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(申请人提供)：威尔逊病(WD)是一种铜代谢的常染色体隐性遗传病。WD的前瞻性筛查已被提出，但目前尚无灵敏、特异的生化遗传分析方法，也无法进行初步的分子分析。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抗的ELISA法已经产生了证据，它为WD的人群筛查提供了基础。在最终验证之后，有计划将这种检测方法商业化，作为人群筛查的试剂盒。生化基因筛查会产生一小部分不明确的结果，而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方法来澄清不明确的结果，并确认推定受影响的患者。WD基因，P型ATPase ATP7B，包含常见的突变，但这些突变是特定种族特有的，患者通常是常见突变和罕见/私人突变的复合杂合子。提出了一种两级基因分型方法。通过使用LightCycler和SimpleProbe化学的特定分析，可以快速识别常见的突变(适用于被检测的人群)。全面的基因扫描利用新开发的染料结合/高分辨率热变性平台来检测异源双链分子。在dsDNA结合染料LC Green I的存在下，使用快速空气驱动的热循环进行PCR。LC Green I的独特性质是在饱和新合成的DNA的浓度时，它不抑制PCR，这是其他dsDNA结合染料所不具备的特性。扩增后，用高分辨热变性对染料饱和的PCR产物进行检测。分析是均匀的，在聚合酶链式反应毛细管中进行，没有聚合酶链式反应后处理。每个样本的分析需要大约90秒。利用空气驱动的PCR、LCGreen I和高分辨率热变性，ATP7B基因，包括编码和邻近剪接位点，在大约1.5小时内扫描异源双链，包括测试样本和对照。显示异源双链证据的聚合酶链式反应产物被测序。染料结合/高分辨率变性为异源双链分析的基因扫描提供了一个廉价且真正用户友好的平台。WD的前瞻性筛查改善了治疗效果和受影响患者的生活质量。根据对无症状患者的铜蓝蛋白分析，基因分型是确认结果的最佳选择。