Assessing the functional characteristics of synonymous and nonsynonymous mutation candidates by use of large DNA constructs.

As we identify more and more genetic changes, either through mutation studies or population screens, we need powerful tools to study their potential molecular effects. With these tools, we can begin to understand the contributions of genetic variations to the wide range of human phenotypes. We used our catalogue of molecular changes in patients with carbamyl phosphate synthetase I (CPSI) deficiency to develop such a system for use in eukaryotic cells. We developed the tools and methods for rapidly modifying bacterial artificial chromosomes (BACs) for eukaryotic episomal replication, marker expression, and selection and then applied this protocol to a BAC containing the entire CPSI gene. Although this CPSI BAC construct was suitable for studying nonsynonymous mutations, potential splicing defects, and promoter variations, our focus was on studying potential splicing and RNA-processing defects to validate this system. In this article, we describe the construction of this system and subsequently examine the mechanism of four putative splicing mutations in patients deficient in CPSI. Using this model, we also demonstrate the reversible role of nonsense-mediated decay in all four mutations, using small interfering RNA knockdown of hUPF2. Furthermore, we were able to locate cryptic splicing sites for the two intronic mutations. This BAC-based system permits expression studies in the absence of patient RNA or tissues with relevant gene expression and provides experimental flexibility not available in genomic DNA or plasmid constructs. Our splicing and RNA degradation data demonstrate the advantages of using whole-gene constructs to study the effects of sequence variation on gene expression and function.

The frequent observation of evidence for nonsense-mediated decay in RNA from patients with carbamyl phosphate synthetase I deficiency.

CPSI deficiency is an inborn error of metabolism caused by mutations in the first, rate-determining enzyme of the urea cycle. Our mutation detection data from this disorder suggest that a significant number of mutant alleles cause RNA instability, most likely through the nonsense-mediated decay pathway. We identified 26 non-consanguinous CPSID patients with an available RNA source (liver tissue or cell line) and screened both genomic DNA and RNA for the identification and classification of mutations. Out of 52 total alleles screened from these patients, 21 (40%) have strong evidence for RNA processing mutations demonstrated by absent/minimal heterozygosity in patient cDNA sequences despite heterozygous genomic changes. These 21 alleles are a heterogenous group primarily composed of splicing defects and frameshifts that form premature termination codons which should subsequently elicit the nonsense-mediated decay pathway. This study provides evidence for the high prevalence of RNA instability mutations in genetic disease and underscores the importance of accounting for them in mutation-screening strategies.

Characterization of genomic structure and polymorphisms in the human carbamyl phosphate synthetase I gene.

Human carbamyl phosphate synthetase I (CPSI) is an essential hepatic enzyme that initiates the urea cycle. Deficiency of this enzyme usually results in lethal hyperammonemia. CPSI is encoded by the CPSI gene located on chromosome 2q35. In the present study, we report the coding sequence and define the intron-exon structure of the human CPSI gene. These data are compared to the previously defined rat CPSI gene structure. This work was generated from direct sequence determination of human genomic DNA (35 introns) and comparison to public domain sequence of anonymous BACs (2 introns). The human CPSI gene spans >120kb of genomic DNA. CPSI has 38 exons and 37 introns, and all adhere to the consensus splicing sequences. Comparison of the human and rat CPSI genes reveals that the nucleotide sequences, amino acid sequences, and intron-exon organizations are highly similar. We report the primers and conditions for screening the human CPSI exonic and bordering intronic sequences. We also screened 100 individuals for polymorphisms in the human CPSI gene and identified 14 polymorphisms in the CPSI message. The knowledge of the CPSI gene structure and the 14 polymorphisms presented in this study will greatly facilitate future molecular studies involving the CPSI gene and the enzyme it encodes.