Genotype Analysis for Diagnosis of Urea Cycle Disorders

描述（申请人提供）：涉及尿素循环的疾病在临床上表现为多种症状，从新生儿高氨血症昏迷到产后精神病。高氨血症是尿素循环缺陷的主要表型，但在尿素循环中区分个体基因缺陷涉及一系列复杂的生化试验。脲原性需要下列基因的产物：n -乙酰谷氨酸合成酶氨甲酰磷酸合成酶1、鸟氨酸转氨基甲酰基化酶、精氨酸琥珀酸合成酶、精氨酸琥珀酸裂解酶、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一个既定部分，但测试的可用性有限。利用熔体分析和PCR反应混合物冻干保存两种创新技术，开发了一种评估尿素循环基因的简化方法。尿素循环基因的突变是罕见的或私有的，需要进行全面的基因分析，这是一个复杂和劳动密集型的过程。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）冻干后装板。使用这些试剂需要用含有待评估DNA样本的水重新悬浮它们，这绕过了复杂的、容易出错的、昂贵的过程，即制定和分配综合基因分析所需的众多反应。该试剂设计用于PCR使用一个共同的条件。扩增后，无需任何pcr后操作，用高分辨率熔体分析。熔体分析识别包含序列变异的区域，这样DNA序列分析就可以选择性地靶向。由于熔体分析对扩增产物无破坏性，鉴定为产生异常熔体分析的产物被回收作为DNA测序模板。制备了尿素循环8个基因的检测板。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范围。结合冻干试剂和熔体分析将使这种复杂的分析，任何分子病理实验室进行。由于尿素循环缺陷在新生儿期可能会迅速致命，快速的周转时间对患者的生存至关重要，拟议的检测小组将加快对受影响患者的诊断。这些检测小组将应用于研究评估常见疾病状态（如器官移植或肝纤维化/肝硬化）中观察到的高氨血症的原因。新生儿筛查通过质谱鉴定代谢物提示尿素循环缺陷，这些分析可以评估这些患者。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的生存至关重要，建议的测定板将有助于诊断。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、接受丙戊酸治疗的患者以及由于各种原因导致分解代谢的患者），必须怀疑尿素循环基因是表现高氨血症的一种手段。提议的测定板提供了一种方法，可以很容易地评估已确定的导致高氨血症的基因。

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.