PB1077 - A novel C238G, PROC mutation in patients with thromboembolism, causes intracellular protein retention

Background: A novel single mutation in exon 8 of PROC in three Thai pediatric patients who presented with thromboembolic events was reported [1]. The mutation analysis demonstrated a substitution of cysteine with glycine at amino acid position 238 (C238G) in a prepro-protein sequence. Although the point of mutation in PROC was identified, the detailed mechanisms on how this novel mutation results in severe protein C (PC) deficiency have not been studied.

Aims: To elucidate how a novel mutation C238G of PC results in a severe PC deficiency in children with thromboembolisms.

Methods: Recombinant wild-type (WT-PC) and C238G-PC were generated in HEK293T by transient transfection. PC in culture supernatant was determined by Western blot (WB) analysis, and the PC antigen level was quantitatively measured by ELISA. In addition, intracellular localization of PC was visualized by an immunofluorescence microscopy.

Results: Transient transfection was successfully done as demonstrated by qRT-PCR (Fig 1A). Analysis of culture supernatant by WB demonstrated the absence of C238G-PC compared with WT-PC and R189W-PC (the other mutation on PC) (Fig 1B). Quantitative analysis of culture supernatant by ELISA showed a significant reduction of C238G-PC antigen compared with WT-PC (37.80 ± 1.53 vs 906.67 ± 10.14, p = 0.0001) (Fig 1C). However, WB analysis of cell lysate revealed a similar level of C238G-PC compared with WT-PC and R189W-PC (Fig 1D). The immunofluorescence microscopy showed that C196G-PC was retained in ER (Fig 2A).

Conclusion(s): C238G mutation of PC causes a protein retention in the ER, ultimately leading to impaired PC secretion. The result from this in vitro analysis was compatible with the phenotypes of Thai pediatric patients carrying this mutation who presented with thromboembolic phenomenon.