Genotype Analysis for Diagnosis of Urea Cycle Disorders

Information

  • Research Project
  • 7159437
  • ApplicationId
    7159437
  • Core Project Number
    R44DK069106
  • Full Project Number
    2R44DK069106-02
  • Serial Number
    69106
  • FOA Number
    PA-06-06
  • Sub Project Id
  • Project Start Date
    7/1/2004 - 20 years ago
  • Project End Date
    8/31/2008 - 16 years ago
  • Program Officer Name
    MCKEON, CATHERINE T
  • Budget Start Date
    9/1/2006 - 18 years ago
  • Budget End Date
    8/31/2007 - 17 years ago
  • Fiscal Year
    2006
  • Support Year
    2
  • Suffix
  • Award Notice Date
    8/31/2006 - 18 years ago
Organizations

Genotype Analysis for Diagnosis of Urea Cycle Disorders

[unreadable] DESCRIPTION (provided by applicant): 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, MgCl-2, 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. [unreadable] [unreadable] [unreadable]

IC Name
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
  • Activity
    R44
  • Administering IC
    DK
  • Application Type
    2
  • Direct Cost Amount
  • Indirect Cost Amount
  • Total Cost
    375000
  • Sub Project Total Cost
  • ARRA Funded
  • CFDA Code
    847
  • Ed Inst. Type
  • Funding ICs
    NIDDK:375000\
  • Funding Mechanism
  • Study Section
    ZRG1
  • Study Section Name
    Special Emphasis Panel
  • Organization Name
    IDAHO TECHNOLOGY
  • Organization Department
  • Organization DUNS
  • Organization City
    SALT LAKE CITY
  • Organization State
    UT
  • Organization Country
    UNITED STATES
  • Organization Zip Code
    84108
  • Organization District
    UNITED STATES