Novel Strategies for Development of a Multiplexed Test with Expanded Content for Spinal Muscular Atrophy

开发具有扩展内容的脊髓性肌萎缩症多重测试的新策略

• 批准号: 9909806
• 负责人: Huiping Zhu
• 金额: $ 18.24万
• 依托单位: ASURAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909806
• 关键词: Address; Adult; African American; Age of Onset; Algorithms; Alleles; Anterior; Asians; Biological Assay; Capillary Electrophoresis; Cell Line; Cells; Cessation of life; Chemistry; Classification; Clinical; Computer software; Copy Number Polymorphism; Counseling; Cyclic GMP; DNA; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Disease Progression; Early Diagnosis; Family history of; Genes; Genetic; Genotype; Goals; Horns; Hour; In Vitro; Individual; Laboratories; Methods; Modeling; Molecular; Motor Neurons; Mutation; Nature; Parents; Pathogenicity; Patients; Performance; Phase; Population; Prenatal care; Process; Proteins; Reporting; Resolution; Respiratory Failure; Running; SMN2 gene; Sampling; Severities; Severity of illness; Signal Transduction; Spinal Muscular Atrophy; Spinal cord grey matter structure; Symptoms; Technology; Testing; Tube; Underserved Population; Variant; Whole Blood; base; carrier status; cost effective; cost effectiveness; design; digital; experience; fluorophore; follower of religion Jewish; genetic element; genetic variant; improved; infant death; instrument; loss of function mutation; molecular diagnostics; motor function improvement; next generation sequencing; novel; novel diagnostics; novel strategies; outcome forecast; patient stratification; prospective; prospective test; screening; signal processing; skeletal muscle wasting; survival motor neuron gene; synthetic construct

Summary The overall goal of this project is to develop a cost-effective multiplexed diagnostic test for spinal muscular atrophy (SMA) to rapidly and accurately analyze multiple classes of disease-causing, disease-modifying DNA variants, and carrier status markers. Ultimately, the proposed test will have a far-reaching impact by expanding the identification and stratification of patients who may benefit from new treatments, and by improving the detection of SMA carriers. The proposed test will detect an expanded panel of variants that are not assessed by conventional testing. The broader content is important because age of onset, disease severity and progression correlations with the nature of the SMN1 mutations, and the compensatory and modifying effects of SMN2 and other genes. The expanded SMA test will not only extend the molecular testing to benefit more prospective parents and patients with atypical symptoms including adult onset cases, but will also address SMA testing needs of underserved populations. The expanded SMA test panel will build upon Asuragen’s AmplideX® PCR/CE SMN1/2 Kit (RUO and CE- IVD) and take advantage of a novel strategy Multiplexed & Efficient Resolution of Genetic Elements (MERGE)- PCR to assess: 1) copy number variants (CNVs) of SMN1 and SMN2, 2) pathogenic mutations in SMN1, 3) disease-modifying mutations, and 4) silent carrier markers. We will also incorporate a novel gene-tagged genotyping strategy to determine whether a mutation is in the SMN1 or SMN2 gene. The specific aims are: Aim 1. Design and optimize individual assays for a unified multiplex MERGE-PCR/CE test to detect multiple classes of disease-causing, disease-modifying DNA variants and carrier markers. Aim 2. Develop algorithms to support assay signal deconvolution and enable accurate genotype classification of CNV, SNV, and INDEL signals from multiple fluorophore channels. Aim 3. Develop and verify a single-tube multiplex PCR-based test. In Phase II, we will advance the technology into a cGMP kit development process, enable market-ready software for automatic variant calling, and develop controls and standards for an in vitro diagnostic product. The project will benefit from Asuragen’s years of experience optimizing multiplexing PCR chemistries to develop and commercialize high-performance diagnostic PCR/CE assays.

概括 该项目的总体目标是开发一种具有成本效益的脊髓肌肉综合诊断测试 萎缩 (SMA) 可快速、准确地分析多类致病、缓解疾病的 DNA 最终，拟议的测试将通过扩展产生深远的影响。 识别和分层可能受益于新疗法的患者，并通过改善 SMA 携带者的检测 拟议的测试将检测未评估的一组扩展变异。 由于发病年龄、疾病严重程度和进展，更广泛的内容很重要。 与 SMN1 突变的性质以及 SMN2 和 SMN2 的补偿和修饰作用的相关性 扩大的 SMA 检测不仅会扩大分子检测范围，使更多的潜在患者受益。 父母和有非典型症状的患者，包括成人发病病例，但也将讨论 SMA 测试 服务不足人群的需求。 扩展的 SMA 测试面板将基于 Asuragen 的 AmplideX® PCR/CE SMN1/2 套件（RUO 和 CE- IVD）并利用遗传元件多重高效解析（MERGE）的新策略 - PCR 评估：1) SMN1 和 SMN2 的拷贝数变异 (CNV)，2) SMN1 的致病性突变，3) 疾病缓解突变，4) 沉默载体标记，我们还将纳入一种新的基因标记。 确定 SMN1 或 SMN2 基因中是否存在突变的基因分型策略具体目标是： 目标 1. 设计和优化统一多重 MERGE-PCR/CE 测试的单独测定，以检测多种 致病、改善疾病的 DNA 变异和载体标记的类别。 目标 2. 开发算法支持检测信号反卷积并实现准确的基因型分类 来自多个荧光团通道的 CNV、SNV 和 INDEL 信号。 目标 3. 开发并验证基于单管多重 PCR 的测试。 在第二阶段，我们将将该技术推进到 cGMP 试剂盒开发流程中，为市场做好准备 用于自动变异调用的软件，并为体外诊断产品开发控制和标准。 该项目将受益于 Asuragen 多年优化多重 PCR 化学物质的经验，以开发和 将高性能诊断 PCR/CE 检测商业化。