Highly Multiplexed Sensitive Specific & Automated Test for Neurogenetic Disorders

描述(由申请人提供)：常规人类基因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，最近通过实施新的强大的分子诊断技术，如Southern杂交、定量PCR、微阵列和其他技术，诊断服务的范围大大扩大，尽管它们要么耗时，要么限于单个区域，要么需要另一种方法获得批准。随之而来的是，基因诊断数据的大量可获得性和对改善患者管理的要求大幅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家庭酿造分析。因此，现在迫切需要高度多元化的遗传异常诊断分析系统。不幸的是，目前的突变检测技术，特别是基因Dsis定量技术，对多路复用能力有严格的限制，而且执行起来不划算。为了应对这一挑战，Nesher Technologies Inc.(NTI)建议开发一种具有成本效益的、基于解决方案的、高度多元化的、超敏感和特定的、定量、快速和全自动的基于基因组异常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&gt；1000例异常)。NTI已为加州大学洛杉矶分校(UCLA)单分子生物物理实验室(由Shimon Weiss教授领导)开发的具有精致单井多路复用潜力的革命性超灵敏生物检测技术授予知识产权。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)识别分子被标记上不同颜色的荧光染料。两种或三种颜色的重合共焦检测构成了一个积极的目标检测事件，允许对溶液中扩散的分子进行分子识别，并同时自由检测多个目标。在第一阶段的资助期内，我们将通过同时区分不同的微突变、1.4-Mb CMT1A基因座的复制和缺失以及单核苷酸多态(SNPs)来展示可行性，这些微突变、复制和缺失以及单核苷酸多态(SNPs)可以导致两种常见的周围神经疾病，夏科-玛丽-牙(CMT)病和遗传性压力性瘫痪(HNPP)。我们的具体目标是：1.在选定的患者样本中，分别检测染色体17p11.2-12上与正常剂量基因(如β-肌动蛋白)相比的外周髓鞘蛋白22(PMP22)的三个编码外显子的复制和缺失，以及CMT1A区域的三个已知SNP的单独检测。2.在选定的患者样本中对上述像差进行多路检测。3.分析250个存档的患者样本(包括典型和非典型的CMT1A重复和HNPP缺失)，并与在CMT和HNPP诊断的质量控制参考实验室常规使用的自制定量聚合酶链式反应和多重连接依赖探针扩增(MLPA)方法进行比较。针对基因剂量变异和突变检测的高度多重、灵敏和特异、定量、低成本自动化测试的拟议开发从根本上突破了当前技术的极限，允许从单个患者样本中同时检测和量化多个遗传异常。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异常(S)，这些测试将克服当前诊断方法的局限性，并显著改进遗传性神经遗传疾病的实验室诊断。这将极大地方便患者管理，并为医生在许多神经遗传性疾病的启动和监测方面提供更好的指导，例如与人类基因组异常相关的发育障碍、精神发育迟缓、运动障碍、言语问题等。

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.