FMR 1-SLS: Improving Fragile X diagnosis using amplification-free single locus ta

描述（由申请人提供）：FMR 1基因中三核苷酸重复CGG的扩增导致FMR 1蛋白表达失调，并导致许多严重疾病，如认知障碍、自闭症、卵巢衰竭和进行性神经系统疾病。超过150万美国人携带扩增的重复区域，1000万人有症状或家族史需要检测扩增。由于感兴趣区域的100%GC含量和观察到的重复长度的临床范围，先前的测序方法缺乏通读该重复的能力。DNA片段大小的方法，无论是Southern印迹或PCR后，毛细管电泳，用于分类个体具有正常，前突变或完全突变等位基因。在《脆弱的X》中， 前突变类别代表具有其自身推荐和临床结果的不同基因型，因此这些方法中由固有片段大小不准确引起的错误分类对患者的治疗是有害的，导致不适当的或甚至是破坏性的指导。该基因座通常在重复长度上表现出镶嵌性，最近已显示其影响疾病的严重程度，并且还预测药物反应（或缺乏药物反应）。这些方法缺乏检测临床上重要的镶嵌水平的灵敏度。AGG序列对CGG重复基序的中断大大减少了突变前携带者后代中完全突变状态的进展，但是今天使用的基于大小的方法不能传达关于这些特征的任何信息。Pacific Biosciences的单分子实时（SMRT（R））测序能够读取几乎任何DNA序列背景（包括CGG的三核苷酸重复序列），产生平均长度近5000个碱基的读数，并内在地产生对研究和临床诊断重要的区域甲基化状态的信息。SMRT测序对FMR 1的应用已经被证明用于PCR扩增子，但是为了避免PCR产生的假重复长度，需要在不使用PCR的情况下靶向基因座的手段来将该方法转化为临床。因此，该计划的I期目标是：i）证明来自取自血液的< 20 μ g gDNA的未扩增DNA的至少100个读数，ii）显示因此可以分析突变的整个临床范围，iii）显示以下能力： 检测嵌合体个体中的次要等位基因和iv）显示这些等位基因的甲基化状态可以使用动力学信息SMRT测序产率来确定。第二阶段的目标是优化检测方法，开发所需的信息学工具，在300人一致性研究的背景下证明检测方法，然后将技术转移到加州大学戴维斯分校CLIA实验室，以开发为临床诊断检测方法，供临床医生使用 在第三阶段开始时。

DESCRIPTION (provided by applicant): Expansion of the trinucleotide repeat CGG in the FMR1 gene causes dysregulation of FMR1 protein expression and results in a host of serious conditions, from cognitive impairment, autism, ovarian failure, and progressive neurological disorders. Over 1.5 million Americans harbor expanded repeat regions, and 10 million are indicated by symptoms or family history to be tested for the expansion. Because of the 100% GC content of the region of interest and the clinical range of observed repeat lengths, prior sequencing methods have lacked the ability to read through this repeat. DNA fragment sizing methods are used today, either Southern Blotting or PCR followed by capillary electrophoresis, to categorize individuals as having normal, premutation or full mutation alleles. In Fragile X, the premutation category represents a distinct genotype with its own recommendations and clinical outcome, thus misclassifications caused by inherent fragment sizing inaccuracies in these methods are detrimental to the treatment of patients, leading to inappropriate or even damaging guidance. This locus often exhibits mosaicism in the repeat length, which has recently been shown to affect the severity of disease and also to be predictive of drug response (or lack thereof). These methods lack the sensitivity to detect clinically important levels of mosaicism. Interruptions of the CGG repeat motif by AGG sequences greatly reduce progression to full mutation status in offspring of premutation carriers, but the size-based methods used today don't convey any information about those features. Pacific Biosciences' Single Molecule, Real-Time (SMRT(R)) Sequencing has the ability to read through virtually any DNA sequence context (including trinucleotide repeats of CGG), produces reads averaging almost 5000 bases in length, and intrinsically produces information about the methylation status of the region important to research and clinical diagnosis. Application of SMRT sequencing to FMR1 has already been demonstrated for PCR amplicons, but to avoid the spurious repeat lengths produced by PCR, means of targeting the locus without the use of PCR are required for translation of this method to the clinic. Therefore the Phase I aims of this program are: i) to demonstrate at least 100 reads from unamplified DNA from < 20 ug of gDNA taken from blood, ii) show that the entire clinical range of mutations can thus be analyzed, iii) show the ability to detect minor alleles in mosaic individuals and iv) show that the methylation status of these alleles can be determined using the kinetic information SMRT sequencing yields. The goal of Phase II is to optimize the assay, develop the required informatics tools, prove the assay in the context of a 300-person concordance study and then transfer the technology to the UC Davis CLIA lab for development into a clinical diagnostic test which will be made available to clinicians at the start of Phase III.