Highly Multiplexed Sensitive Specific & Automated Test for Neurogenetic Disorders

高度多重 敏感 特异性

• 批准号: 7326915
• 负责人: ARMIN H REITMAIR
• 金额: $ 20.68万
• 依托单位: NESHER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7326915
• 关键词: Address; Affinity; Antibodies; Applications Grants; Archives; Area; Binding; Biological Assay; Biophysics; Blood capillaries; Body Fluids; Charcot-Marie-Tooth Disease; Chromosomes; Code; Color; Complex; Count; DNA; DNA Sequence Rearrangement; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Services; Diffuse; Disease; Dyes; Energy Transfer; Event; Exclusion; Exons; Fluorescence; Fluorescence Spectroscopy; Funding; Gene Dosage; Genes; Genetic; Genomics; Goals; Head; Home environment; Hospitals; Housing; Human Genetics; Human Genome; Immobilization; Inherited; Institution; Intellectual Property; Label; Laboratories; Laboratory Diagnosis; Lasers; Leadership; Licensing; Ligation; Measurement; Mental Retardation; Methodology; Methods; Molecular; Molecular Diagnostic Techniques; Monitor; Movement Disorders; Mutation; Mutation Detection; Myelin Proteins; Noise; Numbers; Paralysed; Patients; Peripheral; Peripheral Nervous System Diseases; Personal Satisfaction; Phase; Physicians; Polymerase Chain Reaction; Proteins; Pulsed-Field Gel Electrophoresis; Quality Control; RNA; Range; Rate; Research; Resolution; Sample Size; Sampling; Sequence Analysis; Signal Transduction; Single Nucleotide Polymorphism; Small Business Technology Transfer Research; Solutions; Sorting - Cell Movement; Source; Speech; Standards of Weights and Measures; System; Systems Analysis; Techniques; Technology; Testing; Time; Today; Tooth structure; Training; Variant; base; beta Actin; capillary; cost; developmental disease; follow-up; hereditary neuropathy; high throughput analysis; improved; interest; method development; neurogenetics; pressure; prototype; response; single molecule; southern hybridization; tool; virtual

DESCRIPTION (provided by applicant): The focus of the routine human genetic diagnostic laboratory has traditionally been confined to detection of rather large genomic rearrangements. However, the scope of diagnostic services has recently expanded dramatically through the implementation of new powerful molecular diagnostic techniques like Southern hybridization, quantitative PCR, microarrays, and others, although they are either time consuming, restricted to single regions, or need a second method for approval. Concomitantly, the vast availability of genetic diagnostic data and the requirement for improved patient management has drastically increased demand for rapid laboratory diagnosis, forcing many diagnostic labs to develop their own, non-standardized home-brew assays. Thus, there is now an urgent need for highly multiplexed diagnostic analysis systems for genetic aberrations. Unfortunately, current technologies for mutation detection and especially for gene dosis quantification put strict limits on multiplexing capabilities and do not perform cost-effectively. In response to this challenge, Nesher Technologies Inc. (NTI) proposes to develop a cost-effective, solution-based, highly multiplexed, ultrasensitive and -specific, quantitative, rapid, and fully automated analysis system for neurogenetic disorders based on genomic aberrations (with an ultimate capacity of >1,000 aberrations per standard patient sample). NTI has licensed the intellectual property for a revolutionary ultrasensitive biodetection technology with exquisite single well multiplexing potential, which was developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss). It is based on 3-color alternating laser excitation (3c-ALEX) single molecule fluorescence spectroscopy, whereby two (or three) recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two or three colors constitutes a positive target detection event, allowing molecular identification of diffusing molecules in solution and detection of numerous targets freely simultaneously. Over the Phase I funding period we will demonstrate feasibility by simultaneously distinguishing different micro mutations, duplication and deletion of the 1.4-Mb CMT1A locus as well as single nucleotide polymorphisms (SNPs), that can cause two frequent peripheral neuropathies, Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with liability to pressure palsies (HNPP). Our specific aims are: 1. Separate detection of duplication and deletion of three coding exons of peripheral myelin protein 22 (PMP22) on chromosome 17p11.2-12 compared to a normal dosage gene (e.g. beta-actin), as well as separate detection of three known SNPs in the CMT1A region, in selected patient samples. 2. Multiplexed detection of the above mentioned aberrations in selected patient samples. 3. Analysis of 250 archived patient samples (including typical and atypical CMT1A duplications and HNPP deletions), and comparison to a home-brew qPCR- and a multiplex ligation dependent probe amplification (MLPA)-based assay routinely used at the quality control reference lab for CMT and HNPP diagnostics. The proposed development of a highly multiplexed, sensitive and -specific, quantitative, low-cost automated test for both gene dosage variation and mutation detection radically pushes the limits of current technologies by allowing simultaneous detection and quantification of multiple genetic aberrations from a single patient sample. By quickly and accurately identifying the disease-causing aberration(s) among the multitude of possibilities according to the patient's presentation, these tests will overcome limitations of current diagnostic methodologies and dramatically improve laboratory diagnosis of inherited neurogenetic disorders. This will greatly facilitate patient management and offer physicians superior guidance for initiation and monitoring of therapies in context with many neurogenetic diseases, e.g. developmental disorders, mental retardation, movement disorders, speech problems etc., associated with aberrations of the human genome.

