| dc.description.abstract | Carbamyl phosphate synthetase I (CPSI) is the liver-specific enzyme that catalyzes the first and rate-determining step of the urea cycle. Mutations in the CPSI gene cause CPSI Deficiency (CPSID), an autosomal recessive disease characterized by hyperammonemia. During the course of this project, many patient mutations likely causing RNA instability were identified. These genomic variants were present in patient genomic DNA but not detectable in patient RNA, indicating RNA degradation perhaps due to nonsense mediated decay (NMD) or splicing errors. This large number of suspected RNA instability mutations suggests CPSID is a good model to understand the prevalence of this mutation type in genetic disease. However, the ability to study a heterogeneous set of mutations in such a large gene has suffered from the lack of an appropriate model system. A goal of this project was to develop a novel model system for testing the functional consequences of these potential RNA instability mutations. Using two homologous recombination strategies, a BAC clone containing the full CPSI gene was modified to contain the pEHG vector and the CMV promoter upstream of CPSI. These modifications allow for stable transfection, selection and ubiquitous expression in eukaryotic cells. Patient mutations were then incorporated into the resulting BECC model system and these constructs were subsequently tested to assay for splicing changes as well as NMD. These mutations included the intronic substitutions c.654-3T>G and c.1210-1G>T, the exonic c.1893T>G mutation which directly creates a nonsense codon, and c.2388C>A which is a translationally silent exonic mutation with unknown function. Extensive control assays show that the BECC model system properly expressed wild-type CPSI, while each mutant shows decreased expression as measured by quantitative RT/PCR assays. To determine if this decrease in expression was due to NMD, it was inhibited by siRNA mediated knockdown of UPF2. Following NMD inhibition, relative expression levels of each mutant were increased, indicating these mutations cause degradation via the NMD pathway. In addition, cryptic splice sites activated by intronic mutations were identified. This project is significant because using CPSID as a model, it shows the prevalence of RNA instability mutations in a severe metabolic disease and provides a new model system to test any mutation, irrespective of type or location, for functional consequences including the activation of aberrant splicing and nonsense-mediated decay. | |
