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遗传的铜代谢疾病。已提出对 WD 进行前瞻性筛查，但尚无灵敏且特异的生化遗传测定，且初步分子分析也不可行。 WD 可能是儿童慢性肝病最常见且最可预防的原因。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 ELISA 测定法已产生证据，表明它为 WD 的群体筛查测定法提供了基础。经过最终验证后，计划将该测定法作为人群筛查试剂盒商业化。生化基因筛查检测产生一小部分模棱两可的结果，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模棱两可的结果并确认可能受影响的患者。 WD 基因（P 型 ATP 酶 ATP7B）包含常见突变，但这些突变是特定种族群体所特有的，并且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 2 层基因分型测定。使用 LightCycler 和 SimpleProbe 化学方法进行特定测定，可以快速识别常见突变（适合所测定的人群）。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双链分子。 PCR 是在 dsDNA 结合染料 LCGreen I 存在的情况下使用快速空气驱动热循环进行的。 LCGreen I 的一个独特特性是浓度使新合成的 DNA 饱和，它不会抑制 PCR，这是其他 dsDNA 结合染料所不具备的品质。扩增后，通过高分辨率热变性分析染料饱和的 PCR 产物。分析是均质的，在 PCR 反应毛细管中进行，无需 PCR 后处理。每个样本的分析大约需要 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（包括编码位点和相邻剪接位点）的异源双链体，其中包括测试样本和对照。对显示异源双链体证据的 PCR 产物进行测序。染料结合/高分辨率变性为通过异源双链分析进行基因扫描提供了一个廉价且真正用户友好的平台。 WD 的前瞻性筛查可提高受影响患者的治疗效果和生活质量。基因分型是根据无症状患者的铜蓝蛋白分析来确认结果的最佳选择。