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Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
Two nonsense mutations cause protein C deficiency by nonsense-mediated mRNA decay
Chun-jie Luan1, Wei Shen2, Zhen Yu1
1Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
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.
Insights
Nonsense mutations in the protein C gene (PROC) can cause protein C deficiency by triggering nonsense-mediated mRNA decay (NMD). This study confirms NMD as a key mechanism in PROC deficiency pathogenesis.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Protein C deficiency is a genetic disorder linked to mutations in the protein C gene (PROC).
- Over 10% of PROC mutations involve nonsense or frameshift mutations leading to premature termination codons.
- The precise molecular mechanisms underlying these mutations in protein C deficiency pathogenesis are not fully understood.
Purpose of the Study:
- To investigate the role of nonsense-mediated mRNA decay (NMD) as a mechanism contributing to protein C deficiency.
- To elucidate how specific PROC mutations impact mRNA and protein levels through NMD.
Main Methods:
- Genomic DNA sequencing of the PROC gene to identify mutations.
- Construction of recombinant plasmids expressing wild-type (wt) and mutant EGFP-protein C (EGFP-PC) cDNA.
- Transfection of human embryonic kidney cells with plasmids and analysis of EGFP-PC and UPF1 mRNA and protein expression via qPCR and Western blot.
Main Results:
- Identification of novel heterozygous nonsense mutations (p.Trp247*) and compound heterozygous mutations (p.Phe181Val and p.Arg199*) in PROC.
- Cells transfected with mutant EGFP-PC plasmids showed significantly reduced mRNA and protein levels compared to wild-type.
- Inhibition of translation (cycloheximide) or UPF1 (siRNA) increased EGFP-PC mRNA and protein expression in cells with mutant plasmids.
Conclusions:
- Specific PROC nonsense mutations (p.Trp247* and p.Arg199*) activate the NMD pathway.
- Nonsense-mediated mRNA decay is a significant mechanism leading to protein C deficiency.
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