Molecular basis of inherited protein C deficiency results from genetic variations in the signal peptide and propeptide regions

• Published in: Journal of thrombosis and haemostasis 2023-11, Vol.21 (11), p.3124-3137
• Main Authors: Cao, Qing, Hao, Zhenyu, Li, Cheng, Chen, Xuejie, Gao, Meng, Jiang, Nan, Liu, Hongli, Shen, Yan, Yang, Haiping, Zhang, Shujuan, Yang, Aiying, Li, Weikai, Tie, Jian-Ke, Shen, Guomin
• Format: Article
• Language: English
• Subjects: aberrant pre-mRNA splicing; Humans; inherited protein C deficiency (PCD); missense mutation; Mutation; Mutation, Missense; pathogenic mechanism; Protein C Deficiency; Protein Sorting Signals - genetics; RNA Precursors - genetics; RNA Precursors - metabolism; RNA Splicing; RNA, Messenger - genetics; signal peptide and propeptide; thrombophilia
• ISSN: 1538-7836; 1538-7933; 1538-7836
• DOI: 10.1016/j.jtha.2023.06.021

Inherited protein C deficiency (PCD) caused by mutations in protein C (PC) gene (PROC) increases the risk of thrombosis. Missense mutations in PC’s signal peptide and propeptide have been reported in patients with PCD, but their pathogenic mechanisms, except mutations in R42 residue, remain unclear. To investigate the pathogenic mechanisms of inherited PCD caused by 11 naturally occurring missense mutations in PC’s signal peptide and propeptide. Using cell-based assays, we evaluated the impact of these mutations on various aspects such as activities and antigens of secreted PC, intracellular PC expression, subcellular localization of a reporter protein, and propeptide cleavage. Additionally, we investigated their effect on pre-messenger RNA (pre-mRNA) splicing using a minigene splicing assay. Our data revealed that certain missense mutations (L9P, R32C, R40C, R38W, and R42C) disrupted PC secretion by impeding cotranslational translocation to the endoplasmic reticulum or causing endoplasmic reticulum retention. Additionally, some mutations (R38W and R42L/H/S) resulted in abnormal propeptide cleavage. However, a few missense mutations (Q3P, W14G, and V26M) did not account for PCD. Using a minigene splicing assay, we observed that several variations (c.8A>C, c.76G>A, c.94C>T, and c.112C>T) increased the incidence of aberrant pre-mRNA splicing. Our findings suggest that variations in PC’s signal peptide and propeptide have varying effects on the biological process of PC, including posttranscriptional pre-mRNA splicing, translation, and posttranslational processing. Additionally, a variation could affect the biological process of PC at multiple levels. Except for W14G, our results provide a clear understanding of the relationship between PROC genotype and inherited PCD. •The mechanisms of protein C (PC) deficiency caused by mutations in PC’s signal peptide and propeptide remain unclear.•We systemically studied the pathogenic mechanisms of these mutations using cell-based assays.•These variations have varying effects on the biological process of PC to cause PC deficiency.•Understanding the mechanisms helps to evaluate pathogenic risk and inheritance patterns.