描述（由申请人提供）：常规人类遗传诊断实验室的重点传统上仅限于检测相当大的基因组重排。然而，最近通过实施新的强大分子诊断技术（如Southern杂交、定量PCR、微阵列等），诊断服务的范围急剧扩大，尽管这些技术要么耗时、仅限于单一区域，要么需要第二种方法获得批准。与此同时，基因诊断数据的大量可用性和改进患者管理的要求大大增加了对快速实验室诊断的需求，迫使许多诊断实验室开发自己的非标准化自制检测方法。因此，现在迫切需要针对遗传畸变的高度多重诊断分析系统。不幸的是，当前的突变检测技术，特别是基因剂量定量技术对多重分析能力施加了严格的限制，并且不具有成本效益。为了应对这一挑战，Nesher Technologies Inc. (NTI) 提议开发一种经济高效、基于解决方案、高度多重、超灵敏和特异性、定量、快速和全自动的神经遗传疾病分析系统，该系统基于基因组畸变（每个标准患者样本的最终容量> 1,000 个畸变）。 NTI 已获得革命性超灵敏生物检测技术的知识产权授权，该技术具有精湛的单井多重潜力，该技术是在加州大学洛杉矶分校单分子生物物理实验室（由 Shimon Weiss 教授领导）开发的。它基于三色交替激光激发 (3c-ALEX) 单分子荧光光谱，其中两个（或三个）识别分子用不同颜色的荧光染料标记。两种或三种颜色的重合共焦检测构成了阳性目标检测事件，允许对溶液中的扩散分子进行分子识别并同时自由地检测大量目标。在第一阶段资助期间，我们将通过同时区分不同的微突变、1.4-Mb CMT1A 基因座的重复和缺失以及单核苷酸多态性 (SNP) 来证明可行性，这些突变可能导致两种常见的周围神经病：腓骨肌萎缩症 (CMT) 和容易导致压力性麻痹的遗传性神经病 （HNPP）。我们的具体目标是： 1. 在选定的患者样本中，与正常剂量基因（例如β-肌动蛋白）相比，单独检测染色体 17p11.2-12 上外周髓磷脂蛋白 22 (PMP22) 的三个编码外显子的重复和缺失，以及单独检测 CMT1A 区域中的三个已知 SNP。 2.对所选患者样本中的上述畸变进行多重检测。 3. 分析 250 个存档的患者样本（包括典型和非典型 CMT1A 重复和 HNPP 缺失），并与质量控制参考实验室常规用于 CMT 和 HNPP 诊断的自制 qPCR 和多重连接依赖性探针扩增 (MLPA) 检测进行比较。拟议开发一种高度多重、灵敏、特异性、定量、低成本的自动化测试，用于基因剂量变化和突变检测，通过允许同时检测和量化单个患者样本中的多个遗传畸变，从根本上突破了当前技术的极限。通过根据患者的表现快速准确地识别多种可能性中的致病异常，这些测试将克服当前诊断方法的局限性，并显着改善遗传性神经遗传性疾病的实验室诊断。这将极大地促进患者管理，并为医生在许多神经遗传疾病（例如神经系统疾病）的治疗启动和监测方面提供卓越的指导。与人类基因组畸变有关的发育障碍、智力低下、运动障碍、言语问题等。