Genotype Analysis for Diagnosis of Urea Cycle Disorders

尿素循环障碍诊断的基因型分析

• 批准号: 7159437
• 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
• 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/7159437
• 关键词: ammonia; carbamoyl phosphate synthetase deficiency; clinical research; diagnosis design /evaluation; enzyme deficiency; genetic disorder diagnosis; genetic techniques; genotype; inborn metabolism disorder; lyophilization; mass spectrometry; metabolism disorder diagnosis; molecular genetics; molecular pathology; orphan disease /drug; polymerase chain reaction; rapid diagnosis; urea cycle

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.

描述（由申请人提供）：涉及尿素周期的疾病在临床上表现为症状，这些症状是新生儿高症昏迷到产后精神病。高氨比是尿素周期缺陷的主要表型，但在尿素周期内区分个体基因缺陷涉及一系列复杂的生化测试。尿素发生需要以下基因的产物：N-乙酰基谷氨酸合成酶碳酰磷酸合成酶1，鸟氨酸经钙基酰基酶，精氨酸糖酸合成酶，精氨酸糖酸酯裂解酶，精氨酸酶，精氨酸酶，小核核酸骨质甲酸酯术和甲状腺粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒粒贴粒粒粒粒粒粒贴粒粒粒粒粒贴；基于基因的分析是尿素周期缺陷的诊断方案的确定部分，但测试的可用性有限。使用2种创新技术，融化PCR反应混合物的融化分析和冷冻干燥的保存，开发了一种评估尿素周期基因的简化方法。尿素周期基因中的突变是罕见或私有的，需要全面的基因分析，这是一个复杂且劳动密集型的过程。 PCR试剂（缓冲液，MGCL-2，引物，LCGREEN染料，TAQ聚合酶）完全分析尿素循环基因被冷冻到板中。使用这些试剂要求将其重悬于含有的水的水中，并评估了正在评估的DNA样品，这些DNA样品绕过了艰苦的，容易发生的误差和昂贵的制定和分发综合基因分析所需的反应的昂贵过程。这些试剂是为PCR设计的，使用常见条件。放大后，没有进行任何PCR后操作后，通过高分辨率融化分析来分析板。熔体分析标识了包含序列变体的区域，以便将DNA序列分析选择性靶向。由于熔体分析对扩增产物无损，因此恢复了被鉴定为产生异常熔融曲线的产品作为DNA测序模板。测定面板是为尿素周期的8个基因准备的。分析尿素周期的基因一直是专业参考实验室和研究方案的权限。结合冻干试剂和熔体分析将使这种复杂分析能够由任何分子病理实验室进行。由于尿素周期缺陷在新生儿时期可能会迅速致命，因此快速转折对于患者的生存至关重要，拟议的测定面板将加快受影响患者的诊断。这些测定面板将应用于评估在常见疾病状态（例如器官移植或肝纤维化/肝硬化）中观察到的高氨血症原因的研究。通过质谱法筛查的新生儿筛查确定了尿素周期缺陷的代谢产物，这些测定方法可以评估这些患者。当观察到新生儿高症血症并提出的测定面板将促进诊断时，迅速识别尿素周期缺陷对于患者的存活至关重要。在几种常见疾病状态（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出于各种原因是分解代谢的患者）和尿素周期的基因必须被认为是一种表现出来的手段，在几种常见的疾病状态下观察到高氨血症。拟议的测定面板提供了一种容易评估基因的手段，这些基因已确立，可以促进高症血症。