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

描述（由申请人提供）：FMR1基因中三核苷酸重复CGG的扩增导致FMR1蛋白表达失调，并导致一系列严重疾病，包括认知障碍、自闭症、卵巢衰竭和进行性神经系统疾病。超过150万美国人患有扩大的重复区域，1000万人有症状或家族史，需要接受扩大的检测。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读取该重复的能力。今天使用的DNA片段分级方法，要么是Southern Blotting，要么是PCR，然后是毛细管电泳，将个体分类为具有正常、预突变或完全突变的等位基因。在《脆弱的X》中

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.