Highly Multiplexed Sensitive Specific & Automated Test for Neurogenetic Disorders

高度多重 敏感 特异性

• 批准号: 7501963
• 负责人: ARMIN H REITMAIR
• 金额: $ 20.84万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7501963
• 关键词: Address; Affinity; Antibodies; Applications Grants; Archives; Area; Bar Codes; 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局限于检测相当大的基因组重排。然而，诊断服务的范围最近通过实施新的强大的分子诊断技术如Southern杂交、定量PCR、微阵列等而急剧扩大，尽管它们要么耗时，要么限于单个区域，要么需要第二种方法才能获得批准。与此同时，遗传诊断数据的大量可用性和对改善患者管理的需求急剧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测定法。因此，现在迫切需要用于遗传畸变的高度多重诊断分析系统。不幸的是，目前的突变检测技术，特别是基因剂量定量技术对多重能力有严格的限制，并且不具有成本效益。为了应对这一挑战，Nesher Technologies Inc. (NTI)提出开发一种基于基因组畸变的用于神经遗传性疾病的具有成本效益的、基于溶液的、高度多路复用的、超灵敏的和特异性的、定量的、快速的和全自动的分析系统（每个标准患者样本具有> 1，000个畸变的最终容量）。NTI已获得革命性的超灵敏生物检测技术的知识产权许可，该技术具有精致的单井多路复用潜力，由加州大学洛杉矶分校单分子生物物理实验室（由Shimon韦斯教授领导）开发。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聚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鉴定并同时自由地检测许多目标。在I期资助期间，我们将通过同时区分不同的微突变，1.4 Mb CMT 1A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，Charcot-Marie-Tooth（CMT）疾病和遗传性神经病易患压力麻痹（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，分别检测染色体17p11.2-12上外周髓鞘蛋白22（PMP 22）的三个编码外显子的重复和缺失，以及分别检测CMT 1A区域中的三个已知SNP。2.对选定患者样本中上述畸变的多重检测。3.分析250份存档患者样本（包括典型和非典型CMT 1A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和基于多重连接依赖性探针扩增（MLPA）的测定进行比较。所提出的开发用于基因剂量变化和突变检测的高度多重的、灵敏的和特异性的、定量的、低成本的自动化测试，通过允许从单个患者样品同时检测和定量多种遗传畸变，从根本上推动了当前技术的极限。通过根据患者的表现在众多可能性中快速准确地识别致病畸变，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，并为医生提供上级指导，用于在许多神经遗传性疾病的背景下启动和监测治疗，例如发育障碍、智力迟钝、运动障碍、言语问题等，与人类基因组畸变有关。