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

FMR 1-SLS：使用无扩增单基因座 ta 改善脆性 X 诊断

基本信息

批准号：
8591962
负责人：
STEPHEN WHITFIELD TURNER
金额：
$ 14.92万
依托单位：
PACIFIC BIOSCIENCES OF CALIFORNIA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-16 至 2014-07-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8591962
关键词：
Adult Affect Algorithms Alleles American Autistic Disorder Behavioral Biological Assay Blinded Blood CGG repeat CGG repeat expansion Capillary Electrophoresis Categories Child Clinic Clinical Clinical Research Cognitive Collaborations Complex Coupled DNA DNA Sequence Development Diagnosis Diagnostic Diagnostic tests Disease Exhibits FMR1 Gene FXTAS Family Fragile X Mental Retardation Protein Fragile X Premutation Fragile X Syndrome Gait Ataxia Gel Gene Mutation Genes Genetic Genetic Risk Genomics Genotype Goals Gold Guanine + Cytosine Composition Impaired cognition Impairment Individual Informatics Inherited Intellectual functioning disability Interruption Kinetics Length Letters Measurable Methodology Methods Methylation Minor Mosaicism Mothers Mutation Nerve Degeneration Outcome Ovarian Patients Persons Pharmaceutical Preparations Phase Premature Menopause Procedures Protocols documentation Reading Recommendation Recording of previous events Reporting Research Sampling Scientist Sequence Analysis Severity of illness Southern Blotting Symptoms Technology Technology Transfer Testing Time Translations Trinucleotide Repeat Expansion Trinucleotide Repeats Turner&apos s Syndrome United States Ursidae Family Validation Woman base clinical Diagnosis improved interest medical schools men nervous system disorder neurodevelopment novel novel diagnostics offspring ovarian failure programs protein expression public health relevance response single molecule tool transmission process

项目摘要

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.

描述（由申请人提供）：FMR1 基因中三核苷酸重复 CGG 的扩展导致 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、卵巢功能衰竭和进行性神经系统疾病。超过 150 万美国人拥有扩大的重复区域，其中 1000 万人的症状或家族史表明需要接受扩大检测。由于感兴趣区域的 100% GC 含量以及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。如今，人们使用DNA片段大小测定方法，即Southern印迹法或PCR，然后进行毛细管电泳，将个体分类为具有正常、前突变或完全突变等位基因。在《脆弱X》中，前突变类别代表了一种独特的基因型，具有自己的建议和临床结果，因此，这些方法中固有的片段大小不准确导致的错误分类不利于患者的治疗，导致不适当甚至破坏性的指导。该基因座经常在重复长度中表现出嵌合现象，最近已被证明会影响疾病的严重程度，并且还可以预测药物反应（或缺乏药物反应）。这些方法缺乏检测临床重要嵌合水平的灵敏度。 AGG 序列对 CGG 重复基序的中断极大地减少了前突变携带者后代向完全突变状态的进展，但目前使用的基于大小的方法并不能传达有关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），生成平均长度近 5000 个碱基的读数，并本质上生成有关 DNA 序列的信息。该区域的甲基化状态对于研究和临床诊断很重要。 SMRT 测序在 FMR1 上的应用已在 PCR 扩增子中得到证实，但为了避免 PCR 产生的虚假重复长度，需要在不使用 PCR 的情况下靶向基因座的方法，以便将此方法转化为临床。因此，该计划的第一阶段目标是：i) 证明从血液中提取的 < 20 ug gDNA 中至少获得 100 个未扩增 DNA 的读数，ii) 表明可以分析整个临床范围的突变，iii) 表明有能力检测嵌合个体中的次要等位基因，并且 iv) 表明这些等位基因的甲基化状态可以使用 SMRT 测序产量的动力学信息来确定。第二阶段的目标是优化检测，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，然后将技术转移到加州大学戴维斯分校 CLIA 实验室，开发成临床诊断测试可供临床医生使用在第三阶段开始时。