SBIR-STTR Award

Urea Cycle Disorders (UCDs) are metabolic diseases disrupting ureagenesis. Unequivocal and rapid diagnosis of UCDs is difficult because hyperammonemia, the primary biochemical phenotype, is not specific to UCDs being observed in broad categories of metabolic deficiencies including fatty acid oxidation defects, lactic acidosis, and organic acidemias. Clinical presentation includes poor feeding, vomiting, and lethargy symptoms leading to misdiagnosis as sepsis. Differential diagnosis of individual urea cycle enzyme defects is complex. The urea cycle contains 6 core genes: N-acetylglutamate synthetase, carbamyl phosphate synthetase, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase, and arginase. OTC deficiency is the most frequently observed UCD and molecular genetic analysis is an established tool for diagnosis. Dye-Binding/High-Resolution Thermal Denaturation (DB/HRTD) is a chemistry that rapidly assesses a PCR product for sequence aberration and was first applied using a single sample instrument the HR-1. A prototype instrument, the LightScanner, allows DB/HRTD to be performed in 96 or 384 well plates enabling concurrent melting and analysis of all samples in a plate. Herein, it is proposed to demonstrate DB/HRTD as a simple means to rapidly assess the 6 core genes of the urea cycle. Specific Aim 1 seeks to validate a preliminary gene-scanning assay for the OTC gene. Specific Aim 2 proposes to design, develop, and provide preliminary validation of a gene-scanning assay for the N-acetylglutamate synthetase gene, defects in which cause NAGS deficiency. Specific Aim 3 proposes to use in situ analysis to unequivocally identify common polymorphisms by means of unlabeled probe chemistry within the context of gene scanning analysis. Specific Aim 4 will demonstrate gene scanning reagents (buffer, dye, dNTPs, oligonucleotides, enzyme) are stabilized by freeze drying and following re-suspension perform in a manner comparable to freshly prepared reagents. Rapid diagnosis of UCD is critical to patient survival. Genetic analysis for urea cycle defects is not widely available yet its utility as a component of the diagnostic regimen is recognized and documented. Stable freeze-dried reagents support rapid genotyping, to strengthen biochemical and clinical evidence in diagnosis of candidate patients. A plate-based system is compatible with block thermalcyclers and existing systems for automated plate loading

Diseases involving the urea cycle are clinically manifest by symptoms diverse as neonatal hyperammonemic coma to postpartum psychosis. Hyperammonemia is the primary phenotype of urea cycle defects but differentiating individual gene deficiencies within the urea cycle involves a complex series of biochemical tests. Products of the following genes are required for ureagenesis: N-acetylglutamate synthetase carbamyl phosphate synthetase 1 , ornithine transcarbamylase , argininosuccinate synthetase , argininosuccinate lyase , arginase, mitochondrial ornithine transporter and mitochondrial glutamate/aspartate transporter. Gene- based analysis is an established part of the diagnostic regimen for urea cycle defects but availability of testing is limited. Using 2 innovative technologies, melt profiling and freeze-dried preservation of PCR reaction mixtures, a simplified means to assess genes of the urea cycle is developed. Mutations in urea cycle gene are rare or private, necessitating comprehensive gene analysis, which is a complex and labor-intensive process. PCR reagents (buffer, MgCl2, primers, LCGreen dye, taq polymerase) to fully analyze urea cycle genes are freeze-dried into plates. Using these reagents requires they be resuspended with water containing the DNA sample being evaluated which bypasses the painstaking, error prone, and costly process of formulating and distributing the numerous reactions required for comprehensive gene analysis. The reagents are designed for PCR using a common condition. After amplification and without any post-PCR manipulation, the plate is analyzed by high resolution melt profiling. Melt profiling identifies regions containing sequence variants such that DNA sequence analysis is selectively targeted. As melt profiling is non-destructive to the amplification product, the product identified as producing an aberrant melting profile is recovered to serve as DNA sequencing template. Assay panels are prepared for the 8 genes of the urea cycle. Analyzing the genes of the urea cycle has been the purview of specialized reference labs and research protocols. Combining freeze-dried reagents and melt profiling will enable this complex analysis to be performed by any molecular pathology laboratory. As urea cycle deficiencies can be rapidly fatal in the neonatal period, fast turn around time is critical to patient survival and the proposed assay panels will expedite diagnosis of affected patients. These assay panels will have application to research assessing the cause of hyperammonemia observed in common disease states such as organ transplant or liver fibrosis/cirrhosis. Newborn screening by mass spectrometry identifies metabolites suggesting urea cycle defects and these assays can assess these patients. Rapidly identifying urea cycle defects is critical to patient survival when neonatal hyperammonemia is observed and the proposed assay panels will facilitate diagnosis. Hyperammonemia is observed in several common disease states (hepatitis, liver fibrosis or cirrhosis, organ or bone marrow transplant patients, patients undergoing chemotherapy, patients receiving valproic acid, and patients who for various reasons are catabolic) and gene of the urea cycle must be suspect as a means by which this is manifest. The proposed assay panels provide a means to readily assess gene that are well established to contribute to hyperammonemia.

Thesaurus Terms:
Ammonia, Diagnosis Design /Evaluation, Enzyme Deficiency, Genetic Disorder Diagnosis, Genotype, Inborn Metabolism Disorder, Metabolism Disorder Diagnosis, Molecular Genetics, Rapid Diagnosis, Urea Cycle Carbamoyl Phosphate Synthetase Deficiency, Lyophilization, Molecular Pathology, Orphan Disease /Drug Clinical Research, Genetic Technique, Mass Spectrometry, Polymerase Chain Reaction