Genotype Analysis for Diagnosis of Urea Cycle Disorders

描述(申请人提供)：尿素循环障碍(UCDs)是一种扰乱尿失禁的代谢性疾病。UCDs的明确和快速诊断是困难的，因为主要的生化表型高氨血症并不是UCDs在包括脂肪酸氧化缺陷、乳酸酸中毒和有机酸血症在内的多种代谢缺陷中所特有的。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、氨基甲酰磷酸合成酶、鸟氨酸转氨甲基酶(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公认的诊断工具。染料结合/高分辨率热变性(DB/HRTD)是一种快速评估PCR产物序列异常的化学方法，首次使用单一样品仪器HR-1进行应用。原型仪器LightScanner允许在96或384孔板中执行DB/HRTD，从而能够同时熔化和分析板中的所有样品。在此，建议将DB/HRTD作为一种快速评估尿素循环的6个核心基因的简单方法。特定目的1试图验证OTC基因的初步基因扫描分析。特定目的2建议设计、开发并初步验证N-乙酰谷氨酸合成酶基因的基因扫描分析，该基因扫描分析中的缺陷会导致NAGS缺乏。具体目标3建议在基因扫描分析的背景下，通过未标记的探针化学方法，使用原位分析来明确地识别常见的多态。具体目标4将展示基因扫描试剂(缓冲液、染料、dNTPs、寡核苷酸、酶)通过冷冻干燥和随后的再悬浮以与新制备的试剂相媲美的方式稳定下来。UCD的快速诊断对患者的生存至关重要。尿素循环缺陷的遗传分析并不广泛，但它作为诊断方案的一个组成部分的效用已被认识并被记录在案。稳定的冻干试剂支持快速基因分型，以加强在候选患者诊断中的生化和临床证据。基于板材的系统与块式热循环器和用于自动板材装载的现有系统兼容。

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.