Genotype Analysis for Diagnosis of Urea Cycle Disorders

描述（由申请人提供）：尿素循环障碍（UCDs）是一种破坏尿素发生的代谢性疾病。明确和快速诊断ucd是困难的，因为高氨血症，主要的生化表型，并不是ucd所特有的，在广泛的代谢缺陷中观察到ucd，包括脂肪酸氧化缺陷，乳酸酸中毒和有机酸血症。临床表现包括进食不良、呕吐和嗜睡症状，导致误诊为败血症。个别尿素循环酶缺陷的鉴别诊断是复杂的。尿素循环包含6个核心基因：n -乙酰谷氨酸合成酶、氨甲酰磷酸合成酶、鸟氨酸转氨基甲酰基酶（OTC）、精氨酸琥珀酸合成酶、精氨酸琥珀酸裂解酶和精氨酸酶。OTC缺乏症是最常见的UCD，分子遗传学分析是一种成熟的诊断工具。染料结合/高分辨率热变性（DB/HRTD）是一种快速评估PCR产物序列畸变的化学方法，首次应用于单一样品仪器HR-1。原型仪器LightScanner允许在96或384个孔板中进行DB/HRTD，从而可以在一个孔板中同时熔化和分析所有样品。在此，我们提出证明DB/HRTD是一种快速评估尿素循环的6个核心基因的简单方法。特异性目标1旨在验证OTC基因的初步基因扫描测定。Specific Aim 2建议设计、开发并提供n -乙酰谷氨酸合成酶基因的基因扫描检测的初步验证，该基因缺陷导致NAGS缺乏。具体目标3建议使用原位分析，通过基因扫描分析背景下的未标记探针化学手段明确识别共同多态性。特异性目标4将演示基因扫描试剂（缓冲液，染料，dNTPs，寡核苷酸，酶）通过冷冻干燥稳定，然后以与新鲜制备的试剂相当的方式重新悬浮。快速诊断UCD对患者的生存至关重要。尿素循环缺陷的遗传分析尚未广泛应用，但其作为诊断方案组成部分的效用已得到认可和记录。稳定的冻干试剂支持快速基因分型，加强候选患者诊断的生化和临床证据。基于板的系统与块热循环器和现有的自动板加载系统兼容。

DESCRIPTION (provided by applicant): Urea Cycle Disorders (UCDs) are metabolic diseases disrupting ureagenesis. Unequivocal and rapid diagnosis of UCDs is difficult because hyperammonemia, the primary biochemical phenotype, is not specific to UCDs being observed in broad categories of metabolic deficiencies including fatty acid oxidation defects, lactic acidosis, and organic acidemias. Clinical presentation includes poor feeding, vomiting, and lethargy symptoms leading to misdiagnosis as sepsis. Differential diagnosis of individual urea cycle enzyme defects is complex. The urea cycle contains 6 core genes: N-acetylglutamate synthetase, carbamyl phosphate synthetase, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase, and arginase. OTC deficiency is the most frequently observed UCD and molecular genetic analysis is an established tool for diagnosis. Dye-Binding/High-Resolution Thermal Denaturation (DB/HRTD) is a chemistry that rapidly assesses a PCR product for sequence aberration and was first applied using a single sample instrument the HR-1. A prototype instrument, the LightScanner, allows DB/HRTD to be performed in 96 or 384 well plates enabling concurrent melting and analysis of all samples in a plate. Herein, it is proposed to demonstrate DB/HRTD as a simple means to rapidly assess the 6 core genes of the urea cycle. Specific Aim 1 seeks to validate a preliminary gene-scanning assay for the OTC gene. Specific Aim 2 proposes to design, develop, and provide preliminary validation of a gene-scanning assay for the N-acetylglutamate synthetase gene, defects in which cause NAGS deficiency. Specific Aim 3 proposes to use in situ analysis to unequivocally identify common polymorphisms by means of unlabeled probe chemistry within the context of gene scanning analysis. Specific Aim 4 will demonstrate gene scanning reagents (buffer, dye, dNTPs, oligonucleotides, enzyme) are stabilized by freeze drying and following re-suspension perform in a manner comparable to freshly prepared reagents. Rapid diagnosis of UCD is critical to patient survival. Genetic analysis for urea cycle defects is not widely available yet its utility as a component of the diagnostic regimen is recognized and documented. Stable freeze-dried reagents support rapid genotyping, to strengthen biochemical and clinical evidence in diagnosis of candidate patients. A plate-based system is compatible with block thermalcyclers and existing systems for automated plate loading.