Genotype Analysis for Diagnosis of Urea Cycle Disorders

尿素循环障碍诊断的基因型分析

基本信息

批准号：
7282572
负责人：
Steven F Dobrowolski
金额：
$ 37.5万
依托单位：
BIOFIRE DEFENSE, LLC.
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2008-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7282572
关键词：
Affect Algorithms Alleles Amino-acid N-acetyltransferase Biochemical Biochemical Genetics Biological Assay Biological Preservation Bone Marrow Transplantation Buffers Bypass Carbamyl Phosphate Child Cirrhosis Clinical Research Clinical Services Collaborations Coma Complex Condition DNA DNA Sequence DNA Sequence Analysis Data Defect Diagnosis Diagnostic Disease Dyes Enzymes Evaluation Freeze Drying GLAST Protein Genes Genetic Polymorphism Genotype Hepatitis Hereditary Disease Hyperammonemia Idaho Individual Institution Laboratories Lead Ligase Liver Fibrosis Magnesium Chloride Mass Spectrum Analysis Medical Medical center Mitochondria Molecular Genetics Mutation Neonatal Neonatal Screening Newborn Infant Organ Organ Transplantation Ornithine Carbamoyltransferase Patients Personal Satisfaction Phenotype Polymerase Chain Reaction Postpartum Period Predisposition Process Protocols documentation Psychotic Disorders Rare Diseases Reaction Reagent Research Resolution Sampling Scanning Series Site Specificity Symptoms Taq Polymerase Technology Testing Time Today Transplant Recipients Treatment Protocols Validation Valproic Acid Variant Water arginase argininosuccinate lyase argininosuccinate synthase aspartate-glutamate carrier base chemotherapy design genetic analysis innovative technologies melting molecular pathology ornithine transporter tool urea cycle validation studies

项目摘要

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, 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.

描述（由申请人提供）：涉及尿素循环的疾病在临床上表现为新生儿高氨血症昏迷和产后精神病等多种症状。高氨血症是尿素循环缺陷的主要表型，但区分尿素循环内的个体基因缺陷涉及一系列复杂的生化测试。尿素生成需要以下基因的产物：N-乙酰谷氨酸合成酶氨甲酰磷酸合成酶 1 、鸟氨酸转氨甲酰酶、精氨琥珀酸合成酶、精氨琥珀酸裂合酶、精氨酸酶、线粒体鸟氨酸转运蛋白和线粒体谷氨酸/天冬氨酸转运蛋白。基于基因的分析是尿素循环缺陷诊断方案的既定部分，但测试的可用性有限。利用 PCR 反应混合物的熔解分析和冻干保存这两种创新技术，开发了一种评估尿素循环基因的简化方法。尿素循环基因突变是罕见或私密的，需要进行全面的基因分析，这是一个复杂且劳动密集型的过程。用于全面分析尿素循环基因的 PCR 试剂（缓冲液、MgCl2、引物、LCGreen 染料、taq 聚合酶）被冷冻干燥到平板中。使用这些试剂需要用含有待评估 DNA 样本的水重新悬浮，从而绕过了制定和分配综合基因分析所需的众多反应的艰苦、容易出错且成本高昂的过程。这些试剂设计用于使用常见条件的 PCR。扩增后且无需任何 PCR 后操作，即可通过高分辨率熔解图对板进行分析。熔解图谱可识别含有序列变异的区域，从而选择性地靶向 DNA 序列分析。由于熔解图谱对扩增产物没有破坏性，因此回收被鉴定为产生异常熔解图谱的产物以用作 DNA 测序模板。针对尿素循环的 8 个基因准备了检测组。分析尿素循环的基因一直是专门参考实验室和研究方案的职责范围。结合冻干试剂和熔体分析将使任何分子病理学实验室都能进行这种复杂的分析。由于尿素循环缺陷在新生儿期可能迅速致命，因此快速周转时间对于患者的生存至关重要，并且拟议的检测组合将加快对受影响患者的诊断。这些检测组合将适用于评估在器官移植或肝纤维化/肝硬化等常见疾病状态下观察到的高氨血症原因的研究。通过质谱法进行新生儿筛查可识别提示尿素循环缺陷的代谢物，这些检测可以评估这些患者。当观察到新生儿高氨血症时，快速识别尿素循环缺陷对于患者的生存至关重要，并且所提出的检测组合将有助于诊断。高氨血症见于几种常见疾病状态（肝炎、肝纤维化或肝硬化、器官或骨髓移植患者、接受化疗的患者、接受丙戊酸的患者以及因各种原因分解代谢的患者），并且必须怀疑尿素循环基因作为体现这一点的一种手段。所提出的检测组提供了一种容易评估已确定导致高氨血症的基因的方法。