of the PROC gene were amplified by PCR, and the products were directly sequenced. The conservation and pathogenicity of the mutations, as well as changes in the spatial structure of PC proteins before and after mutations, were analyzed using ClustalX-2.1-win, online bioinformatics software, and PyMOL. The function of the mutant proteins was detected using the thrombin generation assay (TGA). Recombinant PC was ectopically expressed in HEK293T cells, with mRNA levels quantified by RT-qPCR. The recombinant protein was further characterized using Western blotting, ELISA, and immunofluorescence microscopy. Proband A and B, aged 39 and 63 respectively, are both diagnosed with deep vein thrombosis (DVT) in both lower limbs and pulmonary embolism (PE). Two missense mutations, p.Arg440Cys and p.Trp444Arg, were identified in the probands. Bioinformatics and protein modeling analyses revealed that the two mutations probably affected the normal function of PC. The thrombin generation assay revealed impaired thrombin generation capacity in both probands, with proband B showing more severe impairment. In vitro expression experiments demonstrated that p.Trp444Arg do not significantly affect mRNA expression levels of PC protein compared to wild-type, but result in lower PC: Ag content and protein expression in the supernatant and higher levels in the lysate. These two mutations may be the causes of reduced PC in two independent Chinese families. Notably, this is the first reported instance of the p.Trp444Arg mutation.