Highly Multiplexed Sensitive Specific & Automated Test for Neurogenetic Disorders

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的分子诊断技术，如南方杂交、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，要么仅限于单一区域，要么需要第二种方法获得批准。同时，遗传诊断数据的大量可用性和对改善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，目前迫切需要高度多路复用的遗传畸变诊断分析系统。不幸的是，目前的突变检测技术，特别是基因剂量定量技术，严格限制了多路复用能力，而且成本效益不高。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经济高效、基于解决方案、高度多路、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（每个标准患者样本的最终容量为1000个畸变）。NTI已经获得了一项革命性的超灵敏生物检测技术的知识产权，该技术具有精致的单孔复用潜力，该技术是由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3色交替激光激发（3c-ALEX）单分子荧光光谱，其中两个（或三个）识别分子被不同颜色的荧光染料标记。两种或三种颜色的同步共聚焦检测构成了一个积极的目标检测事件，可以对溶液中的扩散分子进行分子识别，同时自由地检测多个目标。在I期融资期间，我们将通过同时区分1.4 mb CMT1A位点的不同微突变、重复和缺失以及单核苷酸多态性（snp）来证明可行性，这些微突变可导致两种常见的周围神经病变，沙科-玛丽-图斯病（CMT）和遗传性神经病变伴压力性麻痹（HNPP）。我们的具体目标是：1。与正常剂量基因（如β -肌动蛋白）相比，在选定的患者样本中分别检测到17p11.2-12染色体上外周髓鞘蛋白22 （PMP22）的三个编码外显子的重复和缺失，以及在CMT1A区域单独检测到三个已知的snp。2. 在选定的患者样本中对上述畸变进行多路检测。3. 分析了250个存档的患者样本（包括典型和非典型CMT1A重复和HNPP缺失），并与自制qPCR和基于多重连接依赖探针扩增（MLPA）的检测方法进行了比较，该方法通常用于CMT和HNPP诊断的质量控制参考实验室。提出的开发一种高度复用、敏感和特异性、定量、低成本的基因剂量变化和突变检测自动化测试，通过允许同时检测和定量来自单个患者样本的多种遗传畸变，从根本上突破了当前技术的极限。通过快速准确地根据患者的表现在众多可能性中识别致病畸变，这些测试将克服当前诊断方法的局限性，并显著提高遗传性神经遗传疾病的实验室诊断。这将极大地促进患者管理，并为医生在许多与人类基因组畸变相关的神经遗传疾病（如发育障碍、智力迟钝、运动障碍、语言问题等）的启动和监测治疗提供卓越的指导。

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.