Two nonsense mutations cause protein C deficiency by nonsense-mediated mRNA decay

• Published in: Thrombosis research Vol. 135; no. 4; pp. 733 - 738
• Main Authors: Luan, Chun-jie, Shen, Wei, Yu, Zhen, Chen, Lei, Gu, Yi, Tang, Lin-yun, Wang, Zhu-gang, Dai, Letian, Gu, Ming-min
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 01.04.2015
• Subjects: Codon, Nonsense - immunology; Hematology, Oncology and Palliative Medicine; Humans; Mutation; Nonsense mutation; Nonsense-mediated mRNA decay (NMD); Premature termination codons (PTCs); PROC; Protein C deficiency; Protein C Deficiency - genetics; RNA, Messenger - genetics; RNA, Small Interfering - genetics; Transfection; PROC; Premature termination codons (PTCs); Nonsense-mediated mRNA decay (NMD); Protein C deficiency; Nonsense mutation
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/j.thromres.2015.01.022

Abstract Introduction Protein C deficiency is a genetic disorder caused by mutations in the protein C gene ( PROC ). More than 10% of nonsense and frameshift mutations carrying premature termination codons have been identified in PROC , but the exact molecular mechanisms of these mutations on the pathogenesis of protein C deficiency remain unclear. Objective The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) can be a mechanism accounting for protein C deficiency. Methods PROC of genomic DNA was amplified and sequenced. Recombinant plasmids expressing wild-type (wt) and mutant EGFP-protein C (EGFP-PC) cDNA were constructed and transiently transfected into human embryonic kidney cells using lipofectamine. Expression of mRNAs and proteins of EGFP-PC and NMD factor UPF1 were analyzed by qPCR and Western blot. Results DNA sequencing revealed a novel heterozygous nonsense mutation (p.Trp247*) in patient 1 and two compound heterozygous mutations (p.Phe181Val and p.Arg199*) in patient 2. Expression studies showed that cells transfected with the mutant plasmids expressed significantly lower levels of EGFP-PC mRNAs and proteins compared to cells transfected with the wt plasmid. A translation inhibitor cycloheximide and UPF1 small interfering RNA (UPF1 siRNA) significantly increased mRNA or protein expression of EGFP-PC in cells transfected with the mutant plasmids. Conclusion Two PROC nonsense mutations (p.Trp247* and p.Arg199*) trigger NMD, resulting in protein C deficiency.