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的扩展会导致FMR1蛋白表达的失调，并导致许多严重的疾病，从认知障碍，自闭症，卵巢衰竭和进行性神经神经系统疾病。超过150万的美国人港口扩大了重复区域，1000万个症状或家族史表明了要进行扩张的测试。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，因此先前的测序方法缺乏阅读此重复的能力。当今，使用DNA碎片大小的方法，无论是南部印迹还是PCR，然后是毛细管电泳，将个体归类为正常，预先征用或全突变等位基因。在脆弱的x中预先享有的类别代表了一种独特的基因型，其自己的建议和临床结果，因此这些方法固有的碎片尺寸不准确而引起的错误分类对患者的治疗有害，从而导致不适当甚至有害指导。该基因座经常在重复长度上表现出镶嵌性，最近已证明会影响疾病的严重程度，并且可以预测药物反应（或缺乏药物反应）。这些方法缺乏检测临床重要水平的镶嵌水平的敏感性。通过AGG序列对CGG重复基序的中断大大降低了预先载体后代的完整突变状态，但是当今使用的基于尺寸的方法并不能传达有关这些功能的任何信息。 Pacific Biosciences的单分子，实时（SMRT（R）测序）能够读取几乎所有DNA序列上下文（包括CGG的三核苷酸重复），可产生读取几乎5000个基础的长度，并且本质上产生有关研究和临床诊断的甲基化状态重要的信息。 SMRT测序在FMR1中的应用已经证明了PCR扩增子，但是为了避免PCR产生的虚假重复长度，不使用PCR靶向基因座的手段才能将此方法转换为诊所。因此，该程序的I阶段目标是：i）表明从血液中<20 ug的gdna <20 ug的dna中至少读取了100次读取，ii）表明，可以分析整个突变的临床范围，iiii iii）显示了能够的能力。检测镶嵌个体中的小等位基因，iv）表明可以使用动力学信息SMRT测序屈服确定这些等位基因的甲基化状态。第二阶段的目的是优化测定，开发所需的信息学工具，证明在300人的一致性研究的背景下证明该测定法，然后将技术转移到UC Davis Clia实验室中，以开发开发临床诊断测试在第三阶段开始时。