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）：涉及尿素循环的疾病在临床上表现为新生儿高氨性昏迷至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，但区分尿素循环中的个体基因缺陷涉及一系列复杂的生化测试。以下基因的产物是尿素生成所需的：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、氨基琥珀酸合成酶、氨基琥珀酸裂解酶、谷胱甘肽酶、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一个既定部分，但检测的可用性有限。使用2项创新技术，PCR反应混合物的熔融分析和冷冻干燥保存，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的或私人的，需要全面的基因分析，这是一个复杂和劳动密集型的过程。将用于充分分析尿素循环基因的PCR试剂（缓冲液、MgCl-2、引物、LCGreen染料、taq聚合酶）冷冻干燥至平板中。使用这些试剂需要将它们与含有待评估的DNA样品的水重悬，这绕过了配制和分配综合基因分析所需的众多反应的艰苦、容易出错和昂贵的过程。试剂设计用于使用通用条件的PCR。扩增后，在没有任何PCR后操作的情况下，通过高分辨率熔解曲线分析平板。解链谱鉴定含有序列变体的区域，使得DNA序列分析被选择性地靶向。由于解链谱对扩增产物是非破坏性的，因此回收鉴定为产生异常解链谱的产物以用作DNA测序模板。为尿素循环的8个基因制备测定组。分析尿素循环的基因一直是专业参考实验室和研究方案的范围。结合冷冻干燥试剂和熔融分析将使这种复杂的分析能够由任何分子病理学实验室进行。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患者生存至关重要，拟议的检测试剂盒将加快受影响患者的诊断。这些检测板将应用于评估在常见疾病状态（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。新生儿筛查质谱鉴定代谢物提示尿素循环缺陷，这些测定可以评估这些患者。当观察到新生儿高氨血症时，快速识别尿素循环缺陷对患者生存至关重要，并且所提出的检测面板将有助于诊断。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、接受丙戊酸的患者和因各种原因分解代谢的患者）中观察到高氨血症，必须怀疑尿素循环基因是一种表现方式。拟定的检测试剂盒提供了一种方法，可轻松评估已确定可导致高氨血症的基因。