title: Two nonsense mutations cause protein C deficiency by nonsense-mediated mRNA decay.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  Two PROC nonsense mutations (p.Trp247* and p.Arg199*) trigger NMD, resulting in protein C deficiency.
authors: Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Chun-jie
authors: CJ
authors: Luan
authors: Department of Laboratory Medicine, Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.
authors: Wei
authors: W
authors: Shen
authors: Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Zhen
authors: Z
authors: Yu
authors: Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
authors: Lei
authors: L
authors: Chen
authors: Department of Laboratory Medicine, Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.
authors: Yi
authors: Y
authors: Gu
authors: State Key Laboratory of Medical Genomics, Research Centre for Experimental Medicine, Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors: Lin-yun
authors: LY
authors: Tang
authors: Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; State Key Laboratory of Medical Genomics, Research Centre for Experimental Medicine, Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors: Zhu-gang
authors: ZG
authors: Wang
authors: Centre for Haemostasis and Thrombosis, Haemophilia Reference Centre, St Thomas Hospital, London SE1 7EH, UK.
authors: Letian
authors: L
authors: Dai
authors: Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. Electronic address: gumm@sjtu.edu.cn.
authors: Ming-min
authors: MM
authors: Gu
mesh_terms: D018389:Codon, Nonsense / Q000276:immunology*; D006801:Humans; D009154:Mutation; D020151:Protein C Deficiency / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D018389:Codon, Nonsense; D012333:RNA, Messenger; D034741:RNA, Small Interfering
keywords: Nonsense mutation; Nonsense-mediated mRNA decay (NMD); PROC; Premature termination codons (PTCs); Protein C deficiency