Analysis of ATP7B in Screening for Wilson Disease

ATP7B 在威尔逊病筛查中的分析

• 批准号: 6788663
• 负责人: Steven F Dobrowolski
• 金额: $ 9.94万
• 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: 2004-05-01 至 2004-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6788663
• 关键词: biomarker; biotechnology; computer assisted diagnosis; computer data analysis; diagnosis design /evaluation; early diagnosis; ferroxidase; gene mutation; genetic polymorphism; genetic screening; genetic susceptibility; genotype; hepatolenticular degeneration; high throughput technology; human genetic material tag; human tissue; inborn metabolism disorder diagnosis; intermolecular interaction; liver disorder diagnosis; nucleic acid quantitation /detection; nucleic acid sequence; patient oriented research; pediatrics; polymerase chain reaction

DESCRIPTION (provided by applicant): Wilson Disease (WD) is an autosomal recessive disorder of copper metabolism. Prospective screening for WD has been proposed however a sensitive and specific biochemical genetic assay was not available and primary molecular analysis was not feasible. WD may be the most frequent and most preventable cause of chronic liver disease in children. Wilson Disease is treatable and serious symptoms can be avoided if a diagnosis is made early. A recently developed ELISA assay using specific monoclonal antibody to ceruloplasmin has generated evidence that it provides the basis of a population screening assay for WD. Following a final validation, there are plans to commercialize this assay as a kit for population screening. Biochemical genetic screening assays generate a small protion of equivocal results and patients detected presymptomatically require confirmation. Genotyping provides an effective means by which to clarify equivocal results and confirm putativly affected patients. The WD gene, P-type ATPase ATP7B, contains common mutations however these are specific to given ethnic groups and patients are most often compond heterozygotes for a common mutation and a rare/private mutation. A 2-tiered genotyping assay is proposed. Common mutations (appropriate to the population assayed) are rapidly identified with specific assays using the LightCycler and SimpleProbe chemistry. Comprehensive gene scanning employs the newly developed dye binding/high resolution thermal denaturation platform to detect heteroduplex molecules. PCR is performed using rapid air-driven thermalcycling in the presence of the dsDNA binding dye LCGreen I. A unique property of LCGreen I is at concentrations saturating newly synthisized DNA, it does not inhibit PCR, a quality not shared by other dsDNA binding dyes. Following amplification, dye saturated PCR product is assayed by high resolution thermal denaturation. Analysis is homogeneous, performed in the PCR reaction capillary, with no post-PCR processing. Analysis requires approximately 90 seconds/specimen. Utilizing air driven PCR, LCGreen I, and high resolution thermal denaturation, the ATP7B gene, including coding and adjoining splice sites, is scanned for heteroduplexes in approximately 1.5 hours, which includes test specimens and controls. PCR products showing evidence of heteroduplexes are sequenced. Dye binding/high resolution denaturation provides an inexpensive and truly user-friendly platform for gene scanning by heteroduplex analysis. Prospective screening for WD improves efficacy of treatment and quality of life for affected patients. Genotyping is the best option to confirm results based upon ceruloplasmin analysis in asymptomatic patients.

描述（由申请人提供）：威尔逊病（WD）是一种常染色体隐性铜代谢疾病。WD的前瞻性筛查已被提出，但没有一种敏感和特异性的生化遗传检测方法，初级分子分析也不可行。WD可能是儿童慢性肝病最常见和最可预防的原因。威尔逊病是可以治疗的，如果及早诊断，可以避免严重的症状。最近开发的一种使用铜蓝蛋白特异性单克隆抗体的酶联免疫吸附试验已产生证据，表明它为WD的人群筛查试验提供了基础。在最终验证之后，计划将该检测作为人群筛查试剂盒进行商业化。生化遗传筛选分析产生了一小部分模棱两可的结果，并且在症状前检测到的患者需要确认。基因分型提供了一种有效的手段，通过它来澄清模棱两可的结果和确认推定受影响的患者。WD基因，p型atp酶ATP7B，包含常见突变，但这些突变是特定于特定种族的，患者通常是常见突变和罕见/私人突变的复合杂合子。提出了一种双层基因分型分析方法。使用LightCycler和SimpleProbe化学试剂，可以快速识别常见突变（适用于所测群体）。综合基因扫描采用新开发的染料结合/高分辨率热变性平台检测异双相分子。PCR是在dsDNA结合染料LCGreen I的存在下使用快速空气驱动热循环进行的。LCGreen I的独特性质是在浓度饱和新合成的DNA时，它不会抑制PCR，这是其他dsDNA结合染料所没有的品质。扩增后，染料饱和PCR产物通过高分辨率热变性检测。分析是均匀的，在PCR反应毛细管中进行，没有PCR后处理。分析每个样品大约需要90秒。利用空气驱动PCR， LCGreen I和高分辨率热变性，在大约1.5小时内扫描ATP7B基因，包括编码和相邻的剪接位点，以寻找异源双工，其中包括测试样本和对照。PCR产物显示异质双链的证据进行测序。染料结合/高分辨率变性提供了一个廉价和真正用户友好的平台，基因扫描通过异双工分析。WD的前瞻性筛查提高了患者的治疗效果和生活质量。对于无症状患者，基因分型是确认基于铜蓝蛋白分析结果的最佳选择。