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）是一种铜代谢的常染色体隐性疾病。已经提出了对WD的前瞻性筛查，但是没有可用的敏感和特定的生化遗传测定法，并且一级分子分析不可行。 WD可能是儿童慢性肝病最常见，最可预防的原因。威尔逊病是可以治疗的，如果提早做出诊断，可以避免严重的症状。最近使用特定的单克隆抗体的ELISA测定法产生了证据，表明它提供了WD的种群筛选测定的基础。在最终验证之后，有计划将此测定法商业化为人口筛查的套件。生化遗传筛查测定法产生了模棱两可的结果的少量，并且被检测到的患者需要确认。基因分型提供了一种有效的手段来阐明模棱两可的结果并确认受皮质受到影响的患者。 WD基因P型ATPase ATP7B包含常见的突变，但是这些突变特异于给定的族裔，并且患者通常构成常见突变和罕见/私有突变的杂合子。提出了2层基因分型测定。使用LightCycler和Simple Probe Chemistry迅速鉴定出常见的突变（适用于种群测定的人群）。全面的基因扫描采用新开发的染料结合/高分辨率热变性平台来检测异源分子。在存在DSDNA结合染料LCGREEN I. LCGREEN I的独特特性的浓度是饱和新合成的DNA的浓度下，它不抑制PCR，它不抑制PCR，它不抑制PCR，它不会抑制其他DSDNA结合染料，它不会抑制PCR。放大后，通过高分辨率热变性来测定染料饱和PCR产物。分析是均匀的，在PCR反应毛细管中进行，没有PCR后处理。分析大约需要90秒/标本。利用空气驱动的PCR，LCGREEN I和高分辨率热变性，ATP7B基因（包括编码和毗邻的剪接位点）在大约1.5小时内被扫描以进行异质双链化，其中包括测试样品和控件。测序显示杂化证据的PCR产品。染料绑定/高分辨率变性为通过异源分析提供了一个廉价且真正的用户友好平台，用于扫描基因扫描。对WD的前瞻性筛查可提高受影响患者的治疗和生活质量的功效。基因分型是根据无症状患者的Ceruloplasmin分析确认结果的最佳选择。