Exon 2 skipping eliminates γ-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency
| Autor: | Kevin W. Hallgren, Kathleen L. Berkner, Haitao Zhang, Mark A. Rishavy, Kurt W. Runge |
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| Rok vydání: | 2018 |
| Předmět: |
Gene isoform
RNA Splicing Mutant 030204 cardiovascular system & hematology medicine.disease_cause Article Factor IX 03 medical and health sciences Exon 0302 clinical medicine Blood Coagulation Disorders Inherited medicine Humans Genetic Predisposition to Disease Blood Coagulation Gene Editing Mutation Chemistry Homozygote RNA Hematology Exons Pyruvate carboxylase HEK293 Cells Phenotype Biochemistry Carbon-Carbon Ligases RNA splicing Protein carboxylation CRISPR-Cas Systems Protein Processing Post-Translational |
| Zdroj: | J Thromb Haemost |
| ISSN: | 1538-7836 |
| Popis: | Essentials A carboxylase mutation that impairs splicing to delete exon 2 sequences was previously reported. We found that the mutant was inactive for vitamin K-dependent (VKD) protein carboxylation. An incomplete splicing defect likely accounts for VKD clotting activity observed in the patient. The results indicate the importance of proper carboxylase embedment in the membrane for function. Background Mutations in the γ-glutamyl carboxylase (GGCX), which is required for vitamin K-dependent (VKD) protein activation, can result in vitamin K clotting factor deficiency (VKCFD1). A recent report described a VKCFD1 patient with a homozygous carboxylase mutation that altered splicing and deleted exon 2 (Δ2GGCX). Only Δ2GGCX RNA was observed in the patient. Objectives Loss of exon 2 results in the deletion of carboxylase sequences thought to be important for membrane topology and consequent function. Carboxylase activity is required for life, and we therefore tested whether the Δ2GGCX mutant is active. Methods HEK 293 cells were edited by the use of CRISPR-Cas9 to eliminate endogenous carboxylase. Recombinant wild-type GGCX and recombinant Δ2GGCX were then expressed and tested for carboxylation of the VKD protein factor IX. A second approach was used to monitor carboxylation biochemically, using recombinant carboxylases expressed in insect cells that lack endogenous carboxylase. Results and conclusions Δ2GGCX activity was undetectable in both assays, which is strikingly different from the low levels of carboxylase activity observed with other VKCFD1 mutants. The similarity in clotting function between patients with Δ2GGCX and these mutations must therefore arise from a novel mechanism. Low levels of properly spliced carboxylase RNA that produce full-length protein would not have been observed in the previous study. The results suggest that the splicing defect is incomplete. Δ2GGCX RNA has been detected in normal human liver, and has been designated carboxylase isoform 2; however, Δ2GGCX protein was not observed in normal human liver. The lack of activity and protein expression suggest that isoform 2 is not physiologically relevant to normal VKD protein carboxylation. |
| Databáze: | OpenAIRE |
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